Fermention Technology

Fermentation Technologies

FERMENTATION TECHNOLOGIES

FERMENTATION TECHNOLOGIES MSC-BIOTECHNOLOGY

over view

Unit III

Fermentation technologies are the most prestigious bioanalytical technique in the study of biotechnology. It is very important for all the biotechnologist to understand the concept of fermentation.It paves way for quality research in industry. This subject is further linked to some of the other key streams of science like bioengineering, microbiology, biophysics, biochemistry, biotechnology, mathematics etc.

Kinetics of Fermentation This unit is linked to biochemical engineering branch of science The kinetics of various fermentation techniques will be discussed.

The students will learn the following salient aspects of the

Unit IV

subject

Products of Fermentation The entire unit deals with the protocols needed to produce

Unit I

various commercial products like Ethanol, antibiotics, enzyme, beverages and various fermented food products

Introduction to Fermentation Technolgy In this unit the student will learn the historical perspectives of fermentation. This unit can be linked to the basic media

Unit V

preparation experiments in micrbiology.Preparation of media

Biosafety & Future of Fermentation Technology

for fermentation will be taught.Various techniques related to strain improvement and assay procedures will be discussed.All the necessary pre requisites for fermentation will be detailed.

The knowledge of biohazards in fermentation is a pre requisite before handling fermentor based experiments. Students need to update themselves on the current trends and future prospects in fermentation Therefore the main objective of this unit

Unit II

is to increase the awareness of students in current areas of research in fermentation.

Structure and Working of a Fermentor In this unit the student will learn all about fermentor design, about bioreactors and its instrumentation. Principles of sterilization, aeration, agitation, mass and heat transfers will also be discussed. Techniques related to down stream processing and product recovery will be explained.

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FERMENTATION TECHNOLOGIES

SYLLABUS

Unit I

•

To review the history of fermentation process and to understand all the necessary infrastructure for fermentation

Introduction to Fermentation Technolgy: Brief history of fermentation process, Fermentation Media, Screening, Scale up and scale down, Inoculum preparation, Assay techniques, Strain improvement techniques.

techniques

•

Study fermentor design and downstream processing

•

Interpreting the kinetics of fermentation

•

Understand the protocols needed to produce various fermented products

Unit II •

Structure and Working of a Fermentor

Understand the biohazards in fermentation. Updating with current and future trends in fermentation technology

Design of a fermentor, Design of a bioreactor, Sterilization, Aeration and agitation, Mass & Heat transfer, Instrumentation and control, Product recovery and downstream processing

Outcomes and Assessment Criteria Outcomes

Unit III

Assessment criteria To achieve each outcome a student must demonstrate the ability to :

Kinetics of Fermentation:

1 Review the historical developments in fermentation technologies

-

Understand the history of fermentation

Growth Kinetics in fermentation, Kinetics of fed batch

-

Enumerate the steps involved in the preparation of media for fermentation

fermentation, Kinetics of continuous Fermentation.

-

Discuss different types improvement techniques techniques

-

describe the parts of a fermentor

-

Explain the principles of sterilization, Aeration, agitation, and mass- heat transfer

-

Discuss the crucial steps involved in recovery of desired products and downstream processing

-

To understand the effective use of computers in fermentation technologies

-

Discuss Growth kinetics

-

Interpret the kinetics of fed batch and continuous fermentation

2 Understand the intricate details of fermentor design

UNIT IV Products of Fermentation Microbial biomass production, enzyme, Antibiotic and steroid

of and

strain assay

fermentations, Food & Beverage fermentation, Ethanol production from conventional and non-conventional substrates, Industrial wastewater treatment, Bioenergy production.

UNIT V

3 Undertake the study of fermentation kinetics

4 To understand all the steps involved in the production of various useful fermented products by fermentation technologies

- Describe the steps involved in the production of Microbial biomass ,enzyme, Antibiotic and steroid -Understand Food & Beverage fermentation -

Assess ethanol conventional and substrates

Biohazards in fermentation, Containment in fermentation and

-

Analyze industrial waste water treatment

downstream processing, Patent and secret processes, Fermenta-

-

Discuss bioenergy production.

-

Discuss the biohazards and safety measures observed in fermentation industry

-

Study the recent and future prospects in fermentation industry

Biosafety & Future of Fermentation Technology

tion economics, Future of fermentation technology. Students must ensure to achieve the following learning

5 Review various biohazards involved in fermentaion.Study the future prospects in fermentation technologies

outcomes during the semester:

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production from non conventional

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FERMENTATION TECHNOLOGIES

FERMENTATION TECHNOLOGIES

BERC

CONTENT Unit No.

Lesson No.

Unit 1

Page No.

Lesson Plan

v

Course Requirement

ix

Introduction to Biosensors Lesson 1

Introduction to fermentation process

1

Lesson 2

Fermentation Media

6

Lesson 3

Screening

13

Lesson 4

Scale up and scale down

23

Lesson 5

Inoculum preparation

27

Lesson 6

Assay technique

31

Lesson 7

Strain Improvement

40

Unit 2

Structure and Working of a Fermentor

Lesson 8

Design of a fermentor

49

Lesson 9

Design of a bioreactor

56

Lesson 10

Sterilization

63

Lesson 11

Aeration and agitation

71

Lesson 12

Mass and heat transfer

75

Lesson 13

Instrumentation & Control I

86

Lesson 14

Instrumentation & Control II

91

Lesson 15

Product recovery & downstream processing

95

Lesson 16

Use of computers in fermentation

102

Unit 3

Kinetics of Fermentation

Lesson 17

Growth kinetics in fermentation

108

Lesson 18

Fed batch fermentation

112

Lesson 19

Continuos fermentation

119

Unit 4

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Topic

Products of Fermentation

Lesson 20

Microbial biomass production

122

Lesson 21

Microbial enzyme production

127

Lesson 22

Microbial enzyme production

136

Lesson 23

Beer and wine fermentataion

146

Lesson 24

Ethanol fermentataion

154

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FERMENTATION TECHNOLOGIES

BERC

FERMENTATION TECHNOLOGIES

Lesson 25

Microbial production of health care products I

159

Lesson 26

Microbial production of health care products II

164

Lesson 27

Industrial wastewater treatment

168

Lesson 28

Bioenergy

175

Unit 5

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Biosafety and Future of Fermentation Technology

Lesson 29

Biohazards in fermentation

180

Lesson 30

Containment in fermentation

184

Lesson 31

Containment in downstream processing

188

Lesson 32

Patent and secrete processes

194

Lesson 33

Fermentation economics

200

Lesson 34

Future of Industrial fermentations

206

Glossary

214

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FERMENTATION TECHNOLOGIES

REQUIREMENT

Class Participation •

The student should come forward to interact with his

•

Contribution to the syndicate to prove team spirit

•

Contribution of syndicates will be assessed individually and

classmates in order to share his thoughts.

•

group wise

To understand the subject better it is very important that there is a healthy classroom discussions.

•

•

Performance of syndicate members will be judged 3-4 times during the semester.

•

This is a great opportunity for introvert students to get rid of their fear to face the audience.

The weightage of evaluation methods are

1. Class room assessment – 30 % 2. Assignment and projects - 20 %

Expectations from Students

3. End sem exam -50 %

•

Regularity is the key to make continuous evaluation a success

•

Students are expected to read the lesson plan regularly so that they can participate well in classroom discussions.

•

Students should come prepared for the class discussion

•

Extra reference and awareness of latest trends is required

•

Students are expected to discuss any relevant doubts openly during the interaction session.

•

Performance in end semester examination

Class room participation will help the students to improve their communication skills

•

•

Syndicate

•

Syndicate is a group of students who will take up the given task and work as a team.

•

The whole class will be divided into syndicates for evaluation.

•

Syndicate member will submit the assignment individually

The current areas of research related to the topic chosen for

Assignments

discussion should be given more emphasis

•

After completion of each learning outcome (or maximum two), different assignments will be given for each syndicate

Need of their preparation

•

For healthy and interactive discussion

Syndicate solution

•

Exchange of thoughts and ideas and to analyze the topic

•

The students of a particular syndicate should discuss the assignment as a group and arrive at a common solution

from various perspectives

•

To enrich their knowledge

Individual Submission

•

To make them realize, how the preparation helps them to

•

participate in group discussion

However each student should submit assignments individually.

•

Method of evaluation

In the assignment they can put their views separately in addition to the combined solution of the team.

•

Attendance: 75% attendance is compulsory

•

Expression of ideas without any fear or inhibition

•

Participation in group discussions, presentations and

•

The student will have an added advantage if he has provided the latest information from books, journals and web site.

projects. Presentation and quality of assignments.

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FERMENTATION TECHNOLOGIES

•

The student is given a chance to assess himself (self

Fermentation Kinetics

assessment) and also will be evaluated by the peers.

www.np.edu.sg/~dept-bio/biochemical_ engineering/lectures bioferm1_main.htm

Group Presentation

•

Syndicates will have to present their work before the class.

•

The instructor as well as the peers will evaluate presentation.

•

The final assessment will be done considering both the

www.np.edu.sg/~dept-bio/questions/ fermentation_1 ferm1_mcq.html www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=106562 www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=380741

evaluations.

www.asbcnet.org/Journal/ abstracts/search/1995

Group Project

bc1995a15.htm

•

Each syndicate will be given a particular project.

•

Different projects will be assigned to different syndicates.

Fermentor

•

After completion of the project, the syndicates are expected

www.electrolab.biz

to give a presentation on that project.

www.labkorea.com/products/fermenter/fermenter.html www.bakker.org/cfm/webdoc12.htm

End Semester Examination

• •

www.thefreedictionary.com/Fermenter

All the students have to appear in the end semester examination.

www.mrc-dunn.cam.ac.uk/facilities/fermenter.html

At the end of the semester, the cumulative percentage will be

Bioreactors

calculated from the continuous evaluation performance, assignments, projects and end semester examination performance.

www.labx.com www.electrolab.biz www.frtr.gov/matrix2/section4/4-42.html

Useful References

www.bellcoglass.com/us/bioreactors.shtml

1. Bailey and Ollis : Biochemical Engineering Fundamentals

www.nbsc.com/ferm_eq/ferm.htm

2. Casida L.E.: Industrial Microbiology 3. Hambelton,P., Melling J.&Salusbury T.T.: Biosefty in

Fermented Food Products www.aomori-tech.go.jp/hiro/en/k_hakko.html

Industrial Biotechnology

www.healingcrow.com/ferfun/ferfun.html

4. Jogdand S.N. : Enviromental Biotechnology 5. Michael J. Waites, Neil L. Morgan, John S. Rockey & Gary Higton(2001): Industrial Microbiology an Intoduction.

www.cplpress.com/contents/C1315.htm store.blackwell-professional.com/0813800188.html

6. Prescott & Dun’s : Industrial Microbiology

www.fbfc.com/scoop/feb01/lacto.html

7. Stanbury P.F. , Whitaker A.,& Hall S.J.: Principal of

Beer and wine fermentation

fermentation technology (Second Edition)

www.homebrewit.com/aisle/1190

Useful Links for Study

www.sp.uconn.edu/~ns166vc/Notes/Beer.html

seafood.ucdavis.edu/iufost/lee.htm

www.beveragebusiness.com/art98/bryson0203.html

seafood.ucdavis.edu/iufost/creative.htm

www.leeners.com/ferment.html

www.metkinen.fi/

www.homecraft.on.ca/instructions.htm

www.cplpress.com/contents/C1315.htm www.fz-juelich.de/ibt/ferm/ferm.html

Industrial microbiology www.microbes.info/resources/Industrial_Microbiology/ www.cas.muohio.edu/~stevenjr/ mbi630

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Future of Industrial Microbiology

www.slic2.wsu.edu:82/hurlbert/ micro101/pages

www.life.umd.edu/classroom/ bsci424/BSCI223WebSiteFiles

Chap19.html

Chapter28.htm

www.brockportmicrobiology.com/

dwb.unl.edu/Teacher/NSF/C11/C11Links/ ww.gch.ulaval.ca

www.sc.mahidol.ac.th/scbt/ academics/research_areas/IM.htm

7Eagarnier/hur_c20.htm

bioresearch.ac.uk/browse/mesh/C0021262L0021262.html

highered.mcgraw-hill.com/sites/0072320419/ student_view0

www.transgalactic.com/publications/

chapter1/study_outline.html

p_industrial_microbiology.htm

www.biocareers.org.uk/IT3cl.htm

methanogens.pdx.edu/boone/ courses/BI480/Lectures

Antibiotic Fermentation

BI480Lec15.html

www.fda.gov/ohrms/dockets/ac/ 02/briefing

Enzyme Technology

3841B1_05_PFIZER/sld033.htm

www.lsbu.ac.uk/biology/enztech/

www.fda.gov/ohrms/dockets/ac/ 02/briefing

www.irl.cri.nz/get/biocat/

3841B1_05_PFIZER/tsld033.htm

www.aetltd.com/

books.cambridge.org/0521304903.htm

www.biores-irl.ie/biozone/enzymes.html enzymes.novo.dk/enzymes/enzyme-technology.html

Cell growth in Fermentation www.spectroscopymag.com/spectroscopy/ article articleDetail.jsp?id=86260 www.np.edu.sg/~dept-bio/questions/ fermentation_1 ferm1_mcq.html www.blackwell-synergy.com/links/ doi/10.1111/j.1365 2672.2004.02331.x/abs/

Bioenergy Production oasys2.confex.com/acs/228nm/techprogram/S12991.HTM www.ieabioenergy.com/events/Brazil2002/bg.php www.ieabioenergy.com/events/Brazil2002/ www.envirofacs.org/bioenergy.pdf www.fsa.usda.gov/pas/publications/ facts Bioenergy03QA.pdf

Biohazards in Fermentation www.ipu.ac.in/BTBA115.htm www.cerl-fsi.com/biohaz.htm www.ehs.ucsf.edu/Manuals/BSM/BSMEntireDoc.htm

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FERMENTATION TECHNOLOGIES

industrialmicro630.html

LESSON 1: AN INTRODUCTION TO FERMENTATION PROCESS

In this lecture, you will learn

• • •

What is fermentation? What are its types? Products of fermentations Use of recombinant technology in fermentations

What is Fermentation? Fermentation has always been an important part of our lives: foods can be spoiled by microbial fermentations, foods can be made by microbial fermentations, and muscle cells use fermentation to provide us with quick responses. Fermentation could be called the staff of life because it gives us the basic food, bread. But how fermentation actually works was not understood until the work of Louis Pasteur in the latter part of the nineteenth century and the research which followed. Fermentation is the process that produces alcoholic beverages or acidic dairy products. For a cell, fermentation is a way of getting energy without using oxygen. In general, fermentation involves the breaking down of complex organic substances into simpler ones. The microbial or animal cell obtains energy through glycolysis, splitting a sugar molecule and removing electrons from the molecule. The electrons are then passed to an organic molecule such as pyruvic acid. This results in the formation of a waste product that is excreted from the cell. Waste products formed in this way include ethyl alcohol, butyl alcohol, lactic acid, and acetone-the substances vital to our utilization of fermentation

What is The Role of Fermentation in Industry? In industry, as well as other areas, the uses of fermentation progressed rapidly after Pasteur’s discoveries. Between 1900 and 1930, ethyl alcohol and butyl alcohol were the most important industrial fermentations in the world. But by the 1960s, chemical synthesis of alcohols and other solvents were less expensive and interest in fermentations diminished. Questions can be raised about chemical synthesis, however. Chemical manufacture of organic molecules such as alcohols and acetone rely on starting materials made from petroleum. Petroleum is a nonrenewable resource; dependence on such resources could be considered short-sighted. Additionally, the use of petroleum has associated environmental and political problems. The worldwide interest in microbial fermentations is once again growing especially with reference to renewable resources and microbial biocatalysts. Plant starch, cellulose from agricultural waste, and whey from cheese manufacture are abundant and renewable sources of fermentable carbohydrates. Additionally these materials, not utilized, represent solid waste that must be buried in dumps or treated with waste water. What Other Benefits Microbial Fermentations Offer? Microbial fermentations have several other benefits. For one, they don’t use toxic reagents or require the addition of 2.521

intermediate reagents. Microbiologists are now looking for naturally occurring microbes that produce desired chemicals. In addition, they are now capable of engineering microbes to enhance production of these chemicals. In recent years, microbial fermentations have been revolutionized by the application of genetically-engineered organisms. Many fermentations use bacteria but a growing number involve culturing mammalian cells. Some examples of products currently produced by fermentation are listed in Tables 1 and 2 . Products Produced by Fermentation Table 1.1 Fermentations by Naturally-Occurring Organisms

PRODUCT

APPLICATION

ORGANISM

Bacitracin Chloramphenicol Citric acid Erythromycin Invertase Lactase Neomycin Pectinase Penicillin Riboflavin Streptomycin Subtilisins Tetracycline

Antiobiotic Antiobiotic Food flavoring Antibiotic Candy Digestive aid Antibiotic Fruit juice Antibiotic Vitamin Antibiotic Laundry detergent Antibiotic

Bacillus subtilis (bacterium) Streptomyces venezuelae (bacterium) Aspergillus niger (fungus) Streptomyces erythaeus (bacterium) Saccharomyces cerevisiae (fungi) Escherichia coli (bacterium) Streptomyces fradiae (bacterium) Aspergillus niger (fungus) Penicillium notatum (fungus) Ashbya gossypii (fungus) Streptomyces griseus (bacterium) Bacillus subtilis (bacterium) Streptomyces aureofaciens (bacterium)

Table 1. 2 Fermentations by Genetically Engineered Organisms PRODUCT B. growth hormone Cellulase H. growth hormone Human insulin Monoclonal antibodies Ice-minus Sno-max t-PA Tumor necrosis factor

APPLICATION Milk production(cows) Cellulose Growth deficiencies Diabetics Therapeutics Prevents ice on plants Makes snow Blood clots Dissolves tumor cells

ORGANISM Escherichia coli (E. coli) E. coli E.coli E. coli Mammalian cell culture Pseudomonas syringae Pseudomonas syringae Mammalian cell culture E.coli

How Does Fermentation Work in Biotechnology? In the pharmaceutical and biotechnology industries, fermentation is any large-scale cultivation of microbes or other single cells, occurring with or without air. In the teaching lab or at the research bench, fermentation is often demonstrated in a test tube, flask, or bottle-in volumes from a few milliliters to two liters. At the production and manufacturing level, large vessels called fermentors or bioreactors are used. A bioreactor may hold several liters to several thousand liters. Bioreactors are

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FERMENTATION TECHNOLOGIES

Learning Objectives

UNIT-1 AN INTRODUCTION TO FERMENTATION TECHNOLOGY

FERMENTATION TECHNOLOGIES

equipped with aeration devices as well as nutrients, stirrers, and pH and temperature controls.

First we will see the microbial biomass as the fermentation product. The commercial production of microbial biomass may be divided into two major processes: the production of yeast to be used in the baking industry and the production of microbial cells to be used as human or animal food (single-cell protein). Bakers’ yeast has been produced on a large scale since the early 1900s and yeast was produced as human food in Germany during the First World War. However, it was not until the 1960s that the production of microbial biomass as a source of food protein was explored to any great depth. As a result of this work, a few large-Scale continuous processes for animal feed production were established in the 1970s. These processes were based on hydrocarbon feedstocks which could not compete against other high protein animal feeds, resulting in their closure in the late 1980s (Sharp, 1989). However, the demise of the animal feed biomass fermentations was balanced by ICI pic and Rank Hovis McDougal establishing a process for the production of fungal biomass for human food. This process was based on a more stable economic platform and appears to have a promising future.

This topic has been discussed in more details in one of the subsequent lessons.

At Genentech, Inc., for example, in order to get a product from fermentation, fermentation scientists develop media and test growth conditions. Then, a scale-up must be done to reproduce the process at a large volume. During production, technicians monitor temperature, pH, and growth in the bioreactors to ensure that conditions are optimum for cell growth and product. Bioreactors are used to make products such as insulin and human growth hormone from genetically engineered microorganisms as well as products from naturally-occurring cells, such as the food additive xanthan. The products being developed by the biotechnology industry have enormous implications for our future health and wellbeing. All of the exciting discoveries in current biotechnical research and its applications will, of course, have repercussions within human history. Science and politics have always interacted, in both direct and indirect ways.

What are the Various Products Manufactured using Fermentations? There are five major groups of commercially important fermentations: (i) Those that produce microbial cells (or bio mass) as the product. (ii) Those that produce microbial enzymes (iii) Those that produce microbial metabolites. (iv) Those that produce recombinant products. (v) Those that modify a compound which is added to the fermentation - the biotransformation process.

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Exercis Study the different types of fermentations where single cell proteins and mycoproteins are produced. Then write briefly what are the limitations of using microorganisms as human food. Use the space provided to express your views. Exercise Find out what do you mean by the term ‘probiotics’. Briefly mention what benefits probiotics can offer in human beings, animals and birds. Write your views below. Let us now see how microbial enzymes can be produced by fermentations. Microbial enzymes are most widely used in the food and beverage industries and to less extent in clinical and analytical laboratories and as protease detergents in washing powders. The most economical and convenient method of producing these enzymes is by microbial fermentations. Bacillus stearothermophilus produces amylases as secondary metabolites, but most other microbes produce enzymes as primary metabolites, during exponential growth. Table 1.3 Some commercially valuable microbial secondary compounds Secondary Metabolite Actinomycin Cephalosporin Penicillin Streptomycin Cyclosporin Bestatin Gibberellin

Commercial significance/application Antitumour Antibiotic Antibiotic Antibiotic Immunosuppressant Cancer treatment Plant growth regulator

Most of the enzymes in industrial use are extracellular proteins produced by Aspergillus sp. or Bacillus sp. and include alphaamylase, beta-glucanase, cellulase, dextranase, lactase, lipase,

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Table 1.4 Some commercially important microbial primary metabolites

There are other enzymes required for non-industrial uses. These are intracellular and are produced in much smaller amounts. Some examples are- catalase, asparaginase, cholesterol oxidase, beta-galactosidase, and glucose oxidase and glucose phosphate dehydrogenase. These enzymes have to be greatly purified to homogeneity. They can be produced in kilogram quantities from a few thousands of liters of culture suspension. Also, for isolating these enzymes, cells have to be lysed. One of the first examples of the industrial use of immobilized enzymes was to produce large volumes of high fructose corn syrup (HFCS; isoglucose). HFCS serves as a good substitute for invert sugar (glucose + fructose) and is prepared from pure glucose syrups with a dextran content of over 95%. Its most important uses are the soft drinks and canning industry where saccharose can be totally replaced. Other important markets are the dairy products, confectionary and baked foods. Enzyme production is closely controlled in micro-organisms and in order to improve productivity these controls may have to be exploited or modified. Such control systems as induction may be exploited by including inducers in the medium, whereas repression control may be removed by mutation and recombination techniques. Also, the number of gene copies coding for the enzyme may be increased by recombinant DNA techniques.

Exercise Find out about the following: a) Microbial enzymes used for detergent applications b) Microbial enzymes used for treatment of animal feed (clue: phytase) c) Significance of invertase in confectionary industry and in sugar industry ( clue: Microbially Induced Sugar Inversion ) Briefly write your answers in the space provided: All right. Now about the microbial metabolites which are produced by fermentations. First let us see what metabolites are. During exponential growth, microbial cultures produce such essential metabolites as amino acids, nucleotides, proteins, lipids, and carbohydrates. They also produce certain by-products of energy-yielding metabolism such as ethanol, butanol and acetone. Both these’ categories of metabolites are referred to as the primary metabolities. Some examples of commercially important primary metabolites are listed in Table 1.4

Primary Metabolite Ethanol

Producing microbes Saccharomyces cerevisiae

Commercial application Production of alcoholic beverages

Acetone, butanol Lysine

Clostridium acetobutyricum Corynebacteriu m glutamicum Corynebacteriu m glutamicum Xanthomonas spp.

Solvent

Glutamic acid Polysaccharide

Feed supplement Flavour enhancement Food industry, enhance oil recovery

Ok. Now tell me about the microbial products where recombinant technology is used. The advent of recombinant DNA technology has extended the range of potential fermentation products. Genes from higher organisms may be introduced into microbial cells such that the recipients are capable of synthesizing ‘foreign’ proteins. These are called heterologous proteins. A wide range of microbial cells have been used as hosts for such systems including E.coli, Saccharomyces cerevisiae and filamentous fungi. Products produced by such genetically engineered organisms include interferon, insulin, human serum albumin, factor VIII and somatostatin. Important factors in the design of these processes include the secretion of the product, minimization of the degradation of the product and control of the onset of synthesis during the fermentation, as well as maximizing the expression of the foreign gene.

•

Sometimes microbial systems can be effectively used not for the production of any particular product, but for the transformation of a compound into a structurally similar, higher-value compound. The conversion of ethanol into acetic acid (vinegar) is the oldest such process; more modern processes involve the production of much more highly valuable substances than vinegar. Various kinds of chemical reactions can be catalyzed to convert a cheaper compound into an expensive product. In many instances the cells or isolated enzymes may be immobilized to improve the efficiency of the reaction. Fermentations are generally carried out in huge fermentors. That means all the fermentations are in liquid phase, isn’t it? Not necessarily. Whilst most industrial processes are indeed carried out in liquid media, there are some, and important ones, which employ a solid medium. These are the solid state fermentations.

•

The growth and metabolism of microorganisms on moist solid substrates lacking free water is called solid state fermentation (SSF). They differ in this respect from submerged fermentations where free water is present.

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FERMENTATION TECHNOLOGIES

pectinase, proteases and others. The extent of purification required for most of these enzymes is minimal, and they can be produced in tons without serious problems.

FERMENTATION TECHNOLOGIES

Historically, SSF processes have been more popular in Oriental, Asian and African countries whereas submerged fermentations were popular in Europe. The presence of some moisture (about 15%) is necessary for SSP to occur but there should be no free water. Though many microbes can grow on solid substrates, only filamentous fungi can grow to a significant extent in the absence of free water. These fungi can even penetrate within the solid substrate. Bacteria and yeasts grow on solid substrates having moisture levels ranging between about 30-70% (such as compost). Single-celled organisms usually require free water. Thermophilic bacteria grow mainly in the first stage of composting. Lactic acid bacteria grow in ensiling processes. Yeast grows on solid substrates symbiotically with other microbes in composting, ensiling, and some industrial SSF processes. . SSF has two kinds of applications: (1) Socio-economic and (2) profit-economic. Some examples of the former category include composting of waste and municipal refuse, ensiling of grasses and lignocellulosic materials, and upgrading the quality of food. These processes are fairly simple, not requiring aseptic techniques, and are mediated by naturally-occurring microbial flora of the substrate. The examples of profit-economic category are the production of enzymes, organic acids, mould-ripened cheeses, edible mushrooms and fermented foods. All these products generate profits.

•

Can you give some example where solid state fermentations are used?

Some notable applications of SSF in industry are stated below: 1. Fermented Foods: Thousands of kinds of fermented foods are being produced industrially in Japan, Korea, China and other Oriental countries. Miso, shoyu, ontjam, khimchi, beer, tempeh, and fermented fish and meat are good examples, Fermentation often makes the food more nutritious, more digestible, safer, or having better flavour. It also tends to preserve the food, increasing its shelf life and lowering the need for refrigeration. Fermentation can be applied to all kinds of foods. Eight classes of fermented food may be recognized, viz., beverages, cereal products, dairy products, fish products, fruit and vegetable products, legumes, meat products, and starchy products. Of these, dairy products, cereal products, and beverages are the most common. Beer is produced from cereal grains which have been malted, dried and ground into fine powder. The powder is washed in warm water. Fermentation of the washed powder is mediated either by bottom yeast (e.g., Saccharomyces uvarom) or by a top-yeast (S. cerevisiae). The final product (beer) has up to about 8% alcohol. Grapes can be directly fermented by yeasts to wine. 2. Cereals Products: Three popular cereals are wheat, rice and maize. Bread is the commonest type of fermented cereal product. Wheat dough is fermented by S. cerevisiae along with some lactic acid bacteria. Idli, dosa, vada, dhokla and papadam are some common Indian examples of fermented cereal foods. These use mixtures of wheat and legume

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flours, which are fermented by Streptococcus, Pediococcus, and Leuconostoc. 3. Dairy Products: Cheeses constitute one of the largest groups of fermented dairy products, besides yogurt. Cheese is formed when the casein in milk is coagulated as the pH drops to 4.5. This happens when acid is produced by the lactic acid bacteria which convert the milk lactose to lactic acid. Streptococcus lactis is the principal microorganism involved. Yogurt is made from milk by lactic acid bacteria, a mixture of Lactobacillus bulgaricus and Streptococcus thermophilus. 4. Fruits and vegetables Products: Fermentation of fruits and pickles is usually carried out alongwith the addition of salt and acid for their preservation. In these products, saltresistant lactic acid bacteria initially of Leuconostoc species and Lactobacillus brevis, soon to be replaced by Lactobacillus plantarum and Pediococcus spp. Some coliform bacteria (Escherichia coli), Enterobacter spp. and Klebsiella spp are also involved. With the release of acids and drop in pH, yeastsbecome prominent. 5. Enzymes: Large quantities of the enzyme Koji are produced in Asian countries. Koji consists of moulded solid substrate for Use in food fermentations. Koji contains mixtures of different enzymes such as alpha-amylases, proteases, maltase, sucrase, lipase, phosphatase deaminase, and cellulase. Different proportions of the various enzymes yield specific types of Koji for specific foods. 6. Organic Acids: Citric acid is being industrially produced by SSF. Itaconic acid and gallic acid can also be so produced. For citric acid, Aspergillus niger is grown on moistened wheat or rice bran at pH 4.0-5.0 at 28C for about a week. Citric acid from the fermented solids is then leached using hot water and the extract so obtained is subjected to further downstream processing. 7. Mushrooms: The quality and flavour of mushrooms produced by SSF are better than those by submerged fermentations. Composting of the substrates, spawn preparation, and mushroom cultivation all involve SSF. Two mushrooms being widely produced by SSF are: the Button mushroom- Agaricus bisporus, and Shiitake Lentinus edodes. In addition, some other genera of edible fungi cultivated in various parts of the world include Volvariella, Pleurotus and Tremella. The compost for mushroom cultivation is traditionally prepared from mixtures of wheat straw and horse manure. Phase I of composting involves the wetting and thorough mixing of the materials in long stacks (about 2 meters in cross section) on a concrete yard. The stacks are shaken and re-stacked at 2-3 day intervals over about 2 weeks. The temperature in the stacks can rise to 80°C by microbial oxidation of organic material. Microbial activity enriches the nitrogen content of the substrate, and a complex called nitrogen-rich lignin-humus complex which is rich in nutrients, is formed. At the start of the composting phase, such fungi as Absidia cylindrospora, Mucor hiemalis, M. mucedo, Thamnidium elegans and Zygorhynchus moelleri are common and active.

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They are soon outcompeted by Aspergillus nidulans and some yeasts. Later, thermophilic species of Rhizomucor. Hunicola and Chaetomium become dominant. During early phase, several mesophilic bacteria, e.g., Flavobacterium, Pseudomonas and Serratia are also present. With the rise in compost temperature, these tend to be replaced by thermophilic, spore-forming bacteria. After 1-2 weeks, Phase-II (pasteurization) commences. During this, the pests and diseases of mushrooms are eliminated /minimized by placing the Phase-I compost in trays or shelves in bulk pasteurization tunnels and exposed to heat aerobically. During this second phase, thermophilic fungi lower the content of ammonia and improve the quality of the compost. Species of Aspergillus, Chaetomium, Thermophilus and other fungi play important role in PhaseII. Some actinomycetes are also important in this context. The compost is then inoculated with mushroom spawn which is a culture of mushroom mycelium on moist, autoclaved cereal grains. Rye and millets are the grains of choice for growing the mycelium. The colonized compost is then “capped” or “cased” to a depth of 3-5 cm with a layer of peat neutralized with limestone. Mushroom mycelium readily colonizes this casing layer. The casing layer induces fruit formation in large numbers. The fruits (sporophores) are finally harvested. 8. Cheeses: Throughout the world, cheeses are ripened by fungi through SSF to impart distinct flavours. Soft cheese (Camembert) is formed by growing Penicillium camembertii on the surface of curd cake. When P. roquefortii grows through the body of raw, processed curd, marbled cheeses such as green and blue-veined varieties are formed. 9. Fodder Preservation: Ensiling of straw and fodder plants involves the growth of naturally occurring lactic acid producing bacteria on grasses or straw. Lingnocellulosic materials are upgraded and preserved for the utilization at times when fresh fodder is scarce. 10.Insecticides: Diverse microbial insecticides and pesticides are produced commercially by SSF. Two good examples are Biotrol XK and Metauino. 11. Biodegradation and pollution control: Undesirable, pollution materials can be destroyed by SSF. Sludge farming involves soil solid substrate on which organic biotransformations are affected either by promoting indigenous microbes or by supplying specific culture.

Notes

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LESSON 2: FERMENTATION MEDIA-SOME PRIMARY CONSIDERATIONS Learning Objectives In this lecture, you will learn

• • •

Nutritional requirements Media requirements – major and minor

Media composition Having learned about what fermentations are, let us now move towards the fermentation media. What, in the first place, are the fermentation media? Fermentation media is a blend of natural and synthetic substrate specifically designed to promote the growth and production of the fermentation product. Thus, a fermentation medium must support the growth of the desired organism AS WELL AS the production of the fermentation product. This is especially important because many times the production of the desired metabolite is not directly linked with the growth of the organism. Ok, so first the growth of the organisms. What are the basic requirements for the growth of any living organism? We all need a carefully balanced diet for normal growth and metabolism. Microbes and animal cells are no exceptions. For all heterotrophic organisms, the general nutritional requirements could be remembered using a simple formula:

•

C. HOPKINS, Canteen Manager Where, C stands for Carbon, H for Hydrogen, O for Oxygen, P for Phosphorous, K for Potassium, I for iron, N for Nitrogen, S for Sulfur and, Canteen Manager stands for Calcium and Magnesium! Thus these are the ten elements required for growth by any heterotrophic organisms. In addition to this, many fastidious organisms require specific growth factors like vitamins for their growth and production of the desired metabolite. The fermentation medium therefore must contain ALL these ingredients IN THE RIGHT PROPORTION! So the fermentation medium must contain all above constituents. Will the composition of all fermentation media be the same then? No. The particular composition of a fermentation medium can be simple to complex depending on the particular microorganism and its fermentation. Autotrophic microorganisms require only the simplest of inorganic media. They require a few inorganic salts, water, and a nitrogen source; their carbon requirement is fulfilled by the carbon dioxide of the air or by carbonates. Thus, from simple inorganic nutrients, autotrophs are able to synthesize all of the complex organic compounds required to sustain life and to allow growth and multiplication of their cells, and they meet their total energy requirements by oxidation of some particular inorganic

•

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component of their medium. At the opposite end of the scale are the highly fastidious microorganisms, such as some of the lactic acid bacteria, which lack the ability to synthesize many of their sustenance and growth requirements. These organisms require the presence of many types of simple to complex preformed nutrients in the medium, and they must have an organic carbon supply to provide for synthesis of cell substance and release of metabolic energy. These are the two extremes and, obviously, microorganisms exist with requirements intermediate between these extremes. However, in addition to these considerations, fermentation growth conditions impose a metabolic stress on microorganisms, as for instance, the high aeration rates and high substrate levels commonly employed, so that additional nutrients and growth factors may be required as compared to the usual laboratory culture of the organisms. Thus, enzyme systems that normally are not limiting factors in metabolic sequences, because of a lack of sufficient levels of coenzymes or for other reasons, may become limiting under the stresses of fermentation growth: under these conditions more complex media are required than would normally be employed. What other parameters the fermentation medium should satisfy? In addition, the fermentation medium must satisfy several other conditions like

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1. Ability to produce the maximum yield of product or biomass per unit substrate used. 2. Ability to produce the maximum concentration of product or biomass. 3. Ability to promote the product formation at the maximum rate. 4. Ability to produce the minimum of undesired products. 5. To cause minimal problems during media preparation and sterilization 6. Retain consistent quality and readyavailability throughout the year. 7. To cause minimal problems in other aspects of the production process particularly aeration and agitation, extraction, purification and waste treatment. Plus, the designed fermentation medium must facilitate easy scale up from the laboratory to the pilot scale, and subsequently to the industrial scale. A medium with a high viscosity will also need a higher power input for effective stirring. This, in turn, affects the profitability of the fermentation process. In addition, several other factors like pH variation, foam formation, change in the oxidation-reduction potential and the morphological form of the organism must be taken into account before arriving at the composition of a fermentation medium. The ease with which the end product could be recovered and the

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The crystallized sugar is then separated from its mother liquor, and the mother liquor is further concentrated to allow recovery of additional crops of crystalline sugar. This procedure is repeated several times until crystallization inhibitors accumulate to such a concentration that further recovery of sucrose is not economical. At this point, the mother liquor still contains approximately 52 percent total sugars calculated as sucrose (30 percent sucrose, and 22 percent invert sugars) and is known as black-strap molasses. When this molasses is used as a fermentation medium component, it is considered to contain 50 percent fermentable sugars. Refinery blackstrap molasses is a similar product that differs from black-strap molasses only in that it is the residual mother liquor that has accumulated in the recrystallization refining of crude sucrose.

Finally, the fermentation medium has to be economically viable. Therefore, most fermentation media employ industrial and/ agricultural by products as their major ingredient. Some typical examples are cane molasses, beet molasses, sulfite waste liquor, cereal grains, corn steep liquor, soya bean meal, slaughter-house waste etc. While the incorporation of these ingredients considerably reduces the cost of production, in many cases this may not be possible because of one or more of the above mentioned reasons. This is especially true in case of fermentations where recombined organisms are used as they are likely to be nutritionally more demanding than their native counterparts.

•

Tell me more about molasses.Beet and cane molasses are by-products of the sugar industry. These molasses are the concentrated syrups or mother liquors recovered at any one of several steps in the sugar-refining process, and different names are applied to the molasses depending on the particular step from which it was recovered. Of these, blackstrap molasses prepared from sugar cane normally is the cheapest and the most used sugar source for industrial fermentations. In the commercial production of sugar, the juice from crushed sugar cane is concentrated to allow crystallization of its sucrose. In addition to sucrose, blackstrap molasses contains small amounts of complex polysaccharides and invert sugars. The presence of the invert sugars is attributed to the action of the “invertase” enzyme present in the original cane juice. Blackstrap molasses also contains various noncarbohydrate materials. Thus, dark colored, nitrogen-containing; polymeric substances result from “browning,” a reaction of the sugars with amino acids because of the heat and alkali used in processing. Inorganic ions are present in high concentrations and include most of the ions of the original cane juice which were concentrated till the mother liquor during sugar crystallization. Calcium also is present, being added during processing. Organic-acid constituents include aconitic, malic, citric, lactic, formic, acetic, and propionic acids. The nitrogencontaining compounds (other than the polymeric forms) are mainly amino acids and, particularly, aspartic and glutamic acids resulting from the deamidation of the asparagine and glutamine of the cane juice. A few heat and alkali stable vitamins are present, such. as myo-inositol. niacin, pantothenic acid, riboflavin, and small amounts of biotin. Also present are organic phosphorus compounds such as inositol hexaphosphate and its calcium-magnesium salt known as “phytin.” The overall compositions of the various molasses differ according to the specific geographic areas of production. This is particularly true for their contents of certain metal ions and, in fact, for certain fermentations, such as that for citric acid, the molasses is pretreated with cation exchange resins or potassium ferrocyanide before use so as to remove interfering cations.

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High-test or invert molasses contains approximately 70 to 75 percent sugar, and it is produced in a manner different from that previously described. The whole cane juice is partially inverted to prevent sugar crystallization; that is, the sugar is partially hydrolyzed to monosaccharides with heat and acid then neutralized and concentrated without the removal of any of the sugar. Thus, high-test or invert molasses contains much of the original sugar of the cane juice, although it has been partially hydrolyzed to D-glucose and D-fructose. It is preferred to blackstrap molasses because of the lower shipping charges on a sugar concentration basis and because of its lower levels of nonfermentable solids including salts and unfermentable sugars. In blackstrap molasses, the unfermentable sugars result from the action of heat on the sugars, particularly fructose, during the refining process. High-test molasses is produced only during years of sugar cane overproduction and, hence, its availability at any one time may be somewhat questionable.

•

And how are beet molasses different from cane molasses? Beet molasses are produced by procedures resembling those for sugar cane. However, beet molasses may be limiting in biotin for yeast growth so that Ii small amount of cane blackstrap or other source of biotin should be added for growth of these microorganisms. “Hydrol” is a molasses resulting from the manufacture of crystalline dextrose from corn starch. It contains approximately .60 percent sugar, but it also contains a relatively high salt concentration that must be considered if this molasses is to be used as a medium component.

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Any other industrial by product that can be used as a substrate for fermentation? Good that you asked. Corn steep liquor and Sulfite waste liquor are used for many industrial fermentations. Corn steep liquor is the water extract by-product resulting from the steeping of corn during the commercial production of corn starch, gluten, and other corn products. The used or spent steep waters are concentrated to approximately 50 percent solids, and this concentrate, known as corn steep liquor, is used in the commercial manufacture of feedstuffs and as a medium adjunct in the fermentation industry. It was first extensively employed in fermentation media for the

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treatment of effluent generated out of this process also needs careful consideration.

FERMENTATION TECHNOLOGIES

manufacture of penicillin. Of the 50 percent solids of corn steep liquor, approximately half is lactic acid. The rest includes amino acids, glucose and other reducing sugars, salts, vitamins, and precursors such as those for the penicillin molecule. Although corn steep liquor does contain this high lactic acid content, the acid is not necessarily utilized by microorganisms during growth in industrial fermentation processes. The high lactic acid content of corn steep liquor results from the growth of lactic acid bacteria and of mycoderma, which are film forming, asporogenous, yeast like fungi. Thus, the lactic acid is not a component of corn but results from a natural fermentation of the corn steep liquor. In other words, corn steep liquor in itself actually is a natural fermentation product and, as such, it can vary greatly in composition for lots from a single supplier, or between lots received from various suppliers. This variation in composition, at times, can lead to poor reproducibility of an industrial fermentation. Thus, if the corn steep liquor is supplying certain medium components (such as a particular amino acid, vitamin, or precursor) at low but critical levels, it may be necessary to determine the specific level of this compound as it is present in each lot of corn steep liquor is to be used.

•

Ok. Any other by product? Then there is sulfite waste liquor. Sulfite waste liquor is the spent sulfite liquor from the paper-pulping industry. It is the fluid remaining after wood for paper manufacture is digested to cellulose pulp with calcium bisulfite under heat and pressure and, as such, it presents a serious disposal problem for the paper-pulp manufacturers. Disposal in streams, as is the usual practice, causes stream pollution, and in several states legislation has been enacted against this method of disposal. Sulfite waste liquor can be employed as a dilute fermentation medium, being used in the production of ethanol by Saccharomyces cerevisiae and in the growth of Torula uti/is cells for feed. The economics of these fermentations dictate that the fermentation plant is located in close proximity to the pulping operation so that the cost of transporting the waste liquor is not a factor. The waste liquor contains 10 to 12 percent solids of which sugars make up about 20 percent. Thus, sulfite waste liquor is a dilute sugar solution containing approximately 2 percent sugar. These sugars include the hexosc5 D-glucose, D-galactose and Dmannose, and the pentoses D-xylose and L-arabinose. However, the relative amounts of these sugars present in sulfite waste liquor depend, to some extent, on the woods being digested, with soft woods being higher in hexoses and hardwoods higher in pentoses. This is important if yeast such as Saccharomyces cerevisiae is to be employed as the fermentation organism, since it uses only hexoses. Torula utilis, however, can ferment both hexoses and pentoses. In any event, regardless of which type of organism is being considered, the sugars of sulfite waste liquor cannot be fermented directly; the free sulfur dioxide or sulfurous acid of the waste liquor must first be removed by steam stripping or precipitation with lime.

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Wood-waste residues hydrolyzed by acid provide sugars similar to those of sulfite waste liquor. The hydrolyzed material is partially neutralized and filtered before use in a fermentation medium. Thus, wood wastes are a virtually untapped source for fermentation carbohydrate nutrients.

Formulation of a Fermentation Medium–What is our objective? Unless the end product of fermentation is the biomass itself, we are not interested in the growth of organisms during fermentation. We would ideally prefer to have the minimum of growth and the maximum of product synthesis. Unfortunately, this is not always possible. In a fermentation process, therefore, attempts are made to keep the cell growth at its bare minimum. How to formulate a fermentation medium? Medium formulation is an essential stage in the design of successful laboratory experiments, pilot-scale development and manufacturing processes. The constituents of a medium must satisfy the elemental requirements for cell biomass and metabolite production and there must be an adequate supply of energy for biosynthesis and cell maintenance. The first step to consider is an equation based on the stoichiometry for growth and product formation. Thus for an aerobic fermentation: Carbon + Oxygen + Nitrogen + Other requirements = biomass + products + CO2 + H2O + heat We should be able to express this equation in quantitative terms. i.e. it should be possible to calculate the minimal quantities of nutrients which will be needed to produce a specific amount of biomass and it should be possible to calculate substrate concentrations necessary to produce required product yields. But it is not always easy to quantify all the factors very precisely. • Then how do we know how much of which nutrient is needed? I will tell you that. First, different organisms will have different nutritional requirements. This is quite elementary. Secondly, since we want to keep the biomass to the minimum, we have to know the basic composition of the organisms being used so that the fermentation medium can be designed accordingly. Based on the general composition of commonly used cells in fermentation, we can formulate the fermentation medium. • Are there any other considerations to be made? Yes. We will see what are the various parameters to be considered with reference to individual constituents of the medium. Let us start from water, which is the commonest of all the ingredients in a fermentation medium. Clean water of consistent composition is therefore required in large quantities from reliable permanent sources. When assessing the suitability of a water supply it is important to consider pH, dissolved salts and effluent contamination. The mineral content of the water is very important in brewing, and most critical in the mashing process, and historically influenced the location of breweries and the types of beer produced. Hard waters containing high CaS04 concentrations are better for the English Burton bitter beers and Pilsen type

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•

What is the next nutrient that should be considered? After water let’s learn about the Carbon and Energy source. Most industrial micro-organisms are chemo-organotrophs; therefore the commonest source of energy will be the carbon source such as carbohydrates, lipids and proteins. Some micro-organisms can also use hydrocarbons or methanol as carbon and energy sources. The rate at which the carbon source is metabolized often influences the formation of biomass or production of primary or secondary metabolites. Fast growth due to high concentrations of rapidly metabolized sugars is often associated with low productivity of secondary metabolites. At one time the problem was overcome by using the less readily metabolized sugars such as lactose but many processes now use semi-continuous or continuous feed of glucose or sucrose. Alternatively, carbon catabolite regulation might be overcome by genetic modification of the producer organism.

•

If this is the range of carbohydrates available, how do we select the right substrate? What we want to produce from fermentation often tells us what we can use to produce that. In other words, the main product of a fermentation process will often determine the choice of carbon source, particularly if the product results from the direct dissimilation of it. In fermentations such as ethanol or single-cell protein production where raw materials are 60 to 77% of the production cost, the selling price of the product will be determined largely by the cost of the carbon source. But most companies involved in the business of fermentation are continuously looking out for alternative substrates which could be used as a carbon sources. This enables a company to use alternative substrates, depending on price and availability in different locations, and remain competitive. Up to ten different carbon sources have been or are being used by Pfizer Ltd for an antibiotic production process depending on the geographical location of the production site and prevailing economics. The purity of the carbon source may also affect the choice of substrate. For example, metallic ions must be removed from carbohydrate sources used in some citric

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lagers, while waters with high carbonate content are better for the darker beers such as stouts. The reuse of water in media also must be considered. ICI, a giant SCP producer, realized that very high costs would be incurred if fresh purified water was used on a once through basis, since operating at a cell concentration of 30 g biomass (dw) dm3 would require 2700 X lO6 dm3 of water per annum. Laboratory tests to simulate the process showed that the Methylophilus methylotrophus could be grown successfully with 86% continuous recycling of supernatant with additions to make up depleted nutrients. This approach was therefore adopted in the full scale process to reduce capital and operating costs and it was estimated that water used on a once through basis without any recycling would have increased water costs by 50% and effluent treatment costs 10 fold.

The method of media preparation, particularly sterilization, may affect the suitability of carbohydrates for individual fermentation processes. It is often best to sterilize sugars separately because they may react with ammonium ions and amino acids to form black nitrogen containing compounds which will partially inhibit the growth of many microorganisms. Starch, when heated in the sterilization process, gelatinizes, giving rise to very viscous liquid so that only concentrations of up to 2% can be used without modification. The local regulations and the prices of raw material also affect the choice of carbon source.

•

The carbon source then must be easily degradable, sterilizable and cheap, right? Right! And that laves us with one obvious choice – carbohydrates. It is common practice to use carbohydrates as the carbon source in microbial fermentation processes. The most widely available carbohydrate is starch obtained from maize grain. It is also obtained from other cereals, potatoes and cassava. Maize and other cereals may also be used directly in a partially ground state, e.g. maize chips. Starch may also be readily hydrolyzed by dilute acids and enzymes to give a variety of glucose preparations (solids and syrups). Hydrolyzed cassava starch is used as a major carbon source for glutamic acid production. Barley grains may be partially germinated and heat treated to give the material known as malt, which contains a variety of sugars besides starch. Malt is the main substrate for brewing beer and lager in many countries. Malt extracts may also be prepared from malted grain. Sucrose is obtained from sugar cane and sugar beet. It is commonly used in fermentation media in a very impure form as beet or cane molasses which are the residues left after crystallization of sugar solutions in sugar refining. Molasses is used in the production of high-volume /low-value products such as ethanol, SCP, organic and amino acids and some microbial gums. Molasses or sucrose also may be used for production of higher value/low-bulk products such as antibiotics, specialty enzymes, vaccines and fine chemicals The cost of molasses will be very competitive when compared with pure carbohydrates. However, molasses contains many impurities and molasses-based fermentations will often need a more expensive and complicated extraction/purification stage to remove the impurities and effluent treatment will be more expensive because of the unutilized waste materials which are still present. Some new processes may require critical evaluation before the final decision is made to use molasses as the main carbon substrate Corn steep liquor is a by-product after starch extraction from maize. Although primarily used as a nitrogen source, it does contain lactic acid, small amounts of reducing sugars and complex polysaccharides. Certain other materials of plant origin, usually included as nitrogen sources, such as soybean

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Nitrogen. Most industrially used micro-organisms can utilize inorganic or organic sources of nitrogen. Inorganic nitrogen may be supplied as ammonia gas or ammonium salts Ammonia has been used for pH control and as the major nitrogen source in a defined medium for the commercial production of human serum albumin by Saccharomyces cerevisiae. Ammonium salts such as ammonium bisulphate will usually produce acid conditions as the ammonium ion is utilized and the free acid will be liberated. On the other hand nitrates will normally show an alkaline drift as they are metabolized. Ammonium nitrate will first cause an acid drift as the ammonium ion is utilized, and nitrate assimilation is repressed. When the ammonium ion has been exhausted, there is an alkaline drift as the nitrate is used as an alternative nitrogen source.

meal and Pharmamedia, contain small but significant amounts of carbohydrates.

•

That was about carbohydrates. Any other substrate? Sure. Vegetable oils (olive, maize, cotton seed, linseed, soya bean, etc.) may also be used as carbon substrates, particularly for their content of the fatty acids, oleic, linoleic and linolenic acid, because costs are competitive with those of carbohydrates. A typical oil contains approximately 2.4 times -the energy of glucose on a per weight basis. Oils also have a volume advantage as it would take 1.24 dm3 of soya bean oil to add 10 kcal of energy to a fermentor, whereas it would take 5 dm3 of glucose or sucrose assuming that they are being added as 50% w /w solutions. Ideally, in any fermentation process, the maximum working capacity of a vessel should be used. Oil based fed-batch fermentations permit this procedure to operate more successfully than those using carbohydrate feeds where a larger spare capacity must be catered for to allow for responses to a sudden reduction in the residual nutrient level Oils also have antifoam properties which may make downstream processing simpler, but normally they are not used solely for this purpose.

One exception to this pattern is the metabolism of Gibberella fujikuroi. In the presence of nitrate the assimilation of ammonia is inhibited at pH 2.8-3.0. Nitrate assimilation continues until the pH has increased enough to allow the ammonia assimilation mechanism to restart.Organic nitrogen may be supplied as amino acid; protein or urea. In many instances growth will be faster with a supply of organic nitrogen, and a few microorganisms have an absolute requirement for amino acids. Amino acids are commonly added as complex organic nitrogen sources which are non-homogeneous, cheaper and readily available. In lysine production, methionine and threonine are obtained from soybean hydrolysate since it is relatively inexpensive. Other proteinaceous nitrogen compounds serving as sources of amino acids include cornsteep liquor, soya meal, peanut meal, cotton-seed meal, Distillers’ solubles, meal and yeast extract. Chemically defined amino acid media devoid of protein are necessary in the production of certain vaccines when they are intended for human use.

Pfizer antibiotic process operated with a range of oils and fats on a laboratory scale. In the UK, when both technical and economic factors are considered, soybean oil or rapeseed oil is the preferred substrates. Glycerol trioleate is known to be used in some fermentations where substrate purity is an important consideration. Methyl oleate has been used as the sole carbon substrate in cephalosporin production.

•

Carbohydrates, oils. Anything else? There has been considerable interest in hydrocarbons. Development work has been done using n-alkanes for production of organic acids, amino acids, vitamins and cofactors, nucleic acids, antibiotics, enzymes and proteins. Methane, methanol and n-alkanes have all been used as substrates for biomass production. In processes where the feedstock costs are an appreciable fraction of the total manufacturing cost, cheap carbon sources are important. In the 1960s and early 1970s there was an incentive to consider using oil or natural gas derivatives as carbon substrates as costs were low and sugar prices were high. On a weight basis n-alkanes have approximately twice the carbon and three times the energy content of the same weight of sugar. Although petroleum-type products are initially impure they can be refined to obtain very pure products in bulk quantities which would reduce the amount of effluent treatment and downstream processing. At this time the view was also held that hydrocarbons would not be subject to the same fluctuations in cost as agriculturally derived feedstock because it would be a stable priced commodity and might be used to provide a substrate. The scenario, however, has changed now. Now, not only the agricultural derivatives are cheaper to petroleum products, but their prices are steadier, enabling the fermentation to be economically more predictable.

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Ok and the next nutrient?

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Ammonia, its salts, urea, proteins, amino acids… again, how do we select the right source? Ammonia or ammonium ion is the preferred nitrogen source. Not only because it is inexpensive but also because of the fact that nitrate reductase, an enzyme involved in the conversion of nitrate to ammonium ion, is repressed in the presence of ammonia. In fungi that have been investigated, ammonium ion represses uptake of amino acids by general and specific amino acid permeases. In Aspergillus nidulans, ammonia also regulates the production of alkaline and neutral proteases. Therefore, in mixtures of nitrogen sources, individual nitrogen components may influence metabolic regulation so that there is preferential assimilation of one component until its concentration has diminished. It has been shown that antibiotic production by many micro-organisms is influenced by the type and concentration of the nitrogen source in the culture medium. Antibiotic production may be inhibited by a rapidly utilized nitrogen source and may start only after most of the nitrogen has been consumed. In shake flask media experiments, salts of weak acids (e.g. ammonium succinate) may be used to serve as a nitrogen source and eradicate the source of a strong acid pH change

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In specific processes the concentration of certain minerals may be very critical. It has been suggested that in streptomycin fermentation, an important function of calcium salts in fermentation media was to precipitate excess inorganic phosphates. Thus the addition of calcium indirectly improved the yield of streptomycin. The inorganic phosphate concentration also influences production of bacitracins, citric acid (surface culture), ergot, monomycin, novobiocin, oxytetracycline, polyenes, ristomycin, rifamycin Y, streptomycin, vancomycin and viomycin.

The use of complex nitrogen sources for antibiotic production has been common practice. They are thought to help create physiological conditions in the trophophase which favour antibiotic production in the idiophase. For example, in the production of polyene antibiotics, soybean meal has been considered a good nitrogen source because of the balance of nutrients, the low phosphorus content and slow hydrolysis. It has been suggested that this gradual breakdown prevents the accumulation of ammonium ions and repressive amino acids. These are probably some of the reasons for the selection of ideal nitrogen sources for some secondary metabolites.

Of these nine, the concentrations of manganese, iron and zinc are the most critical in secondary metabolism. In every secondary metabolic system in which sufficient data has been reported, the yield of the product varies linearly with the logarithmic concentration of the ‘key’ metal. The linear relationship does not apply at concentrations of the metal which are either insufficient, or toxic, to cell growth. Some of the primary and secondary microbial products whose yields are affected by concentrations of trace metals greater than those required for maximum growth are given in Table 4.11.

In gibberellin production the nitrogen source has been shown to have an influence on directing the production of different gibberellins and the relative proportions of each type.

Chlorine does not appear to playa nutritional role in the metabolism of fungi. It is, however, required by some of the halophilic bacteria. Obviously, in those fermentations where a chlorine-containing metabolite is to be produced the synthesis will have to be directed to ensure that the nonchloro-derivative is not formed. The most important compounds are chlortetracycline and griseofulvin. In griseofulvin production, adequate available chloride is provided by the inclusion of at least 0.1 % KCI , as well as the chloride provided by the complex organic materials included as nitrogen sources. Other chlorine containing metabolites are caldriomycin, nomidulin and mollisin.

Other pre-determined aspects of the process can also influence the choice of nitrogen source. It has been shown that the optimum concentration of available nitrogen for griseofulvin production showed some variation depending on the form of inoculum and the type of fermentor being used. Obviously these factors must be borne in mind in the interpretation of results in media-development programmers. Some of the complex nitrogenous material may not be utilized by a micro-organism and create problems in downstream processing and effluent treatment. This can be an important factor in the final choice of substrate.

•

Many media cannot be prepared or autoclaved without the formation of a visible precipitate of insoluble metal phosphates. The problem of insoluble metal phosphate(s) may be eliminated by incorporating low concentrations of chelating agents such as ethylene diamine tetraacetic acid (EDTA), citric acid, polyphosphates, etc., into the medium. These chelating agents preferentially form complexes with the metal ions in a medium. The metal ions then may be gradually utilized by the microorganism In many media, particularly those commonly used in large scale processes, there may not be a need to add a chelating agent as complex ingredients such as yeast extracts or proteose peptones will complex with metal ions and ensure gradual release of them during growth.

Those were the major requirements. Any minor ones? Well, I wouldn’t call them minor because they are also equally important. But they could be termed All micro-organisms require certain mineral elements for growth and metabolism. In many media, magnesium, phosphorus, potassium, sulphur, calcium and chlorine are essential components, and because of the concentrations required, they must be added as distinct components. Others such as cobalt, copper, iron, manganese, molybdenum and zinc are also essential but are usually present as impurities in other major ingredients. There is obviously a need for batch analysis of media components to ensure that this assumption can be justified, otherwise there may be deficiencies or excesses in different batches of media. When synthetic media are used the minor elements will have to be added deliberately. As a consequence of product composition analysis, it is possible to estimate the amount of a specific mineral for medium design, e.g. sulphur in penicillins and cephalosporins, chlorine in chlortetracycline. The concentration of phosphate in a medium, particularly laboratory media in shake flasks, is often much higher than that of other mineral components. Part of this phosphate is

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•

That’s all that is needed, isn’t it? Well, almost. There is one more class of nutritional requirements that’s quantitatively insignificant but qualitatively extremely significant.

•

Vitamins, is it? Yes. Actually these are called trace elements and growth factors.

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being used as a buffer to minimize pH changes when external control of the pH is not being used.

due to chloride or sulphate ions which would be present if ammonium chloride or sulphate were used as the nitrogen source. This procedure makes it possible to use lower concentrations of phosphate to buffer the medium. High phosphate’ concentrations inhibit production of many secondary metabolites

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The role of trace elements in medium formulation can be significant. Cultured cells normally require Fe, Zn, Cu, Se, Mn, Mo and V. These are often present as impurities in other media components. It has been found that if the number of trace elements were increased, insulin, transferrin, albumin and liposomes were not needed in a serum-free hybridoma medium. They included AI, Ag, Ba, Br, Cd, Co, Cr, F, Ge, J, Rb , Zr, Si, Ni and Sn as well as those previously mentioned. Some micro-organisms cannot synthesize a full complement of cell components and therefore require preformed compounds called growth factors. The growth factors most commonly required are vitamins, but there may also be a need for specific amino acids, fatty acids or sterols. Many of the natural carbon and nitrogen sources used in media formulations contain all or some of the required growth factors. When there is a vitamin deficiency it can often be eliminated by careful blending of materials. It is important to remember that if only one vitamin is required it may be occasionally more economical to add the pure vitamin, instead of using a larger bulk of a cheaper multiple vitamin sources. Calcium pantothenate has been used in one medium formulation for vinegar production. In processes used for the production of glutamic acid, limited concentrations of biotin must be present in the medium). Some production strains may also require thiamine. And there are buffers, precursors and inhibitors to be added into the fermentation medium.

•

Buffers are for the control of pH, aren’t they? They are. They are the substances that resist sudden change in pH of the medium. The control of pH may be extremely important if optimal productivity is to be achieved. A compound may be added to the medium to serve specifically as a buffer, or may also be used as a nutrient source. Many media are buffered at about pH 7.0 by the incorporation of calcium carbonate (as chalk). If the pH decreases the carbonate is decomposed. Obviously, phosphates which are part of many media also play an important role in buffering. However, high phosphate concentrations are critical in the production of many secondary metabolites. The balanced use of the carbon and nitrogen sources will also form a basis for pH control as buffering capacity can be provided by the proteins, peptides and amino acids, such as in corn-steep liquor. The pH may also be controlled externally by addition of ammonia or sodium hydroxide and sulphuric acid.

•

And what are precursors and inhibitors? Some chemicals, when added to certain fermentations, are directly incorporated into the desired product. Probably the earliest example is that of improving penicillin yields. A range of different side chains can be incorporated into the penicillin molecule. The significance of the different side chains was first appreciated when it was noted that the addition of corn-steep liquor increased the yield of penicillin from 20 units /cm-3 to 100 units/ cm - 3. Corn-steep liquor was found to contain phenylethylamine which was preferentially incorporated into the penicillin molecule to

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yield benzyl penicillin (Penicillin G). Having established that the activity of penicillin lay in the side chain, and that the limiting factor was the synthesis of the side chain, it became standard practice to add side-chain precursors to the medium, in particular phenylacetic acid. It was soon found out that the addition of phenylacetic acid and its derivatives to the medium were capable of both increasing penicillin production threefold and to directing biosynthesis towards increasing the proportion of benzyl penicillin from 0 to 93% at the expense of other penicillins. Phenylacetic acid is still the most widely used precursor in penicillin production. Some important examples of precursors are given in Table When certain inhibitors are added to fermentations, more of a specific product may be produced, or a metabolic intermediate which is normally metabolized is accumulated. One of the earliest examples is the microbial production of glycerol. Glycerol production depends on modifying the ethanol fermentation by removing acetaldehyde. The addition of sodium bisulphite to the broth leads to the formation of the acetaldehyde bisulphite addition compound (sodium hydroxy ethyl sulphite). Since acetaldehyde is no longer available for re-oxidation of NADH2, its place as hydrogen acceptor is taken by dihydroacetone phosphate, produced during glycolysis. The product of this reaction is glycerol-3-phosphate, which is converted to glycerol. The application of general and specific inhibitors is illustrated in Table. In most cases the inhibitor is effective in increasing the yield of the desired product and reducing the yield of undesirable related products. A number of studies have been made with potential chlorination inhibitors, e.g. bromide, to minimize chlortetracycline production during tetracycline fermentation. Inhibitors have also been used to affect cell-wall structure and increase the permeability for release of metabolites. The best example is the use of penicillin and surfactants in glutamic acid production. The majority of enzymes which are of industrial interest are inducible. Induced enzymes are synthesized only in response to the presence in the environment of an inducer. Inducers are often substrates such as starch or dextrins for amylases, maltose for pullulanase and pectin for pectinases. Some inducers are very potent, such as isovaleronitrile inducing nitralase. Substrate analogues that are not attacked by the enzyme may also serve as enzyme inducers. Most inducers which are included in microbial enzyme media are substrates or substrate analogues, but intermediates and products may sometimes be used as inducers. For example, maltodextrins will induce amylase and fatty acids induce lipase. However, the cost may prohibit their use as inducers in a commercial process. One unusual application of an inducer is the use of yeast mannan in streptomycin production. During the fermentation varying amounts of streptomycin and mannosidostreptomycin are produced. Since mannosidostreptomycin has only 20% of the biological activity of streptomycin, the former is an undesirable

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It is now possible to produce a number of heterologous proteins in yeasts, fungi and bacteria. These include proteins of viral, human, animal, plant and microbial origin. However, heterologous proteins may show some degree of toxicity to the host and have a major influence on the stability of heterologous protein expression. As well as restricting cell growth as biomass the toxicity will provide selective conditions for segregant cells which no longer synthesize the protein at such a high level. Therefore, optimum growth conditions may be achieved by not synthesizing a heterologous protein’ continuously and only inducing it after the host culture has grown up in a vessel to produce sufficient biomass. In cells of S. cerevisiae where the Gall promoter is part of the gene expression system, product formation may be induced by galactose addition to the growth medium which contains glycerol or low nonrepressing levels of glucose as a carbon source. One commercial system that has been developed is based on the ale A promoter in Aspergillus nidulans to express human interferon a2. This can be induced by volatile chemicals, such as ethylmethyl ketone, which are added when biomass has increase to an adequate level and the growth medium contains a non-repressing carbon source or low non-repressing levels of glucose. Methylotrophic yeasts such as Hansenula po/ymorpha and Piehia pastoris may be used as alternative systems because of the presence of an alcohol oxidase. During growth on methanol, which also acts as an inducer, the promoter is induced to produce about 30% of the cell protein. In the presence of glucose or ethanol. it is undetectable. Expression systems have been developed with P. pastoris for tumour necrosis factor, hepatitis B surface antigen and a-galactosidase. Hepatitis B surface antigen and other heterologous proteins can also be expressed by H. polymorpha.

•

And what about the air? Don’t the organisms require any air? Sure they do. All aerobic fermentations require oxygen. Oxygen, although not added to an initial medium as such, is nevertheless a very important component of the medium in many processes, and its availability can be extremely important in controlling growth rate and metabolite production.

•

Suppose we want to carry out a fermentation using two different media but the same organism. Will the oxygen requirements be different? They could be. The composition of the medium may influence the oxygen availability in a number of ways. The culture may become oxygen limited because sufficient oxygen cannot be made available in the fermentor if certain substrates, such as rapidly metabolized sugars which lead to a high oxygen demand, are available in high concentrations. This, in some cases can be corrected by making fine

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adjustments in the aeration and agitation speeds. In other cases, when there is the possibility of oxygen limitation due to fast metabolism, it may be overcome by reducing the initial concentration of key substrates in the medium and adding additional quantities of these substrates as a continuous or semi-continuous feed during the fermentation. It can also be overcome by changing the composition of the medium, incorporating higher carbohydrates (lactose, starch, etc.) and proteins which are not very rapidly metabolized and do not support such a large specific oxygen uptake rate. The individual components of the medium can influence the viscosity of the final medium and its subsequent behaviour with respect to aeration and agitation. Highly viscous media will need special consideration with respect to the availability of oxygen to the cells they harbour.Polymers in solution, particularly starch and other polysaccharides, may contribute to the rheological behaviour of the fermentation broth. As the polysaccharide is degraded, the effects on rheological properties will change. Allowances may also have to be made for polysaccharides being produced by the micro-organism. In most microbiological processes, foaming is a problem. It may be due to a component in the medium or some factor produced by the micro-organism. The most common cause of foaming is due to proteins in the medium, such as cornsteep liquor, Pharmamedia, peanut meal, soybean meal, yeast extract or meat extract. These proteins may denature at the air-broth interface and form a skin which does not rupture readily. The foaming can cause removal of cells from the medium which will lead to autolysis and the further release of microbial cell proteins will probably increase the stability of the foam. If uncontrolled, then numerous changes may occur and physical and biological problems may be’ created. These include reduction in the working volume of the fermentor due to oxygen exhausted gas bubbles circulating in the system, changes in bubble size, lower mass and heat transfer rates, invalid process data due to interference at sensing electrodes and incorrect monitoring and control . The biological problems include deposition of cells in upper parts of the fermentor, problems of sterile operation with the air filter exits of the fermentor becoming wet, and there is danger of microbial infection and the possibility of siphoning leading to loss of product. There are five patterns of foaming in fermentations

1. Foaming remains at a constant level through-out the fermentation. Initially it is due to the medium and later due to microbial activity. 2. A steady fall in foaming during the early part of the fermentation, after which it remains constant. Initially it is due to the medium but there are no later effects caused by the micro-organism. 3. The foaming falls slightly in the early stages of the fermentation then rises. There are very slight effects caused by the medium but the major effects are due to microbial activity.

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product. The production organism Streptomyces griseus can be induced by yeast mannan to produce, B-mannosidase which will convert mannosidostreptomycin to streptomycin.

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4. The fermentation has a low initial foaming capacity which rises. These effects are due solely to microbial activity.

Unfortunately, the concentrations of many types of antifoam which are necessary to control fermentations will reduce the oxygen-transfer rate by as much as 50%; therefore antifoam additions must be kept to an absolute minimum. There are also other antifoams which will increase the oxygen-transfer rate. If the oxygen-transfer rate is severely affected by antifoam addition then mechanical foam breakers may have to be considered as a possible alternative. Undoubtedly, though, foam control in industry is still an empirical art. The best method for a particular process in one factory is not necessarily the best for the same process on another site. The design and operating parameters of a fermentor may affect the properties and quantity of foam formed.

5. A more complex foaming pattern during the fermentation which may be a combination of two or more of the previously described patterns. If excessive foaming is encountered there are three ways of approaching the problem: 1. To try and avoid foam formation by using a defined medium and a modification of some of the physical parameters (pH, temperature, aeration and agitation). This assumes that the foam is due to a component in the medium and not a metabolite. 2. The foam is unavoidable and antifoam should be This is the more standard approach.

used.

•

3. To use a mechanical foam breaker. Antifoams are surface active agents, reducing the surface tension in the foams and destabilizing protein films by (a) hydrophobic bridges between two surfaces, (b) displacement of the absorbed protein, and (c) rapid spreading on the surface of the film.

Next comes the optimization of the medium. Optimization, in simple terms, is to decide the exact concentration of different media ingredients which will give the highest production of the desired fermentation product. It is important to remember that the optimum conditions required for the production of the desired metabolite and those for the optimum growth of the organism need not be the same. In most cases, they are not. Therefore, it becomes necessary to fine-tune the composition of fermentation medium to suit the maximum production of the desired product. Different combinations and sequences of process conditions need to be investigated to determine the growth conditions which produce the biomass with the physiological state best constituted for product formation.

Ideal antifoam should have the Following Properties

1. Should disperse readily and have fast action on the existing foam. 2. Should be active at low concentrations. 3. Should be long acting in preventing new foam formation. 4. Should not be metabolized by the microorganism. 5. Should be non-toxic to the micro-organism 6. Should be non-toxic to humans and animals 7. Should not cause any problems in the extraction and purification of the production and purification of the product.

Medium optimization by the classical method of changing one independent variable (nutrient, antifoam, pH, temperature, etc.) while fixing all the others at a certain level can be extremely time consuming and expensive for a large number of variables. Imagine a medium containing 15 ingredients which is equivalent to 15 variable parameters. If we keep on changing each of the medium parameter one by one keeping all others constant, an extremely large number of experiments will have to be carried out. An industry will have neither the resources nor the time to conduct those many experiments. When more than five independent variables are to be investigated, the Plackett-Burman design may be used to find the most important variables in a system, which is then optimized in further studies.

8. Should not cause any handling hazards. 9. Should be inexpensive. 10. Should have no effect on oxygen transfer. 11. Should be heat sterilizable. The following compounds which meet most of these requirements have been found to be most suitable in different fermentation processes: 1. Alcohols; stearyl and octyl decanol. 2. Esters. 3. Fatty acids and derivatives, particularly glycerides, which include cottonseed oil, linseed oil, soy-bean oil, olive oil, and castor oil, sunflower oil, rapeseed oil and cod liver oil. 4. Silicones. 5. Sulphonates. 6. Miscellaneous; Alkaterge C, oxazaline, poly-propylene glycol. These antifoams are generally added when foaming occurs during the fermentation. Because many antifoams are of low solubility they need a carrier such as lard oil, liquid paraffin or castor oil, which may be metabolized and affect the fermentation process .

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All right. Now after we have formulated a medium, what next?

That was about the general fermentation media. Then there are some special media.

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What are special media? Special media are the ones that either use special or uncommon media ingredients or are used for special purposes like cultivation of animal cells. Mammalian cell lines have been cultured in vitro for 40 years. Initially, animal cells were required for vaccine manufacture but they are now also used in the production of monoclonal antibodies, interferon, etc. The media initially used for this purpose contained about 10% serum (foetal calf or calf) plus other organic and inorganic components. Since this

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The serum is a very complex mixture containing approximately 1000 components including inorganic salts, amino acids, vitamins, carbon sources, hormones, growth factors, haemoglobin, albumin and other compounds. However, most of them do not appear to be needed for growth and differentiation of cell lines which have been tested.

their use may be very limited and not very cost effective. The growth of Chinese hamster cell lines in a protein-free medium formulated from amino acids,’ vitamins, organic compounds and inorganic salts has been demonstrated. All kinds of cells used for fermentations must be grown in isotonic solutions and at pH values most suitable for cellular growth and, more importantly for the production of the desired metabolite. That was about fermentation media. Now find out the composition of commonly used fermentation media and the fermentations they are used for.

Notes

Serum is used extensively in production media for animal cell culture to produce recombinant proteins and antibody based products for in vivo use in humans. At present the regulations. governing the quality of serum which can be used for manufacturing processes vary considerably from country to country. However,’ FDA approval of a process will be essential to market a product in the USA and therefore regulate the quality of serum which can be used. Serum tested by approved laboratories should be free of bacterial, viral or BSE (bovine sporangiform encephalitis) contamination and other components should be within strictly defined limits. Serum of this standard is needed for the cell culture media which is used to maintain the cell culture stocks as well as the production media. The cost of foetal calf serum, US$190/ dm3 in Europe, makes serum free media attractive economic alternatives, but it would take a number of years to develop suitable serum free media. The absence of the many unutilized components in serum will also simplify purification of potential products produced in such media. However, these process changes would need approval by the FDA or other regulatory bodies before a product could be marketed using a modified process.

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Do you mean to say that we can now grow animal cells without using serum? Yes we can. These are the serum free media where serum is replaced by albumin, insulin, transferrin, ethanolamine, selenium and l3-mercaptoethanol.

The advantages of removing serum from media include: 1. More consistent and definable medium composition to reduce batch variation. 2. Reduction in potential contamination to make sterility easier to achieve. 3. Potential cost savings because of cheaper replacement components. 4. Simplifying downstream’ processing because the total protein content of the medium has been reduced. In addition the serum free media we can have protein free media. The elimination of proteins seems an attractive objective. However, the design of such media is difficult and

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pioneering work it has been possible to develop a range of serumfree media. These media contain carbohydrates, amino acids, vitamins, nucleic acids, etc, dissolved in high purity water. Media costs are therefore considerably higher than those for microbial cells. At a 1000 dm3 scale the medium costs may account for 40% of the unit costs, and serum may be 80% of the medium cost.

FERMENTATION TECHNOLOGIES

LESSON 3: SCREENING

Learning Objectives In this lecture, you will learn

• • • •

What is screening? What are its types? Primary screening Secondary screening Improvement in industrial microorganism

Screening may be defined as the use of highly selective procedures to allow the detection and isolation of only those microorganisms of interest from among a large microbial population. Thus, to be effective, screening must in one or a few steps allow the discarding of many valueless microorganisms, while at the same time allowing the easy detection of the small percentage of useful microorganism that are present in the population. The concept of screening will be illustrated by citing specific examples of screening procedures that are or have been commonly employed in industrial research programs. In each instance, except for that of the crowded – plate technique, a natural microbial source such as soil is diluted to provide a cell concentration such that aliquots spread, sprayed, or applied in some manner to the surfaces of agar plates, will yield colonies not touching neighboring colonies. Which are the selective substrates for the isolation of actinomycetes and antibiotic-producing actinomycetes? Substrates selective for Actinomycetes

Starch Humic acid (0.1 %) Methanol Calcium Substrates selective for antibiotic-producing actinomycetes

Alanine Potassium Vitamins Cobalt 6CO2 + 6H2O + 3) The oxidation of ethanol, which predominates when fermentative substrates are not available or in very limited supply. The cells attain a maximum specific growth rate of about 0.2 hr-1 with a high biomass yield of about 0.6-0.7 g dry mass per gram ethanol consumed, a low respiratory quotient of about 0.7, and an energy yield of about 6-11

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Enzymatic Assays Enzymatic assays are highly specific, and they will quantitatively detect minute amounts of a fermentation product, as well as differentiate between biologically active and inactive forms of a compound. An enzyme preparation (from a commercial source, a microbial culture, or other enzyme source) is incubated with a sample of culture broth so as to cause some enzyme-mediated change in the fermentation product, such as a partial decomposition with consequent formation of a measurable product. For example, L-glutamic acid in a small sample of fermentation broth can be assayed by adding washed cells of certain strains of Escherichia coli which contain the enzyme “glutamic acid decarboxylase.” Toluene also is added to this mixture to liberate the enzyme from the cells, and the assay is carried out at a pH of 5. One mole of CO2 is liberated from each mole of glutamic acid. The CO2, which is only poorly soluble in water at this pH value, is evolved to the atmosphere as the gas, and it is measured by manometric means such as with a Warburg respirometer . Toluene is not required if the Escherichia coli cells are first carefully dried in vacuo over CaCl2 or dried by several washings with cold acetone (an acetone powder). Enzymatic assays must be carefully tested to determine that they actually function properly under the specific conditions being employed. A known amount of the pure chemical product is added as an internal standard to one sample of a typical fermentation broth, but not to another sample. The assay results should quantitatively reflect the amount of added chemical when the assay values for the two samples are compared. If this is not the case, several possibilities should be checked. The pH or temperature at which the assay is being carried out may not be optimal for the enzyme, or the enzyme may be inactive under these conditions. There may be compounds in the sample of fermentation broth, such as metals or alternate substrates, which inhibit the enzyme or compete for its active sites. The enzyme may be inherently unstable or may be unstable under the conditions of the assay. Enzyme from a microbial cell source may have been produced in only small quantities or not at all, since special growth conditions often are required for good production of a given enzyme. Enzyme from a commercial source may have been improperly stored; these preparations should be obtained from a reliable source and stored dry in the cold without being allowed to become too old before use. The specificity of the enzyme may be either too little or too great. Obviously, an enzyme that attacks compounds in the culture broth other than the fermentation product will provide erroneous results. Enzymes are usually specific for stereoisomers. For example, in a mixture of D- and L-isomers of glutamic acid only one of the isomers, usually the L-form, will be degraded. However, a microbial cell enzyme source may contain additional enzymes known as “racemases” which racemize either the D- or L-isomer to a mixture of the isomers, a phenomenon that also can cause erroneous assay results. The toluene possibly may not release the enzyme from the cells, or it may be inhibitory to the released enzyme. If this proves to be the case, N-butanol or chloroform often can be substituted for the toluene and, in certain instances, the enzyme can be released from the cells by

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ATP per mole of ethanol metabolized. The stoichiometry of this reaction is: C2H5OH + 3O2 ———————> 2CO2 + 3H2O + Utilization of glycerol, a non-fermentable carbon source, by Saccharomyces cerevisiae is is repressed by glucose. After the depletion of any faster growth-supporting substrate, the enzymes necessary for the utilization of glycerol are induced, and an exponential growth phase on glycerol will follow a diauxic lag phase.

Experimental Conditions The temperature of the water bath surrounding the fermentor will be controlled at 30°C, and the impellers inside each fermentor will be operated at 400 rpm. The pH will be monitored and controlled at pH 5, the optimum for yeast growth. One of the two batch fermentor will have glucose as the only initial carbon source at a concentration of 5 g/l. The other batch fermentor will have two initial carbon sources glucose at 1 g/l and glycerol at 4 g/l. The continuous culture will begin as a batch culture. After the cells grow to sufficient concentration in the mid-exponential phase, the feed inlet and outlet pumps will be switched on to start the continuous culture, at an intermediate dilution rate of about 0.15 hr1. Oxygen will be sparged into this fermentor at constant rate of 10 l/min.

Introduction to Lab Procedures You will monitor the growth characteristics of the yeast in the batch fermentors in three ways - by measuring the concentration of cells, by determining the concentration of the carbon substrates glucose and/or glycerol, and by assaying for the amount of ethanol present at regular intervals throughout the batch culture. Yeast cell concentration can be determined indirectly by measuring the optical density (absorbance) of a culture sample. You will take a sample of the culture medium from the fermentor and read its absorbance using a spectrophotometer. Up to a certain cell density, the concentration of yeast cells (gdw/l) in the sample is proportional to the absorbance reading on the spectrophotometer. The calibration curve correlating cell concentration with absorbance deviates from a linear correlation at high cell densities. Because of this, it’s a good idea to dilute any of your high OD samples (that may be on the non-linear portion of the curve) by a known dilution factor to confirm that the measured OD values fall on the linear portion. You’ll use an automatic glucose analyzer to determine the glucose concentration in the culture samples and estimate how much glucose the cells have consumed. The glucose analyzer determines the amount of glucose in your samples according to the following reactions:

A membrane in the analyzer contains the glucose oxidase enzyme, and the analyzer senses the electron flow generated by the H2O2 when it is oxidized at the platinum anode. The current generated is proportional to the glucose concentration in the culture sample and the analyzer has been calibrated to give the glucose concentration directly. The ethanol concentration is determined using a simple and quick chemical assay. This assay is based on the following reaction:

The amount of NADH produced in this reaction is proportional to the amount of ethanol added as a substrate. This quantity of NADH is determined spectrophotometrically at 340 nm. The Ethanol Assay Reagent that you will use for this assay contains NAD and Alcohol Dehydrogenase. The concentration of glycerol in the cell-free culture medium will be analyzed by high performance liquid chromatography. The HPLC method will also provide measurements of glucose and ethanol in the liquid, in addition to glycerol. The dissolved oxygen concentration in the culture medium is monitored in situ by a galvanic probe containing a silver anode and a lead electrode in an acetate electrolyte. The dissolved oxygen from the culture medium permeates the membrane and initiates an electrochemical reaction. The current generated is proportional to the dissolved oxygen concentration in the culture medium and is calibrated with the maximum solubility of oxygen in the culture medium when sparged with pure oxygen. The changes in the dissolved oxygen concentration with fermentation time in each fermentor is recorded and plotted directly on the computer screen. The oxygen is controlled by computer activation of valves, which allow either pure oxygen or pure nitrogen to be bubbled into the fermentor. The “per” value indicated on the computer monitor is the percentage of time that the valve is open to pure oxygen. The remaining percentage of the time, the valve is open to pure nitrogen. You can regulate the flow rate of these gases to the fermentor using the rotameter on the front of the fermentation control apparatus.

2.Experimental Procedures A.Determining Cell Concentration

1) Zeroing the spectrophotometer. Set the wavelength to 630 nm. Using the knob on the left, set the reading to 0% transmission when the chamber is empty; using the knob on the right, set the reading to 100% transmission when the chamber contains a test tube with about 4 ml of pure medium. 2) Determining cell concentration. First flush out the sample tube for your group’s fermentor by taking an 8-10 ml sample, which you will then discard. Take another 8-10 ml sample from your group’s fermentor and gently mix. Take about 4 ml from your sample tube and transfer it to a glass test tube. Clean the outside of the test tube with ethanol, insert it into the spectrophotometer, and record the

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B) Glucose Measurement

Take your undiluted yeast sample tube and insert the thin plastic sample tubing from the glucose analyzer into it. If the analyzer is in the “Standby” mode, hit the “Run” key; after the machine has calibrated, hit the “Sample” key. If the analyzer is not in the “Standby” mode, just hit the “Sample” key. The glucose concentration in the medium from your sample will print out in about one minute. Record this value.

performed at the end of the batch cultures and provided in the next class period). C) Interpret these two graphs in light of the background information on yeast metabolic pathways and the “cybernetic” principle that cells choose to grow at the fastest possible rate. Specifically, discuss why the cell mass, glucose, and ethanol concentration profiles look as they do for each batch fermentor. D)Briefly discuss the mechanisms of metabolic competition between the three pathways for the sustained oscillations that you may have observed in the continuous fermentation.

Notes

C) Ethanol Aassay

1) Take 0.5 ml from your undiluted sample, pipette it into an eppendorf tube, and spin at 14,000 rpm for 5 minutes. 2) Add 2 ml of Ethanol Assay Reagent to each of three cuvettes. 3) Add 10 µl of pure medium to the first cuvette; this is your Blank. Cover with Parafilm and mix gently. 4) Add 10 µl of the Ethanol Standard (.08% w/v) to the second cuvette; this is your Standard. Cover with Parafilm and mix gently. 5) Add 10 µl of sample (from the supernatant from step a) above) to the third cuvette; this is your Sample. Cover with Parafilm and mix gently. 6) Incubate cuvettes at room temperature for 10 minutes. 7) Clean the outside of the cuvettes, and then read the absorbance of each at 340 nm; use the Blank to zero the spectrophotometer. If the Sample absorbance is > 1.700, dilute the supernatant from your sample 1:4 and perform the assay again using this diluted supernatant. Don’t redo the Blank and Standard. 8) Calculate the ethanol concentration for your sample using the following formula:

Record this Value Results

A) Draw a graph of: a) b) c)

logarithm of cell concentration vs. time glucose/glycerol concentration vs. time ethanol concentration vs. time for the two batch fermentors.

B) Determine the specific growth rate and the yield coefficient (gram dry weight of cells produced per gram of carbon source consumed) for each growth phase in the two batch fermentors. (The calibration between the absorbance reading and the dry cell mass concentration of the yeast cells will be

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absorbance reading. If the absorbance reading is greater than 0.25, a typical limit of linear correlation between the absorbance and cell mass concentration, dilute the sample with a known amount of pure medium, and measure the absorbance again to check if the absorbance reading is on the linear portion of the calibration curve. Record the time you take the sample along with the absorbance reading in the linear range as well as the dilution factor.

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LESSON 7: STRAIN IMPROVEMENT

Learning Objectives

colonies producing antibiotic activity. The ability of a colony to exhibit antibiotic activity is indicated by the presence of a Zone of growth of inhibition surrounding the colony. Such a colony is subcultured to a similar medium, and purified by streaking before making stock cultures. It is necessary to carry on further testing to confirm he antibiotic activity associated with a microorganism, since the zone of inhibition surrounding,the colony,may sometimes be due to other causes.

In this lecture, you will learn

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Introduction Screening Technique Strain development Preservation of microorganisms Preparation of inoculum

We have seen about the basic screening techniques. Once we have isolated a particular microbial strain, attempts are made to improve upon the productivity of the organism. Let’s see how it can be done. It is highly desirable to use a production strain possessing the following four characteristics: (I) It should be a high-yielding strain. (ii) It should have stable biochemical characteristics. (iii) It should not produce undesirable substances. (iv) It should be easily cultivated on a large-scale.

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What are the different scanning techniques? Well, we have seen some of them earlier. Let’s see them in details. Both detection and isolation of high-yielding species from the natural source material, such as soil, containing a heterogeneous microbial population is called screening. There are many screening techniques. Usually screening programmes include primary screening and secondary screening.

Primary Screening This consists of some elementary tests required to detect and to isolate new microbial species exhibiting the desired property. With antibiotic producers, primary screening programmes serve to remove worthless microorganisms on basis of relatively simple, fundamental criteria. The important selection criteria are the activity of antibiotics in vitro and possibly in vivo, against a small number of the most important test organisms. Primary screening is also needed in the case of other useful microbial species (e.g. microorganisms capable of producing organic acids, amines, vitamins etc.). The evaluation of the primary screening in industrial research programmes may be made by citing some specific examples of screening procedures as under:

The Crowded Plate Technique The crowded plate technique is the simplest screening technique employed in detecting and isolating antibiotic producers. It consists of preparing a series of dilution of the soil or other source material for the antibiotic producing microorganisms,followed by spreading of dilution on the nutrient agar plates. The agar plates having 300 to 400or more colonies per plate are considered since they are helpful in locating the

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The crowded plate technique has a limited application, since it merely provides information regarding the inhibitory activity of a colony against the unwanted microbes that may be present by chance on the plate. Therefore, the technique has been improved upon by introducing the use of a ‘test-organism’. In this modified technique, agar plates, which give well-isolated colonies (roughly 100 to 300 colonies per plate) after incubation, are flooded with a suspension of the test organism. Then the plates are subjected to further incubation to allow the growth of the test organism. The formation of inhibitory zones around certain colonies indicates their antibiotic activity. The diameters of the zones of inhibition are measured in millimeters, to obtain a rough approximation of the relative amounts of antibiotic(s) produced by various colonies. The colonies of the antibiotic producers must be isolated and purified before further testing.

Auxanography This technique is largely employed for detecting microorganisms able to produce growth factors (e.g. amino acids and vitamins) extracellularly. The two major steps of the technique are as under: (A) Preparation of First Plate

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A filter paper strip (15 x 12cm) is put across the bottom of a peal dish in such a way that the two ends pass over the edge of the dish.

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A filter paper disc of petri dish size is placed over paper strip on the bottom of the dish.

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The nutrient agar is poured on the paper disc in the dish and allows solidifying.

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Microbial source material such as soil is subjected to dilution such that aliquots on plating will produce well-isolated colonies.

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Plating of aliquots properly diluted soil sample is done.

(B) Preparation of Second Plate

1. A minimal medium lacking the growth factor under consideration is seeded with the test organism. 2. The seeded medium is poured on the surface of fresh Petri dish. 3. The plate is allowed to set.

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The growth factor(s) produced by colonies present on the surface of the first layer of agar can diffuse into the lower layer of agar containing the test organism. The zones of stimulated growth, of the test organism around the colonies are an indication that they produce growth factor(s) extracellularly. Productive colonies are sub-cultured and are further tested.

Enrichment Culture Technique This technique was designed by a soil microbiologist, Beijerinck, to isolate the desired microorganisms from a heterogeneous microbial population present in soil. Either medium or incubation conditions are adjusted so as to favour the growth of the desired microorganisms. On the other hand, unwanted microorganisms are eliminated, or develop poorly since they do not find suitable growth conditions in the newly created environment. Today, this technique has valuable tool in many screening programmes meant for isolating industrially important strains. Generally it consists of the following steps: • Nutrient broth containing an unusual substrate (e.g. Cellulose powder) is inoculated with the microbial source material (e.g. soil) and incubated. • A small portion of inoculum from step (1) is plated onto a solid medium having the same composition. Well isolated colonies appear after incubation Suspected colonies from plate of step (2) are sub-cultured on fresh media and they are also subjected to further testing. An example of screening of enzyme producing microorganisms may be cited. Micro-organisms excreting alkaline_ proteases may be detected from the soil as under: Soil is subjected to-serial dilution. • All soil dilutions are heated at 80 0C for 10 minutes. This treatment kills vegetative cells but spores remain unaffected. • The plating of heat treated samples is done by spreading the samples (usually 01 ml) from dilution onto the surface of nutrient agar containing casein at pH 10-12. • The colonies surrounded by a clear zone are sub-cultured. Use of an Indicator Dye The pH indicating dyes may be employed in some screening method for detecting microorganisms capable of producing organic acids or amines, since a pH indicating dye undergoes colour changes according to its pH. Such dyes (e.g. neutral red, bromothymol blue etc.) are added poorly buffered nutrient agar media. The change in the colour of a dye in the vicinity of the colony suggests the capability of colonial cells to produce either organic acid(s) or amines depending upon the nature of reaction. Such colonies are subcultured to make stock cultures. However, further testing is needed, since inorganic acids or bases are also potential metabolic products of microbial growth. In other words, these methods are not fool proof.

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Secondary screening Secondary screening is strictly essential in any systematic screening progmmme intended to isolate industrially useful microorganisms, since primary screening merely allows the detection and isolation of microorganisms that possess potentially interesting industrial applications. Moreover primary screening does not provide much information needed in electing really useful microorganisms in fermentation processes. This can be realized by a careful of the following points associated with secondary screening: 1. It is very useful in sorting out microorganisms that real commercial value from many isolates obtained during primary screening. At the same time, microorganisms that have poor applicability in a fermentation process are discarded. It is advisable to discard poor cultures as soon as possible, since studies involve much labour and high expense. 2. It provides information wheather the produced by microorganism is a new one or not. Paper, thin layer, or other chromatographic techniques may accomplish this. The compound under consideration is compared with previously known compounds. 3. It gives an idea about the economic position of the fermentation process involving the use of a newly discovered culture. Thus, one may have a comparative study of informtion about the solubility of the product in various organic solvents is made available. This knowledge is useful in the recovery and the subsequent purification of the product. 4. This process with processes that are known, so far as the economic status picture is concerned. 5. It helps in providing information regarding the product yield potentials of different isolates. Thus this is useful in selecting efficient culture for the fermentation processes. 6. It determines the optimum conditions for the growth or accumulation of a product associated with a particular culture. 7. It provides information pertaining to the effect of different components of a medium. This is valuable in designing the medium may be attractive so far as economic consideration is concerned. 8. It detects gross genetic instability in microbial cultures. This type of information is very important, since microorganisms tending to undergoes mutation or alteration in some way may lose their capability for the maximum accumulation of the fermentation products. 9. It gives information about the number in single fermentation. Additional major or 10.Chemical, physical and biological properties of a product are also determining during secondary screening. Moreover, it reveals whether a product produced in the culture broth occurs in more than one chemical form, 11.It reveals whether the culture is homofermentative or heterofermentative.

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The agar in the first plate as prepared in step (A), is carefully and aseptically lifted out with the help of tweezers and a spatula and, placed, without inverting, on the surface of the second plate as prepared in major steps (B)

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12.Demrmination of the structure of the product is done. The product may have a simple, complex, or even a macromolecular structure. 13.With certain types of products (e.g. antibiotics) determinations of the toxicity for animal, plant or men are made if they are to be used for therapeutic purposes. 14.It reveals whether micro-organisms are capable or a chemical change or of even destroying their own fermentation products. For instance, microorganisms that produce the adaptive enzyme, decarboxylase can remove carbon dioxide from the amino acid, raving behind an organic amine. 15.It tells us something about the chemical stability of the fermentation product. Thus, secondary screening gives answers to many questions that arise during the final sorting out of industrially useful microorganisms. This is accomplished by performing experiments on agar plates, in flasks or small bioreactors containing liquid media, or by a combination of these approaches. A specific example of antibiotic producing Streptomycin species may be taken for an under the sequence of events during a screening programme. Those streptomycetes able to produce antibiotics are detected and isolated in a primary screening programme. These streptomycetes exhibiting antimicrobial activity are subjected to an initial secondary screening where their inhibition spectra are determined. A simple, ‘giant-colony’, technique is used to do this. Each of the streptomycal isolates is streaked in a narrow band across the center of the nutritious agar plates. Then, these plates are incubated until growth of a streptomycete occurs now, the test organisms are streaked from the edges of the plates upto but not touching the streptomtycete growth Further screening is carried out employing liquid media in flasks, since such studies give more information than that which can be obtained on agar media. At the same time, it is advisable to use accurate assay techniques (e.g. paper disc-agar diffusion assay) to exactly determine the amounts of antibiotic present in samples of culture fluids. Thus, each of the streptomycete isolates is studied by using several different liquid media in Erlenmeyer flasks provided with baffles. These streptomycete cultures are inoculated into sterilized liquid media. Then, such seeded flasks are incubated at a constant temperature. Usually, such cultures are incubated at near room temperature. Moreover, such flasks are aerated by keeping them on a mechanical shaker, since the growth of streptomycetes and production of antibiotics occur better in aerated flasks than in stationary ones. Samples are withdrawn at regular intervals under aseptic conditions and are tested in a quality control laboratory. Important tests to be carried out include: (i) Checking for contamination, (ii) Checking of pH, (iii) Estimation of critical nutrients, (iv) Assaying of the antibiotic, and (v) Other determinations, if necessary.

The result of the above tests points out which medium is the best for antibiotic formation, and which stage the antibiotic yields are greatest during the growth of the culture on the various media. After performing all necessary routine tests in the screening of an actually useful streptomycete for a fermentation process, other additional determinations are made. They are: 1. Screening of fermentation media through the exploitation of which the highest antibiotic yields may be obtained. 2. Determination of whether the antibiotic is new. 3. Determination of the number of antibiotics accumulated in the culture broth is made. 4. Effect of different bio parameters on the growth of streptomycete culture, fermentation process and accumulation of antibiotic. 5. Solubility picture of antibiotic in various organic solvents. also, it is to be determined weather antibiotic is adsorbed by adsorbent materials (e.g. ion-exchange resin or activated carbon). This knowledge is essential in the recovery and purification of an antibiotic from the fermented broth. 6. Toxicity tests are conducted on mice or other laboratory animals. An antibiotic is also tested for the adverse effects if any, on man, animal or plant. 7. The streptomycete culture is characterized and is classified up to species 8. Further studies are made on a selected individual streptomycete culture. For example, mutation and other genetic studies for strain improvement are carried out. In conclusion, tests are designed and conducted in such a way that production streptomycete strains may be obtained with least expenses. Similar screening and analytical techniques could be employed for the isolation of microbial isolates important in the production of other industrial chemical substances.

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What are the different strain improvement techniques and how are they carried out?

It is highly desirable that the industrial fermentation process should be made more & more economical. This largely depends upon the efficiency of the production strain involved in the fermentation process. Therefore, a person interested in starting a fermentation industry or in competing with other industries must procure an efficient strain. Thus it is clear that, the use of a high-yielding strain in any fermentation process is the most critical factor. Usually, newly isolated strains obtained by screening techniques are not so efficient as could be used in industrial fermentation process. Therefore such strains require improvement, so far as the yield of a particularly desired compound is concerned this is accomplished by producing the mutant fermentation strains with the help of physical or, chemical methods. These mutants may be grouped into two major categories: (i) auxotrophic mutants, and (ii) mutants resistant to analogues.

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1. A mutant strain of Corynebacterium glutamicum can excrete about 60 g of lysine per litre in a medium based on glucose and minerals. This mutant strain needs homoserine. On the other hand wild strain of this bacterium does not need homoserine and fails to excrete lysine. This can be well explained by the schematic illustration as shown in Figure

Fig. 7.2 Branched biosynthetic pathway and control of lysine biosynthesis in conybacterium glutamicum

Fig 7.1 Regulatory system of iso-enzymes involved in a branched biosynthetic pathway. There are two main regulatory mechanisms that differ from each other. There may three distinct iso-enzymes (a, b and c capable of effecting the first reaction in the pathway (A to B). And each may be inhibited or repressed by one of the three end products. With the multivalent or concerted regulatory mechanism, repression is only apparent if all the three end products are present together as illustered in Figure 7.2.

In the case of a wild strain, there is a common biosynthetic pathway to the biosynthesis of lysine and threonine, for the first few reaction steps. This pathway is subject to feed-back inhibition by a mixture of lysine and threonine controlling the activity of aspartate kinase. But a mutant strain requiring homoserine can no longer synthesize threonine. Moreover, feed-back inhibition no longer occurs, and lysine gets accumulated in the medium. Optimum production of lysine takes place in a medium containing 400microgram of homoserine per ml., and a high concentration of biotin (20 microgram). It maybe shown that inhibition due threonine is increased by methionine. Methionine reacts competitively with a regulatory site on aspartate kinase. The ratio of threonine to methionine also plays an important role as shown in the following Table 7.3 Table 7.3 Specific inhibition of lysine production of cornybacterium glutamine

Here is to be noted that, there is only a single enzyme for the reaction A to B. It should also be noted that there is sometimes a cumulative action of the three end products as shown in Table Table 7.2 Feed-back control by end products in branched biosynthetic pathway

Different types of industrially important mutants have been summarized as under:

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2. It should be noted that in the wild strains of Escherichia coli, the biosynthetic pathway to lysine is the same, but its regulatory mechanism is different. In this case, there are three aspartate kinases, each separately controlled by either lysine,

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Micro-organisms, usually, have regulatory mechanisms that control the amount of metabolites synthesized. Therefore, micro-organisms cannot synthesize excess of the metabolites over-limiting the cells’ requirements. Obviously, suppression of these regulatory mechanisms is necessary to develop the strains for higher yields of the desired metabolites. Microbial cultures which have multivalent mechanisms, concerted repression or feed-back inhibition may be used for strain improvement. Subsequently, search is made for mutants, which have lost the ability to synthesize one of the end products capable of feedback inhibition or repression. This may be explained by considering a situation where three end products (E.P.1, E.P.2 and E.P.3) are synthesized via a branched biosynthetic pathway from an intermediary metabolite A as shown in the following figure:

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methionine or threonine. This regulatory system may be represented by Figure 3.1 E.P.3 being lysine. There is also feed-back inhibition of dihydropicolinate synthetase by lysine.

selection of a mutant strain may be done at the following two stages:

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3. Those mutant strains in which one of the enzymes of a biosynthetic pathway is missing are also valuable strains, since they may be employed in the production of an intermediary metabolite of that particular pathway. This may be exemplified by a mutant strain of Corynebacterivm glutamicum. The biosynthesis of amino acid, arginine, occurs by a biosynthetic pathway as illustrated in the following figure:

The analogue of an amino acid, threonine, is added during the preparation of a nutrient agar medium-that is poured into a sterile petri-dish. Then, the medium is allowed to solidify at an angle shown under:

When the wedge has set, a second layer of the same medium, without analogue is poured onto it and allowed to set with the plate level as under: After some time, diffusion of an analogue into the upper

fig 7.4 The biosynthetic pathway for arginine and its regulatory mechanism in conybacterium glutamicum Now, a mutant strain which has lost the enzyme acting on ornithine will excrete that amino acid so long as just sufficient arginine is provided for growth, without enough being present to cause feed back inhibition. Optimum production of ornithine occurs in a medium containing 200 microgram of arginine/ml and 5 microgram of biotin per litre. Moreover, this medium should be rich in glucose and ammonium salts. 4. There are some mutant strains with enzymes that offer resistance to feed-back control. Looking to the regulatory mechanism of feed-back inhibition, interaction between the end product and the regulatory site of an enzyme changes the enzyme configuration. Subsequently, the on become non-functional. A mutant strain may be produced having the enzyme with an altered regulatory site. Such an altered regulatory site fails to interact with the inhibitor. Therefore, feed-back inhibition does not take place. 5. It is also possible to use an analogue in the selection of industrially important stains. An analogue can interact with the regulatory site associated with feed-back inhibition. Such an analogue often exerts toxic effect. And, this toxicity eliminates all sensitive mutant cells in a population. For example, alpha-amino beta-hydroxyvaleric acid is the analogue of threonine. By the use of this antimetabolite the

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layer of the medium takes place. As a result of this, there is development of a Concentration gradient at the surface. Now, a culture, previously treated with a mutagen, is spread on the surface of this medium. Then selection of any mutants offering resistance to high concentrations is done.

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Lastly, a search is to be made to find out resistant mutants capable of producing threonine. This may be accomplished by inoculating the mutants, as point cultures, onto an agar medium seeded with a threonine dependent culture. The growth of seeded culture (i.e., threonine requiring culture) around each colony of threonire excreting mutant strain may occur. The diameter of the zone of seeded culture growth depends upon the quantity of threonine produced by the mutants. Thus, analogue- resistant mutant strains excreting higher yields of threonine may be obtained. Using the above technique, a mutant strain of Brevibacterium flavum capable of excreting threonine upto 12.6g. per litre is obtained.

6. Mutant strains may, sometimes, undergo reversion since mutations are not always stable. As a result of this, revertants may develop in the microbial population of a mutant strain. The revertant strain possesses an enzyme different from that which has been lost due to the previous mutation. Also, the enzyme is not sensitive to feed-back inhibition. This may be exemplified by the threonine deaminase of a revertant strain of the bacterial genus Hydrogenomonas. The situation is represented as under;

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(ii)The auxotrophic mutant required isoleucine for growth and multiplication. (iii) The revertant strain did not require isoleucine. In addition to that, it also produced this amino acid as shown in the following figure:

provided the medium contains lactose as the sole source of carbon. (4)It is possible to isolate constitutive mutants which offer resistance against toxic substances. The enzyme, for which the mutants arc constitutive destroys the toxic substances present in the environment. For example, when a culture of the photosynthetic bacterium, is repeatedly exposed to 0.1 M 25 per cent of the surviving mutants are constitutive mutants for the production of catalase (5) Use of toxic antimetabolites for selecting constitutive mutants having ability to produce an increased yield of involved in the bio-synthesis of the metabolite concerned, may be made. For example, mutants of Lactobacillus casei resistant easel resistant to dichloromethoprotein excrete eighty times more thymidylate synthetase than those of the parent culture. Apart from different methods for inducing high-yielding mutant strains, there is also another method for obtaining high-yielding strains. In this case, the genetic constitution of the microbial cells is changed. This is accomplished by transferring all, or part of the DNA to the recipient culture from the donor culture. Again, there are many techniques for the transfer of genetic material. They have been listed as under:

fig 7.5 Biosynthesis of isoleucine from threonine 7. Constitutive mutants are also important in a fermentation industry, since they may be used to produce increased yields of particular enzymes. These mutants produce particular enzymes the absence of inducing substrates or other substrates that offer resistance to catabolite repression. There are numerous techniques for selecting these mutants. Some simple methods have been briefly discussed here: (1)The microbial cells are cultured on a medium containing a carbon source with the following two characteristics: (i) It should not act as an reducing substrate for a particular enzyme. (ii)It should serve as a substrate for the same particular enzyme. For example, phenyl beta-galactoside may be used for selecting constitutive mutants for the excretion of beta-galactosidase. By limiting the concentration of an inducing substrate (Lactose) during contineous cultivation of a culture, a constitutive mutant of Escherichia coli has been obtained. This mutant strain produces the enzyme. Beta-galactosidase, as 25 per cent of total protein. (2)The microbial culture is cultivated in a cyclic manner, alternatively with and without and substrate. For example, the microbial culture may be grown in the presence of glucose and then of lactose in a cyclic manner. After a certain number of cycles, the bacterial population will contain an increased proportion of constitutive mutants. (3)Use of inhibitors of the inducer in a medium may also be made in the selection of constitutive mutants. For example, 2-nitrophenyl beta-fucoside is an inhibitor of lactose and may be used in selecting mutants constitutive for the production of beta-galactosidase. Thus, mutants of Escherichia coli produce beta-galactosidase without induction,

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(i) Transformation (ii) Transduction (iii) Lysogeny (iv) Conjugation, and (v) Parasexuality.

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How are the microorganisms preserved?

All practicing microbiologists have felt the need to preserve the viability of micro-organisms with which they work. In addition, all the cultural characteristics of a culture, as they were at the time of preservation, must be conserved. The nature of work being done will determine whether the preservation requirement is only very short-term (e.g. a few days) or for an unlimited time period (e.g. many years). Long-term preservation of a culture is required if a culture is to be deposited in one of the service culture collections the view to preserving something of scientific value for perpetuity”. Many methods of preservation for microorganisms have been developed. Here, it is to be noted that there exist different types of micro-organisms (bacteria, viruses, algae, protozoa, yeasts and moulds). Therefore, there are two criteria for selecting a method of preservation for a given culture. They are: (i) The period of preservation desired, and (ii)

The nature of a culture to be preserved.

With the increasing importance of micro-organisms to industry (e.g. in biochemical and antibiotic production, bio-assay, as spoilage microbes, and the like), human, animal and plant pathologists, geneticist, taxonomists and teachers have felt the need for culture collections. There are several large public service collections. These serve as repositories for cultures and as sources of their distribution.

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(I) The wild strain did not require the amino acid, isoleucine. Moreover, it did not produce this amino acid.

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The best known of these are the Central Bureau voor Schimmelcultures (C.B.S.), founded in 1906, the American Type Culture Collection (A.T.C.C.), founded in 1925 and the collection of the Common wealth Mycological Institute (CMI.), founded in 1947. Several other countries are developing their own national collections, and there are large collections belonging to industrial concerns as well as specialized government departments. However, any biologist dealing with living material must at least temporarily maintain his own cultures during the course of his studies and preserve them until they are ready for depositing them in one of these major collections. This depositing of important strains is most desirable as in the past; many organisms which have been the subject of intensive investigation have been discarded at the end of the work or on the death of the biologist. Thus, much valuable material has been lost. There are three basic aims in maintaining and preserving the micro-organisms. They are: (i) to keep cultures alive (ii) uncontaminated, and (iii) as healthy as possible, both physically and physiologically, preserving their original properties until they are deposited in any major collection (i.e. unchanged in their properties). For very long-term preservation, involving stocks of the strains (as opposed to single specimens of each strain) and where withdrawals from stocks are regularly made a fourth aim is to have adequate stocks and appropriate systems for replenishing stocks when necessary. This fourth aim is very much the concern of service culture collections, of course, but he other three are the concern of any maintenance and preservation programme. The running of the collection and the methods of maintenance used are designed to minimize the following hazards to which cultures are exposed: (I) By repeated transfer selection can occur, either of a mutant strain or of a purely vegetative non-sporulating form. The transfer should, therefore, be done as far as possible by an expert with an eye lot the wild strain. However, the fewer transfers made, the less is the risk. (ii) Some strains, sometimes, tend to become attenuated under the artificial conditions of culture. Others deteriorate to wet slimy disintegrated mycelium or spores. Simmons (1963) suggests that this may be due to virus infections and there is considerable evidence to support his theory. (iii)The maintenance processes to which the micro-organism (e.g. the fungus) is subjected are selective, and only adaptive strains survive. These may have somewhat a typical characteristic

(v) Adequate documentation of the strains must be made. In a culture collection of tong standing the strains may well survive several generations of microbiologists, so to assist in maintaning them in their original condition a clear description of the cultural characteristics supported by dried cultures should be provided at the time of depositing them.

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What is serial subculturing & how is it done?

This is the simplest and most common method of maintaining microbial cultures. Microbes are grown on agar slants and are transferred to fresh media before they exhaust all the nutrients or dry out. An exception to this is aerobic Streptomyces spp. where drying-up of the medium has been found successful, provided the initial growth showed the production of aerial hyphae. The drying-up of the medium appeared to encourage good sporulation and the preserved specimen became simply a dried out strand of agar coated with spores which remained viable for a few years at room temperature. For some microbial cultures, no other methods have been found satisfactory, but for the majority of species other methods are available. There are several factors to be borne in mind while choosing a suitable medium. Solid media should be chosen in preference to liquid media, as growth of a contaminant can be more readily observed. However, bacteriophages are often successfully maintained as suspensions in liquid media. Also, anaerobes, especially Clostridium spp., are frequently maintained in a liquid medium (e.g. Robertson’s cooked meat). Some technicians prefer stab cultures for maintenance. But there do not appear to be any published data to show these to be any better than slope cultures. Obviously, if the microorganism is oxygen sensitive, a stab culture would be suggested as an extra safeguard while handling on the bench. While a rich medium may give the best initial growth for heterotrophs. it may also run the risk of accumulating toxic end-products of metabolism. Therefore, the best medium for growth may not necessarily be the best for maintenance and preservation of microorganisms. Besides a suitable substratum, other factors affect the growth of cultures for storage. They are: light intensity, temperature, humidity, standard growth conditions, method of transfer, culture vessel and storage. The time period appropriate for subculture may range from a week to even a few years. Under normal conditions cultures have to be re-grown at fairly frequent intervals (e.g. every four, six or eight months). With a large collection, this requires much labour. Moreover, there is a risk of occurring hazards as discussed previously, everytime a culture is handled. To cut down the frequency of handling of the cultures, it is, therefore desirable to prolong the intervals between subculturing. There are various means to accomplish this (e.g. cold storage and mineral oil storage).

(iv) Cultures are subject to contamination, infection with mites and adverse conditions, temperature, light, humidity, etc., are responsible for their contamination. The latter may arise through breakdown of apparatus, or by incomplete understanding of the organism.

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(iii) The method is especially advantageous when working with unstable variants where occasional transfers to fresh media or growth in mass cultures results in changes in the developmental stages of the strains. Ok, now tell me about freeze drying. Lyophilization is the most satisfactory method of long-term preservation of microorganisms. It is universally used for the preservation of bacteria, viruses, fungi, sera, toxins, enzymes and other biological materials. While, it offers a convenient technique for preserving a large number of cultures, it is by no means the perfect method for storing yeasts with completely unchanged characteristics.

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How are the cultures preserved with the help of mineral oil? This method of preservation is a modification of serial subculture technique. It was first extensively used by Bueli and Weston (1947). Of 2000 fungus strains maintained under oil for 10 years at the C.M.I., only forty-five were lost.

This method is cheap and easy, since it does not require special skills or apparatus such as a centrifuge, dessiccator, or vacuum pump. The steps involved in this method are: (i) First of all, inoculation of the agar slant contained in a screwcap tube with a given culture is practised. (ii)Inoculated agar slant is subjected to incubation until good growth appears. (iii)Using sterial technique, a healthy agar slant culture (from above step) is covered with sterile mineral oil to a depth of about 1 cm. above the top of the agar slant. If a short slant of agar is used, less oil is required. (iv)Finally, oiled culture from step (iii) can be stored at room temperature. But better viability is obtained when stored at lower temperatures. The oil used should be of good quality. British pharmacopoeia medicinal paraffin oil of specific cavity 0.865 to 0.890 is quite satisfactory. Sterilization of oil at the CMI is done in Mc Cartney bottles for 15 minutes at 15 lb/in2. The covering of the culture with oil prevents drying out. The oil allows slow diffusion of gases so growth continues at a reduced rate. This may induce change due to adaption to growth in oil. Some fungus isolates appear stable and survivals of over 20 years have been obtained at the CMI. The depth of oil of 1 cm. is fairly critical (Fennel, 1960), as the oxygen transmission by layers of mineral oil in excess of 1 cm. becomes less favourable. If less oil is used, strands of mycellium may be exposed which allows the cultures to dry out. If McCariney bottles are used the rubber liners should be removed from the metal caps as the oil tends to dissolve the rubber and this can be toxic 10 the cultures. This method has the following advantages: (5) Practically all bacterial species or strains tested live longer under oil than in the control tubes without oil. Some bacterial species have been preserved satisfactorily for 15 to 20 years.

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fig 7.6 Lyophilization process for preservation of cultures Raper and Alexander1942 first applied the process of lyophilization to microfungi on a large scale. They were successful in processing the cultures at the N.R,R.L. (Northern Regional Research Laboratory) at Peoria. Lyophilization is perhaps the most popular form of suspended metabolism. It consists of drying cultures or a spore suspension from the frozen state under reduced pressure. This can be accomplished several Ways. There are various kinds of equipment available to do this. Major steps involved in this technique are (Fig. 3.6): (i) A cell or spore suspension is prepared in a suitable protective medium (at the Commonwealth Mycological Institute 10% skimmed milk and 5% inositol in distilled water is found suitable). (ii) Using a sterile technique, the suspension from (i) is distributed in small quantities into glass ampoules. (iii) The ampoules are connected with a high vacuum system usually incorporating a desiccant (e.g. phosphorous pentoxide, silica gel or a freezing trap), and immersed into a freezing mixture of dry ice and alcohol. (iv) The vacuum pump is turned on and the ampoules are evacuated till drying is complete after which they may be sealed off. The details of the methods used vary from one laboratory to another.

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(ii) Transplants may be prepared when desired without affecting the preservation of the stock cultures.

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Factors affecting the viability of freeze-dried cultures include: (I) Chemical composition of the protective (suspending) medium: (ii) Addition of certain compounds to the culture suspension before freeze-drying to give protection to the culture against the toxicity exerted by moisture and oxygen when stored in unsealed ampoules; (iii)Sealing the ampoules after freeze-drying to stop access of oxygn and moisture; (iv)Insufficient elimination of oxygen and moisture on the survival rate of freeze-dried culture; and (v) Storage temperature of freeze-dried and sealed cultures. This method possesses the following advantages: (i) As the ampoules are sealed there is no risk of contamination or infection with mites. (ii) There is less opportunity for cultures to undergo changes in characteristics (i.e. they remain unchanged during storage period). (iii)Owing to the small size of glass ampoules, hundreds of lyophilized cultures can be stored in a small storage space. In addition to this, the ampoules’ small size makes them ideal for postage. (v) Lyophilization cuts down the number of transfers.

Notes

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UNIT- 2 AN INTRODUCTION TO FERMENTATION TECHNOLOGY

LESSON 8: FERMENTOR DESIGN

I. There are two types of fermentors with reference to their size

In this lecture, you will learn

a) Pilot Plant Fermentors

Requirements of a fermentor design

B) Large Fermentor

Types of fermentors Solid state fermentations The main function of fermentor is to provide a controlled environment which allows an efficient growth of cells and product formation. The modern fermentor is designed as a sophisticates unit having the properties and instrumentation necessary to develop and operate a variety of fermentation process.

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What are the essential requirements of a fermentor?

Any fermentor has to satisfy some basic requirements like: 1. The vessel must be strong enough to withstand the pressure of large volumes of aqueous medium. 2. The vessel should not corrupt the fermentation product or contribute toxic ions to the growth medium. 3. Usually pure culture is used in the fermentor. Therefore, provision for control of or prevention of growth of contaminating microorganisms must be provided. 4. If the fermentation is aerobic then provision must be made for rapid incorporation of sterile air into the medium in such a manner that oxygen of this air is dissolved. This is utilized by microorganism that evolves CO2 which must be removed through a different flushing system. 5. Some form of starring should be available. 6. The fermentor should provide facilities for the intermittent addition of anti foam agent as demanded by the foaming state of the medium. 7. The fermentor should posses a temperature control for the effective growth of the organisms. 8. The fermentor should also posses a mechanism for detecting pH values of culture medium and mechanism to adjust the pH according to the microbial growth. 9. There must be a drain in the bottom of the fermentor or some mechanism provided for removing the completed fermentation broth from the tank.

What are the various types of fermentors? Fermentors could be classified on the basis of many parameters. These parameters are like size, type of fermentation, application of the fermentation etc. Fermentor is available in varying sizes. These sizes are based on the total capacity of the fermentor. Fermentors can be classified according to their size and mode of culture design.

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Pilot fermentors are used in large state studies of fermentation. They have a size of twenty five to hundred gallons and can go unto 2000 gallons of total volume. Large fermentor is used in industrial production of fermentation product of microbial cells. They have size of 5000 to 10,000 gallons. II. The second type of classification has two types of fermentor. 1. Submerged cultured designed. 2. Solid substrate fermentors.

1.Submerged Culture Design The example for this design a) STR system b) Tower fermentor, c) Activated sludge process.

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Learning Objectives

FERMENTATION TECHNOLOGIES

A) Str System It is the most widely used bio reactor for aerated fermentation. It exhibits reliability and flexibility. It has a modified aerated system. Example for STR system is frings acetator used in vinegar production.

3. They are also used in vinegar production and alcohol fermentation. 4. Tower ferment or operate as continuous system with bottom entry feed and top exit. 5. It has high biomass retention.

In this design air is drawn in and distributed via high speed hollow body turbine rotor connected to an air suction pipe. The aerator is self aspirating.

One Type of lower fermentor is loop airlift bioreactor it has an internal or external drafts tube is baffled. It increases mixing by forcing a directional flow of bulk liquid. Another type of tower fermentor is ICT pressure cycle reactor. Here air is introduced at the base of the fermentor and forced into solution by hydrostatic pressure of the bottom. It is used in SCP production. Loop air lift bioreactor is used in methanol production.

C.Activated Sludge Process 1. It is another example of submerged culture design. 2. It is used in the municipal treatment of waste water. 3. This design has variation from other submerged fermentor it has an oblong deep activated tank at one end to which the inoculated waste water are introduced b.Tower Fermentor 1. Air is introduced at the base of fermentor. 2. They are used in citric acid production using pellets of Aspergillus Niger and Candia guylliermondi. 3. They are also used in vinegar production and alcohol fermentation. 4. Tower ferment or operate as continuous system with bottom entry feed and top exit. 5. It has high biomass retention. 1. Air is introduced at the base of fermentor. 2. They are used in citric acid production using pellets of Aspergillus Niger and Candia guylliermondi.

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2. Soild Substrate Fermentor

4. Aeration is accomplished by a fast moving ceramic disc and air nozzle to provide finally dispersed air bubble with consequent solution of oxygen in liquid phase.

FERMENTATION TECHNOLOGIES

Fig. Activated sludge units (Abson and Todhunter,1967)

5. Fresh alcoholic stock solution is added from a concurrently operating fermentor.” 6. When the alcoholic reaches 0.5% fresh alcohol is added automatically. 7. The completes fermentation broth is with drawn

They include 1. Slow continuous agitation system like the rotating drums. 2. Tray system. 3. Air flow system. a) The rotating drums usually equipped with an inlet and outlet for circulation of humidified air and often contain baffles to agitate the contents. b) Tray fermentors holding 1 to 2 inch deep layers of substrate are stalked in chambers usually force aerated with humidified. c) In forced air circulation chamber bed temperature is monitored and the appropriate temperature adjustment is made to the recycling flow.

Advantages 1. Superior productivity. 2. Simpler Technique 3. Low capital investment 4. Reduced energy requirement 5. Low waste water out put 6. No foam problems

Solid State Fermentors

Limitations 1. Heat build up 2. Bacterial contamination 3. Difficulty of controlling substrate moisture level. Operation Example: Aerator used in vinegar production 1. Provide high aeration levels. 2. The agitator can pump or cycle its own air and a compressor is not required. 3. Aerator operates in a semi batch manner.

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Batch Fermentor Batch fermentors are used to carry out micro-biological processes on a batch basis. There are a number of steps involved. These are associated with the development of microorganisms from a stock culture, and include agar slope and shake-flask stages. Thereafter, this is followed by ‘seed’ and production stages. Size Batch fermentors are available with varying capacities. The capacity of the tank may range from a few hundred to several thousand gallons. The capacity of the fermentor is usually stated on the basis of the total volume capacity of the same. Thus, small laboratory fermentors, pilot-plant fermentors and larger or production fermentors may be available. Small

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laboratory fermentors are in the size range of 1 to 2 litres with a maximum upto 12 to 15 litres. Pilot plant fermentors have a total volume of 25 to 100 gallons upto 2000 gallons total volume. Larger fermentors range from 5,000 or 10,000 gallons total volume to approximately 1,00,000 gallons. Still larger sized fermentors are rarely employed. These are spherical (Horton spheres) with a size range of 2,50,000 to 5,00,000 gallons total capacity.

Of

blades are available a Of blades are available and are used according to the requirements. The shaft passes through a bearing in the lid of fermentation tank. It is rotated with the help of an electric

Batch Fermentors Actually, the working volume in a fermentor is always less than that of the total volume. In other words, a ‘head space’ is left at the top of the fermentor above the aqueous medium. The reason for keeping a head space is to allow aeration, splashing and foaming of the aqueous medium. This head space usually occupies a fifth to a quarter or more of the volume of the fermentor. pH Control

pH Control is achieved by acid or alkali addition, which is controlled by an auto-titrator. The autotitrator, in turn, is connected to a pH probe. Temperature Control

Temperature control is achieved by a water jacket around the vessel. This, is often supplemented by the use of internal coils, in order to provide sufficient heat-transfer surface. Agitation

The agitating device consists of a strong and straight shaft to which impellers are fitted. An impeller, in turn, consists of a circular disc to which blades are fitted with bolts. Different types of blades are available, and are -used according to the requirements. The shaft passes through a bearing in the lid of the fermentation tank. It is rotated with the help of an electric motor mounted externally at the top of the tank. Usually, the speed of the agitator is varied with the help of adjustable pulleys and belts connected with the motor. In some cases, where the agitator is directly driven, impeller action is varied by the use of different types of impeller blades. Recently, the impeller of small fermen-tors is moved by a magnetic coupling to a motor mounted externally at the bottom of the tank. More-over, the height of the impeller blades above the bottom of the ferment or is adjustable according 10 ounces desire. The liquid medium is thrown up towards the walls of the fermentor while rotating the impeller blades at a high speed. This results in the formation of a vortex which is eliminated, usually, by four equally spaced baffles attached to the walls of the fermentor.

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of blades are available, and are used according to the requirements. The shaft passes through a bearing in the lid of the fermentation tank. It is rotated with the help of an electric motor mounted externally at the top of the tank. Usually, the speed of the agitator is varied with the help of adjustable pulleys and belts connected with the motor. In some cases, where the agitator is directly driven, impeller action is varied by the use of different types of impeller blades. Recently, the impeller of small fermen-tors is moved by a magnetic coupling to a motor mounted externally at the bottom of the tank. More-over, the height of the impeller blades above the bottom of the ferment or is adjustable according to one’s desire. The liquid medium is thrown up towards the walls of the fermentor while rotating the impeller blades at a high speed. This results in the formation of a vortex which is eliminated, usually, by four equally spaced baffles attached to the walls of the fermentor.

Aeration Usually, the aerating device consists of a pipe with minute holes, through which pressurized air escapes into the aqueous medium in the form of tiny air bubbles. This aeration device is called a sparger. The size of the holes in a sparger ranges from 1/64 to 1/32 of an inch or larger. Holes smaller than this require too high an air pressure for economical bubble formation. One should always remember that the smaller the air bubbles, the greater- is the bubble surface area. Subsequently, it is more likely that the oxygen of that air would

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FERMENTATION TECHNOLOGIES

pass across the bubble boundary and dissolved in the aqueous medium. However, small air bubbles require higher air pressure for their formation through the fine holes. It is desirable to adjust the size of the air bubbles to give the greatest possible aeration without greatly increasing the overall cost of the fermentation process. The reason for this is that sterile air is a costly item for large scale fermentation. Figure 7.4(a) shows the conventional type of ring sparger where the orifices face upwards, just below -the impeller. When working with mycelium-forming microbial fermentations with heavy mycelia and involving long fermentation times, clogging problems can become serious. The second reason for the clogging of the orifices is occasional power failures, when the cel1ulose particles settle down by gravity to plug the orifices. These problems have been overcome by a simple modification by changing the direction of orifices by a full 1800 (Fig.7.4(b)). As a result, the orifices now face downwards, and fermenta-tions run successfully. Time

The time required for a batch fermentation varies from hours to weeks, depending upon the conversion being attempted, and the conditions used (Rhodes and Fletcher, 1966). Throughout this time, contamination must be avoided and the vessel contents must be agitated and their temperature controlled.

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Designing

The design problem associated with a deep tank fermentor lies in the specification of the size of the vessel, the process time, the initial reactant (substrate) concentrations required, the holdup volume of microbial mass per unit volume of a fermentor, of micro-organisms, the power and aeration requirements and the area of heat-transfer surface.

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1. Air –pressure regulator 2. Araotameter 3. Glass-wool air filter 4. Millipore air filter 5. Check valve 6. Air sparger 7. Heating and cooling 8. pH electrode 10. Centrifugal pump 11. Thermistor probe 12. Silica gel bed 13. Diaphragm pump 14. Paramagnetic oxygen analyzer 15. Recorder

Continuous Stirred-tank Fermentor A continuous stirred tank fermentor is not different from the batch fermentor except insofar as feed and overflow device are added. The fundamental difference lies in the fact that the content of the vessel are at steady state. Steady-state conditions can be achieved by operation on either ‘chemostatic’ or ‘turbidostat’ principles. The former involves adjusting of the flow-rate to the fermentor to an appropriate and constant value and allowing the micro-organism, substrate and biochemical product concentrations to attain their natural levels.

The ‘Turbidostat’ requires an experimental determination of the turbidity (Le. an indirect measurement of the microbial concentration). This is then used to control the flow-rate. Both these methods have been employed in practice, though the former is obviously the simpler from every view point. Consequently, it is the one used in operations other than those on a laboratory scale. The immediate consequence of the steady state condition is, that for satisfactory economic opera-tion the environmental conditions selected for the fermentor have to lead to acceptable yields of microbial and biochemical products. This single feature demands depth of knowledge of the physio-logical and biochemical factors (which influence the microbial activity) far in excess of that demanded by batch fermentations (where detailed empirical procedures for fermentor operation are usually developed over a period of years). Once a small yield of product has been achieved in a batch fer-mentation, this can be 54

enhanced on a developmental basis (Demain, 1971). In contrast, for conti-nuous operation detailed knowledge is demanded of all the relationships between the rate of reaction and the operating variables. A further complication arises in that ‘natural’ media, media obtained from complex natural sources, in particular farm products or other fermentations e.g. molasses and corn steep-liquor, as opposed to specially prepared chemical substrate solutions, are the norms in the fermentation industries. Therefore, the determination of the effects of all the concentration vari-ables is scarcely feasible. The use of such media results in a further restraint, since the concentration, on a relative basis, .are fixed and this makes it difficult to consider a variety of inlet compositions as part -of the design exercise. Detailed application of batch data to a continuous system that presents significant difficulties, and these are scarcely made easier by the physiological and biochemical changes known to occur in the micro-organisms themselves during the period of a batch fermentation (Herbert, 1961). The most successful continuous systems to date have been those employing yeasts and bacteria, in which the desired products are the cells or primary metabolites, compounds that form the chemical ‘inventory’ Of a microbe, (e.g. enzymes and amino acids), or some product clearly associated with growth or energy producing mechanisms (e.g. the production of alcohol). A comment made by Righelato and Elsworth (1970) is worth noting, ‘...if they wish to adopt continuous methods, industrial researchers must be prepared to carry out their own empirical research as they have done with batch cultures, perhaps developing both new strains and media’. In fact, there appears to be no reason why any fermentation cannot be achieved in a continuous process, provided that the eco-nomic justification can be made, and the will to achieve it exists. The most widely used continuous process based on the CSTF is the activated sludge process (Ainsworth, 1970). It is used in the waste water treatment industry. About this design, we have already seen.

In continuous processing the autocatalytic (a reaction in which one of the products of the reaction increases the overall rate of a reaction) nature of microbiological reactions takes on a further signi-ficance. This is because the presence of one of the products, additional micro-organisms, enhances the overall rate of reaction. In the absence of micro-organisms no reaction can take place. Therefore, it is essential to retain at least a portion within the fermentor. It follows that if the flow rate is raised to a high value, then all the micro-organisms will be swept from the fermentor, and the conversion will cease. This phenomenon is commonly known ‘.Wash-out’. Obviously, if micro-

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10. Washing vessel 11. Carbon dioxide inlet 12. Heat exchanger (cooler)

13. Reservoir 1. Finishing inlet fig.: Flow sheet of the contineous, single stage fermentation for the production of beer (Coutts,1958,1961).

Both these methods have been successfully applied under industrial conditions for the production of bee (Coutts,1958,1961) as given in figure. The latter method is a normal feature of the activated sludge process as shown in fig following figure A constant inflow of microorganisms obviates the washout problem. It also result in increased productivity from the higher total hold-up of microorganisms within the fermentor.

Wort Inflow 1.a Control Valve 2. Fermentor 3. Impeller 4. Beer outflow 4a.Control valve 5. Sedimentation vessel 6.Yeast recirculation 7. Pump 8. Recirculation control valve 9. Clear beer outflow

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organisms are fed to the fermentor simultaneously with the substrate feed, the problems associated with wash-out are abated, and the reaction proceeds normally. Operation under such conditions requires a continuous flow of micro-organisms identical with those within the fermentor. The logical source of these is the effluent stream, as this contains micro-organisms and nutrients in the same biochemical and physiological conditions, as those within the fermentor. The effluent may be passed through either a centrifuge or a sedimentation tank. This operation produces a concentrated microbial suspension for recycling to the fermentor..

FERMENTATION TECHNOLOGIES

LESSON 9: DESIGN AND WORKING OF A BIOREACTOR

Learning Objectives Control of bioreactor parameters

The three basic modes of bioreactor operation are batch, fedbatch and continuous. The control issues related to each of these will be discussed in later sections.

Mathematical models for cell growth

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In this lecture, you will learn

Feedback control systems and optimal control theory

As we know, the functioning of a bioreactor can be divided into three major steps, viz. upstream processing, bioreactions and downstream processing. The optimal performance of the upstream processing can not be achieved without the knowledge of the state of the system and on control algorithms that can optimize the process. Controls of bioprocesses is challenging, particularly in batch and fed-batch bioreactors, due to high degree of nonlinearity (meaning that nonlinear differential equations are required for mathematical modeling); and their potential for instability when they involve high-yield mutant or recombinant microorganism. These problems are further complicated by the scarcity of on-line real -time sensors and realistic models that capture the intricate complexities of biological systems.

Hi there. Have you seen an industry? I mean a resllybig industry? What are the different departments it has? First and foremost, it has the production unit, which is the heart of the industry. Then there are the utility services which supply the different resources required for production. Then there are administrative and control systems. Then there is security system. So on and so forth. Have you seen a biochemical industry? An industry where numerous complicated biochemical reactions are carried out with clockwork precision. An industry that is highly efficient consumes a minimum of resources and generates very low effluent? Oh yes, you have. All of us have. These remarkable industries are our microscopic living cells. All plant, animal and microbial cells can be considered as microscopic biochemical factories. Materials such as carbon, nitrogen, oxygen and others are brought into the cell and converted within the cell via hundreds of reactions to the various constituents of the cell as well as to biochemical products, which may be retained or transported back into the environment outside the cell. Metabolic activities inside the cell are regulated at various levels both inside and outside the cell. Moreover, biological activity of the cell is extremely sensitive to the environment it is exposed to. Because of this multi - level complex regulation, by an engineering point of view, it is of the most importance to understand the nutritional and environmental factors affecting cell metabolism. This is especially important during the designing of a bioreactor.

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An engineer is always interested in consistently producing large quantities of product of interest over long periods of time. The best way to achieve this goal will be to grow the cells in a bioreactor where the cellular activity can be controlled efficiently.

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Ok, what are the different parameters that need to be considered during the design of a bioreactor? Well, what makes the bioreactors different from the chemical reactors is the presence of the living microorganisms inside the bioreactor. It is extremely important to gather the knowledge about the state of the bioreactor prior to design and implementation of any control system for the reactor. The complete state of the biochemical reactor can be assessed by knowing the parameters like physical parameters, chemical (extracellular) parameters, biochemical (intracellular), and biological parameters. We will see them one by one. First the physical parameters. The important physical parameters for the operation of a bioreactor are agitation power, agitation speed, broth volume, color, expanded broth volume (density), foaming, gas flow rate, gas humidity, heat generation rate, heat transfer rate, liquid feed rate, liquid level, mass, osmotic pressure, pressure, shear rate, tip speed, temperature, turnover time, and viscosity. Many of these parameters have important implications in the control of bioreactors.

What as a Bioreactor and How is it Designed? A bioreactor, in simple words, is an equipment in which the growth and metabolism of cells takes place. Common example of a bioreactor is a fermentor. All animal and plant tissue culture growth assemblies are also bioreactors. It is extremely important that the environment in a bioreactor remains most suitable for the growth and, more importantly, the metabolite production of the cells. As we have seen before these two sets of conditions need not be the same and, in many cases, they are not.

How do we achieve the control of various process parameters in the bioreactors?

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That’s a long list. Now what are the chemical parameters? The list giving the different parameters that define the chemical environment inside the reactor are amino acids, carbon dioxide (gas), cation level, conductivity, inhibitor, intermediate(s), ionic strength, malliard reaction products, nitrogen (free and total), nutrient composition,

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advantage that the nonlinear model may better represent the process over a significant range of state values, whereas the linear empirical model resulted from the former approach may not be reliable for process states away from the state at which model is identified. This is particularly important in case of batch and fed – batch fermentations in which the process state changes significantly during operation. The disadvantage of the latter approach is that the available controller - design tools are less developed for the nonlinear models.

The biochemical parameters are the intracellular parameters that indicate the metabolic state of the cell at any given time during the cell growth. These include amino acids, ATP/ADP/AMP, carbohydrates, cell mass composition, enzymes, intermediates, NAD/NADH, nucleic acids, total protein, and vitamins.

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And what are the biological parameters? Biological parameters characterize the bioreactor in terms of what is happening inside the reactor at the cellular level. The list includes age/age distribution, aggregation, and contamination, degeneration, doubling time, genetic instability, morphology, mutation, size/size distribution, total cell count and viable cell count.

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All right. Now which are the most important control parameters and how do we develop strategies for their optimization?

This is a very important question. All the bioreactors used at the present time use control strategies for three basic environmental factors: pH, temperature and dissolved oxygen. Invariably, these control implementations are achieved via regulation of flow rate of acid/base, flow rate of fluid through the cooling coil, and agitation respectively. Needless to say, these three parameters are extremely important for optimal cellular activity. But they alone do not guarantee the maximum productivity, which is the objective for most of the industrial fermentations. We will now see how this can be achieved. Before attempting to understand the details of the control strategies used for bioprocesses, we should be familiar with the common features in the field of controls. One of the integrated feature in any control system is control algorithm. The control algorithm is that part of the control system that takes the available measurements and level of process understanding and decides on the best way to influence the process with the available manipulated variable to achieve the desired objective. A control system can not be implemented unless the process under consideration is understood. An efficient way of understanding the process is a mathematical model of the process. A good process model is an invaluable tool to develop a control algorithm. It is not implied that controllers can not control poorly understood processes; indeed, that is often their function. However, an expensive, and time – consuming trial and error adjustment of the control algorithm is required in that case. A common approach to obtain a simple, empirical model for controller design is to make small step changes in the inputs and observe the dynamic behavior of the outputs. One can then obtain a linear, time – invariant process model in a straightforward fashion. A more fundamental approach is to formulate mass and energy balances for different components, resulting in a set of nonlinear ordinary differential equations. This approach has an 2.521

There are three modes of bioreactor operation: batch, fedbatch and continuous. The proper choice of the control algorithm will be dictated by the performance objective, process model, available measurements, and manipulated variables. We will see what all are the various tools to develop control algorithms Now let’s see some considerations about the bioreactor as a multivariable system with nonlinear dynamics

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That is quite a mouthful. What does it mean? The two main characteristics that are important to know before designing a control system for a bioreactor are: Multivariable system: that is the system involves many variables.

Nonlinear Dynamics The control of a bioreactor is complicated by the fact that nonsteady state behavior is nonlinear. This has several consequences. Hysteresis is often observed. For example, a step increase in reactor feed rate in case of CSTBR (continuously stirred tank bioreactor) will result in a transient that will be different than when the corresponding equivalent step decrease in feed rate back to initial conditions is made. Moreover, multiple steady states are often observed for identical feed conditions, and in certain cases, exotic dynamics like limit cycles, oscillatory transients, long time lags may be exhibited. The reasons for the above mentioned behaviors are ultimately related to the complexities of living cells. Finally, many of the important variables which are desirable for monitoring and control are only measurable with large time lags or not measurable at all. This gives scope for accurate mathematical models and/or state estimation techniques. Fortunately, simple models and single input - single output feedback loops are available and work well in many cases. • How do we develop a mathematical model for cell growth in a batch reactor? The simplest way to model cell growth will be to consider an unstructured, unsegregated model for cell growth. For this kind of model, rx = dX/dt = mX where, rx = rate of cell generation (g/l-hr) X = cell concentration (g/l) m = specific growth rate (hr-1) The most commonly used expression that relates the specific growth rate of the cell to the substrate concentration is Monod’s equation, which is given as

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oxygen, pH, phosphorous, precursor, product, redox and substrate.

dX/dt = (m - D)X m = m maxS/(Ks + S)

where D = dilution rate = F/V (hr-1)

where, m = specific growth rate (hr ) mmax = maximum specific growth (hr-1) S = substrate concentration (g/l) KS = saturation constant for substrate (g/l) One should note that Monod’s equation is empirical and does not have any mechanistic basis. The equation is only valid for an exponentially growing culture under condition of balanced growth. The equation does not fair well in transient conditions. Despite its simplicity and no fundamental basis, it works -1

A balance on the substrate yields the following equation FS - FSf + V(dS/dt) = rsV (5) where, F = volumetric flow rate (l/hr) S = cell concentration inside the bioreactor and in the outlet stream (g/l) Sf = substrate concentration in the feed (g/l) V = reactor volume (l) rs = rate of substrate consumption (g/l-hr) A yield parameter (Yx/s) is defined that relates the amount of cell mass produced per amount of substrate consumed, and is mathematically represented as Yx/s = mass of cells produced/mass of substrate consumed = rx/-rs (6) Combining equations (1), (5), and (6) yields dS/dt = D(Sf - S) - mX/Yx/s

(7)

The CSTBR (continuous stirred tank bioreactor) is now completely described by equations (4) and (7) with m given by equation (2). At steady state (with fixed Sf and D), the following are the values for m (specific growth rate), S (substrate concentration) and cell concentration (X) m=D

(8)

Figure 1. Monod’s growth curve surprisingly well in a large number of steady state and dynamic situations. This characteristic has important implications in control of bioreactors.

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Ok, and how do we study the dynamics of continuous bioreactor? For a continuously fed bioreactor, the cells are continuously supplied substrate at growth limiting level, and hence they remain in the exponential phase. Since the cells remain in the exponential phase, Monod’s equation can be applied. A cell balance on the reactor can be written as FX - FXf + V(dX/dt) = r x where, F = volumetric flow rate (l/hr) X = cell concentration inside the reactor and in the outlet stream (g/l) Xf = cell concentration in the feed (g/l) V = reactor volume (l) rx = rate of cell generation (g/l-hr) For a sterile feed (Xf = 0), and noting that the reaction rate can be written in terms of the specific growth rate (rx = mX), equation (3) can be reduced to

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S = DKS/(mmax D) X = Yx/s (Sf - S)

(9) (10)

There are a few characteristics of an open-loop CSTBR that are conceptually different from that of a chemical reactor which are important to know before any control system for a bioreactor can be designed. Figure 2 shows that D must be less than

mmax for a realistic value of S to be achieved. The same conclusion can Figure 2. Relationship between dilution rate and specific growth rate for a steady state CSTBR be derived by looking at the steady state solution of equation (4). The two solutions are equation (8) and X=0

(11)

The corresponding substrate concentration is S=Sf

(12)

Equation (11) and (12) define a situation called washout. This situation is encountered whenever the value of dilution rate equals or exceeds mmax. A rigorous discussion of washout would point to the fact that whenever m (Sf), i.e., m evaluated at Sf , is less than ?max, then the critical dilution rate for washout will occur at D = m (Sf), and not at D = mmax. The control algorithm should be completely aware of this unproductive state. For the given set of equations, numerical solution is required since the system is described by two coupled nonlinear differential equations, i.e., equations (4) and (7). Linear control theory can be applied in only a limited sense, i.e., only near the steady state when the system model is literalized. Start up is an important consideration as well. The general procedure in the start up avoiding washout would be to initiate cell growth in a batch mode until the exponential phase is reached. At this point, the sterile feed would be started with a dilution rate such that D < m (Sf). A non washout steady state would be reached after a transient phase. Additionally, the multiplicity and stability of the steady states should be studied for a continuous bioreactor.

What is this? Though the control loop of a CSTBR is simple, the system is complicated by the presence of multiple steady states and the stability considerations of these steady states. Let us see this aspect in details. As already implied, the control design of a biological reactor described by equation (4) and (7) should take into account the nonlinear nature of these differential equations. Multiple critical points are common with nonlinear systems. This has been shown earlier in the discussion of washout. A systematic approach to an efficient control design will involve 1. calculation of the number of steady states 2. characterization of the nature of the steady states with respect to their stability 3. design of appropriate control loops based on the results from step 1 and step 2 First, the calculation of multiple steady states .

Once the governing equations describing the system are in place, the steady states are found by replacing all time derivatives by zero. This can be done by inspection and algebric solution. For high order or complex models, a nonlinear root finding technique should be employed. Secondly, the stability of a steady state . A steady state is stable if, for initial conditions near the steady state, all transients converge to it. If the transients diverge, steady state is called unstable. The diverging transients always end at some other stable state. Stability analysis of a steady state would involve whether the steady state under consideration is stable or not and the information about state - to - state transitions in case of unstable steady states. The information about the stability and local dynamics of the steady states is accomplished through linear stability analysis. It should be borne in mind that the results of the linear stability analysis are good only near the steady state. For general (nonlocal) behavior and information about state - to - state transitions, generation of the phase plane is suitable. Lastly, before designing the closed - loop continuous bioreactor, one should understand the open - loop CSTBR fully since the scope of closed - loop CSTBR will be given only by the knowledge about the open - loop CSTBR.

What are feedback control systems? Feedback control is an action by which PID controllers as well as the controllers based on advanced control strategies implement their control action. In feedback control system, the controlled variable is measured and compared to the setpoint. Subsequently, an error signal is generated by subtracting the setpoint from the value of the controlled variable. Then the controller calculates the appropriate corrective action, to be implemented by the manipulated variable, by using the value of the error signal. In feedback control applications, the most widely used form for the control algorithm is PID controller equation given below p(t) = p + Kc [ e(t) + (int (e(t)dt))/T1 + TDde(t)/dt ] (24) where, p(t) = controller output p = bias, which is set at the desired output when the error signal is zero e(t) = controlled variable’s error int (e(t)dt) = integral of e(t) Kc = controller gain T1,TD = controller parameters It should be borne in mind that the feedback control can cause oscillations in closed - loop systems. Depending on the situation, these oscillations may or may not damp out quickly. In case where the oscillations may persist, the closed loop system is said to be unstable. This undesirable behavior can

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usually be eliminated by proper adjustments of the PID controller constants While the single-loop PID controller is satisfactory in many process applications, its performance is not satisfactory in many processes, e.g., the ones with slow dynamics, frequent disturbances, or multivariable interactions. In that case, various strategies like cascade control, time - delay compensation, and feedforward control can be employed to improve the performance. Excellent review about these techniques has been given by Fordyce et al.

balance equations, whose quantities can be measured directly or indirectly.

•

There are several types of advanced controllers. The most effective control system can be visualized as the one with an advanced digital control system using a model predictive controller. One of the most beneficial results of computer technology revolution has been the ease in process control. With all the modern hardware and software in place, it is extremely simple to solve complex mathematical models on line. State estimation techniques make the heart of these advanced controllers. Let me tell you more about these State estimation techniques. Briefly, state estimation techniques are the techniques of obtaining complete and accurate characterization of a process’s current condition from an incomplete, noisy set of process measurements. These techniques are absolutely essential to modern control of fermentation systems, due to the unavailability of adequate on – line sensors for many biochemical process states.

How do we achieve the feedback control of multivariable systems ? Many processes contain a number of manipulated variables and controlled variables. Such a process is called multivariable system. A multivariable control system can be treated as a control system comprising of several single - loop controllers. The techniques discussed earlier can only be used if the interactions between these controllers are not strong. If such is not the scenario, the controllers need to be detuned to reduce oscillations. A better approach is to utilize multivariable control techniques such as optimal control.

•

Yes. There are component balancing technique is used to provide on - line estimates of the fermentor state variables which then are used by the control algorithm, filtering techniques to generate growth rate estimates, integration of the rates can be performed to estimate other states such as biomass concentration. This approach does not work well in the practice since all real measurements are associated with error. This issue is addressed by classical state estimation theory and particularly in this case by filtering techniques. Optimization and control of a bioreactor can best be accomplished with a model – predictive control technique. In this technique, a dynamic model of the process calculates the response as a function of initial conditions, input, and/or setpoint changes. Future compensation is computed to maximize a profit function or to optimize a control function such as the sum of squares of the off - set residuals.

·What is optimal control theory? Optimal control theory provides an appealing framework for controller analysis and design since it treats very general models and can account for state and manipulated variable directly. Therefore, optimal control theory is an excellent tool for controller design for nonlinear, strongly interacting, and multivariable fermentors that can not be treated with simple multiloop PID controllers. Further, we must know about adaptive control. The control algorithms that are discussed in the feedback control system sections are tuned based on either time invariant models or trial and error techniques. This poses serious problem in case of fermentation processes since these processes are highly nonlinear systems with poorly understood dynamics and time - varying parameters and a linear controller with constant tuning parameters may not be suitable for it. This situation necessitates a need for changing the controller tuning parameters to achieve satisfactory system performance. An obvious approach to fulfill this need is to adapt the controller based on the present operating conditions. This approach is appropriately known as adaptive control. As already indicated, in adaptive control system, the controller learns about the process by acquiring data from the process and keeps on updating the control model. A parameter estimator monitors the process and estimates the process dynamics in terms of the parameters of a previously defined mathematical model of the process. A control design algorithm is then used to generate controller coefficients from these estimates, and the controller sends the required control signals to the devices controlling the process. An extremely important feature of an adaptive controller is the structure of the model used by the parameter estimator to analyze estimates of process dynamics. The process can be described by a set of mass

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What are the more advance types of controllers?

•

How are the controllers designed? The features that distinguish the biochemical reactors from the chemical reactors,as seen before are

1. Several of the crucial variables can not be directly measured quickly or easily. Quite often a time delay, which is larger than the system time-constants, is associated with their measurement. Therefore, a mathematical model must be used in place of feedback information. 2. Linear system analysis is mostly not applicable in case of bioreactors, especially for evaluation of long term response, since biochemical reaction systems usually are nonlinear. Hence, numerical solution of differential equations is required. 3. As already pointed out, the fed-batch reactor operation does not have a true steady state. In this case, evaluation of the state variables will locate the position of the system on a trajectory through the operational cycle. Since these state variables can not be measured online, the estimation of state becomes an important element of optimization and control of the reactor.

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What are the potential problems in bioprocess - control designing? The difficulty of implementing a feedback control is three fold. First, response of oxygen sensors tends to be slower than many of the processes they monitor. Second, sensors are generally not available for measurement of substrate with rapid dynamics for feedback application. And third, there is also generally not available a sensor for the biomass concentration without which the state of the system can not be estimated. Let us study a Case: Implementation of a control process to maximize the enzyme (protease) production by a marine bacterium, Teredinobacter Turnirae in a high cell density fedbatch fermentation process

Objective To design a control algorithm to maximize the protease productivity by Teredinobacter Turnirae in a fed-batch fermentation process. System description

1. The cells are grown aerobically in the presence of a single carbon source, i.e., sucrose. 2. The protease is an extracellular and growth - associated product.

consumption rate (rs(g/l-hr)), oxygen consumption rate (ro(g/lhr)), and enzyme production rate (rp(g/l-hr)) are given by rx = mX

(26)

rs = - (mX)/Yx/s - amX/YP/ S

ro = - (mX)/Yx/o - amX/YP/ O

(28)

rp = amX

(29)

where, X = cell concentration (g/l) Yx/s = gms of cells produced/gms of substrate consumed YP/S = gms of enzyme produced/gms of substrate consumed Yx/o = gms of cells produced/gms of oxygen consumed YP/O = gms of enzyme produced/gms of oxygen consumed a = gms of enzyme produced/gms of cells produced Eq. (29) results from the fact that the enzyme is a growth associated product. For a fed batch reactor in which a substrate at concentration Cs (g/l) is introduced at a rate of F (l/hr), the dynamic mass balance equations for biomass (X), substrate (S), oxygen (CL), enzyme (P(g/l)), and volume of liquid (V(l)) in bioreactor are given by

3. Preliminary experiments show that the enzyme production is repressed by the presence of excess sucrose.

dX/dt = rx - (F/V)X

Assumptions

dS/dt = rs + F/V(Cs - S)

1. The cells exhibit balanced growth.

(30) (31)

dCL/dt = ro + KLa(C - CL) * L

2. The reactor is considered a well - mixed system. 3. Sucrose and oxygen are limiting nutrients.

5. There are no nutritional requirements for the maintenance of the cells. 6. Yx/s, Yx/o, YP/S, YP/O, Ks, Ko, mmax, and a are constant. 7. The critical concentration of oxygen, i.e., below which oxygen becomes limiting, is 10%.

Mathematical Description The growth of the cells can be represented by a model of Monod’s type for two limiting substrates, i.e. sucrose and oxygen in this case. According to this model, the specific growth rate of the bacterium can be written as a function of the concentrations of sucrose (S) and oxygen (CL): m = mmax (S/(S + Ks))(CL/(CL + Ko)) (25) where m = specific growth rate (hr-1) mmax = maximum specific growth rate (hr-1) S = substrate concentration (g/l) Ks = saturation constant for substrate (g/l) CL = oxygen concentration (mg/l) Ko= saturation constant for oxygen (mg/l) Neglecting the endogenous metabolism, the expressions for biomass production rate (rx(g/l-hr)), substrate

(32)

dP/dt = rp - (F/V)P

4. Monod’s equation is valid to represent the dependency of m on S.

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(27)

(33)

dV/dt = F

(34)

where, KLa = volumetric gas - liquid mass transfer coefficient CL* = equilibrium solubility of oxygen in broth Using eqs. 26, 30, 34, and using the relation for the total cell mass, i.e., M(g) = XV, the following equation can be derived dM/dt = mM

(35)

If the cells grow at a constant m during the fed - batch fermentation, then the expression for M is given by M = Moexp(mt)

(36)

Where, Mo = total cell mass before starting the feed (g) = XoVo where, Xo = biomass concentration before starting the feed (g/l) Vo = volume of the fermentor before starting the fermentor (l) t = elapsed time since beginning of the feed Similar expressions to eqs. 35 and 36 for total enzyme mass (Mp) can be derived by using eqs. 29, 33, and 34, which are given by dMp/dt = amM p

(37)

Mp = Mpoexp(mt)

(38)

Where, Mpo = total cell mass before starting the feed (g) Hence, m should be kept as high as possible to maximize enzyme productivity (g/l-hr). The desired value of m which

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should be maintained in the reactor is mmax, which is the physiological high limit for the specific growth rate of the cell. The high specific growth rate of cells, however, has to be constrained due to the limited oxygen transfer capacity in fermentation system and the reduced enzyme production at high m. The latter constraint arises because of the fact that high concentration of sucrose results in reduced enzyme production. The value of the desired m can be found out by doing the shake - flask experiments at different substrate concentrations. The value of m corresponding to optimum substrate concentration for the enzyme production would be the desired value of m to be maintained in the fed - batch operation.

the user. Finally, an output is given from the nodes on the output layer. “Knowledge based” control of fermentation processes is an exciting area of research devoted to solving control problems associated with the uncertainties and nonquantitative nature of biochemical systems. The use of fuzzy logic is most attractive for fermentation processes because of the poorly understood dynamics but with a good amount of process information accumulated as experienced knowledge of a human operator.

Notes

An optimal cycle for this fed batch fermentation would involve the initial feed profile to maintain the constant m. The flow rate of sugar solution, F, required for the growth of cells at a desired specific growth rate (m) can be calculated by solving eqs. 27, 31, and 36, and is given as F = (mXoVoexp(mt)/Cs)((1/Yx/s) + (a/YP/S)) (39) The following assumptions are made while deriving the above relationship 1. Quasi - steady state w.r.t substrate is assumed to exist throughout the fed - batch fermentation span where cells grow at constant m, that leads to the derivative dS/dt reducing to 0. 2. Cs >> S. This profile should continue till the system reaches the critical oxygen concentration. Afterwards, the oxygen concentration would be controlled at 10% of its maximum solubility. The feed profile now would be changed accordingly to control oxygen concentration at this desired level. This feed profile can be numerically calculated by using eqs. 26,27,28,31,32, and 34. What lies Ahead?

Neural network has received a great deal of attention over the past decade among scientists and engineers in all the disciplines. The term “neural network” resulted from artificial intelligence, that aims at understanding and modeling brain behavior. A neural network can be considered as an expert computing system comprising of a number of simple, highly interconnected nodes or processing elements, in which information is processed by their dynamic state response to external inputs. The goal of a neural network is map a set of input patterns onto a corresponding set of output patterns. This mapping is achieved by neural network system by first learning from a series of past examples defining sets of input and output correspondences. The network then applies what it has learned to a new input to predict the appropriate output. A typical neural network architecture consists of various nodes that are interconnected via links that are used that are use to transfer the information. The neural network has an input layer, at least one (normally 1 to 3) hidden layer, and an output layer. The whole network can be viewed as a “black box” into which specific inputs (each corresponding to a specific node) are sent. This information is processed through the interconnections between nodes, but the entire processing step is hidden from

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Learning Objectives

Sterilization of the culture Media

In this lecture, you will learn

Nutrient media as initially prepared contain a variety of different vegetative cells and spores, derived from the constituents of the culture medium, the water and the vessel. These must be eliminated by a suitable means before inoculation. A number of means are available for sterilization, but in practice heat is the most often used mechanism. A number of factors influence the success of heat sterilization: the number and type of microorganisms present, the composition of the culture medium, the pH value, and the size of the suspended particles. Vegetative cells are rapidly eliminated at relatively low temperatures such as 600C for 5-10 minutes, but for destruction of spores, temperatures of 1210C are needed during 15 minutes.

• • •

Sterilization of culture media Kinetics of sterilization Heat exchanger designs

Introduction Hello students, now that we have seen about the fermentation medium, let’s see about another important aspect of the fermentation process viz. the sterilization. It would not be difficult to imagine the reasons behind the need to sterilize the fermentation medium, equipments and the air used for sparging. During the course of fermentation, we are interested in the growth of the desired organism only and hence, as far as possible, all contaminants should be kept at bay. This may not be always possible and indeed, some fermentations are found to be contaminated especially during the later stages. In some fermentations, the cost of sterilization becomes prohibitive especially if the final selling price of the product is low. But in virtually all fermentation processes, it is mandatory to have contamination free seed cultures at all stages, from the preliminary culture to the fermentor. What will happen if the fermentation is carried out under non sterile conditions? 1. Loss of productivity because of contaminant growth overtaking that of the desired organism.

During heat sterilization there is always the possibility of destroying ingredients in the medium. Apart from the degradation of heat-labile components, also contributes to the loss of nutrient quality during sterilization. A common phenomenon is the occurrence of the Maillard-type browing reactions, which cause discoloration of the medium as well as loss of nutrient quality. These reactions are normally caused by carbonyl groups, usually from reducing sugars, interacting with amino groups from amino acids and proteins. Separate sterilization of the carbohydrate component of the medium may be necessary to prevent such reactions.

•

Filter sterilization is often used for all components of nutrient solutions, which are heat sensitive. Sugars, vitamins, antibiotics or blood components are examples of heat-labile components which must be sterilized by filtration.

2. Total displacement of the desired organism by contaminants especially in continuous fermentations. 3. Contamination of the final product/s by the contaminating organisms especially when the final product is cell mass. ( e.g. SCP )

Most nutrient media are presently sterilized in batch volumes in the bioreactor at 1210C. Approximate sterilization times can be calculated from the nature of the medium and the size of the fermentor. Not only the nutrient media, but also the fitting, valves and electrodes of the fermentor itself must be sterilized. Therefor, actual sterilization times are significantly longer than calculated ones and must be empirically determined for the specific nutrient solutions in the fermentor. Smaller fermentors are sterilized in an autoclave while larger fermentors are sterilized by indirect or direct steam injection.

4. Interference in the final recovery process due to contamination 5. Contamination of an antagonistic / pathogenic organism could result into rapid death of the host organism bringing the fermentation to a complete halt.

•

All right. Now, what are the major considerations to be made during the sterilization operations? Ideally, all the inputs required for the fermentation should retain a state of absolute sterility all the time. First, there is the fermentor vessel to be sterilized. Fermentors can be sterilized either by destroying the viable microorganisms by a physical procedure such as filtration. Then we have to sterilize the culture media and the incoming and outgoing air. Additionally, attention has to be paid to the appropriate construction of the bioreactor for sterilization and for prevention of contamination during fermentation. We will now see these operations in details.

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What’s the alternative then, to heat sterilization?

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·Ok. That was about the sterilization of media. How do we then sterilize the air that is used for the fermentations? Most fermentations are operated under high aeration and the air supplied to the fermentor must be sterilized. The number of particles and microorganisms in air varies greatly depending on the location, air movement, and previous treatment of the air. On the average, outdoor air has 10100,000 particles per m3 and 5-2,000 microorganisms per

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LESSON 10: STERILIZATION

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m3. Of these, 50% are fungus spores and 40% are Gramnegative bacteria.

There have been some attempts to commercialize enzymatic sterilization of air. The basic concept is to bring microorganisms and viruses into contact with enzymes that attack nucleic acids. Viruses are destroyed by passage through a web of surfaces coated with deoxyribonuclease enzymes.

Fermentors generally work with aeration rates of 0.5-2 vvm (air volume/liquid volume per minute). The methods available for sterilizing gases include filtration, gas injection (ozone), gas scrubbing, radiation (UV) and heat. Of these, only filtration and heat are practical. Can you tell me why the other methods are not practical? Use the space below to express your thoughts.

•

Tell me more about the sterilization of air by filtration. The volume of air handled during aerobic fermentations is very high. Very large compressors are used, and at least two are required so that one can be down for maintenance. In the past, air filters were columns that approached diameters of one-fourth of the fermenter diameter. The packing was slag wool that lumped up with repeated use, fiberglass that broke down because of repeated thermal expansion and contraction, or beads of carbon that sometime underwent spontaneous combustion and melted the column. Carbon packing works fairly well but is too bulky. Currently, there is a pronounced trend to use of membrane filters in a cartridge configuration for air sterilization to obtain excellent performance with units of relatively small size.

•

You mentioned about the design of the fermentor affecting the process of sterilization. How? The fermentor design should not encourage contamination at any stage. There should be a minimum number of openings in the fermentor to favor maintenance sterility. Small openings must be made leak proof with O-rings, larger openings with flat gaskets. Whenever a movable shaft penetrates the fermentor wall, special problems of sterility maintenance should be solved. The material of construction of the fermentor should permit easy and repeated sterilization. The fermentor vessel should have minimum dead and inaccessible areas.

Moisture is bad for all methods of air sterilization and may help microorganisms to pass. A membrane pore size of 0.2 to 0.3 micrometers is recommended. Hydrophilic membranes should not be used because moisture held tightly in the pores is not dislodged unless there is quite high-pressure drop across the membrane. Moisture tends to drain from hydrophobic membranes and collect in a sump. The units are modular and housed in a shell with a manifold. Sizing is based on the number of cartridges needed.

•

Can we use filters for the air that is going out of the fermentors?

Remember,

As a rule, the air going out of all fermentations should be free of living organisms, microbial products or spores. Because of all the problems mentioned above, filtration is not a reliable method for the control of organisms in the air that is leaving the fermentor. Air leaving a vessel in which pathogenic organisms are cultured is sterilized by heating. Air in a room for culturing microorganisms may be exposed to ultraviolet light to reduce the number of potential contaminants. Ultraviolet light penetrates poorly through glass, so organisms in shake flasks are not killed. It is also necessary to regularly replace the ultraviolet source if its continued efficacy is desired. Usually, a single light switch turns on white light before a person enters, and the Ultraviolet goes on when the person flips the switch on leaving. There are also ultraviolet lights mounted in flow devices for water sterilization, but quartz bulbs or enclosures are needed to get out of the altering of Ultraviolet wavelengths. Such devices are also plagued by turbidity in the water and by dirt forming on the transparent surfaces. 64

•

·Understood. Now how do we carry out the sterilization operation as such?

a) For all sterilization calculations, we are considering the total no. of organisms present in the volume of the medium to be sterilized, not the concentration b) The minimum number of organisms needed to contaminate a batch is one, regardless of the volume of the batch. c) Any system is either sterile or non sterile. Nothing like partial sterility exists. The destruction of micro-organisms by steam (moist heat) may be described as a first-order chemical reaction and, thus, may be represented by the following equation: -dN/dt = kN

(1)

Where; N t is the number of viable organisms present,

t is the time of the sterilization treatment, k is the reaction rate constant of the reaction, or the specific death rate.

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Nt/No = e –kt

(2)

equations (3) and (5), the following expression may be derived for the he at sterilization of a pure culture at a constant temperature:

Where;

In N0/Nt = A.t.e –E/RT

No is the number of viable organism present at the start of the sterilization

Deindoerfer and Humphrey used the term In No/lV, as a design criterion for sterilization, which has been variously called the Del factor, Nabla factor and sterilization criterion represented by the term V. Thus, the Del factor is a measure of the fractional reduction in viable organism count produced by a certain he at and time regime. Therefore:

Treatment, Nt is the number of viable organisms present after a treatment period t, On taking natural logarithms, equation (2) is reduced to: In( Nt/No) = -kt

(3)

It is be seen that viable organism number declines exponentially over the treatment period. A plot of the natural logarithm of Nt/No against time yields a straight line, the slop of which equals –k. This kinetics description makes two predictions which appear anomalous: 1. An infinite time is required to achieve sterile conditions (i.e. Nt =0) 2. After a certain time there will be less than one viable cell present. Thus, in this context, a value of Nt is less than one is considered in terms of the probability of an organism surviving the treatment. For example, if it were predicted that a particular treatment period reduced the population to 0.1 of a viable organism, this implies that the probability of one organism surviving the treatment is one in ten. This may be better expressed in practical terms as a risk of one batch in ten becoming contaminated by one organism.

V=In ( N0/Nt ) But, In( N0/Nt ) = kt And kt = A.t.e –(E/RT) Thus v=A.t.e–(E/RT) (8) On rearranging, equation (8) becomes, In t = E/RT + IN (V/A) (9) Thus, a plot of the natural logarithm of the time required to achieve a certain V value against the reciprocal of the absolute temperature will yield a straight line, the slope of which is dependent on the activation energy. It is clear that the same degree of sterilization (V) may be obtained over a wide range of time and temperature regimes; that is, the same degree of sterilization may result from treatment at a high temperature for a short time as from a low temperature for a long time.

•

•

where E is the activation energy, R is the gas constant, T is the absolute temperature. On integration equation (4) gives: K= Ae –E/RT

(5)

Where A is the Arrhenius constant. On taking natural logarithms, equation (5) becomes: In k = In A - E/RT. (6) From equation (6) it may be seen that a plot of In k against the reciprocal of the absolute temperature will give a straight line. Such a plot is termed an Arrhenius plot and enables the calculation of the activation energy and the prediction of the reaction rate for any temperature. By combining together

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These calculations would work fine for a heat stable medium wherein no degradation or interaction will take place between the media ingredients. But what about natural or crude media? This is a very valid point. When we are using a crude medium, for example, molasses, the thermal degradation of its various components becomes extremely significant. Sugars, especially are prone to quick thermal degradation. Extended sterilization periods often drastically reduce the nutritive value of crude fermentation media, mainly due the thermal degradation of sugars. Interestingly, when the same medium is autoclaved only briefly, its nutritive value is found to increase.

As with any first-order reaction, the reaction rate increases with increase in temperature due to an increase in the reaction rate constant, which, in the case of the destruction of microorganisms, is the specific death rate (k). Thus, k is a true, constant only under constant temperature conditions. The relationship between temperature and the reaction rate constant was demonstrated by Arrhenius and may be represented by the equation: d Ink/dT =E/RT2 (4)

(7)

Why should this happen? This is because of a ‘cooking effect’ that makes more nutrients from the crude media available after they are briefly exposed to heat and pressure. Take the example of saccarification of starch. When heated under pressure, starch is hydrolysed to oligosaccharides and sugars, thereby improving its degradability. Subsequent heating, however, results in browning and charring of starch thereby making it less degradable and less nutritive. The reactions between the carbonyl groups from the reducing sugars and the amino groups from amino acids and proteins also result in reduction of nutritive value of the media. Certain amino acids, vitamins and proteins will also suffer from thermal degradation thereby making the medium less nutritive. These problems can be generally solved by separately sterilizing the heat sensitive ingredients using gentler methods of sterilization like filtration.

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On integration of equation (1) the following expression is obtained:

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Depending on the type of fermentation, there are two different types of sterilizations. What are those?

•

•

See, the two basic types of fermentations from the operational point of view are batch and continuous fermentation. Accordingly, sterilization can also be classified into batch type and continuous sterilization. First let’s see about batch sterilization. A1though a batch sterilization process is less successful in avoiding the destruction of nutrients than a continuous one, the objective in designing a batch process is still to achieve the required probability of obtaining sterility with the minimum loss of nutritive quality. ‘The highest temperature which appears to be feasible for batch sterilization is 121°C so the procedure should be designed such that exposure of the medium to this temperature is kept to a minimum. This is achieved by taking into account the contribution made to the sterilization by the heating and cooling periods of the batch treatment. What do we need to know when we are designing a batch sterilization process?

•

cooling, the holding time may be calculated to give the required overall Del factor.

(i) A profile of the increase and decrease in the temperature of the fermentation medium during the heating and cooling periods of the sterilization cycle.

How do we do that? The relationship between Del factor, the temperature and time is V = A. (. e-(E/RT). However, during the heating and cooling periods the temperature is not constant and, therefore, the calculation of V would require the integration of equation (5.8) for the time-temperature regime observed. Deindoerfer and Humphrey (1959) produced integrated forms of the equation for a variety of temperature-time profiles, including linear, exponential and hyperbolic. However, the regime observed in practice is frequently difficult to classify, making the application of these complex equations problematic. The time axis is divided into a number of equal increments, t1,t2,t3, etc., Richards suggesting 30 as a reasonable number. For each increment, the temperature corresponding to the mid-point time is recorded. The total Del factor of heating up period is equivalent to the um of Del factors of the midpoint temperatures for each time increment. The value of Del factor corresponding to each time increment may calculate from the equations: V1= k1t,

(ii) The numbers of microorganisms originally present in the medium.

V2= k2t,

(iii) The thermal death characteristics of the ‘design’ organism. As explained earlier this may be Bacillus stearothermophilus or an alternative organism relevant to the particular fermentation.

The sum of the Del factors for all the increments will then equal the Del factor for the eating –up period. The Del factor for the cooling-down period may in a similar fashion.

Knowing the original number of organisms present in the fermenter and the risk of contamination considered acceptable, the required Del factor may be calculated. A frequently adopted risk of contamination is 1 in 1000, which indicates that Nt should equal 10-3 of a viable cell. It is worth reinforcing at this stage that we are considering the total number of organisms present in the medium and not the concentration. If a specific case is considered where the unsterile broth was shown to contain 1011 viable organisms, then the Del factor may be calculated, thus: v = In (1011 /10-3) v = In 1014 = 32.2. Therefore, the overall Del factor required is 32.2. However, the destruction of cells occurs during the heating and cooling of the broth as well as during the period at 121°C, thus, the overall Del factor may be represented as: Voverall = Vheating + Vholding + Vcooling Knowing the temperature-time profile for the heating and cooling of the broth (prescribed by the characteristics of the available equipment) it is possible to determine the contribution made to the overall Del factor by these periods. Thus, knowing the Del factors contributed by heating and

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V3 = k3t, etc.

What are the different methods of batch sterilization? The batch sterilization of the medium for fermentation may be achieved either in the fermentation vessel or in a separate mash cooker. The major advantages of a separate medium sterilization vessel may be summarized as: 1. One cooker may be used to serve several fermenters and the medium may be sterilized as the fermenters are being c1eaned and prepared for the next fermentation, thus saving time between fermentations. 2. The medium may be sterilized in a cooker in a more concentrated form than would be used in the fermentation and then diluted in the fermenter with sterile water prior to inoculation. This would allow the construction of smaller cookers. For example, in the case of alcohol fermentation, where dilute molasses is used as the substrate, the concentrated molasses could be sterilized in the mash cooker and it can be diluted with sterile water in the fermentation vessel. 3. In some fermentation, the medium is at its most viscous during sterilization and the power requirement for agitation during sterilization is very high. If the sterilization is to be carried out in the fermentation vessels themselves, the power requirement would be much more. In such cases, it would be

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4. The fermenter would be secure from the corrosion which may occur with the fermentation medium at high temperature.

exchanger, is held in the coil, and passes back through the heat exchanger, heating more unsterile medium while becoming cool itself, as it is collected in a sterile fermenter.

•

The name is self explanatory, isn’t it? A heat exchanger is an instrument in which two fluids come in indirect contact with each other exchanging their heat content. In other words, one fluid loses heat and the other gains it. To further simplify, one fluid gets heated and the other gets cooled.

The major disadvantages of a separate medium sterilization vessel may be summarized as: 1. The cost of constructing a batch medium sterilizer is much the same as that for the fermenter. 2. If a cooker serves a large number of fermenters complex pipe work would be necessary to transport the sterile medium, with the inherent dangers of contamination.

•

In a heat exchanger, there is a cold stream and a hot stream. The two streams are separated by a thin, solid wall. The wall must be thin and conductive in order for heat exchange to occur. Yet the wall must be strong enough to withstand any pressure by the fluid. Copper seems to be one of a common choice for construction. Here is a simple flow diagram showing how heat transfers in a heat exchanger.

All right. Now tell me about continuous sterilization. Batch sterilization wastes energy and can overcook the medium. Batch sterilization uses steam or direct firing to elevate the temperature, and then cooling water stops the process and brings the material back toward room temperature. Both the heat and the cooling water are spent with no opportunity for energy recovery. Large volumes should be passed continuously through heat exchangers for energy economy with the hot, treated fluid heating the cold, incoming feed. The advantages offered by continuous sterilization include very short heating up times, suitability for media containing suspended solids, low capital costs, easy cleaning and low maintenance and high steam utilization efficiency. The steam requirements in case of continuous sterilization would be more uniform throughout the duration of the sterilization. The application of continuous sterilization would also simplify process control and reduce the time required for sterilization. . Shorter sterilization time means less thermal degradation of medium The disadvantages include the possibility of foaming and the condensation of steam in the medium diluting it. The application of continuous sterilization demands high steam requirements in a shorter period of time than batch sterilization. Since steam is actually dispersed in media, steam must be clean to avoid contamination .these issues must be addressed to in case of continuous sterilization. One method of continuous sterilization injects steam into the medium (no heat exchanger). The medium stays in a loop for a predetermined holding time until the entire medium is sterile. Better heat economy comes from substituting heat exchangers for direct steam injection. Instead of having a cold water stream to cool the sterile media, the lower temperature unsterile media stream absorbs heat from the warm stream, cooling the sterile media.

How does a heat exchanger work? A heat exchanger utilizes the fact that heat transfer occurs when there is a difference in temperature.

3. Mechanical failure in a cooker supplying medium to several fermenters would render all the Fermentors temporarily redundant. The provision of contingency equipment may be prohibitively costly.

•

What is a heat exchanger?

This flow arrangement is called co-current. If the direction of one of the stream is reversed, the arrangement is called counter-current flow. Here are the temperature profiles along the heat exchanger. Note that the temperature profiles are different for co-current flow and for counter-current flow. Air or water cooled radiators of a car is one of the commonest examples of heat exchanger. Can you think of other common examples of heat exchange? Feel free to use the space provided below to express your thoughts.

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·What does a heat exchanger looks like? Depending on the structural assembly, there are many types of heat exchanger. Shell and tube exchanger, plate heat exchanger etc. are some of the common examples.

•

What is a shell and tube type exchanger? When the flow in a heat exchanger is countercurrent (i.e. against each other), the outlet temperature of the stream being heated can approach the temperature of the hot stream to be cooled. Countercurrent heat exchanger provides more effective heat transfer. Most of the industrial heat exchangers are counter-current flow design. There is an attempt to show this in the sketch. There are gradients on the shell side as well.

A system for continuous sterilization has a holding coil for detention long enough to kill all of the microorganisms. The medium from a make up vessel flows through the

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economical if the sterilization is carried out in a smaller kettle.

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Shell and Tube Exchangers are common throughout the chemical process industries for heat economy. Many tubes go from a header on one side to a header on the other. The other fluid is in the space outside the tubes. Hot streams exchange energy with colder streams so that the thermal energy of the hot streams is not wasted. Furthermore, companies are not allowed to discharge hot streams that can damage the environment, so removing heat from a waste hot stream is important. The colors in this sketch are supposed to represent heat gradients. Red is hottest, and yellow is coolest. Note that the outlets cannot reach exactly the same temperature as the inlet of the other stream except when there is infinite surface for heat transfer. The sketch is a little misleading for the shell temperatures because there should be a left to right gradient when the inlet and outlet are at different ends. The Shell and tube exchanger is not as well suited to continuous sterilization as the plate-frame type of exchanger.

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·What is a plate heat exchanger? Plate Heat Exchangers utilize corrugated plates stacked between a fixed and a movable pressure plate. The corrugation patterns alternate for maximum operating pressures. As virtually all of the material is used for heat transfer, Plate Heat Exchangers can have large amounts of effective heat transfer surface in a small footprint. It is not uncommon that a Plate Heat Exchanger will have the same thermal capacity as a Shell & Tube five times larger. The following diagrams give an idea about the designing and working of plate heat exchangers.

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On what does the performance of a heat exchanger depend? To maximize the performance of a heat exchanger means saving money, especially if the process is built for a longterm project. Here are some ways to improve the performance of a heat exchanger:

1. heat transfer area 2. fluid flow velocity 3. temperature gradient These suggested ways of improvements are based on the equation for heat transfer rate of a heat exchanger, which is:

Q=U*A*dTlm Where, Q = Heat transfer rate between the fluids U = Overall heat transfer coefficient

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High velocity creates high shear stress in flow. Some proteins or cell structures are very delicate. They cannot withhold such force and they will be destroyed. The whole batch can be ruined.

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And the temperature gradient. What is it and how does it affect the heat transfer efficiency? Temperature gradient is certainly an important part of heat transfer. It is the driving force for heat transfer. If we can introduce fluids with greater temperature difference into the heat exchanger, the heat transfer rate (Q) will be greater. If we go back to the temperature profiles of the co-current and counter flow, we can see that the driving force is great for cocurrent at the beginning but decreases drastically as it moves along the heat exchanger. The counter-current flow provides relatively consistent driving force and therefore performs better than co-current flow.

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OK. Now about the actual sterilization process using the heat exchangers. How is it done? Look at the diagram below. This clearly shows how the fermentation broth is first heated in the heat exchanger and then sterilized in the holding coils.

This design would work only with an exchanger with infinite heat transfer area because there is no driving force for heat transfer as the temperatures for the two streams approach closely. A real design would have another small exchanger to raise the temperature to the set point after the main exchanger has done all it can do. There is no need for a cooler before entering the fermenter because it has a jacket or coils for temperature control that can easily handle this load.

As the cooling fluid velocity increases, the cooling fluid is able to dissipate heat more effectively. The data have shown that it is the case. Although increasing flow velocity can give more effective heat transfer, it may not be a good idea in some bio-process. 2.521

Heat economy is not important for a small pilot plant unit for continuous sterilization, so direct steam injection is simpler. A heat exchanger is then needed with cooling water to bring the medium back down quickly to a temperature at which it is not over cooked. High temperatures for short times are used in preparing nutrient media for industrial fermentations and in pasteurizing milk, because this causes less damage to biochemicals than more prolonged times at lower temperatures. This exploits the temperature effects on activation energies because bacterial

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A = Heat transfer area dTlm = Log mean temperature difference of the system • How does the heat transfer area affect the performance of the heat exchanger? I will give you an example. Imagine two towels of the same size and same fabric. Both are dipped in water and allowed to get wet thoroughly. Now both these towels hold the same degree of moisture. One towel is fully spread over a stand whereas the other one is folded into a ball. Which one of them will dry first and why? The heat transfer area (or contact area) is directly proportional to the heat transfer rate. If the heat transfer area increases, heat transfer rate increases as well. The towel which is well spread has a larger surface area as compared to the one which is folded into a ball and hence loses heat faster and consequently dries faster. A common way to increase heat transfer area is adding fins to the surface. It is cheap to put fins to the heat transfer area but fins also increase fouling, especially in bio-process. • The speeds with which the fluids flow through the exchanger also affect the rate of heat transfer, right? Yes. The importance of the fluid flow in a heat exchanger is that it changes the overall heat transfer coefficient, U. The data obtained from the heat transfer experiment shows that the velocity of the cooling fluid is directly proportional to the overall transfer coefficient. The following is a plot of 1/U vs. 1/ V0.8 during one of the runs during the lab experiment.

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killing is affected by a temperature change more than is heat destruction of biochemicals. The main purpose of the heat exchanger in a bio-process is sterilization. There are other ways to kill unwanted organisms (contaminants), such as using chemicals and filtration. However, using heat energy seems to be the best way to sterilize feed before entering to the reactor.

Exercise 1. Visit an industrial unit near your campus. Study the different types of heat exchangers. Find out about the common problems encountered in the operation of a heat exchanger. 2. Learn about the terms condenser and cooler. Find out how they differ from heat exchanger. 3. Learn about the different metals used in the manufacturing of heat exchangers. Find out more about heat transfer efficiency, heat transfer rate, heat transfer coefficient and theoretical calculation of heat transfer between two fluids.

Notes

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Learning Objectives

3. the factor which will influence the rate of oxygen transfer into solution

In this lecture, you will learn

• • •

The requirement for oxygen in fermentation processes

•

The quantification of oxygen transfer and Products of fermentations

How do we calculate the requirement of oxygen in a fermentation process? The stoichiometry of respiration gives an appreciation of the problem of oxygen supply; it gives no indication of an organism’s true oxygen demand as it does not take into account the carbon that is converted into biomass and products. To predict the oxygen demand for fermentation a number of workers give following equation given in Table

The factor which will influence the rate of oxygen transfer into solution

Hello students; How many of you are interested in Yoga? It is an ancient Indian science of achieving complete control over body and mind. One of the most popular techniques involved in Yoga is the Pranayama, where you scientifically learn to control your breathing. Even otherwise, one of the indices of your overall health is how long can you hold your breath. If you can do it for 25 seconds or more, you are probably not having any serious health problem. Microorganisms, unfortunately, are deprived of this privilege of Yogic science and most of them require a continuous supply of oxygen for their growth and metabolism. The majorities of fermentation processes are aerobic and, therefore, require the provision of oxygen. If the stoichiometry of respiration is considered, then the oxidation of glucose may be represented as: C6HI2O6 + 6O2 = 6H2O + 6CO2 Thus, 192 grams of oxygen are required for the complete oxidation of 180 grams of glucose. However, both components must be in solution before they are available to a micro-organism and oxygen is approximately 6000 times less soluble in water than glucose (a fermentation medium saturated with oxygen contains approximately 7.6 mg dm-3 of oxygen at 30°C). Thus, it is not possible to provide a microbial culture with all the oxygen it will need for the complete oxidation of the glucose (or any other carbon source) in one addition. Therefore, a microbial culture must be supplied with oxygen during growth at a rate sufficient to satisfy the organisms’ demand. The oxygen demand of an industrial fermentation process is normally satisfied by aerating and agitating the fermentation broth. However, the productivity of many fermentations is limited by oxygen availability and, therefore, it is important to consider the factors which affect a fermentors efficiency in supplying microbial cells with oxygen. In this lesson, we will see 1. the requirement for oxygen in fermentation processes, . 2. the quantification of oxygen transfer and

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From theses determination it maybe seen that a culture’s demand for oxygen is very much dependent on the source of carbon in the medium. Thus, the more reduced the carbon source, the greater will be the oxygen demand.

•

From Darlington’s and Johnson’s equations it may be seen that the production of 100 grams of biomass from hydrocarbon requires approximately three times the amount of oxygen to produce the same amount of biomass from carbohydrate .

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Darlington’s, Johnson’s and Mateles’ equations only include biomass production and do not consider product formation

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whereas Cooney’s and Righelato’s equations consider product formation

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Ryu and Hospodka (1980) used Righelato’s approach to calculate that the production of 1g penicillin consumes 2.2 g of oxygen.

However, it is inadequate to base the provision. of oxygen for a fermentation simply on an estimation of overall demand, because the metabolism of the culture is affected by the

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LESSON 11: AERATION AND AGITATION

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concentration of dissolved oxygen in the broth. The effect of dissolved oxygen concentration on the specific oxygen uptake rate (Q02, mmoles of oxygen consumed per gram dry weight of cells per hour) has been shown to be of the MichaelisMenten type, as shown in Fig. 1.

Bartholomew et at. (1950) represented the transfer of oxygen from air to the cell, during fermentation, as occurring in a number of steps: 2. The transfer of oxygen from an air bubble into solution. 3. The transfer of the dissolved oxygen trough the fermentation medium to the microbial cell. 4. The uptake of the dissolved oxygen by the cell These workers demonstrated that the limiting step in the transfer of oxygen from air to the cell in Streptomyces griseus fermentation was the transfer of oxygen into solution. These findings have been shown to be correct for non-viscous fermentations but it has been demonstrated that transfer may be limited by either of the other two stages in certain highly viscous fermentations. The difficulties inherent in such fermentations are discussed later in this chapter. The rate of oxygen transfer from air bubble to the liquid phase may be described by the equation: dCL/dt = KLa(C*-CL) where;

Fig.4.1 The effect of dissolved oxygen concentration on Qc of a microorganisms From Fig 1 it may be seen that the specific oxygen uptake rate increases with increase in the dissolved oxygen concentration up to a certain point (referred to as Ccrit) above which no further increase in oxygen uptake rate occurs. Some examples of the critical oxygen levels for a range of micro-organisms are given in Table

C is the concentration of dissolved oxygen in the fermentation broth(mmolesdm 3 ), dt is time (hours), dC L/ dt is the change in oxygen concentration over a time period, i.e. the oxygen transfer rate (mmoles O2 dm-3 h-1), is the mass transfer KL is the mass transfer coefficient (cm h -1) a is the gas/liquid interface area per liquid volume (cm2cm-3) and C* is the saturated dissolved oxygen concentration (mmols dm3 ) K L may be considered as the sum of the reciprocals of the resistances to the transfer of oxygen from gas to liquid. (C* - CL) may be considered as the ‘driving force’ across the resistances.

Thus, maximum biomass production may be achieved by satisfying the organism’s maximum specific oxygen demand by maintaining the dissolved oxygen concentration greater than the critical level. If the dissolved oxygen concentration were to fall below the critical level then the cells may be metabolically disturbed. However, it must be remembered that it is frequently the objective of the fermentation technologist to produce a product of the micro-organism rather than the organism itself and that metabolic disturbance of the cell by oxygen starvation may be advantageous to the formation of certain products. Equally, provision of dissolved oxygen concentration far rather than the critical level may have no influence on biomass production, but it may stimulate product formation. Thus, aeration conditions necessary for the optimum production of a product may be different from those favouring biomass productions. How can we quantify the oxygen transfer?

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It is extremely difficult to measure both KL and ‘a’ in a fermentation and, therefore, the two terms are generally combined in the term KLa, the volumetric mass-transfer coefficient, the units of which are reciprocal time(h-1). The volumetric mass-transfer coefficient is used as a measure of the aeration capacity of a fermenter. The larger the KLa, the higher the aeration capacity of the system. The KLa value will depend upon the design and operating conditions of the fermenter and will be affected by such variables as aeration rate, agitation rate and impeller design. These variables affect ‘KL’ by reducing the resistances to transfer and affect ‘a’ by changing the number, size and residence time of air bubbles. It is convenient to use KLa as a yardstick of fermenter performance because, unlike the oxygen-transfer rate, it is unaffected by dissolved oxygen concentration. However, the oxygen transfer rate is the critical criterion in fermentation and, as may be seen from equation 1, it is affected by both KLa and dissolved oxygen concentration. The dissolved oxygen concentration reflects the balance between the supply of dissolved oxygen by sign the fermentor and the oxygen demand of the organism. If the KLa

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•

What are the factors affecting KLa values in fermentation vessel?

A number of factors have been demonstrated to affect the KLa value achieved in a fermentation vessel. Such factors include:

and aerate. The flow pattern of bubbles through a bubble column reactor is dependent on the gas superficial velocity (cm second -1). At gas velocities of below 1-4 cm second-I the bubbles will raise uniformly through the medium (Van’t Riet and Tramper, 1991) and the only mixing will be that created in the bubble wake. This type of flow is referred to as homogeneous. At higher gas velocities bubbles are produced unevenly at the base of the vessel and bubbles coalesce resulting in local differences in fluid density. The differences in fluid density Create circulatory currents and flow under these conditions is described as Heterogeneous as shown in following Figure 4.3

1. the air-flow rate employed 2. the degree of agitation 3. the rheological properties of the culture broth 4. the presence of antifoam agents

.

First we will see the effect of air-flow rate on KLa . This will have to be seen in cases of mechanically agitated reactors and non mechanically agitated reactors.

Mechanically Agitated Reactors The effect of air flow rate on KLa .values in conventional agitated systems is illustrated in Fig. 4.2

Fig. 4.3 The effect of air flow rate on the flow pattern in stirred vessel (A) Non-aerated, (B to F) increasing air flow rates Flooding in a bubble column is the situation when the air flow is such that it blows the medium out of the vessel. This requires superficial gas velocities approaching 1 m second -1 which are not attainable on commercial scales (Van’t Riet and Tramper, 1991). The volumetric mass transfer coefficient (KLa) in a bubble column is essentially dependent on the superficial gas velocity. Heijnen and Van’t Riet (1984) reviewed the subject and demonstrated that the precise mathematical relationship between KLa and superficial gas velocity is dependent on the coalescent properties of the medium, the type of flow and the bubble size. Unfortunately these characteristics are rarely known for a commercial process which makes the application of these equations problematical. However, Van’t Riet and Tramper (1991) claimed that the relationship derived for non-coalescing, non-viscous, large bubbles (6 mm diameter) will give a reasonably accurate estimation for most non-viscous situations:

Fig 4.2 The effect of air flow rate on the KLa of an agitated aerated vessel. The air-flow rate employed rarely falls outside the range of 0.51.5 volumes of air per volume of medium per minute and this tends to be maintained. Constant on scale-up. If the impeller is unable to disperse the incoming air then extremely low oxygen transfer rates may be achieved due to the impeller. Becoming ‘flooded’. Flooding is the phenomenon where the air-flow dominates the flow pattern and is due to an inappropriate combination of air flow rate and speed of agitation.

Non-mechanically Agitated Reactors Bubble columns and air-lift reactors are not mechanically agitated and, therefore, rely on the passage of air to both mix 2.521

KLa = 0.32 (Vcs)0.7 Where, Vcs is the superficial air velocity corrected for local pressure. However, viscosity has an overwhelming influence on KLa in a bubble column which Deckwer et al. expressed as: KLa =CII -0.84 Where; II is the liquid dynamic viscosity (N s m-2). The practical implication of this equation is that bubble columns cannot be used with highly viscous fluids. Van’t Riet and Tramper (1991) suggested that the upper viscosity limit for a bubble column was 100 x 10- 3N s m - 2 at which point the K L a would have decreased 50 fold compared with a reactor batched with water.

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of the fermenter is such that the oxygen demand of the organism cannot be met, the dissolved oxygen concentration will decrease below the critical level (Cerit). If the KLa is such that the oxygen demand of the organism can be easily met the dissolved oxygen concentration will be greater than Cerit and may be as high as 70 to 80% of the saturation level. Thus, the KLa of the fermenter must be such that the optimum oxygen concentration for product formation can be maintained in solution throughout the fermentation.

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Air Lift Reactors The difference between a bubble column and an air-lift reactor is that liquid circulation is achieved in the air-lift in addition to that caused by the bubble flow. The reactor consists of a vertical loop of two connected compartments, the riser and downcomer. Air is introduced into the base of the riser and escapes at the top. The degassed liquid is denser than the gassed liquid in the riser and flows down the downcomer: Thus, a circulatory pattern is established in the vessel - gassed liquid going up in the riser and degassed liquid coming down the downcomer. For a given air-lift reactor and medium KLa varies linearly with superficial air velocity on a log-log scale over the normal range of velocities (Chen, 1990). However, it should be remembered that the circulation in an air-lift results in the bubbles being in contact with the liquid for a shorter time, in a corresponding bubble column. Thus, the KLa obtained in an air-lift will be less than that obtained in a bubble column at the same superficial air velocity, i.e. less than 0.32 (v,.c)O.7. The advantage of the airlift lies in the circulation achieved, but this is at the cost of a lower KLa value. As for a bubble column flooding will not occur within the normal operating superficial air velocities and should not be a problem on a large scale. The degree of agitation has been demonstrated to have a profound effect on the oxygen-transfer efficiency of an agitated fermenter. Banks (1977) claimed that agitation assisted oxygen transfer in the following ways: (j) Agitation increases the area available for oxygen transfer by dispersing the air in the culture fluid in the form of small bubbles. (ii) Agitation delays the escape of air bubbles from the liquid. (iii) Agitation prevents coalescence of air bubbles. (iv) Agitation decreases the thickness of the liquid film at the gas-liquid interface by creating turbulence in the culture fluid. The degree of agitation may be measured by the amount of power consumed in stirring the vessel contents. The power consumption may be assessed by using a dynamometer, by using strain gauges attached to the agitator shaft and by measuring the electrical power consumption of the agitator motor. The assessment of electrical consumption is suitable only for use with large scale vessels.

•

aeration and less torque fluctuations.) may give improved mixing in a xanthan fermentation provided that the polysaccharide concentration is below 25 kg m-3. A novel solution to the problem was proposed by Oosterhuis and Koerts (1987). These workers designed an air-lift loop reactor incorporating a pump to circulate the highly viscous broth. The system was operated on a 4 m-3 scales and proved to be much more efficient than a stirred tank reactor.

•

What is the effect of foam and antifoam agents on oxygen transfer? The high degree of aeration and agitation required in fermentation frequently gives rise to the undesirable phenomenon of foam formation. In extreme circumstances the foam may overflow from the fermenter via the air outlet or sample line resulting in loss of medium and product, as well as increasing the risk of contamination. The presence of foam may also have an adverse effect on the oxygen transfer rate. The presence foam in the region of the impeller may prevent adequate mixing the fermentation broth. Thus, it is desirable to break down a foam before it causes any process difficulties and this may be achieve by the use of mechanical foam breakers or chemical antifoams. However, mechanical foam control consumes considerable energy and is not completely reliable so that chemical antifoam is preferred. All antifoams are surfactants and may have some effect on oxygen transfer. The predominant effect observed by most of workers is that antifoams tend to decrease the oxygentransfer rate. Antifoam causes the collapse of bubbles in foam but they may favour the coalescence of bubbles within the liquid phase, resulting in larger bubbles with reduced surface area to volume ratio and hence reduced rates of oxygen transfer (Van’t Riet and Van onsberg, 1992). Foam formation has a perticular influence on the liquid height in the fermentor at which it is practical to operate. If inadequate space is provided above the liquid level for foam control, then copious amount of antifoam must be used to prevent loss of broth from the vessel. Van’t Riet and Van Sonsberg (1992) observed that, above a critical liquid height, the KLa value decreases dramatically due to the excessive use of antifoams. Thus, it may be more productive to operate a vessel at a lower working volume.

What is the effect of microbial product on aeration? The product of fermentation contributes relatively little to the viscosity of the culture broth. However, the exception is the production of bacterial polysaccharides, where the broth tends to be highly viscous and non-Newtonian. Normally, microbial polysaccharide tends to behave as pseudo plastic fluids. The yield of a polysaccharide make the fermentation particularly difficult because , beyond the certain distance from the impeller, the broth will be stagnant and productivity in these region will be practically zero. Thus, bacterial polysaccharide fermentations present problems of oxygen transfer and bulk mixing. It was concluded that better agitator ( when its pumping direction was upwards rather than downwards resulting in lower power, loss on

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Learning Objectives In this lecture, you will learn •

What is heat transfer?

•

What is mass transfer?

•

Gas-liquid mass transfer

•

Oxygen transfer

•

What is heat and mass Transfer? In the fermentation operations, the transfer of heat and mass is extremely important and generate copious quantities of heat requiring external cooling to maintain constant temperature. In other cases, additional heat needs to be provided to maintain constant temperature ensuring proper growth of microorganisms. The transfer of heat also plays a significant role in downstream processing. Heat and mass transfer in fermentations involves complex mathematical calculations. In order to make the understanding of the topic easier, we will restrict ourselves only to the basics of these calculations. In fermentor design, efficient heat transfer is important in controlling the temperature during sterilization operations and maintaining the required operating temperature throughout the fermentation run. Many fermentations are exothermic. Heat generated in the fermentation is primarily due to metabolic activity of microorganisms and mechanical agitation processes. For most fermentation this heat needs to be dissipated by cooling. Conversely, for fermentations that operate above ambient temperature, such as those involving thermophilic organisms, there needs to be an input of heat. Heat transfer is primarily achieved using an outer jacket surrounding the internal phase or via internal coils. No direct contact exists between the cooling or heating system and the fermentation medium. The heat is conducted through the vessel wall, coils and baffles. These systems are also used to sterilize the vessel and contents before inoculation, by the injection of pressurized steam. Automatic temperature control during the fermentation is accomplished by injecting either cold or hot water into the outer jacket and/or internal coils. In some circumstances alternative cooling media may be used, e.g. glycol.

•

What do we need to learn about mass transfer? It is also important to remember that mass transfer in fermentations refers to the transfer of nutrients in the cells and the release of waste products out of the cells. The transfer of various gases in and out of the cells plays an important role in mass transfer.

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We have seen that the fermentor contains millions or billions of cells per milliliter. As the sources and nutrients, cells, and metabolic products become further separated, the probability increases that some physical-transport phenomena, will influence or even dominate the overall rate of solute processing in the reaction volume under consideration. Indeed, cells and their component catalytic assemblies operate at the maximum possible rate without any serious diffusional limitation. If, in bioprocess circumstances, a richer supply of carbon nutrients is created, evidently the aerobic cell will be able to utilize them fully only if oxygen can also be maintained at a higher concentration in the direct vicinity of the cell. This situation may call for increased gas-liquid mass transfer of oxygen, which has sparingly small solubility in aqueous solutions, to the culture. Evidently, the boundary demarcating aerobic from anaerobic activity depends upon the local bulk-oxygen concentration, the O2 diffusion coefficient, and the local respiration rates in the aerobic region. This line divides the viable from dying cells in strict aerobes such as mold in mycelial pellets or tissue cells in cancer tumors; it determines the depth of aerobic activity near lake surfaces; and it divides the cohabitating aerobes from anaerobic microbial communities in soil particles. Thus, while the modern roots of biological-process oxygen mass transfer began with World War II penicillin production in the 1940s its implications are now established to include many natural processes such as food spoilage via undesired oxidation and lake eutrophication due either to inadequate system aeration by natural oxygen supplies or due to an excessive concentration of materials like as phosphate or nitrate. Other sparingly soluble gases are also of fermentation interest. Methane and other light hydrocarbons have been explored as gaseous substrates for single cell protein production, where, both oxygen and methane must be dissolving continuously at rates sufficient to meet the biological demand. Methane transfer out of solution is important in anaerobic waste treatment, at the metabolic end of which light carboxylic acids (primarily acetic acid are decarboxylated to give the corresponding alkanes. Carbon dioxide is generated in nearly all microbial activity. In spite of its large solubility, the interconversion between gaseous and the various forms of dissolved carbon dioxide (CO2, H2C03, HCO3-,CO3—3) couples its mass transfer rate to the pH variation; this topic figures importantly in controlling the pH of acid-sensitive anaerobic digestors where CO2 and CH4 removal occur simultaneously. Liquid-liquid mass transfer is important in SCP production from liquid hydrocarbon feedstocks, as well as in fermentation recovery operations; e.g., filtered or whole

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LESSON 12: MASS AND HEAT TRANSFER

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broth extraction of pharmaceuticals employing organic solvents. Renewable resource bioconversions, such as the use of cellulosic, hemicellulosic, and lignin fractions of agricultural and forest wastes as fermentation feedstocks, typically involve rate processes (biomass solubilization, liquefaction, hydrolysis) limited by available particulate substrate surface areas and solute diffusion rates. Other topics also involving liquid-solid mass transfer include various sorption and chromatographic methods for product recovery and purification, and liquid phase oxygen transfer to mold pellets or beads and biofilms containing immobilized cells.

As shown in above figure, sparingly soluble gas, usually oxygen, is transferred from a source, say a rising air bubble, into a liquid phase containing cells. (Any other sparingly soluble substrate, e.g., the liquid hydrocarbons used in hydrocarbon fermentations, will give the same general picture.) The oxygen must pass through a series of transport resistances, the relative magnitudes of which depend on bubble (droplet) hydrodynamics, temperature, cellular activity and density, solution composition, interfacial phenomena, and other factors. These arise from different combinations of the following resistances:

Operation at high cell densities may often result in masstransfer limited conditions, as observed in reactors as diverse as laboratory shake flasks or large scale fermentors for penicillin or extracellular biopolymers (xanthan gum) or activated sludge waste plants. The process engineer must, accordingly, know when transport phenomena or biological kinetics are rate-limiting in order to design bioreactors.

1. Diffusion from bulk gas to the gas-liquid interface

Transfer of nutrients from the aqueous phase into the microbial cells during fermentation is relatively straightforward as the nutrients are normally provided in excess. However, oxygen transfer in aerobic fermentations is rather more complex.

5. Transport through the second unmixed liquid region associated with the cells

Some fermentations operate anaerobically, but the majorities are aerobic and require the provision of large quantities of normally sterile air or oxygen that must be dispersed throughout the fermenter. Aeration is also useful for purging unwanted volatile metabolic products from the medium. Compressed air entering a fermenter is usually stripped of moisture and any oil vapours that may originate from the compressor. To prevent the risk of contamination, gases introduced into the fermenter should be passed through a sterile filter. A similar filter on the air exhaust system avoids environmental contamination. Sterile filtered air or oxygen normally enters the fermenter through a sparger system, and air flow rates for large fermentors rarely exceed 0.5-1.0 volumes of air per volume of medium per minute (vvm). To promote aeration in stirred tanks, the sparger is usually located directly below the agitator.

When the organisms take the form of individual cells, the sixth resistance disappears. Microbial cells themselves have some tendency to adsorb at interfaces. Thus, cells may preferentially gather at the vicinity of the gas-bubble-liquid interface. Then, the diffusing solute oxygen passes through only one unmixed liquid region and no bulk liquid before reaching the cell. In this situation, the bulk dissolved O2 concentration does not represent the oxygen supply for the respiring microbes.

2. Movement through the gas-liquid interface 3. Diffusion of the solute through the relatively unmixed liquid region adjacent to the bubble into the well-mixed bulk liquid 4. Transport of the solute through the bulk liquid to a second relatively unmixed liquid region surrounding the cells

6. Diffusive transport into the cellular floc, mycelia, or soil particle 7. Transport across cell envelope and to intracellular reaction site

Similarly, in the microbial utilization of other sparingly soluble substrates such as hydrocarbon droplets, cell adsorption on or near the hydrocarbon-emulsion interface has been frequently observed. The variety of macroscopic physical configurations by which gas-liquid contacting can be effected is indicated in the following figure:

• Ok, tell me about the gas – liquid mass transfer in biological systems

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•

Mechanically Aagitated • In general, we can distinguish fluid motions induced by freely rising or falling bubbles or particles from fluid motions which occur as the result of applied forces other than the external gravity field (forced convection). The 2.521

The oxygen transfer in freely rising fluids and mechanically stirred fluids seems to be different, it is?

Indeed Sparger structure can affect the overall transfer of oxygen into the medium, as it influences the size of the gas bubbles produced. Small bubbles are desirable because the smaller the bubble, the larger the surface area to volume ratio, which provides greater oxygen transfer. However, spargers with small pores that are effective in producing small air bubbles are more prone to blockage and require a higher energy input. Oxygen is only sparingly soluble in aqueous solution and the solubility decreases as the temperature rises. This adds to the other difficulties, particularly those caused by the large volume of the vessel, wherein there will be regions where mixing is less efficient. When high biomass concentrations are used to increase productivity it also creates an enormous demand for oxygen. Consequently, the operation of aerobic processes is generally more demanding, as it is difficult to prevent oxygen from becoming a rate-limiting factor. Oxygen transfer is complex, as it involves a phase change from its gaseous phase to the liquid phase, and is influenced by the following factors. 1 the prevailing physical conditions; temperature, pressure and surface area of air/oxygen bubbles; 2 the chemical composition of the medium; 3 the volume of gas introduced per unit reactor volume per unit time; 4 the type of sparger system used to introduce air into the fermenter; 5 the speed of agitation; or 6 a combination of these factors. During aerobic fermentations molecular oxygen must be maintained at optimal concentrations to ensure maximum productivity. The two steps associated with an oxygen mass balance are the rate at which oxygen can be delivered to the biological system (oxygen transfer rate, OTR) and the rate at which it is utilized by the microorganisms (critical oxygen demand). If the rate of oxygen utilization is greater than the OTR, anaerobic conditions will develop, which may limit growth and productivity. However, attempts can be made to raise the OTR by elevating the pressure, enriching the inlet air with oxygen, and increasing both agitation and airflow rates. The OTR is determined by the driving force, the oxygen gradient, and the resistance to oxygen transfer. Rate of oxygen transfer = driving force Resistance = oxygen gradient (C* - CL)

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distinction is not clear-cut; gas-liquid mixing in a slowly stirred semi batch system may have equal contributions from naturally convected bubbles and from mechanical stirring. The central importance of hydrodynamics requires us to examine the interplay between fluid motions and mass transfer. Before beginning this survey, some comments and definitions regarding mass transfer are in order.

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————

KLa where C* = saturated dissolved oxygen concentration (mmol/ dm3); CL = oxygen concentration at time, t (mmol/dm3); KL = mass transfer coefficient (cm/h), i.e. the sum of reciprocals for the residencies of oxygen transfer from the gaseous to liquid phase; and a = gas-liquid interface area per liquid volume (cm2/ cm3). It should be noted that both KL and a are difficult to measure individually and are usually linked together as KLa, the volumetric mass transfer coefficient (per hour). The driving force for oxygenation is the oxygen gradient (C* CL). Consequently, the rate of oxygenation is faster at low dissolved oxygen concentrations, compared with higher concentrations. However, the overall KL a is not affected. In order for oxygen to transfer from the gaseous phase to an individual cell or site of reaction, it must pass through several points of resistance, as seen in the following figure:

=

KLa(C* - CL)

dt Integration of equation 6.2 gives C* - CL = e-KLat and in terms of natural logarithms, for use in KL a determination below, this is In(C* - CL) = -KLa * t Therefore, KLa is a measure of the aeration capacity of the fermentor and must be maintained above a minimum critical level to supply the oxygen requirements of the organism. Determination of KLa is relatively straightforward and is used to compare fermentors in both scale-up and scale-down. A dynamic method suitable for some vessels involves filling the fermentor under investigation with medium, and fixing specific rates of aeration and stirring. The percentage oxygen saturation can be monitored by using a rapid response polarographic or galvanic oxygen probe. When the dissolved oxygen reaches saturation and a steady state is achieved, the air supply is replaced with nitrogen. Once the oxygen in solution has fallen to a sufficiently low level, usually 10% of its saturation (equilibrium) value, air is reintroduced and the exponential reoxygenation profile is recorded. The KL a for these specific conditions is determined by a semi log plot of the reoxygenation profile, In( C* - CL) against time, the slope of which is the negative of the mass transfer coefficient (-KL a). This procedure can be repeated under different operating conditions, e.g. varying stirrer speed, air flow rate, etc. Once in the liquid, the rate of oxygen acquisition by cells depends on the oxygen gradient between the oxygen in the bulk liquid and at the site of utilization.

1 resistance within the gas film to the phase boundary; 2 penetration of the phase boundary between the gas bubble and bulk liquid; 3 transfer from the phase boundary to the bulk liquid; 4 movement within the liquid; 5 transfer to the surface of the cell; 6 entry into cell; and 7 transport to the site of reaction within the cell. The rate-limiting step (controlling factor) in oxygen transfer is the movement of oxygen from the gaseous phase to the gasliquid boundary layer, particularly for viscous media. Gaseous oxygen molecules move rapidly, due to their kinetic energy. However, to enter the liquid they have to cross this boundary layer at the surface of the bubble. This is composed of a thin layer of oxygen molecules that line the inside of the bubble and a thicker layer of water molecules coating the bubble surface. Diffusion across this boundary is particularly influenced by temperature, solutes and surfactants. The rate of transfer of oxygen from an air bubble to the liquid phase is described by

Movement in the bulk liquid is aided by good mixing. The rate of use by the biological system will be determined by the affinity and saturation characteristics of the terminal oxidase. As microorganisms exhibit different oxygen requirements, the level of aeration necessary will vary from fermentation to fermentation. •

Ok, now, what can we learn about heat transfer?

In biological reactors, heat may be added or removed from a microbial fluid for the following reasons: 1. It is desired to sterilize a liquid reactor feed by heating in a batch or continuous flow vessel. Thus, the temperature desired must be high enough to kill essentially all organisms in the total holding time . 2. If the heat generated in substrate conversion is inadequate to maintain the desired temperature level, heat must be added. For example, the reactor is anaerobic sewage-sludge digestor which operates best between, say, 55 and 60°C ( See figure) 3. The conversion of substrate generates excess heat with respect to optimal reactor conditions for, e.g., maintenance of viable cells, so heat must be removed, as in most microbial fermentation processes. 4. The water content of a cell sludge is to be reduced by drying.

dCL

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Generally the heat required by microbial fermentations is provided by the coils placed in the inside of the fermentor. The presence of such internal piping clearly alters mixing patterns, fluid velocities, and perhaps bubble-coalescence rates. In very large scale systems with large heat loads, such as bacterial growth on methanol in a 1500 m3 reactor, internal coils become inadequate for cooling. Then, circulation through an external heat exchanger, or through an exchanger integral to a loop vessel configuration, is necessary. Here is an example where cooling loads, in concert with other considerations such as required power input for aeration and mixing, dictate a need for bioreactor designs substantially different from traditional agitated tank configurations.

The first three relate to cell viability and metabolism and will therefore be of concern here. The last example is a unit operation, drying The heat is transferred between the bioprocess fluid to or from a second fluid in several ways, i.e., with externally jacketed vessels, coils inserted in a larger vessel, flow through a heat exchanger, or by evaporation or condensation of water and other volatile components of the cell-containing fluid. Examples of such configurations are shown in Fig. 8.16. Temperature fluctuations between atmosphere and thermally stratified lakes and land also clearly involve heat transfer, the resulting temperatures determining the habitable niches for species. The present section focuses on heat transfer in process reactors. Assuming that .transfer rates and changes in other forms of energy are negligible, the fundamental steady-state equation in heat transfer relates the total rate at which heat is generated to its rate or removal through some heat-transfer surface; thus Net generation rate = removal rate = hADT Where D T = characteristic temperature difference between bioprocess and Cooling or heating fluid A = heat-transfer surface area h = overall heat-transfer coefficient As with mass transfer, most of the resistance to heat transfer resides in a relatively quiescent thin fluid near the solid heatingcooling boundary, the bulk fluid being frequently well mixed and thus approximately isothermal. Our main concern is development of an overall energy balance and review of useful predictive formulas for h for various heater-cooler-sterilizer systems of interest. The heat-transfer coefficients for boiling water and condensing vapors (steam, typically) make such fluids convenient in sterilization “reactors”. Where lower temperatures are needed, as in heated anaerobic sludge digestors, a non boiling water stream

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In many small systems there is a heating element, 300 to 400 W capacity being adequate for a 10-dm3 fermenter, and a cooling water supply; these are on or off depending on the need for heating or cooling. The heating element should be as small as possible to reduce the size of the ‘heat sink’ and resulting overshoot when heating is no longer required. In some cases it may be better to run the cooling water continuously at a steady rate and to have the heating element only connected to the control unit. This can be an expensive mode of operation if the water flows directly to waste. In large fermentors, where heating during the fermentation is not normally required, a regulatory valve at the cooling-water inlet may be sufficient to control the temperature. There may be provision for circulation of refrigerated brine if excessive cooling is required. Steam inlets to the coil and jacket must be present if a fermenter is being used for batch sterilization of media. •

What are the recent developments in the field of heat and mass transfer? Since the heat and mass transfer operations are directly linked with the energy inputs, and hence affect the fermentation economics, continuous research goes on these topics. All the developments are beyond the scope of present discussion. It would, however, be sufficient to present the following case study:

Case Study Oxygen transfer is a limiting factor in xanthan fermentation, and has traditionally been overcome by increasing agitation, which results in increased power consumption and reduced process efficiency. Microbubble sparging has been found to be an effective alternative for improvement of oxygen transfer in several fermentation studies. However, microbubble technology has not been applied to xanthan gum fermentation. In a study, microbubble sparging for enhancing oxygen transfer and product yield during xanthan gum fermentation was investigated. . Microbubble properties were evaluated at a range of process conditions, including process times of 2-5 minutes, agitation speeds of 5000-8000 rpm, and surfactant levels of 120-500

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is useful. Viscous liquids exhibit greater heat-transfer resistances than water; as with mass transfer, this is due to lesser degree of bulk-fluid interchange with wall fluid and also to reduced thermal conductivity.

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ppm. Properties measured included gas hold-up and foam stability, biocompatibility of Tween-20 with Xanthomonas campestris microorganisms, microbubble size (by a particle size analyzer), and shear resistance of the microorganisms (by standard plate counts). Fermentation studies were conducted with full air sparging and partially substituted microbubble sparging, in which microbubbles were injected for 30 minutes every six hours after the exponential growth phase (6-L production volume; constant air flow rate of 0.2 vvm). Biomass growth, xanthan production, oxygen uptake (using the dynamic technique) and power consumption were measured for each fermentation run. Microorganisms were found compatible at numerous surfactant levels and further showed considerable resistance to shear conditions in the microbubble generator. Microbubbles with a size of about 145 µm, gas-hold-up of 65%, and foam stability of about 3 minutes resulted at standardized conditions (8000 rpm, 3 minutes,300 ppm surfactant). A comparison of microbubble and air sparging methods showed that partially substituted microbubble sparging increased oxygen uptake by about 50%, and increased xanthan gum yield by about 30%. Results indicate that Xanthamonas Campestris is amenable to conditions encountered within a microbubble generator, and further that microbubble sparging improved oxygen transfer and xanthan gum yield. Biochemical Engineering Mass and Oxygen Transfer

n d.An increase in the reactor temperature. j k l m 5 A fermentation system has a kLa of 3 s-1 and a Co* of 5 ppm of O2. If the bulk liquid is completely depleted of oxygen, then the oxygen transfer rate will equal: the oxygen transfer rate will equal j a.zero k l m n j b.15 mg.l-1.s-1 k l m n j c.10mg.l-1.s-1 k l m n j d.5 mg.l-1.s-1 k l m n 6 Liquid in a stirred tank is to be cooled using a coil through which cooling water is to be pumped. Which of the following is not correct?

j a.The liquid in the tank will cool at a faster rate if the k l m n stirrer speed is increased j b.The inclusion of baffles in the tank will increase the k l m n rate at which the liquid in the tank will be cooled.

7 Which of the following will lead to an increase in the heat transfer out of a stirred tank reactor?

Your Name (optional) Your Email Address (optional) 1 A fermentation system has a kLa of 3 s-1 and a C o of 5 ppm of O2. If the bulk liquid is saturated with oxygen then the oxygen transfer rate will equal *

j a.15 mg.l-1.s-1 k l m n j b.5 mg.l-1.s-1 k l m n

j a.A higher cooling water temperature. k l m n j b.A higher stirrer speed. k l m n j c.Removal of the baffles from the reactor k l m n j d.A slower stirrer speed. k l m n 8 An alkane is being degraded by a Streptomyces sp. The maximum solubility of the alkane in the medium is 5 ppm. The actual dissolved concentration of the alkane in the medium is 1 ppm. From this data

j c.3 mg.l-1.s-1 k l m n j d.0 mg.l-1.s-1 k l m n 2 The rate limiting step in the movement of oxygen from the gas phase in a bubble to the cell is the movement of oxygen molecules through

j a.it can be concluded that the growth rate of the k l m n Streptomyces cells is limited by the rate of mass transfer of the alkane into the fermentation medium j b.it can be concluded that the growth rate of the k l m n Streptomyces cells is limited by the intrinsic kinetic properties of the cells and not by mass transfer

j a.the gas-liquid interface. k l m n j b.the bubble boundary layer.. k l m n j c.the bulk liquid. k l m n j d.the gas phase. k l m n 3 Which of the following is most likely to cause an increase in the rate of oxygen transfer into a particular aerated fermentation system?

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j c.The production of detergent like molecules by the k l m n microbial population.

j d.Faster cooling rates can be achieved by pumping the k l m n cooling water through the coils at a faster rate

Wayne Lee Forday

j c.An increase in stirrer speed k l m n

j a.The addition of oil. k l m n j b.The addition of antifoam. k l m n

j c.Faster cooling rates can be achieved by pumping the k l m n cooling water through the coils at a slower rate

The Quizzes

j a.The addition of antifoams k l m n j b.A increase in temperaturee k l m n

n d.Both b and c j k l m 4 Which of the following will lead to an increase in the oxygen transfer rate in a bioreactor?

j c .it can be concluded that the growth rate of the k l m n Streptomyces cells is neither mass transfer nor kinetically limited j d.no conclusion as to the relative importance of mass k l m n transfer or kinetics on the growth rate of the Streptomyces. 9 The average concentration of oxygen in the boundary layers surrounding the bubbles (Co*) in a reactor is normally determined

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j c.by measuring the steady state concentration of oxygen k l m n in the bulk liquid prior to inoculation of the reactor j d.by measuring the steady state concentration of k l m n oxygen in the bulk liquid after inoculation of the reactor 10 An increase in stirring speed from 300 rpm to 500 rpm was found to increase a reaction rate by 2 fold. This observation suggests that

j a.the reaction was mass transfer limited at 300 rpm k l m n j b.the reaction was mass transfer limited at 500 rpm. k l m n j c.the reaction did not involve any mass transfer step k l m n j d.The movement of molecules in the reactor at 300 k l m n rpm occurred due to diffusion alone. 11 In a particular fermentation system, the saturation concentration of oxygen was found to be 7 ppm. During the operation of the fermenter, microbial activity caused the oxygen level to drop to 1 ppm. Which one of the following conclusions can be drawn from this result?

j a. Microbial growth will be oxygen limited since the k l m n oxygen concentration is less than the saturation concentration. b.. Microbial growth will not be oxygen limited since the oxygen concentration is less than the saturation concentration.

j k l m n

j c.Microbial growth will be oxygen limited since the k l m n saturation concentration of oxygen is less than 8 ppm. j d.It is not possible to make any conclusion with regard k l m n to whether oxygen is the growth limiting nutrient.

j a.increase the tendency of bubbles to coalesce k l m n j b.tend to accumulate in the gas liquid interface k l m n j c.reduce the surface tension of the liquid k l m n j d.All of the above answers are correct k l m n 16 A microbial population in a suspension culture will only be limited by oxygen availability if

j a.the dissolved oxygen concentration is less than the k l m n critical concentration j b.the dissolved oxygen concentration is greater than the k l m n critical concentration j c.dissolved oxygen levels are greater than the saturation k l m n concentration of oxygen n d.always. j k l m 17 The addition of detergents to an aerated bioreactor will increase oxygen transfer rates because detergents j a.enhance bubble coalescence k l m n j b.cause bubbles to expand k l m n j c.discourage bubble coalescence. k l m n j d.increase the surface tension of the liquid k l m n 18 Higher temperatures affect oxygen transfer rates by

j a.increasing kLa but lowering C*o k l m n j b.lowering kLa but increasing C*o k l m n j c.increasing kLa and increasing C *o k l m n j d.lowering kLa but lowering C*o k l m n 19 Increasing the stirrer speed in an aerated bioreactor will increase the oxygen transfer rate by

12 Which of the following represents the slowest step in the transfer of carbon dioxide from the bulk liquid to an air bubble?

j a.increasing shear levels to decrease the bubble size k l m n j b.decreasing the size of the boundary layer surrounding k l m n a bubble

j a.Movement through the gas liquid interface k l m n j b.Movement through the bubble boundary layer k l m n j c.Movement across the cell membrane k l m n j d.Movement through the bulk liquid k l m n 13 Cooling water jackets on fermenters are typically dimpled. The main function of the dimples is to

j c.increasing the rate of movement of oxygen molecules k l m n through the bulk liquid

j k l m n

a.increase the level of turbulence of the cooling water in the jacket

j k l m n

b.increase the residence time of the cooling water in the jacket

j k l m n

c.increase the cooling water-jacket wall boundary layer size

d.All of the above n j k l m 14 Which of the following will have the largest interfacial area per unit volume?

j a.a bubble with a diameter of 1 mm k l m n j b.a bubble with a diameter of 2 mm k l m n j c.a bubble with a diameter of 3mm k l m n j d.a bubble with a diameter of 4 mm k l m n 2.521

n d.All of the above are correct j k l m 20 Increasing the height of an aerated bioreactor will increase the oxygen transfer rate by j a.increasing the partial pressure of oxygen at the base of k l m n the reactor j b.decreasing the saturation concentration oxygen in the k l m n reactor j c.decreasing the gas-holdup k l m n j d.All of the above are correct k l m n 21 In a tall reactor, oxygen transfer rates will be

j a.highest at the surface of the reactor k l m n j b.highest at the base of the reactor k l m n j c.highest in the middle of the reactor k l m n j d.the same throughout the reactor k l m n 22 If oxygen is the rate limiting substrate in a bioreactor, then if at a particular time the oxygen transfer rate is 23 kg.h-1, then the oxygen uptake will be

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15 The addition of antifoams to an aerated bioreactor will decrease oxygen transfer rates because antifoams

j a.with ultra-small dissolved oxygen probes k l m n j b.with laser based photographic systems k l m n

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j a.23 kg.h-1 k l m n j b.less than 23 kg.h-1 k l m n

j c.measuring the steady state oxygen concentration in the k l m n boundary layer using a micro-probe

j c.greater than 23 kg.h-1 k l m n j d.cannot be determined k l m n 23 The oxygen uptake requirements of a microbial population is characterized by the following parameters:µm = 0.2 h-1Ko = 0.2 mg O2.l-1Yo = 0.5 mg dry weight .mg O2Co,crit=0.8 mg.l-1 The required concentration of cells is 1000 mg.l-1 and the saturation oxygen concentration of the medium is 5.8 mg.l1. The required kLa must be greater than:

j a.64 h -1 k l m n j b.32 h-1 k l m n j c.16 h -1 k l m n j d.8 h-1 k l m n 24 If the stirrer speed is too slow, bubbles will accumulate and coalescese under the impeller. This phenomenon is known as a

j a.coalesced impeller k l m n j b.flooded impeller k l m n j k l m n

c.river impeller

n d.dry impeller j k l m 25 A flooded impeller will lead to poor oxygen transfer rates because j a.bubbles tend to coalesce under the impeller k l m n j b.bubbles tend to break down too rapidly under high k l m n shear conditions j c.bubbles tend to move too quicly through the bulk k l m n liquid n d.the cells clog up the surface of the bubble. j k l m 26 The use of pure oxygen instead of air will increase oxygen transfer rates because j a.the saturation concentration of oxygen is higher k l m n j b.the bubble size is smaller k l m n j c.the oxygen transfer coefficient is larger k l m n j d.the partial pressure of oxygen in the gas phase is k l m n lower 27 Henry’s law relates

j a.the partial pressure of oxygen and the saturation k l m n concentration of oxygen in the liquid. j b.the oxygen transfer rate and the bubble size k l m n j c.the oxygen transfer rate and the temperature k l m n j d.the oxygen transfer rate to the partial pressure of k l m n oxygen in the liquid 28 The concentration of oxygen in the bubble boundary layer can be determined by

j a.measuring the steady state oxygen concentration in a k l m n mixed, aerated reactor before inoculation j b.measuring the steady state oxygen concentration in a k l m n mixed, aerated reactor after inoculation

n d.very small laser beams j k l m 29 A higher liquid height will lead to a higher gas hold up. As a result, the oxygen transfer rate will j a.be higher throughout the reactor k l m n j b.be lower throughout the reactor k l m n j c.be higher at the base of the reactor. However the k l m n bubbles can become rich in carbon dioxide as they proceed through the reactor. This can lead to reduced oxygen transfer rates as the bubbles move up through the reactor. j d.be lower at the base of the reactor. However the k l m n bubbles will become rich in oxygen in the upper regions of the reactor leading to higher oxygen transfer rates near the surface. 30 During aeration, a cylindrical reactor had a height of 10 m. Without aeration, the height was 7.5 m. Under these aeration and mixing conditions, the gas hold up in the reactor is

j a.100% k l m n j b.75% k l m n j c.50% k l m n j d.25% k l m n 31 Which of the following is correct with regards to detergent like substances produced by microorganisms during a fermentation?

j a.They will decrease the surface tension of the liquid k l m n j b.They tend to accumulate at the gas liquid interface k l m n j c.They tend to decrease the rate of bubble coalescence k l m n j d.All of the above are correct. k l m n 32 Which of the following is correct with regards to the effect of the concentration of dissolved salts and sugars on the oxygen transfer rate?

j a.The saturation concentration of oxygen in the k l m n medium decreases with the concentration of dissolved salts and sugars, leading to a lower oxygen transfer rate. j b.The saturation concentration of oxygen in the k l m n medium increases with the concentration of dissolved salts and sugars, leading to a lower oxygen transfer rate. j c.The kLa of the medium decreases with the k l m n concentration of dissolved salts and sugars, leading to a lower oxygen transfer rate. j d.The kLa of the medium decreases the medium k l m n increases with the concentration of dissolved salts and sugars, leading to a lower oxygen transfer rate. Submit your answ ers

Mass Transfer considerations in Biotechnology Oxygen transfer

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When considering fermentation systems, why is it that oxygen transfer is considered to be a more important problem than the supply of other nutrients?

•

Oxygen has a much lower solubility in water than sugars and nutrients. For example sugar dissolves up to 500 -600 g per litre while the maximum solubility of oxygen at 1 atm/4oC in pure water is only 8 mg per litre.

•

•

Many cells are very sensitive to dissolved oxygen concentrations. A sudden drop in the dissolved oxygen concentration can cause cells to drastically modify their metabolism or physiology; for example, some cells may die, others may switch to fermentation, others go into a stationary phase. Some Bacillus spp. sporulate when dissolved oxygen concentrations fall, thus ending the vegetative cycle.

The transfer of oxygen is expensive. Both agitation and air compression consume considerable amounts of energy.

•

What are the major rate limiting steps in the transfer of oxygen from bubbles to cells?

•

The movement of oxygen from through the boundary layer around a bubble

•

The movement of oxygen through microbial slimes (when immobilized cultures are being used)

•

The movement of oxygen through the bulk liquid when mixing is poor or when the medium is viscous.

•

How the total interfacial area between bubbles and the bulk is liquid measured?

•

It is generally not measured. Instead the oxygen transfer coefficient (kl) and the interfacial area (a) are combined into a single term referred to as kla, the oxygen transfer coefficient per unit volume.

How is the saturation concentration of oxygen measured? The concentration of oxygen at the start of the boundary layer around a bubble (Co*) is also equal to the saturation concentration of oxygen. From the oxygen transfer equation, dCo/dt = kla x (Co* - Co ) we see that when dCo/dt = 0, the dissolved oxygen concentration in the bulk liquid equals the concentration in the bulk liquid.

Oxygen is a gas. Unlike other compounds you cannot keep add excess oxygen to the medium. Any oxygen that is not transfered into the medium will be wasted.

Unlike sugars and proteins, cells cannot store oxygen. Fresh oxygen must be available for the cells at all times during the fermentation. •

•

The saturation concentration (Co*) is measured by determining the oxygen concentration in the operating fermenter prior to inoculation. In the absence of cellular activity, the medium will rapidly become saturated with oxygen and the dissolved oxygen concentration will equal the saturation concentration of oxygen. •

What factors affect the interfacial area between the bubbles and the bulk liquid?

The interfacial area can be increased in two ways:

•

•

increasing the number of bubbles in the reactor

•

decreasing the bubble diameter

What factors affect the oxygen transfer coefficient? The oxygen transfer coefficient is determined by the rate at which oxygen molecules pass through the boundary layer. This can be increased by reducing the size of the boundary layer and by increasing the rate of movement of molecules through the boundary layer.

Effect

Operating variable

This is achieved by increasing Decreasing the size of turbulence by increasing stirre the bubble boundary impeller size. Increasing turbu increases the rate of movemen layer dissolved oxygen through the

Operating factor

Effect

Impeller design

Radial flow impellers are more effective at breaking up bubbles than radial flow impellers

Stirrer speed

Bubble break-up increases with the stirrer speed. If the impeller speed is too low, bubbles will coalesce underneath the impeller leading to a flooded impeller and poor oxygen transfer.

Air flow rate

Increasing the air flow rate increases the total volume of air in the reactor

Height of the reactor

The gas hold-up increases with height of the reactor, thus increasing the total volume of air in the reactor

Sparger design

Spargers should be designed such that the air is passed through many small holes creating a large number of small bubbles in the reactor

Antifoams (and detergents)

Antifoams accumulate around the gas-liquid interface encouraging bubbles to coalesce, thus reducing the interfacial area

Increasing temperature caus to move faster, thus the rate Increasing the rate of through the boundary layer in movement of oxygen through the bubble Excess antifoam will accumula boundary layer the gas-liquid interface and hin movement of oxygen.

What factors affect the oxygen concentration gradient across the bubble boundary layer? •

The dissolved oxygen concentration gradient across the bubble boundary layer (Co* - Co)is the driving force which •

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•

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pushes the oxygen out of the bubble. This concentration gradient can be increased by increasing the saturation concentration of oxygen (Co*) by increasing the partial pressure of oxygen in gas phase. This can be achieved by: using pure oxygen rather than air

problem with antifoams is that they also reduce the kla of the oxygen transfer system and thus reduce the oxygen transfer rate. •

using taller reactors and thus increasing the total pressure at the base of the reactor.

Antifoams and detergents are called surface active agents because they act at the surface of the liquid. Water molecules are bound together by hydrogen bonding forces. At the same time, water and air do not attract each other and can be thought of as repelling each other. (Water is hydrophilic and air is hydrophobic).

Factors which will decrease the saturation concentration of oxygen include: •

Increased concentrations

•

Higher temperatures

of sugars and salts

Note that cellular activity and thus also increase the oxygen rate. •

will decrease C o transfer

•

How does agitation affect the oxygen transfer rate?

Agitation moves the bubbles sideways and thus increases the gas holdup.

•

In large reactors or when the medium is viscous, agitation plays an important role moving dissolved oxygen molecules through the bulk liquid.

•

Increasing the agitation rate reduces the size of the boundary layer surrounding bubbles and thus increases the oxygen transfer coefficient (k)

•

What is a flooded impeller?

•

The Rushton turbine is the most often used as an impeller in the aerated culture of microbial cells. The impeller consists of a flat disk onto which flat blades are welded. The impeller is designed to increase oxygen transfer rates by breaking up the air bubbles as they rise from the sparge ring. However, if the impeller speed is too slow the bubbles will accumulate and coalesce below the impeller disk. As a result, large bubbles will be formed and the oxygen transfer rate will be very low. This phenomenon is referred to as a flooded impeller and is undesirable for the culture of aerobic organisms. •

•

Preventative measures

Reducing the air flow rate Reducing the addition of detergents (eg. Tween 80, commonly used to harvest fungal spores can be replaced with glycerol)

Antifoams

Antifoams and oils are often added to fermentations. Antifoams are based on silicone oil formulations. Vegetable oils such as peanut oil, whilst not as effective as silicone antifoams are natural products and cause less problems in downstream processing. Physical foam breakers can be used. These include: the application of heat at the surface. This causes the bubbles to expand and thus break. mechanical breakers which physically break the foam.

Physical methods

vibratory systems likwise cause the foam to break. • •

Increasing the size of the disengagement zone in bubble column and air-lift and fluidized bed bioreactors. A draft tube will reduce foam production, turbulence at the surface and thereby assisting in the breakdown of the foam. An air-lift reactor with an internal riser is said to produce less foam than one with an external riser.

What does the expression “bubble coalescence” mean? •

How does a draft tube reduce bubble coalescence? The oxygen transfer rate in air lift reactors is considerable higher than in comparable size bubble column reactors. This is because the draft forces bubbles to move in one direction. This reduces the opportunities for bubbles to meet and coalesce.

Why are antifoams added during a fermentation and what are the problems associated with antifoams? Antifoams are added to prevent the formation of foam. Foam accumulates during aerobic fermentations. The

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How can foam formation be controlled?

These include

Why are “small bubbles” regarded as the secret to a successful fermentation? Small bubbles have a higher surface to volume ratio than large bubbles. This increases the interfacial area for oxygen transfer and thus increases kla and the oxygen transfer rate. Bubble coalescence occurs when bubbles meet. When two bubbles meet, they can form a single bubble. The presence of antifoams in the medium will increase the tendency of bubbles to coalesce. Ionic detergents on the other hand will reduce bubble coalescence.

•

Why do foams form in fermentation systems and what problems do they cause? Foams form in fermenters because cells release detergent like substances. During a fermentation, cells will proteins and other detergent like compounds into the medium. These compounds can include extracellular enzymes and metal binding compounds. As cells die, they release their DNA and intracellular proteins. One indication that a fermentation has ended is the excessive accumulation of foam or an increase in the antifoam requirement. Foams are a problem in aerated fermenters. When the headspace becomes full of foam, the pressure in the reactor will build up. This pressure will try to push the foam out of the fermenter. As a result. air filters will be damaged and in some cases, the covers and ports which are not screwed down can actually be blown off the fermenter. This can cause injury to personnel and can also cause a loss of the fermenter contents and contamination of the surroundings.

Agitation plays four major roles in improving the oxygen transfer rate. The agitation system is used to break apart bubbles and thus increase the interfacial transfer area (a). •

Why are antifoams and detergents called surface active agents?

•

What is Henry’s Law?

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The oxygen transfer rate is given by the formula: OTR = kla (Co* - Co) Therefore increasing the partial pressure of oxygen in the gas phase will increase the saturation concentration of oxygen in the medium (Co*) and thus increase the oxygen transfer rate. The partial presure of oxygen in the gas phase can be increased by increasing the percentage of oxygen in the gas phase, for example by using pure oxygen in air. At one atmosphere, the partial pressure of oxygen in air is 0.21 atm (air contains 21% gaseous oxygen) while the partial pressure of oxygen in pure oxygen is 1 atm. The partial pressure of oxygen in the gas phase can also be increased by increasing the pressure of air introduced into the reactor. If the pressure of the air when it enters the reactor is 10 atm, then the partial presure of oxygen in the gas will be 21 atm. The air pressure can be increased by increasing the height of the reactor. The pressure at the base of the reactor (P) is calculated from the following formula: P=rgh r is the density of the medium, g is acceleration due to gravity and h is the height of liquid in the reactor.

parameters when oxygen is the growth limiting nutrient, in the same way as the general terms Yxs and Ks are used for other growth limiting nutrients. •

What is the critical oxygen concentration? The critical oxygen concentration is the dissolved oxygen concentration below which cell growth becomes oxygen limited; ie. when the dissolved oxygen concentration falls below the critical oxygen concentration, the specific growth rate fall below the maximum spcific growth rate (µm). In practice, the critical dissolved oxygen concentration is rarely determined.

•

How can we determine the oxygen saturation constant (Ko)? Ko is determined in the same way that Ks is determined. For example, by determining the specific growth rate of the organism at different dissolved oxygen concentrations (Co) and then plotting 1/µ against the 1/Co using a Lineweaver Burke transformation.

Alternatively, non-linear regression techniques or other transformations (eg. Eadie Hofstee) could be used to determine both Ko and µm.

Notes

For example, the pressure at the base of a 10 m tall reactor filled with water is approximately 980,000 Pa or 9.7 atm. The partial pressure of oxyge at the base will therefore be approximately 2.0 atm. The saturation concentration of oxygen in the medium can also be increased by decreasing the value of Henry’s number. Henry’s constant will be lower at lower temperatures and lower solute concentrations. •

Why do we assume that the oxygen transfer rate is equal to the oxygen uptake rate when analysing aerobic fermentations? Aeration, even in a reactor which operates in a batch manner, can be viewed as a continuous process. The rate of accumulation of oxygen in the reactor medium is given by dCo/dt = Rate of oxygen transferring into the reactor Oxygen uptake rate All continuous processes proceed towards a steady state and since aeration is a continuous process, if the operating conditions do change. In fact, operating conditions do change in the bioreactor since the number and specific growth rate of the cells will change during the fermentation. However, oxygen transfer and oxygen uptake are very rapid process and the dissolved oxygen concentration will quickly reach a temporary or apparent steady state. Thus at steady state, dCo/dt=0 and the oxygen transfer rate = oxygen uptake rate.

•

What is the relationship between the Yo (the biomass yield from oxygen) and Ko (the oxygen saturation constant) with the Yxs (the biomass yield) and Ks (the Monod saturation constant)? The terms Yo and Ko are used as stoichiometric and kinetic

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Henry’s Law states that saturation concentration of a gas dissolved in a liquid is proportional to the partial pressure of the gas. Cg* = pg / Hg

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LESSON 13: INSTRUMENTATION AND CONTROL I Learning Objectives

function of temperature. Although temperature-resistance relationship is nonlinear, this is not a serious difficulty for the narrow temperature range of interest for most fermentation (25-45°C). Other possible temperature sensors are the platinum resistance sensor, thermometer bulbs (Hg in stainless steel), and thermocouples.

In this lecture, you will learn •

Control of fermentation parameters

•

On line analysis of fermentation parameters

•

Properties of growing cells

The importance of instrumentation and control in a fermentation system is best known to a fermentation system engineer who has to face a situation when the fermentation is going out of control. When a critical parameter is not monitored properly, and starts going haywire, the fermentation faces a major economic and environmental crisis.

Have a look at the picture below which shows different probes used for the measurement of various fermentation parameters.

We have seen repeatedly that the activity and useful lifetime of an enzyme catalyst or cell population depends directly on the catalyst environment. Accordingly, in order to develop and optimize biological reactors and in order to operate them most efficiently, it is critical that the state of the catalyst environment be monitored and controlled and that the response of the catalyst to the environment is determined. Achieving these goals requires three different functions: measurement, analysis of measurement data, and control. In this lesson we will examine currently available reactor instrumentation and its application. After a brief look at the rapidly changing technologies for data acquisition and computing, we shall summarize some of the strategies for data analysis and process control. Although this lesson’s presentation emphasizes bioreactor instrumentation and control, the principles described also apply to downstream processing and to inoculum / feedstock preparation. •

What are the various types of sensors required in fermentation? Measurement of various chemical and physical parameters of the medium and gas is extremely important. Consequently, one of the major goals, if not requirements, of bioreactor data analysis is estimation of cell properties s based on the available physiochemical measurements of the gas streams and the medium. In this section we will concentrate on instrumentation for on-line physical and chemical monitoring of bioreactors.

First, about the sensors of various physical Parameters. The major physical process parameters that influence cellular function and process economics and which can be monitored continuously are temperature, pressure, agitator shaft power, impeller speed, broth viscosity, gas and liquid flow rates, foaming, and reactor contents volume or mass. In small laboratory reactor’s, only temperatures and air-feed flow rates are commonly measured. Pressure measurement and regulation is common on larger fermentors. The most widely used temperature sensor is the thermistor, a semiconductor device which exhibits changing resistance as a 86

Pressure monitoring is important during sterilization, and maintaining a positive reactor head pressure (around 1.2 atm absolute) can aid in preserving asepsis. Pressure also influences gas solubility. In fermentation reactors, diaphragm gauges are usually used to monitor pressure. These produce a pneumatic signal which may be transducer if necessary to an electrical signal. Several different types of measurements can be made to monitor power input in mechanically agitated vessels. A Hall effect wattmeter measures at the drive motor armature the total energy consumed by the agitator. A torsion dynamometer may

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On-line devices for measuring broth viscosity and other rheological properties are not well developed. One possible strategy is measurement of power consumption at several different impeller speeds. Also, a dynamic method has been proposed in which shaft power input is monitored during and after a brief (less than 30 s) shutoff in agitator drive power. Newtonian and non-Newtonian broths have been observed to respond differently during a brief agitation transient. Several different instruments are available for measuring flow rates of gases (Air feed, exhaust gas). The simplest, a variable area flowmeter such as a rotameter, provides visual readout or may be fitted with a transducer to give an electrical output. Thermal mass flowmeters are increasingly popular, especially for lab and pilotscale reactors. In these devices, gas flows through a heated section of tubing, and the temperature difference across this heated section is directly related to mass flow rate. These instruments have accuracies on the order of 1 % of full-scale and are most useful for flow rates less than 500 L/min. Also available are laminar flow measurement devices which determine flow based on differential pressure drop across a matrix device which divides the total flow into multiple parallel capillary flows. Gas flow rate measurements are important since these quantities are used frequently in material balancing calculations.

reliable probe among these is the pH electrode, which is generally a single unit glass-reference electrode design. Electrodes for in situ sterilization must include a housing to provide pressure balance during sterilization or pressurized bioreactor operation. Measurement of medium redox potential is possible using a combined platinum and reference electrode. Combined pH-redox probes are available. While the influence of pH on biochemical kinetics is clearly established and the physical significance of a pH measurement is straightforward, interpretation of redox potential measurements and understanding the relationship between redox potential and cell activity can be difficult. One promising application of redox measurements is in monitoring low contents of dissolved oxygen ( less than1 ppm) in anaerobic processes where product formation may be quite sensitive to Eh’ The various types of dissolved oxygen probes now available are of galvanic (potentiometric) or polarographic (amperometric or Clark) types. These electrodes measure the partial pressure (or activity) of the dissolved oxygen and not the dissolved oxygen concentration. In both designs, an oxygen-permeable membrane usually separates the electrode internals from the medium fluid. Also, both designs share the common feature of reduction of oxygen at the cathode surface.

Liquid flow rates can be monitored with electromagnetic flowmeters, but these are not used widely due to their cost. Occasionally, especially in laboratory scale studies, one relies on a metering or other well-calibrated pump to provide the desired liquid flow rate. Alternatively, liquid can be added to the reactor in discrete doses of well-defined volume or mass. Long-term monitoring of net flow into the vessel may be achieved by continuous weighing of the reactor and its liquid contents using a strain gauge (vessels> 250 L) or scale (smaller vessels). Alternatively, a liquid level sensor based on a capacitance probe may be used to monitor reactor liquid content. Such capacitance probes or a conductance probe may also be used to detect buildup of foam on the top surface of the reactor contents. In some situations an external loop of-circulating broth is used for measurements, to effect product removal and cell recycle, or for heat and/or gas exchange. Here the presence of suspended particulates and changing broth rheology severely complicate liquid flow rate measurement.

•

And how do we sense the chemical parameters in a fermentation medium?

Electrodes which can be repeatedly steam-sterilized in place are now available for pH, redox potential (Eh) and dissolved oxygen and CO2 partial pressures. The most widely used and 2.521

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also be used to measure shaft power input. A disadvantage of both these measurement methods is inclusion of frictional losses in shaft bearings seals. For example, in a study of mixing in a 270-liter fermentor with 200 lits working volume, it was found that 30 % of the energy used by the motor lost between the motor and the internal impeller shaft. This loss factor was observed to be an increasing function of agitator speed. Direct measurement of impeller power input to the reactor fluid may be achieved using balanced strain gauges mounted on the impeller shaft inside the reactor.

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dissolved oxygen sensors is quite long (10-100 s). However, transient probe measurements may still be applied to characterize mass-transfer properties of bioreactors provided the influence of diffusion through the probe membrane is included in the analysis. • ·All right. How about the analysis of other parameters? Steam-sterilizable electrochemical probes for dissolved CO2 partial pressure have been introduced relatively recently. The CO2 probe produced by Ingold, for example, determines PC02 by measuring the pH of a standard bicarbonate solution which is separated from the process fluid by a gas-permeable membrane. Calibration is accomplished by measuring pH after substitution of a reference buffer solution for the bicarbonate solution. Another class of methods for on-line assay of volatile medium components and dissolved gases is based upon immersion of a length of tubing, permeable to the component(s) of interest, in the fluid to be analyzed. Continuous flow of a carrier gas through the tubing sweeps the compounds which penetrate the tubing to a gas analysis device, where the measurement is conducted. This approach suffers from substantial measurement delays (2-10 min) and, therefore, is not optimal for monitoring rapid transients in concentrations.

The reaction at the anode in a galvanic electrode is Pb

Pb ++ + 2 e -

completes the cell from which a small amount of current is drawn to provide a voltage measurement which in turn is correlated to the oxygen flux reaching the cathode surface. In a polarographic type of oxygen electrode, a constant voltage is applied across the cathode and anode Ag + Cl

- AgCl + e -

and the resulting current, which depends on the oxygen flux to the cathode, is measured. Drift caused by accumulation of hydroxyl or metal ions or chloride depletion is a common drawback of both electrode types. External fouling of the membrane surface may also contribute to drift. •

How does the oxygen probe work?

In steady state, the oxygen flux at the cathode depends upon a series of transport steps in which oxygen moves from the bulk liquid to the outer membrane surface, diffuses through the membrane, and finally diffuses through the electrolyte solution to the cathode surface where reaction occurs effectively instantaneously. To the extent that the first step limits the overall transport rate, and thus the oxygen flux to the cathode, the electrode output will depend on fluid properties (e.g., viscosity) and local hydrodynamic conditions near the electrode. For this reason it has been recommended, for example, that the fluid velocity at the tip of a polarographic electrode should be at least 0.55 m/s. Sensitivity of the electrode output to external boundary layer transport can also be reduced by using a less permeable membrane. This approach has the disadvantage of introducing additional time delay in the instantaneous electrode response to transients in dissolved oxygen partial pressures. The characteristic response time of membrane-covered 88

Several biosensors have been developed for assay of specific components in the liquid phase. These are based on coupling the action of immobilized enzymes or cells with an analytical device which detects a particular product of the biocatalyzed reaction. Also studied extensively are enzyme thermisters, in which the heat released by the enzyme-catalyzed reaction is detected by a nearby calorimeter. Other possibilities for biosensor development using immobilized enzymes include enzyme transitors in which reaction products (for example, hydrogen) cause changes in the electronic properties of solidstate devices (for example, silicon chips with an Si02-layer covered with a Pd film). The spectrum of biosensor designs and configurations can be enlarged by considering a broader class of biocatalyzed reactions, including multistep or coupled reactions, to generate the detected component. Immobilized cells provide a convenient means in many cases for transforming the component to be assayed into a suitable detectable compound. Immobilized whole-cell respiratory activity (assay by oxygen electrode) and production or consumption of electro active metabolites by whole cells (assayed by fuel cell electrode or by pH or CO2 electrodes) have been used as the bases for design and application of biosensors containing immobilized whole cells An interesting and promising alternative strategy for formulating specific “affinity sensors” for individual metabolites has been described and developed by J. S. Schultz and coworkers. The requirements for this method are a specific binding agent for the component to be assayed, availability of a suitably labeled component which competes for the same specific binding agent, and a means of separating binding agent from the solution to be assayed. For example, a fiber optic fluorescence probe has been constructed for glucose analysis by immobilizing conconavalin A (Con-A), a protein from jack bean which selectively binds sugars, on the internal surfaces of a

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Glucose + Con-A

Con-A-glucose

FITC-Dextran + Con-A

Con-A-FITC-dextran

Thus, the amount of unbound FITC-dextran, and hence the measured fluorescence emission intensity, is a function of the solution glucose concentration. Interference by other solutes which also bind to the specific binding agent (maltose, sucrose, and fructose to Con-A, for example) pose potential problems for this approach A further concern in use of any sensor employing enzymes, cells, or other biochemicals is deactivation of the sensor during reactor sterilization. Mechanical designs which allow aseptic removal and insertion of the sensor in the reactor interior have now been developed to address this potential problem. Also, as in all sensors which depend on transport of the monitored component through a membrane, membrane fouling by cells or medium components and external mass transport resistance can cause drift or shifts in calibration of the sensor. Ok, now tell me about the gas analysis. The concentration of CO2 in the exhaust gas from a cell reactor is indicative of respiratory or fermentative activity of the organisms and hence is one of the most useful and widely applied measurements in monitoring and controlling a cell bioreactor. CO2 content in bioreactor gas streams is most commonly monitored using an infrared spectrophotometer. The gas sample stream must be dried carefully before entering the instrument to avoid damage to the sample cell windows. Gas stream CO2 concentration may also be measured using thermal conductivity, gas chromatography, or mass spectrometry. •

Gas stream oxygen partial pressure is usually measured using a paramagnetic analyzer. Here too, elimination of water vapor in the sample stream is essential to minimize drift, and the sample stream flow rate must be controlled carefully for consistent measurements. Paramagnetic analyzers are also quite sensitive to small changes in total atmospheric pressure, requiring simultaneous monitoring of barometric pressure for compensation in oxygen analysis. Drift in readings which necessitate on-line recalibration is a frequent occurrence with paramagnetic analyzers when applied to fermentations. Gas chromatography (GC) can be applied to analyze several components of the exhaust gas stream including O2, CO2, CH4 (e.g., in anaerobic methane generation), and H2 (from Hydrogenomonas cultures, for example). Also, by determining the gas phase partial pressure of volatile components such as ethanol, acetaldehyde, and carboxylic acids, GC measurements provide useful information on the status of the fermentation and on the liquid phase concentrations of these compounds. The requirement of intermittent injection of samples (ca. 15 min apart) limits the utility of GC measurements for monitoring process transients.

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Mass spectrometry (MS) is becoming increasing popular for monitoring gas stream composition. Lower-priced instruments are making MS more accessible for research applications, and reliable, robust process instruments have made mass spectrometry more practical for industrial application. MS instruments offer rapid response times less than 1 min), high sensitivity (around 10-5 M detection limit), capability to analyze several components essentially simultaneously, linear response over a broad concentration range, and negligible calibration drift. Because of the expense of MS instruments, it is often desirable to interface the analyzer to several bioreactors and use a computer-controlled switching manifold to cycle sample streams from different reactors into the MS. Often, standard values (20.91 % O2' 0.03 % CO2) are assumed for the feed air composition, but it is sometimes more reliable to measure feed gas composition directly by including a feed gas sample stream in the manifolding arrangement... Of course, the merits of sharing analyzer instrumentation by use of such multiplexing and manifold arrangements are not limited to cases in which mass spectrometry analyzers are applied. Now, how can we find out about the properties of various cells growing in the bioreactor? Unfortunately, there are few instruments for continuous monitoring of cell properties in a bioreactor. The most basic measurement needed is total biomass content or concentration or, better still, active biomass concentration. Although a number of possible methods exist, no approach has yet been invented which provides such data reliably, consistently, and for a broad class of organisms and media. •

Optical methods based upon light absorbance (spectrophotometry) or scattering (nephelometry, reflectance measurement) have been investigated widely. A sample stream from the reactor may be circulated through a spectrophotometer. A potential difficulty here is the nonlinearity between optical density and biomass concentration above O.D. = 0.5 or 0.5 g biomass per lt. Consequently, sample stream dilution or a shorter light path may be used for measurement of dense cultures. Alternatively, probes which can be inserted into the process fluid for optical cell density measurements have been developed. The only continuous monitoring strategy so far developed that provides information on the biochemical or metabolic state of the cell population is in situ fluorometry. Ultraviolet light (366 nm wavelength) is directed into the culture. Excited by this incident UV radiation, reduced pyridine nucleotides (NADH and NADPH) fluoresce with a maximum intensity at approximately 460 nm. The fluorescence emitted from the culture is measured with a suitable detector such as photodiode or photomultiplier. Originally, these measurements were made through quartz windows installed in the walls of laboratory fermentors. The advent of fluorescence probes which can be used in standard electrode ports in fermentation vessels should increase investigations and application of culture fluorescence measurements. Culture fluorescence intensity depends on cell density, average cell metabolic state and fluorescence emissions, and light absorption by the medium. Experiment in particulate-free

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measurement chamber. The chamber, separated from the assayed solution by a dialysis membrane permeable to glucose, also contains dextran labeled with the fluorochrome fluoroscein isothiocyanate (FITC). The membrane used is impermeable to the FITC-dextran which competes with glucose for binding to Con-A:

FERMENTATION TECHNOLOGIES

media have shown that culture fluorescence measurements provide useful information on biomass concentration, oxygen transfer and reactor mixing times, substrate exhaustion, and metabolic transients. For example, Einsele and coworkers compared the dynamics for liquid mixing in a 40-liter working volume ferment or agitated mechanically at 200 rpm with the dynamics of mixing plus glucose uptake by yeast cells. For the first measurement, fluorescence of quinine pulsed into 0.05 M H2SO4 in the reactor was monitored (this solute has approximately the same fluorescent properties as NADH). The results exhibit oscillations representative of a periodic circulation pattern in the vessel and provide clear evidence of significant dynamic delays in achieving new steady-state conditions in the reactor. In all applications of direct optical measurements in cell cultures, a number of potential problems arise which can interfere with interpretation of the measurements. For example, the optical surfaces .in contact with the process fluid may become fouled with cells or medium components. Gas bubbles and particulates in the multiphase reaction fluid may interrupt or interfere with the desired measurement and, in the case of fluorescence, certain medium components or products may fluoresce at the same wavelengths useful for monitoring intracellular state, complicating interpretation of the measurements. However, in view of the importance of determining the biomass concentration and cellular metabolic state for monitoring, control, and optimization of the process, these and other optical methods can be expected to enjoy expanding applications in the future.

Notes

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Learning Objectives In this lecture, you will learn •

Off line control measures

•

Protein & other cell components

•

Plasmid & energy levels of cells

Well, we have seen the various on – line methods employed for the fermentation control. In this lesson we will see some of the off line methods as well as application of computers in fermentation technology. •

What all is included in the off line control of fermentations? Off line control of the fermentations includes measurement principles and methods applied to determine the properties of process fluids, biocatalysts, and biosorbants. Possible methods span the entire spectrum of analytical chemistry, spectroscopy, and biochemistry, making anything approaching a complete presentation impossible in this context. Here, we emphasize certain new methods relating to cell property measurements which have potential for process monitoring and control applications and also provide an overview of other types of commonly applied analyses.

•

First, how do we test the samples drawn from a bioreactor? After withdrawing a sample from a bioreactor or separation unit, a solid-liquid separation is accomplished by centrifugation or filtration in order to remove cells and any other particulate matter from the fluid phase sample. The analyses conducted subsequently, of course, depend upon the particular application; analytical methods which perform satisfactorily for defined medium may not be accurate or appropriate for analyses in undefined medium which may contain interfering components. The desired measurements in a bioreactor are the concentrations of substrates and components influencing rates, and the concentrations of reaction products and inhibitors. For fermentation, analyses of the carbon and nitrogen sources are often desirable. Also, it may be necessary or useful to determine the levels of certain ions such as magnesium or phosphorus in the medium. Products of cellular processes vary over a broad range of chemical complexity and properties, from small organic compounds such as ethanol to more complex structures such as penicillin to biological macromolecules such as enzymes and other proteins. Accordingly, the spectrum of appropriate methods for product assay is extremely broad. Liquid-phase quantitative analysis is based usually upon light refraction (measured with a refractive index detector), absorption of light at a particular wavelength (measured with spectrophotometer) or fluorescence due to excitation at

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one wavelength and subsequent emission at a longer wavelength (measured with a spectrofluorometer). Sugars, for example, do not absorb light strongly and do not fluoresce but do alter solution refractive index. On the other hand, protein and nucleic acids lend themselves to spectrophotometric and spectrofluorometric detection. Fluorescence measurements are usually more sensitive and allow measurement of lower concentrations. However, spectrofluorometers are more expensive than spectrophotometers, making spectrophotometric measurements very popular. In order to avoid interference from other compounds in solution, separation or concentration of the component to be analyzed from other solutes is often necessary. Sometimes this can be accomplished by chemical treatment to decompose or to precipitate the desired interfering compounds. For example, RNA is extracted from cell lysates with HCI04 (perchloric acid) at 37°C and analyzed by the orcinol method for ribose. Interfering sugars are removed during the extraction. Finer-scale separation among related compounds by chromatographic methods is also commonly applied in medium chemical analysis. The basic principle of chromatography is selective retention or retardation of certain compounds by an immobile phase in a column due to preferential attraction of these components for the immobile phase relative to other solutes. For example, in analyzing mixtures of sugars such as maltose and glucose, the different affinities for these two sugars for the primary amino groups on the surface of the support material in a commercially prepared carbohydrate column is used to separate the sugars in an HPLC (high performance liquid chromatography) apparatus. The different sugars emerge from the column at different times, and they may be then detected and quantified separately using a refractive index detector. Many other separations based on HPLC methods are useful in medium analyses. Also, separations accomplished under atmospheric pressure using ion exchange chromatography or size partitioning chromatography are useful in resolving mixtures of related components before their individual quantification. A useful strategy for chemical analysis is selective conversion of the component of interest to a readily measurable product. This is the basis for one of the standard laboratory methods for glucose assay, in which the enzymes glucose oxidase and peroxidase selectively convert glucose to a colored compound which can be assayed spectrophotometrically. Glucose + 2H2O

gluconic acid + 2H202

This reaction is catalysed by the enzyme glucose oxidase.

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LESSON 14: INSTRUMENTATION & CONTROL II

FERMENTATION TECHNOLOGIES

H202 + o-dianisidine (colorless)

oxidized o-dianisidine ( brown)

This reaction is catalyzed by peroxidase. Ion-specific electrodes have become important tools in assaying certain biologically important ions. Cellular nitrogen content can be determined with an ammonia electrode either directly (NH3 or after chemical modification by pH adjustment, NH4+), reduction (NO-2 , NO-3 ), digestion (amino acids), or enzymatic modification (urea). Ion specific electrodes are also available for analysis of many other ions which influence biochemical structure and function including potassium, sodium, and calcium. Occasionally, as an alternative to determining the concentration of a particular compound in solution, the measurement determines the compound’s biological activity. Assay of penicillin in fermentation broths by this method has been a standard procedure in the pharmaceutical industry. Here, the size of a zone of dead bacteria around a porous disc soaked with the solution to be assayed provides an indication of penicillin activity in the solution. It is very common to analyze enzyme content by measuring the activity of that enzyme. This functional assay is accomplished by exposing the sample solution to a standard enzyme substrate under standard conditions, then measuring the rate of substrate disappearance, or product appearance, often spectrophotometrically or by fluorescence. An alternative set of analytical procedures is .based upon volatilization of the components of interest and their measurement in the gas phase. This can be done for glucose, for example, by forming its TMS (trimethysilyl) derivative which can be vaporized in the injection chamber of a gas chromatograph and the product detected by a flame ionization detector. Determination of the contents of relatively volatile components such as ethanol, acetone, and butanol in fermentation fluids by this method is quite straightforward. Analytical laboratories which support pilot- and productionscale fermentation facilities often contain one or more automated wet chemical analyzers. These automatically partition, dilute, and process a sample to carry out several chemical analyses. The response time of such instrumentation is 10 to 30 minutes, sufficient in many cases to be useful for monitoring of bioreactions in progress. •

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How do we analyze the composition of cell population?

Analytical methods for cell populations can be categorized in much the same way as were mathematical models for cell population kinetics. Most classical measurements of biochemistry provide population-averaged and thus unsegregated data on the cell population. Measurements of this type can be extended to a very large number of cellular constituents, even to the level of particular proteins, RNA molecules, and DNA molecules and sequences. If the experimental measurement is made on a single-cell basis, or can be used to infer single-cell information, so that the distribution of single-cell properties is obtained, the data may be said to be segregated. We shall first discuss measurements of nonsegregated type. The coarsest of such measurements, after determination of total cell mass or number density, is analysis of the elemental composition of the cells including carbon, hydrogen, and nitrogen. Automatic analyzers such as the apparatus for determination of total nitrogen have been developed for bulk sample assays of this type. Specific ions are also known to play an important role in biological processes, and it is common to see total levels of iron, magnesium, phosphorus, and calcium reported as well. Determination of total protein, total RNA content, total DNA content, and other average macromolecular content of the cells can be accomplished by well-established methods.

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•

What, if any, are the other methods for the analysis of proteins?

Individual proteins can sometimes be analyzed by protein chromatography or by examining the relative intensities of bands obtained by electrophoresis of a protein mixture. Increased resolution and extreme sensitivity to many different protein levels can be achieved by two-dimensional gel electrophoresis in which proteins are separated in one direction on the basis of their size and in a second direction on the basis of their charge. When separated in this way, different proteins tend to move to different spots in the two-dimensional plane, making it possible to identify and quantify a large number of proteins simultaneously. For example, this method was used to study the rates of synthesis of 140 different proteins during growth of the bacterium E. coli. Another basis for analysis of individual proteins is binding of antibodies to a particular region on an individual protein molecule. If antibody is available for the protein of interest, analyses based upon precipitation, detection of radioactively labeled antibodies, or the amount of enzyme activity which can be linked to a particular protein by an antibody [the enzymelinked-immunosorbent assay (ELISA) method] may be used to quantify the amount of the individual protein present. Such methods may also be applied to analyze cellular content of other components or of macromolecular structures against which specific antibodies can be made. Antibody labels are used frequently to determine the existence on a cell surface of particular types of molecules or structures and to quantify in some cases the amount of these components on the cell exterior. Labeling of cell surface compounds and subsequent measurement may be conducted without killing the organisms, a feature which may be useful in screening or selection during strain improvement by mutation. Such methods are also convenient for distinguishing between species in a mixed culture, since organisms usually carry specific surface markers which can be identified separately and quantified with specific antibodies. •

How is the plasmid content of cells determined?

The importance of plasmids as the carriers of the genetic instructions for product synthesis in recombinant organisms makes assay of cellular plasmid content a potentially important measurement. The most rigorous method of plasmid quantification in bacteria is done by isolating all DNA from the organism, then separating plasmid DNA from chromosomal DNA in a cesium chloride gradient using an ultracentrifuge. The relative quantities of chromosomal and plasmid DNA can be examined in several ways. For example, if a radioactive 2.521

preparation of DNA was used, fractions can be collected from the bottom of the tube and analyzed for radioactivity using a scintillation counter. Determination of plasmid DNA content in yeast or animal cells may be accomplished by a hybridization assay using a labeled probe complementary to a nucleotide sequence unique to the plasmid. Alternatively, the gene for a particular enzyme, the activity of which is easy to assay, may be included on the plasmid as a marker, and the activity of this enzyme used to estimate the plasmid content of the organisms. This latter method has been implemented in bacteria, yeast, and animal cell recombinant strains. •

But the above techniques do not directly reflect the metabolic state or the energy levels of the organism, right?

Yes. All of the measurements discussed above provide information on cellular composition and the metabolic state of the organism, but they do not directly indicate the current metabolic state or energetic state of the organism. Measurement of cellular ATP content can be carried out with a Biometer that measures luminescence produced by a reaction requiring ATP and catalyzed by the enzyme luciferase. Since ATP levels change rapidly as a function of cellular environment and metabolic activity, it is necessary that samples of the cell population be quenched rapidly in phosphoric acid in order to preserve their ATP content before this or alternative ATP analyses. Since A TP is absent from nonviable cells, measurements of the ATP level can also be interpreted usefully in some cases as a measure of the metabolically active biomass in the population. High resolution nuclear magnetic resonance (NMR) measurements of 31P have been successfully applied to determine intracellular ATP, ADP, sugar phosphate, polyphosphate, and pH values. Several different microorganisms have been studied in this fashion including the bacteria E.coli and Clostridium thermocellum and the yeasts Saccharomyces cerevisiae, Candida utilis, and Zygosaccharomyces bailii]. In addition, tracer isotopes such as 13C and 15N may be used to observe functioning of intracellular pathways of carbon and nitrogen metabolism via NMR . It has been observed in several fermentations with mycelial microorganisms that the process productivity and kinetics are correlated with the morphological state of the mold or actinomycetes. Direct observation and quantitative monitoring of mycelial morphology is quite difficult and time-consuming since repeated microscopic observations and human or computerized image analysis are involved. It has been observed that the filtration properties of a suspension of mycelia are influenced by the mycelial morphology, and this principle has been used by Wang and collaborators and refined by Lim and colleagues to formulate an ingenious mycelium morphology and biomass probe based upon a batch filtration measurement. A small sample of culture suspension is filtered, and the filtrate volume and cake thickness are monitored continuously. Based upon previously established correlations between filtration characteristics and the morphological properties and density of the particular mold considered, these data provide a basis for

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Population-average cell content of particular proteins can be determined in several different ways. First, for enzymes, activity assays are used to monitor the changes in enzyme levels during process operation. Activity levels of the enzymes associated with acids production decline late in the fermentation, while there is an increase in enzyme activity associated with solvents production late in the batch. Based upon information of this type, alterations in metabolism may be more directly correlated with strain and bioreactor operating parameters in order to optimize the organism and the process conditions.

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intermittent on-line monitoring of the progress of the fermentation. •

How are the characteristics of single celled organisms determined?

There are several different methods available for measuring and characterizing the distribution of single-cell characteristics in a population of single-celled organisms. Microscopic observation can give some approximate indications and, coupled with image analysis methods, quantitative information can be obtained, although gathering data on a sufficiently large number of cells to have a good statistical sample is rather difficult. More suitable for rapid measurements of properties of large numbers of individual cells are flow measurement methods- of which there are two general types. In instruments utilizing the Coulter principle, the volume of individual cells is detected as cells suspended in a sample stream of an electrolyte solution flowing through a small orifice across which resistivity is measured. For spherical particles, the alteration in resistivity across the orifice may be correlated directly with the volume of the spherical particle, allowing many particles to be sized as they flow rapidly through the orifice. Alterations in particle morphology can cause some difficulties in interpretation of the measurements, but still this is a useful approach for obtaining the size distribution in a cell population.

information on the cell population. Data of this type, considering two-parameter measurements as an example, take the form of a surface indicating frequency or relative number of cells as a function of the coordinates in an underlying plane representing the measured quantities.. Thus, the amount of fluorescent product accumulated may be correlated with the existence and even with the number of plasmids in the yeast cell. Based upon this type of measurement, the proportion of cells with and without plasmid in the culture can be very rapidly assayed and further information can be extracted on plasmid replication and segregation in the recombinant strain.

Notes

A richer class of measurements is possible using a flow cytometer. In this instrument, a dilute cell suspension again flows through a measuring section and, in this case, optical measurements are conducted. The cell sample stream is irradiated by a laser or other light source and the light absorption scatter and/or fluorescence is measured on a singlecell basis. Light-scattering measurements may be used to obtain information on the cell size distribution. Since right-angle lightscattering intensity is sensitive to intracellular morphology, this measurement has been applied to monitor the accumulation in individual bacterial cells of refractile particles consisting of the storage carbohydrate polyhydroxybutyrate ( PHB ). Individual cell macromolecular composition has been measured for microorganisms and animal cells by applying specific fluorescent dyes which label the macromolecular pool of interest including total cellular protein, double-stranded RNA and cellular DNA. Accumulation of an intracellular fluorescent product produced under the action of a single enzyme can be monitored on the single-cell level in such an instrument, allowing assay of individual enzyme activity in individual cells, study of in vitro enzyme kinetics and, by cloning the gene for this enzyme on a plasmid, characterization of single-cell plasmid content . Flow cytometry measurements may also be used to differentiate and quantify multiple species in a mixed culture and to detect the presence of contaminant in a fermentation inoculum. Since flow cytometry provides not only average information but gives a distribution of single-cell characteristics in the population, the data is rich and provides detailed insight into the state of the microbial population. Although most applications of flow cytometry to study fermentation processes have involved single-parameter measurements, it is possible to effect simultaneous multiple measurements on individual cells, gaining even further detailed

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Learning Objectives

The extraction and purification of fermentation products may be difficult and costly. Ideally, one is trying to obtain a high-quality product as quickly as possible at an efficient recovery rate using minimum plant investment operated at minimal costs. Unfortunately, recovery costs of microbial products may vary from as low as 15% to as high as 70% of the total manufacturing costs. Obviously, the chosen process, and therefore its relative cost, will depend on the specific product.

In this lecture, you will learn •

An introduction to downstream processing and product recovery

•

Methods in DSP and PR

•

Cell separation and cell disruption techniques

•

What is upstream processing and downstream processing?

•

Industrial fermentation processes can be divided into three major areas, viz. upstream processing (USP), which involves all factors and processes leading to the fermentation. This consists of three main areas. The first relates to aspects associated with the producer microorganism. They include the screening strategy for obtaining a suitable microorganism, industrial strain improvement to enhance productivity and maintenance of strain purity. The preparation of a suitable inoculum and the strain development efforts are also included in the first phase. The second aspect of USP involves fermentation media, especially the selection of suitable cost-effective carbon and energy sources, along with other essential nutrients. This media optimization is a vital aspect of process development to ensure maximization of yield and profit. The third component of USP relates to the fermentation, which is usually performed under rigorously controlled conditions developed to optimize the growth of the organism or the production of a target microbial product.

Why would the recovery and extraction costs be high? At the end of the fermentation, the fermentation broth contains intact micro-organisms, cell fragments, soluble and insoluble medium components and metabolic products including the fermentation product. The desired product may be present at a low concentration. It may also be intracellular, heat labile and easily broken down by contaminating microorganisms. All these factors tend to increase the difficulties of product recovery. To ensure good recovery or purification, speed of operation may be the overriding factor because of the labile nature of a product. The processing equipment must therefore be of the correct type and also the correct size to ensure that the harvested broth can be processed within a satisfactory time limit.

•

What are the various factors affecting DSP and how do we select the correct recovery method?

Down stream processing (DSP) encompasses all processes following the fermentation. It has the primary aim of efficiently, reproducibly and safely recovering the target product to the required specifications (biological activity, purity, etc.), while maximizing recovery yield and minimizing costs. The target product may be recovered by processing the cells or the spent medium depending upon whether it is an intracellular or extracellular product. The level of purity that must be achieved is usually determined by the specific use of the product. Often, a product’s purity will be defined by what is not present rather than what is. Purity of an enzyme, for example, is expressed as units of enzyme activity per unit of total protein. Not only is it important to reduce losses of product mass, but also in many cases retention of the product’s biological activity is vitally important.

Fermentation factors affecting DSP include the properties of microorganisms, particularly morphology, flocculation characteristics, size and cell wall rigidity. These factors have major influences on the filterability, sedimentation and homogenization efficiency. The presence of fermentation byproducts, media impurities and fermentation additives, such as antifoams, may interfere with DSP steps and accompanying product analysis. Consequently, a holistic approach is required when developing a new industrial purification strategy. The whole process, both upstream and downstream factors, needs to be considered. For example, the choice of fermentation substrate influences subsequent DSP. A cheap carbon and energy source containing many impurities may provide initial cost savings, but may necessitate increased DSP costs. Hence overall cost savings may be achieved with a more expensive but purer substrate. Also, adopting methods that use existing available equipment may be more cost effective than introducing more efficient techniques necessitating investment in new facilities.

Once upon a time, this recovery operation was considered less important as compared to the fermentation as such, but no longer. Now that the costs of product recovery and purification could be equal to or even higher to those of the fermentation itself, the product recovery has become extremely important aspect of the fermentation.

The physical and chemical properties of the product, along with its concentration and location, are obviously key factors as they determine the initial separation steps and overall purification strategy. It may be the whole cells themselves that are the target product or an intracellular product, possibly located within an organelle or in the form of

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LESSON 15: PRODUCT RECOVERY & DOWNSTREAM PROCESSING

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inclusion bodies. Alternatively, the target product may have been secreted into the periplasmic space of the producer cells or the fermentation medium. Stability of the product also influences the requirement for any pretreatment necessary to prevent product inactivation and/or degradation. The choice of recovery process is based on the following criteria: 1. Whether the product is intracellular or extracellular

of flocculants can also be attempted for selective removal of components from the broth. Assignment 1: Study any one of the following fermentations. Penicillin fermentation Citric acid fermentation Ethanol fermentation using continuous method Find out what are the latest trends in the recovery methods for that fermentation (Suggestion: look up for various types of industrial flocculants viz. cationic, anionic, and non ionic. Find out if any of them will find applications in the recovery procedure)

2. What is the final concentration of the product in the fermented broth? 3. What are the physical and chemical properties of the desired product?

Use the space provided to write briefly about your findings.

4. Where the product is intended to be used?

Assignment 2:

5. What is the minimum desired purity of the product?

Find out more about the continuous fermentation technologies offered for alcohol production (clue: technologies for this are offered by Praj Industries Ltd and Alfa Laval Ltd, both from Pune}. Compare the downstream processing technology offered by various companies. Write your views on which is the least complicated procedure and why. Use the space below to justify your views.

6. What hazards are involved in the recovery and handling of the product or the fermented broth? 7. What impurities are present in the broth? and finally, 8. What is the market price for the product? The major steps involved in the recovery of a product from the fermented broth can be tabularized as:

•

What are the flow sheets of downstream processing? The various steps involved in the processing of fermented broth can be schematically represented by a flow sheet like flows:

Fermented broth à removal of solids à product isolation à product purification à final product isolation An additional step of cell disruption may be involved in some cases if the desired product is intracellular.

Harvested broth ↓

The main objective of the first stage for the recovery of an extracellular product is the removal of large solid particles and microbial cells usually by centrifugation or filtration. In the next stage, the broth is fractionated or extracted into major fractions using ultrafiltration, reverse osmosis, adsorption/ionexchange/gel filtration or affinity chromatography, liquid-liquid extraction, two phase aqueous extraction or precipitation. Afterwards, the product-containing fraction is purified by fractional precipitation, further more precise chromatographic techniques and crystallization to obtain a product, which is highly concentrated, and essentially free from impurities. Other products are isolated using modifications of this flow-stream.

Filtration of mycelial growth using a rotary filter ↓

Adjust the pH to 5.8 by addition of Calcium Hydroxide ↓

Add sulfuric acid at 60 degree centigrade temperature ↓

Filter on rotary vacuum to remove Calcium Sulfate ↓

Treatment with activated charcoal to remove colour ↓

Attempts to simplify this outline extraction procedure for antibiotic recovery using ‘whole broth’ processing have met with limited success. The technique of whole broth processing includes removal of large particles followed by ion exchange or any other suitable technique. •

↓

Evaporation at 36 degree centigrade ↓

Can we do anything that would reduce the complexities involved in the downstream processing? Yes we can. We can, for example, select an organism that is non-pigment producing or non-capsule producing. We can fine-tune the fermentation conditions so that minimum contaminating metabolites are produced. We can select the exact time for harvesting that would permit maximum and simplest product recovery. We can control the pH of the broth precisely especially after harvesting. We can use gentler methods of cell lysis like use of chemicals or enzymes in place of more harsh methods like ultrasonication. The use

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Treatment with cation and anion exchange resins

Crystals of Citric monohydrate separated in continuous centrifuge ↓

Drying at 50 to 0 degree centigrade •

The recovery of a product may be possible by more than one method. How do we select which of these methods is appropriate?

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Sedimentation is extensively used for primary yeast separation in the production of alcoholic beverages, and in waste-water treatment. This low-cost technology is relatively slow and is suitable only for large floes (greater than 100pm diameter). The rate of particle sedimentation is a function of both size and density. Hence, the larger the particle and the greater its density, the faster the rate of sedimentation. The basis of this method of separation is sedimentation under gravity, which for a spherical particle can be represented by Stokes’ Law:

1. capital costs 2. processing costs 3. throughput requirements 4. yield potential 5. product quality 6. technical expertise available 7. regulatory requirements

Vg = d2p(Ps-Pl)g

8. waste treatment aspects

——————————

9. continuous or batch processing

18n

10.automation

where

11.personnel health and safety •

The basic techniques involved are sedimentation or precipitation, filtration and centrifugation. Employment of filtration aids and flocculants come as additional developments in this field. Before separation, some broths may need conditioning. Broth conditioning techniques are mostly used in association with sedimentation and centrifugation for the separation of cells from liquid media. They alter or exploit some property of a microorganism, or other suspended material, such that it flocculates and usually precipitates. However, in certain cases it may be used to promote flotation. This uses the ability of some cells to adsorb to the gas-liquid interfaces of gas bubbles and float to the surface for collection, which occurs naturally in traditional ale and baker’s yeast fermentations. Certain floc precipitation methods are also used at the end of many traditional beer and wine fermentation processes, where the addition of finings (egg albumen, isinglass, etc.) may be employed to precipitate yeast cells. Major advantages of these techniques are their low cost and ability to separate microbial cells from large volumes of medium. Some organisms naturally flocculate, which can be enhanced by chemical, physical and biological treatments. Such treatments can also be effective with cells that would not otherwise form flocs. Coagulation, the formation of small flocs from dispersed colloids, cells or other suspended material, can be promoted using coagu1ating agents (simple electrolytes, acids, bases, salts, multivalent ions and polyelectrolytes). Organic solvents, non-ionic polymers, protein binding dyes and affinity precipitants are also employed for precipitation. Subsequent flocculation is often aided by inorganic salts (e.g. calcium chloride) or polyelectrolytes. These are high molecular weight, water soluble, anionic, cationic or non-ionic organic compounds, such as polyacrylamide and polystyrene sulphate. •

Vg = rate of particle sedimentation (m/s);

How do we separate microbial cells from the fermented broth?

Exercise: find out more about the various flocculating and sedimentation agents used in the conditioning of fermented broths. Write a short note on the same. Now let us see about sedimentation.

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dp = diameter of the particle (m); Ps - PI = difference in density between the particle and surrounding medium (kg/m3); g = gravitational acceleration (m/s2); and n = viscosity (Pascal seconds (Pa s). Therefore, for rapid sedimentation the difference in density between the particle and the medium needs to be large, and the medium viscosity must be low. •

Ok, now what do we need to know about centrifugation? If instead of simply using gravitational force to separate suspended particles, a centrifugal field is applied, the rate of solid-liquid separation is significantly increased and much smaller particles can be separated. Centrifugation may be used to separate particles as small as O.l mu m diameter and is also suitable for some liquid-liquid separations. Its effectiveness, too, depends on particle size, density difference between the cells and the medium, and medium viscosity. In a centrifuge, the terminal velocity of a particle is, Vc = d 2(Ps-Pl) w 2r _________________ 18 n where, Vc = centrifugal sedimentation rate or particle velocity (m/s); w = angular velocity of the centrifuge (rad/s); and r = distance of the particle from the center of rotation (m) Hence, the faster the operating speed (w) and the greater the distance from the center of rotation, the faster the sedimentation rate (Vc). Centrifuges can be compared using the relative centrifugal force (RCF) or g number (the ratio of the velocity in a centrifuge to the velocity under gravity = w 2 r / g). The choice of centrifuge depends on the particle size and density, and the viscosity of the medium. Higher-speed centrifuges are required for the separation of smaller microorganisms, such as bacteria, compared with yeasts. For example, relatively slow centrifugation effectively recovers residual yeast cells remaining in beer after the bulk has

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You are right. Many times it is a critical choice. Factors that govern the appropriate choice of a method for downstream processing include

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sedimented out. Conversely, an RCF of 20000g may be required to recover suspended bacterial cells, cell debris and protein precipitates from liquid media. •

What are the advantages and disadvantages of centrifuges? There are many. We can rapidly process large volumes in small volume centrifuges. Centrifuges are steam sterilizable, allowing aseptic processing, and there are no consumable costs for membranes, chemicals or filter aids. However, the disadvantages of centrifugation are the high initial capital costs, the noise generated during operation and the cost of electricity. Also, physical rupture of cells may occur due to high shear and the temperature may not be closely controllable, which can affect temperature-sensitive products. Bioaerosol generation is a further major disadvantage, particularly when centrifuges are used for certain recombinant DNA organisms or pathogens. Under these circumstances the equipment must be contained.

•

•

Ok, now let’s see about filtration and the different types of filtration techniques available. The basic filter design employs a porous media that selectively retains the solids and allows free passage of liquids. The rate of filtration decreases with time as the filter starts to clog. Two main types of filters used are:

1. Filter presses: An assembly of alternate porous plates and hollow frames mounted on a support structure. The cell suspension is forced through these filters under pressure. The assembly is then dismantled and the filter cake is separated. These types of filters find applications in removal of cell mass from a fermentation like that of baker’s yeast or removal of protein precipitates.

Are industrial centrifuges different from laboratory scale ones, and if so, how? Centrifuges can be divided into small-scale laboratory units and larger pilot- and industrial-scale centrifuges. Laboratory batch centrifuges include, in ascending order of speed attainable: bench-top, high-speed and ultracentrifuges, capable of applying RCFs of 5000 to 500 000 g. Although industrial batch centrifuges are, available, for most industrial purposes semicontinuous and continuous centrifuges are required to process the large volumes involved. However, the RCFs achieved are relatively low.

Four main types of industrial centrifuge are commonly used. 1 Tubular centrifuges usually produce the highest centrifugal force of 13 000-17 000 g. They have hollow tubular rotor bowls providing a long flow path for the suspension, which is pumped in at the bottom and flows up through the rotor. Particulate material is thrown to the side of the bowl, and clarified liquid passes out at the top for continuous collection. As the particulate material accumulates on the inside of the bowl, the operating diameter becomes reduced. Consequently, there must be periodic removal of solids. 2 Multichamber bowl centrifuges consist of a bowl that is divided by vertically mounted interconnecting cylinders and are capable of operating at 5000-10000g. The liquid feed passes from the center through each chamber in turn, and the smaller particles collect in the outer chambers.

1. Rotary vacuum filters: A hollow perforated drum supporting a filter medium rotating in the tank of continuously agitating suspension. Due to the vacuum, liquid is sucked in and is filtered on the surface of the drum. A scraper installed at the other end of the drum removes the separated solids from the drum. Filter media can be precoated with a filter aid like diatomaceous earth which can be continuously replenished. Examples of these kinds of filters are filters used in the separation of fungal mycelia in antibiotic fermentation.

3 Disc stack centrifuges can operate at 5000-13000 g. The centrifuge bowl contains a stack of conical discs whose close packing aids separation. As liquid enters the centrifuge particulate material is thrown outwards. Particle then travel outwards to the bowl wall where they accumulate. These centrifuges have the facility to discharge the collected material periodically during the operation. 4 Screw decanter centrifuges operate continuously at 1500 to 5000 g and are suitable for dewatering coarse solid materials at high solid concentrations. They are used in sewage systems for the separation of sludge and for harvesting yeast and fungal mycelium.

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Both these types of filters are unsafe from the angle of biosafety, as both are potential generators of aerosols and

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•

below 10000 Da. However, non-spherical proteins may exhibit different exclusion reactions to the membrane. Flat membranes are available, but for larger-scale operations hollow-fiber systems are usually preferred .Several of these ultrafiltration units can be linked together to produce a sophisticated purification system These methods are extensively employed for the purification of proteins, and for separating and concentrating materials. Ultrafiltration is also effective in removing pyrogens (bacterial cell wall lipopolysaccharides), cell debris and viruses from media, and for whey processing.

What decides the choice of filter to be used? There are several factors. The properties of the filtrate, particularly its density and viscosity, the nature of the solid particles, particularly their size and shape, distribution and packing character, the solid: liquid ratio, the need to recover liquid fraction as well, the scale of operation, the need for batch or continuous fermentation, the need for asepsis, the need for pressure or vacuum suction ….. the list seems to be endless!

•

Another variation on the ultrafiltration system is diafiltration, where water or other liquid is filtered to remove unwanted low molecular weight contaminants. This can be used as an alternative to gel filtration or dialysis for removing ammonium sulphate from a protein preparation precipitated by this salt (desalting) for changing a buffer or in water purification.

What is membrane filtration? The science of filtration has made remarkable progress over last few years. It is possible no to separate not only the finest of suspended solids but also the dissolved salts out of the solution. Modern methods of filtration involve absolute filters rather than depth filters. These consist of supported membranes with specified pore sizes that can be divided into three main categories. They are, in decreasing order of pore size, microfiltration, ultrafiltration and reverse osmosis membranes. The suspension to be filtered is pumped across the membrane (cross-/tangential-flow) rather than at a right angle to it, as occurs with conventional filtration methods. This retards fouling of the membrane by particulate materials. Particles whose size is below the membrane ‘cutoff ’ will pass through the membrane to become the ultrafiltrate or permeate, whereas the remainder is retained as the retentate. As filtration progresses, the flux across the membrane can slow due to membrane fouling. This may be caused by the accumulation of a layer of solute molecules on the surface of the membrane, referred to as concentration polarization. The presence of silicon antifoams may have a similar negative effect.

•

Reverse osmosis is used for dewatering or concentration steps and has been employed to desalinate seawater for drinking. In osmosis water will cross a semipermeable membrane if the concentration of osmotically active solutes, such as salt, is higher on the opposite side of the membrane. However, if pressure is applied on the salt side, then reverse osmosis will occur, and water will be driven across the membrane from the salt side. This reversal of osmosis requires a high pressure, e.g. a pressure of 30-40 bar is needed to dewater a 0.6 mmol/L salt solution (note: 1 bar=100kPa=0.987 atm). Consequently, a strong metal casing is required to house this equipment. As the membranes have pore sizes of only 10-2 to 10-4 um diameter, solute molecules can deposit on the surface, causing a large resistance to solvent flow. However, this fouling can be overcome by increasing the turbulence at the surface of the membrane. Various chemicals are also used to prevent and control of fouling of these membranes.

Can you tell me more about these techniques? Sure. Microfiltration is used to separate particles of 10-2 um to 10 um, including removal of microbial cells from the fermentation medium. This method is relatively expensive due to the high cost of membranes, but it has several advantages compared with centrifugation. They include quiet operation, lower energy requirements, the product can be easily washed, good temperature control is possible, containment is readily achieved and no bioaerosols are produced. Consequently, it is suitable for handling pathogens and recombinant microorganisms. Ultrafiltration is similar to microfiltration except that the membranes have smaller pore sizes, and are used to fractionate solutions according to molecular wt, normally within the range 2000-500 000 Da. The membranes have anisotropic structure, composed of a thin membrane with pores of specified diameter providing selectivity, lying on top of a thick, highly porous, support structure. A membrane manufactured with an exclusion size of 100 000 Da, for example, should produce a retentate of proteins and other molecules over 10000 Da and an ultrafiltrate of all molecules

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·

Exercise: find out more about the antifoulant chemicals used in RO systems. Write briefly on their mode of activity, advantages and disadvantages. Use additional sheets if required. If possible make a visit to an industrial RO unit and study its operation. Write a report.

•

What is the next technique used? Well, it is cell disruption. Some target products are intracellular, including many enzymes and recombinant proteins, several of which form inclusion bodies, which are concentrated proteins with incomplete tertiary structure. Therefore, methods are required to disrupt the microorganisms and release these products. The breaking of the cell wall/envelope and cytoplasmic membrane can pose problems, particularly where cells possess strong cell walls. For example, a pressure of 650 bar is needed to disrupt yeast cells, although this may vary somewhat at different times during the growth cycle and depending upon the specific growth conditions.

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allergens and thus are unsuitable for processing toxic products, pathogens or certain recombinant DNA microorganisms and their products.

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General problems associated with cell disruption include the liberation of DNA, which can increase the viscosity of the suspension. This may also affect further processing, such as pumping the suspension on to the next unit process and flow through chromatography columns. A nucleic acid precipitation step or the addition of DNase can help to prevent this problem. If mechanical disruption is used then heat is invariably generated, which denatures proteins unless appropriate cooling measures are implemented. Products released from eukaryotic cells are often subject to degradation by hydrolytic enzymes (proteases, lipases, etc.) liberated from disrupted lysosomes. This damage can be reduced by the addition of enzyme inhibitors, cooling the cell extract and rapid processing. Alternatively, attempts may be made to produce mutant strains of the producer microorganism lacking the damaging enzymes. •

How do we achieve this goal of breaking open the cells, then? Cell disruption can be achieved by both mechanical and nonmechanical methods. The disruption process is often quantified by monitoring changes in absorbance, particle size, total protein concentration or the activity of a specific intracellular enzyme released into the disrupted suspension. First, let’s study about the mechanical cell disruption methods. Several mechanical methods are available for the disruption of cells. Those based on solid shear involve extrusion of frozen cell preparations through a narrow orifice at high pressure. This approach has been used at the laboratory scale, but not for large-scale operations. Methods utilizing liquid shear are generally more effective. The French press (pressure cell) is often used in the laboratory and the high-pressure homogenizers, such as the Manton and Gaulin homogenizer (APV-type mill), are employed for pilot- and productionscale cell disruption. They may be used for bacterial and yeast cells, and fungal mycelium. In these devices the cell suspension is drawn through a check valve into a pump cylinder. At this point, it is forced under pressure (up to 1500 bar) through a very narrow annulus or discharge valve, over which the pressure drops to atmospheric. Cell disruption is primarily achieved by high liquid shear in the orifice and the sudden pressure drop upon discharge causes explosion of the cells.

•

What are the factors that decide the efficiency of cell disruption? The efficiency of disruption is independent of the cell concentration, but is depends on the pressure exerted, the number of cycles through the homogenizer and the temperature. Disruption of yeast cell preparations, for example, typically requires three passes through the pressure cell at 650 bar, whereas wild-type Escherichia coli generally needs 1100-1500 bar. During processing the temperature rises by about 2.2-2.4°C per 100 bar, i.e. by approximately 20 0 C over one pass at 800 bar. Consequently, precooling of the cell preparation is usually essential. The energy input necessary is approximately 0.35kW per 100bar and the throughput is up to 6000 L/h. A problem with this method

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of cell disruption is that all intracellular materials are released. As a result, the product of interest must be separated from a complex mixture of proteins, nucleic acids and cell wall fragments. Some fragments of cell debris are not readily separated, making the solution difficult to clarify. In addition, proteins may be denatured if the equipment is not sufficiently cooled and filamentous microorganisms may block the discharge valve. When used for certain categories of microorganisms, the homogenizers have to be contained to prevent the escape of aerosols. •

How can we carry out cell disruption on a small scale, say, in a laboratory? On a small scale, manual grinding of cells with abrasives, usually alumina, glass beads, kieselguhr or silica, can be an effective means of disruption, but results may not be reproducible. In industry, high-speed bead mills, equipped with cooling jackets, are often used to agitate a cell suspension with small beads (0.5-0.9um diameter) of glass, zirconium oxide or titanium carbide. Cell breakage results from shear forces, grinding between beads and collisions with beads. The efficiency of cell breakage is a function of agitation speed, concentration of beads, bead density and diameter, broth density, flow rate and temperature. Cell concentration is also a major factor (optimum 30-60% dry weight), which is an important difference from the liquid shear homogenizers described above. Maximum throughput in these systems is about 2000 Llh. Ultrasonic disruption of cells involves cavitation, microscopic bubbles or cavities generated by pressure waves. It is performed by ultrasonic vibrators that produce a highfrequency sound with a wave density of approximately 20kilohertz/s. A transducer converts the waves into mechanical oscillations via a titanium probe immersed in the concentrated cell suspension. However, this technique also generates heat, which can denature thermolabile proteins. Rod-shaped bacteria are often easier to break than cocci, and Gram-negative organisms are more easily disrupted than Gram-positive cells. Sonication is effective on a small scale, but is not routinely used in large-scale operations, due to problems with the transmission of power and heat dissipation. Some newer disruption systems are being developed to give improved large-scale disruption, often with integral containment. They include a newly designed ball mill, the CoBall Mill; the Constant Systems high-pressure disrupter, which operates at up to 2700 bar; and two systems with no moving parts, the Microfluidics impingement jet system and the Glass-col nebulizer. The Parr Instruments cell disruption bomb is designed for disrupting mammalian cells. This is a relatively gentle method that works on the principle of nitrogen decompression and does not generate heat. Nitrogen is dissolved in cells under high pressure, and sudden pressure release then causes the cells to burst.

Exercise “Mechanical means of cell disruption with high energy inputs are effective, but pose a great threat to the safety of workers and

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environment, especially in the absence of proper containment processes”. Justify. Now let’s see about the non-mechanical cell disruption methods. Autolysis, osmotic shock, rupture with ice crystals (freezing/ thawing) or heat shock are some of the ‘non violent’ methods used for cell disintegration. . Autolysis, for example, has been used for the production of yeast extract and other yeast products. It has the advantages of lower cost and uses the microbe’s own enzymes, so that no foreign substances are introduced into the product. Osmotic shock is often useful for releasing products from the periplasmic space. This may be achieved by equilibrating the cells in 20% (w/v) buffered sucrose, then rapidly harvesting and resuspending in water at 4°C. Wide ranges of other techniques have been developed for small-scale microbial disruption using various chemicals and enzymes. However, some of these can lead to problems with subsequent purification steps. Organic solvents, usually acetone, butanol, chloroform or methanol, have been used to liberate enzymes and other substances from microorganisms by creating channels through the cell membrane. Simple treatment with alkali or detergents, such as sodium lauryl sulphate or Triton X100, can also be effective. Several cell wall degrading enzymes have been successfully employed in cell disruption. For example, lysozyme, which hydrolyses B 1,4 glycosidic linkages within the peptidoglycan of bacterial cell walls, is useful for lysing Gram-positive organisms. Addition of ethylene diamine tetraacetic acid (EDTA) to chelate metal ions also improves the effectiveness of lysozyme and other treatments on Gram-negative bacteria. This is because EDTA has the ability to sequester the divalent cations that stabilize the structure of their outer membranes. Enzymic destruction of yeast cell walls can be achieved with snail gut enzymes that contain a mixture of B-glucanases. These enzyme preparations are also useful for producing living yeast spheroplasts or protoplasts. The antibiotics penicillin and cycloserine may be used to lyse actively growing bacterial cells, often in combination. Additionally, basic proteins like protamines etc. can also be used for cellular lysis. After the products have been brought into an extractable form, our next job is to recover them in pure form. In the next lesson , we will see how to do that.

Notes

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LESSON 16: USE OF COMPUTERS IN FERMENTATION

Learning Objectives In this lecture, you will learn •

Applications of computers in fermentations

•

Adaptive control

•

Data logging, data analysis & process control

Why Computers? This question is really irrelevant. While we can still imagine a world without computers, it would be a very underdeveloped one. It is therefore not surprising that computers are finding application in the field of fermentation technology as well. There are a number of advantages to be gained by coupling process instruments to digital computers. First, the computer can enhance data acquisition functions in several respects. Improved reliability and accuracy can be obtained by using statistical methods and digital filtering. Readings from several parallel sensors can be compared and analyzed to provide online recalibration and to identify sensor failure. With a computer, the number and sophistication of analysis systems can be increased. For example, a computer-controlled system may take samples automatically, conduct a chromatographic analysis, and interpret the results, using internally stored calibrations or algorithms to give output directly in convenient units. Although simple signal conditioning and correcting operations such as linearization can be done with particular electronic circuits, these functions are readily accomplished using a computer without the need for additional specific hardware. Another advantage of computers with respect to data acquisition is the ability to store large quantities of measured results in digital form which may be accessed conveniently, analyzed, and displayed later. Using computers, data analysis and interpretation can be enhanced greatly. Results of several measurements may be combined to calculate instantaneously quantities such as oxygen utilization rate and respiratory quotient. Advanced Slate and parameter estimation methods may also be applied on-line to provide additional useful information on process status from the limited measurements available. More specifics and some examples of computer applications for data analysis are presented in the next section.

Computers expand opportunities tremendously for improved process control and optimization. One computer can replace many conventional analog controllers and control many individual valuables such as pH and temperature using standard feedback algorithms. Furthermore, more sophisticated multi variable control methods may be implemented easily with a computer. Controlled variables may include derived quantities such as RQ when a computer is applied. Computer methods may be used to evaluate and improve process mathematical models which may then be employed for determining optimum operating conditions and strategies. Then, the computer provides the memory and computation capability to implement the optimization method, such as variation of nutrient feeding rate or pH during a batch fermentation. Operation of a batch process requires a carefully controlled and coordinated sequence of valve openings and closings and pump starts and stops. While all of these functions have been done by

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Before turning to these interesting domains of computer application, we shall examine briefly some of the principles of digital computers and computer interfaces. Our objective here is to introduce some generic concepts and, by example, to illustrate specific realizations of different types of computerprocess configurations. Because improvements and cost reductions in computer hardware and software are proceeding presently at a rapid rate, any specific computer system is probably outdated by the time its description has been published-certainly in book form. Thus, we should view the examples here and in the remainder of the chapter as the kinds of things which can be done, recognizing that, as of this reading, there are probably cheaper, more efficient ways of doing the same thing or something even more effective. The basic components of a digital computer are shown in the block diagram. The central processing unit (CPU) accepts instructions from a stored program through its control unit and performs the indicated arithmetic and logical operations in the arithmetic and logic unit (ALV), using internal registers for short-term storage. Operations in the CPV are controlled and synchronized by an internal quartz oscillator clock. The cycle time of the CPU, which may range from less than 10 to 104 ns (10-9 s), combined with a number of bytes (each byte contains eight bits, a binary number with value 0 or 1) processed per cycle (the word size), determines the speed of computation in the Cpu. For example, microcomputers available in 1980 employed 8-bit words. By 1982 16-bit microcomputers were available, and 32-bit machines were manufactured by several companies in 1984. Memory for storage of program instructions and data is provided in several different forms. Read-only memory (ROM) contains fixed instruction sets such as compilers and interpreter programs, while random access memory (RAM) is used for short-term storage of programs, input data, and computational results. The CPV reads frequently from and writes on the RAM during computer operation. As of the mid-1980s, popular microcomputers had RAM capacities from 64,000 bytes (64 kilobytes or just 64 K) to 512 K and beyond. Additional memory is usually available also in one or more peripheral devices. Common external mass memory devices for use with microcomputers are magnetic tape cassettes (storage 80-300 K, access time -10 s), floppy disks (100-600 K, access time = O.5 s) and hard disks (500-20,000 K, access time = 3 s all numbers as of 1984). The input-control allows the computer to communicate with external peripheral devices. Within the computer, a bus system interconnects the CPU, memory, and input-output control segments. The functional elements described above are common to all computers, but the speed and memory capacity are determined 2.521

by the particular hardware configuration. Based on these parameters, computers are often classified into super computers, mainframe computers, minicomputers, and microcomputers, with this list ordered from largest, fastest, and most costly to smallest, slowest, and least expensive. Definition of the boundaries between these different classes of computers is constantly shifting; to day’s microcomputers have the power of mainframe computers of the 1970s. The availability of tremendous computing capacity at low cost is driving a revolution of new computer uses in consumer products, communications, information processing, scientific instrumentation, and in biotechnology. While computercoupled fermentors were a novelty in the 1960s, we can expect in the not too distant future that almost every bioreactor, analytical instrument, and other bioprocess unit will be monitored and controlled by digital computers. It is possible to prepare sequential programs using a computer based control system. Consider control of the dissolved oxygen concentration in a fermentation broth. This may be changed by altering the agitation rate, the air or gas flow rate, the partial pressure of oxygen in the inlet gas or the total fermentor pressure. In practice, combinations of these variables may be used either sequentially or simultaneously using suitable computer programs. Initially the agitation rate can be increased to respond to decreases in dissolved oxygen concentration. When a predetermined maximum agitation rate is reached, the air flow can be steadily increased to a preset maximum, followed by the third and subsequent stages. Setpoint

Disturbances

Adaptive control

•

How are computer techniques applied in fermentations? Since the initial use of computers in the 1960s for modeling fermentation processes and in process control for production of glutamic acid and penicillin, there have been numerous

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various timers and relays in earlier technology, they may now be managed efficiently by computer. Use of a computer to manage such switching operations during batch process operation becomes essential if we wish to optimize the scheduling of a number of parallel batch processes (e.g., fermentors) which feed sequentially to downstream batch processes (e.g., precipitation, chromatography, and so forth).

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publications on computer applications in fermentation technology. Initially, the use of large computers was restricted because of their cost but reductions in costs and the availability of cheaper small computers has widened interest in their possible applications. The availability of efficient small computers has led to their use for pilot plants and laboratory systems since the financial investment for the online computer amounts to a relatively insignificant part of the whole system. Three distinct areas of computer function were recognized.

1. Logging of Process Data The simplest task for a computer is data logging can be measured by sensors which produce a signal which is compatible with the computer system. Programs have been developed so that by reference to the realtime clock, the signals from the appropriate sensors will be scanned sequentially in a predetermined pattern and logged in a data store. Typically, this may be 2- to 60-second intervals, and the data is printed out on a visual display unit. In preliminary scanning cycles the values are compared with predefined limit values, and deviations from these values result in an error print out, or if more extreme then an alarm may be activated. In the final cycle of a sequence, say every 5 to 60 minutes, the program instructs that the sensor readings are permanently recorded on a print out or in a data store. At the same time as on-line data is being recorded from sensors, analytical data for broth viscosity, microbial growth, substrate and precursor utilization and product formation, which have to be determined separately may be logged into the data store for specific known times. Thus, it is now possible to record data continuously for range of parameters from a number of fermentors simultaneously using minimal manpower provided that capital outlay is made for fermentor with suitable instrumentation coupled with adequate computer facility. Data logging is performed by the data acquisition system which has both hardware and software components. There is an interface between the sensors and the computer. The software should include the computer program for sequential scanning of the sensor signals and the procedure of data storage. 2. Data Analysis (Reduction Of Logged Data) Because a computer can undertake so many calculations very rapidly, it is possible to design programs to analyze fermentation data in a number of ways. A linked main-frame computer may be used for part of this analysis as well as the dedicated small computer. A number of the monitoring systems were described as ‘GatewaySensors’byAibaet al. Gateway sensors are so called because the information they yield can be processed to give further information about the fermentation. The respiratory quotient of a culture may be calculated from the metered gas-flow rates and analyses for oxygen and carbon dioxide leaving a known volume of culture in the fermentor. This procedure was used to monitor growth of Candida utilis

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in a 250-dm3 fermentor, to follow or forecast events during operation. If one defines the fraction of substrate which is converted to product then it is possible to write mass balances for C, H, O and N with the measurement of only a few quantities (O2, CO2, NH3, etc.). All the other quantities can be calculated, including biomass and yield, if the biomass elemental composition is known. This procedure was used for the analysis of a bakers yeast fermentation (Cooney et al., 1977). Biomass production can be regarded as a stoichiometric relationship in which substrate is converted, in the presence of oxygen and ammonia to biomass, carbon dioxide and water: Carbon source-energy + oxygen + ammonium ----> cells + water + carbon dioxide. Data reduction is performed by the data-analysis system, which is a computer program based on a series of selected mathematical equations. The analysed information may then be put on a print out, fed into a data bank or utilized for process control.

3. Process control Process control is also performed using a computer program. Signals from the computer are fed to pumps, valves or switches via the interface. In addition the computer program may contain instructions to display devices or teletypes, to indicate alarms, etc. At this point it is necessary to be aware that there are two distinct fundamental approaches to computer control of fermentors. The first is when the fermentor is under the direct control of the computer software. This is termed Direct Digital Control (DDC) and will be discussed in the next section. The second approach involves the use of independent controllers to manage all control functions of a fermentor and the computer communicates with the controller only to exchange information. This is termed Supervisory Set-Point Control (SSC) and will be discussed in more detail in the Process Control section. It is possible to analyze data, compare it with model systems in a data store, and use control programs which will lead to process optimization. However, process optimization by this method is not a widely used procedure in the fermentation industries at present. It is important to be aware of these different applications, since this will influence the size and type of computer system which will be appropriate for the precise role that it is intended to perform, whether in a laboratory, a pilot plant, or manufacturing plant, or a combination of these three. Arrninger and Moran (1979) recognized three levels of process control that might be incorporated into a system. Each higher level involves more complex programs and needs a greater overall understanding of the process. The first level of control, which is already routinely used in the chemical industries, involves sequencing operations, such as manipulating valves or starting or stopping pumps, instrument recalibration, on-line maintenance and fail-safe shut-down procedures. In most of these operations the time base is at least in the order of minutes, so that high-speed manipulations are not vital. Two

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linear processing units arranged in layers with adjustable connecting strengths (weights).

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applications in fermentation processes are sterilization cycles and medium batching. The next level of computer control involves process control of temperature, pH, foam control, etc. where the sensors are directly interfaced to a computer (Direct Digital Control DOC) . When this is done separate controller units are not needed. The computer program determines the set point values and the control algorithms, such as PID, are part of the computer software package. Better control is possible as the control algorithms are mathematically stored functions rather than electrical functions. This procedure allows for greater flexibility and more precise representation of a process control policy. The system is not very expensive as separate electronic controllers are no longer needed, but computer failure can cause major problems unless there is some manual back-up. The alternative approach is to use a computer in a purely supervisory role. All control functions are performed by an electronic controller where the linked computer only logs data from sensors and sends signals to alter set points when instructed by a computer program or manually. This system is known as Supervisory Set-Point Control (SSC) or Digital SetPoint Control (DSC). When SSC is used, the modes of control are limited to proportional, integral and derivative because the direct control of the fermentor is by an electronic controller. However, in the event of computer failure the process controller can be operated independently. The most advanced level of control is concerned with process optimization. This will involve understanding a process, being able to monitor what is happening and being able to control it to achieve and- maintain optimum conditions. Firstly, there is a need for suitable on-line sensors to monitor the process continuously. A number are now available for dissolved oxygen, dissolved carbon dioxide, pH, temperature, biomass (the bug meter. NADH fluorescence, near infra-red spectroscopy) and some metabolites (mass spectroscopy and near infra-red spectroscopy). All these sensors have been discussed earlier in this chapter. Secondly, it is important to develop a mathematical model that adequately describes the dynamic behaviour of a process. Shimizu (1993) has stressed the vital role which these models play in optimization and reviewed the use of this approach in batch, fed-batch and continuous processes for biomass and metabolites. This approach with appropriate online sensors and suitable model programs has been used to optimize bakers’ yeast production (Ramirez et al., 1981; Shi et al., 1989), an industrial antibiotic process and lactic acid production (Shi et al., 1989). Although much progress has been made in the ability to control a process, few sensors are yet available to monitor online for many metabolites or other parameters in a fermentation broth thus delaying or making a fast response difficult for online control action. Also, it is possible that not all the important parameters in a process have been identified and the mathematical model derived to describe a process may be inadequate. Because of these limitations, an artificial neural network may be used to achieve better control (Karim and Rivera, 1992) .These are highly interconnected networks of non-

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Input layer

Hidden layer

Output

layer

Two-layer neural network (not all the possible interconnections are shown). In simpler neural networks there is one input layer, one hidden layer and one output layer. Unlike recognized knowledge-based systems, neural networks do not need information in the form of a series of rules, but learn from process examples from which they derive their own rules. This makes it possible to deal with non-linear systems and approximate or limited data. When training a neural network the aim is to adjust the strengths of the interconnections (neurons) so that a set of inputs produces a desired set of outputs. The inputs may be process variables such as temperature, pH, flow rates, pressure and other direct or indirect measurements which give information about the state of the process. The process outputs obtained (biomass, product, etc.) produce the teacher signal(s) which trains the network. The difference between the desired output and the value predicted by the network is the prediction error. Adjustments are made to minimize the total prediction error by modifying the interconnection strengths until no further decrease in error is achieved. Commercial computer packages are now available to help to determine which of the input variables to use for training and to determine the optimum number of interconnections and hidden layers (Glassey et al., 1994). This method of control is still at an early stage of development, but it has already been used in a case study on ethanol production by Zymomonas mobilis (Karim and Rivera, 1992), in real-time variable estimation and control of a glucoamylase fermentation) and recombinant Escherichia coli fermentations.

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In industrial systems where a significant amount of on-line and off-line process data may be available, but there are tight time restraints imposed on process optimization, the potential for developing a relatively accurate neural network model within short time scales becomes very attractive.

The accuracy of an ADC will depend on the number of bits (the unit of binary information) it sends to computer. An 8bit converter will work in the range 0-255 and it is therefore able to divide a signal voltage into 256 steps. This will give a maximum accuracy of 100/256, which is approximately 0.4%. However, a l0-bit converter can give 1024 steps with an accuracy of 100/1024, which is approximately 0.1%. Therefore when a parameter is to be monitored very accurately a converter of the appropriate degree of accuracy will be required. The time taken for an ADC to convert voltage signals to a digital output will vary with accuracy, but improved accuracy leads to slower conversion and hence slower control responses. However, cycle times of about 1 second may be adequate in many fermentation systems. It is also important to ensure that the voltage ranges of the sensors are matched to the ADC input range. A digital to analogue converter (DAC) converts a digital signal from the computer into an electrical voltage which can be used to drive electrical equipment, e.g. a stirrer motor. Like the ADC, the accuracy If the DAC will be determined by whether it is 8-bit, O-bit, 12-bit, etc., and will for example determine the size of steps in the control of rpm of a stirrer motor.

•

What are the components of a computer-linked system? When a computer is linked to a fermentor to operate as a control and recording system, a number of factors must be considered to ensure that all the components interact and function satisfactorily for control and data logging. A DDC system will be used as an example to explain computer controlled addition of a liquid from a reservoir to a fermentor. A simple outline of the main components is given in figure. A sensor S in the fermentor produces a signal which may need to be amplified and conditioned in the correct analogue form. At this stage it is necessary to convert the signal to a digital form which can be subsequently transmitted to the computer. An interface is placed in the circuit at this point. This interface serves as the junction point for the inputs from the fermentor sensors to the computer and the output signals from the computer to the fermentor controls such as a pump T attached to an additive reservoir. Digital to analogue conversion is necessary between the interface and the pump T. A sensor will generate a small voltage proportional to the parameter it is measuring. For example, a temperature probe might generate 1 V at 10°C and 5 V at 50°C. Unfortunately, the signal cannot be understood by the computer and must be converted by an analogue to digital converter (ADC) into a digital form.

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The small computer itself is dedicated solely to one or more fermentors. This computer is coupled to a real-time clock, which determines how frequently readings from the sensor(s) should be taken and possibly recorded. The other ancillary equipment linked directly to the computer might include a visual display unit, a data store, a teletype, a graphic display unit, a print out, alarms and a barometer. The small computer is often connected to a large main frame computer for random access, not on a real-time scale, but for long-term data storage and retrieval and for complex data analysis which will not be utilized subsequently in real-time control.

Simplified layout of computer-controlled fermentor with only one control loop shown.

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It is also possible to develop programs so that on-line instruments can be checked regularly and recalibrated when necessary. Swartz and Cooney (1979) were able to routinely recalibrate a paramagnetic oxygen analyzer and an infrared carbon dioxide analyzer every 12 hours utilizing a program which connected a gas of known composition to the analyzers and subsequently monitored the analyzer outputs.

Notes

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UNIT-III GROWTH KINETICS IN FERMENTATION

LESSON 17: FERMENTATIONS KINETICS

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Learning Objectives

•

In this lecture, you will learn Introduction to fermentation kinetics Microbial growth kinetics Michaelis-Menten’s model for cell growth

Introduction •

What is fermentation economics and why do we need to study it? When we want to size or design a fermentor process, we should know how the fermentation will proceed before we start constructing the reactor and just as importantly, before we allocate money for the project. There are a number of steps involved in studying a fermentation process. The process will initially be studied in laboratories using flasks and laboratory scale Fermentors. If the process looks commercially successful, the process will be “scaled-up” to a pilot scale process. During this stage, the process will be looked at in terms of engineering factors such as “mass transfer” and “heat transfer”. Downstream processing will also be looked into. The pilot scale studies will determine the commercial viability of the process and if successful the process will be scaled up to industrial/commercial scale.

Ok. How do we study the fermentation kinetics? One important way of undertaking this task is to describe the major components of the system in terms of mathematical equations i.e. mathematical models.You will have used mathematical models since you were in primary school. For example, to understand how the volume of cube related to the length of its side, the following equation is used: Volume = Length3 When studying chemistry, chemical kinetic equations are used. Just as chemical reactions are described or “modelled” using chemical reaction kinetic equations, fermentation are “modelled” using fermentation kinetics. Here are some examples of kinetic equations used to describe chemical, enzymatic and fermentation systems (using commonly used terminology): First order chemical reaction where [A] is the concentration of a reactant Second oder chemical reaction where [A] is the concentration of a reactant Michaelis Menten Enzyme Kinetics where [S] is the concentration of the enzyme substrate Microbial kinetics based on the Monod equations where X is the concentration of biomass and S is the concentration fo substrate

Some fermentation processes however can involve the complex interactions of biological, chemical and physical factors. To properly investigate fermentation and to be able to predict the effects that these factors play on fermentations, the process needs to be broken down into meaningful units. It is often a great help if the behaviour of the entire fermentation operation is predicted BEFORE the actual fermentation starts. While the laboratory and pilot plant studies offer a fairly reliable picture of how the fermentation is going to behave, the exact engineering aspects of the fermentation could be predicted using what is called the fermentation kinetics.

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Note that all of the above equations describe the rate of change in the concentration of a particular compound. When these differential equations are integrated, the resultant equations predict the concentration of a particular component with respect to time. Using kinetic equations, we can, for example, predict how long it will take to achieve a particular %conversion or how long it will take for a system to stabilize after a change in conditions. Similarly, we can find out the behaviour of batch fermentation by studying the kinetics of batch culture.

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Lag phase Log or exponential phase Post-log phase Stationary phase

If we plot ln [Cells] in the exponential phase against time, we get a straight line, hence then name “log phase”

During this phase, cell growth is slow. Cells adapt to the new environment undergo. Cell growth reaches its maximum rate. The cell numbers increase exponentially Cells continue growing but at a rate less than maximum. Cell numbers no longer increase. Cells may continue metabolizing.

Cell death becomes obvious. Note that cell death occurs throughout the batch growth curve. Cell Death phase growth has either stopped or is so slow that the rate of cell death exceeds the rate of cell growth.

•

·Why do cells go through expotential growth phase? To answer this question, consider a cell that divides every hour:

The Expotential Phase During the exponential phase, cells grow at their maximum rate and cell numbers increase exponentially, i.e. at an increasingly faster rate.

If we start with 1 cell, at 2 hours we will have 2 cells. After 3 hours we will have 4 cells (not 3 cells). After 4 hours, we will have 8 hours and not 4 cells. The more cells we have the faster the population will increase at. Cells thus catalyse their own increase in numbers and cells are often described as “autocatalytic”. In mathematical terms, we can write:

Substituting the proportional sign for a constant (µ), we get

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How? When cells are grown in a batch reactor, they go through a series of stages known as

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The “constant” µ is referred to as the specific growth rate and represents the rate at which the individual cells divide at. Assume µ is a constant and if we integrate between time t0 and time t1 when the concentrations of cells are X0 and X1 respectively we obtain:

graph, which appeared similar to enzymatic rate-substrate relationships defined by Michaelis-Menten’s model:

Whereas Michaelis-Menten’s model is:

The above equation can be manipulated to give:

•

A plot of biomass concentration (X) against time will give the exponential relationship seen during the “exponential phase”:

Where Vmax is the maximum enzyme velocity and KM is a saturation constant.

Monod’s model describing the relationship between the specific growth rate and the growth limiting substrate concentration as:

•

where µm is the maximum specific growth rate and Ks is a saturation constant.

It should be noted that Monod’s model was derived simply from a curve fitting exercise, unlike the Michaelis-Menten relationship which has a mechanistic basis. Despite this Monod’s model is widely used to describe the growth of many organisms. Basically because it does adequately describe fermentation kinetics. What’s more the model can and has been modified describe complex fermentation systems. For example, a commonly used expression describing product inhibition is:

Now let us see what the Monod model is. The major problem of the exponential growth equation is that it does not predict an end to growth in a batch environment. According to this model, not only the whole earth but whole solar system could quickly become covered with bacteria (if they could make the journey). In the 1930’s, Jaques Monod looked at this problem. He performed a number of initial rate experiments and then plotted the specific growth rate against the concentration of the growth-limiting substrate. The result was a Langmuir type

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Using the Monod Model, a simple model microbial growth can be written as:

where Yxs is the biomass yield coefficient.

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The biomass yield coefficient is the efficiency of conversion of substrate to biomass and is calculated as:

Notes

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LESSON 18: FED BATCH FERMENTATIONS

Learning Objectives In this lecture, you will learn •

What are fed batch fermentation? What are its types?

•

Advantages of fed batch fermentations

•

Instrumentation & control in fed batch fermentations.

Well, as we have seen, industrial fermentations are operated as batch, fed batch or continuous cultures. Most are batch processes, which are closed systems with no additions of nutrients following inoculations. Exception to this is of course acid or alkali for pH control and air for aeration. Thus, there is a definite beginning and end of the fermentation. The batch, like a class period, starts at a point and ends at another! A fermentor is loaded, sterilized, inoculated and incubated, during which the inoculated organism follows a typical growth cycle. On completion of the fermentation, the product is harvested and the fermentor is subjected to cleaning and sterilization. Period. •

And in the continuous fermentation, there is a beginning but no end since the fermentation will continue indefinitely, right? Unless of course, contaminations or other such problems force us to terminate it. But there is a third type of fermentation which can, roughly be called as a cross between these two. This is called the fed batch fermentations. Fed batch fermentations include intermittent, continuous or single slug addition of extra nutrients during the course of fermentation. These additions are often made towards the end of a rapid phase of growth of organisms.

•

What is the need for fed batch fermentations? In conventional batch fermentations, considerable time is lost between two batches of fermentations. The non productive time of the fermentor is called the ‘down time’. Obviously, from the economic point of view, this unproductive period should be as less as possible. This can be achieved, at least to some extent, by operating the system on a fed batch basis. Fed batch fermentations can extend the product formation phase and may overcome the problems associated with the use of rapidly metabolizable substrates. This method is also useful in cases where the initial viscosity of the medium is too high to permit higher substrate concentration or where the substrate is toxic to the fermenting organism at high concentration. In some cases, selective cell recycle is also possible with fed batch technique. Fed batch technique is successfully used in production of products like baker’s yeast. So, in a nutshell, the benefits offered by fed batch fermentation are as follows:

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Fed-batch offers many advantages over batch and continuous cultures. From the concept of its implementation it can be easily concluded that under controllable conditions and with the required knowledge of the microorganism involved in the fermentation, the feed of the required components for growth and/or other substrates required for the production of the product can never be depleted and the nutritional environment can be maintained approximately constant during the course of the batch. The production of byproducts that are generally related to the presence of high concentrations of substrate can also be avoided by limiting its quantity to the amounts that are required solely for the production of the biochemical. When high concentrations of substrate are present, the cells get “overloaded”, this is, the oxidative capacity of the cells is exceeded, and due to the Crabtree effect, products other than the one of interest are produced, reducing the efficacy of the carbon flux. Moreover, these by-products prove to even “contaminate” the product of interest, such as ethanol production in baker’s yeast production, and to impair the cell growth reducing the fermentation time and its related productivity. Sometimes, controlling the substrate is also important due to catabolic repression. Since this method usually permits the extension of the operating time, high cell concentrations can be achieved and thereby, improved productivity [mass of product/ volume/time]. This aspect is greatly favored in the production of growth-associated products. Additionally, this method allows the replacement of water loss by evaporation and decrease of the viscosity of the broth such as in the production of dextran and xanthan gum, by addition of a water-based feed. As previously mentioned, fed-batch might be the only option for fermentations dealing with toxic or low solubility substrates. When dealing with recombinant strains, fed-batch mode can guarantee the presence of an antibiotic throughout the course of the fermentation, with the intent of keeping the presence of an antibiotic-marked plasmid. Since the growth can be regulated by the feed, and knowing that in many cases a high growth rate can decrease the expression of encoded products in recombinant products, the possibility of having different feeds and feed modes makes fed-batch an extremely flexible tool for control in these cases. Because the feed can also be multisubstrate, the fermentation environment can still be provided with required protease inhibitors that might degrade the product of interest, metabolites and precursors that increase the productivity of the fermentation. Finally, in a fed-batch fermentation, no special piece of equipment is required in addition to that one required by a

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` A cyclic fed-batch culture has an additional advantage: the productive phase of a process may be extended under controlled conditions. The controlled periodic shifts in growth rate provide an opportunity to optimize product synthesis, particularly if the product of interest is a secondary metabolite whose maximum production takes place during the deceleration in growth. •

•

Ok, now tell me about variable volume fed batch fermentations. As the name implies, a variable volume fed-batch is one in which the volume changes with the fermentation time due to the substrate feed. The way this volume changes it is dependent on the requirements, limitations and objectives of the operator.

So fed batch fermentations is an ideal mode for many products. All advantages and no drawbacks, is it? Unfortunately, no. The science of fed batch fermentations also comes with several disadvantages. Some of them are as follows:

The feed can be provided according to one of the following options:

•

it requires previous analysis of the microorganism, its requirements and the understanding of its physiology with the productivity

(ii) a solution of the limiting substrate at the same concentration as that in the initial medium is added; and

•

it requires a substantial amount of operator skill for the setup, definition and development of the process

•

in a cyclic fed-batch culture, care should be taken in the design of the process to ensure that toxins do not accumulate to inhibitory levels and that nutrients other than those incorporated into the feed medium become limiting, Also, if many cycles are run, the accumulation of non-producing or low-producing variants may result.

•

the quantities of the components to control must be above the detection limits of the available measuring equipment

•

Right. Are there different types of fed batch fermentations, too? Sure there are. Two basic approaches to the fed-batch fermentation can be used: the constant volume fed-batch culture - Fixed Volume Fed-Batch - and the Variable Volume FedBatch. We will see the kinetics of the two types of fed-batch culture subsequently. First let’s see what constant volume or fixed volume fed batch fermentations are. In this type of fed-batch, the limiting substrate is fed without diluting the culture. The culture volume can also be maintained practically constant by feeding the growth limiting substrate in undiluted form, for example, as a very concentrated liquid or gas (ex. oxygen). Alternatively, the substrate can be added by dialysis or, in a photosynthetic culture, radiation can be the growth limiting factor without affecting the culture volume. A certain type of extended fed-batch - the cyclic fed-batch culture for fixed volume systems - refers to a periodic withdrawal of a portion of the culture and use of the residual culture as the starting point for a further fed-batch process. Basically, once the fermentation reaches a certain stage, (for example, when aerobic conditions cannot be maintained anymore) the culture is removed and the biomass is diluted to the original volume with sterile water or medium containing the feed substrate. The dilution decreases the biomass concentration and result in an increase

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(i) the same medium used in the batch mode is added;

(iii) a very concentrated solution of the limiting substrate is added at a rate less than (i), (ii) and (iii) . This type of fed-batch can still be further classified as repeated fed-batch process or cyclic fed-batch culture, and single fed-batch process. The former means that once the fermentation reached a certain stage after which is not effective anymore, a quantity of culture is removed from the vessel and replaced by fresh nutrient medium. The decrease in volume results in a increase in the specific growth rate, followed by a gradual decrease as the quasisteady state is established. The latter type refers to a type of fed-batch in which supplementary growth medium is added during the fermentation, but no culture is removed until the end of the batch. This system presents a disadvantage over the fixed volume fed-batch and the repeated fed-batch process: much of the fermentor volume is not utilized until the end of the batch and consequently, the duration of the batch is limited by the fermentor volume. •

All right. Now are there any special considerations to be made about fed batch fermentations? Yes. First and foremost is the fermentation vessel or equipments used for fermentation. Actually no special piece of equipment is required over the equipment required for batch. However, some considerations should be made over the equipment used for fed-batch fermentation. The vessels, particularly those used for the acid and base control, must be constructed from a non-toxic, corrosionresistant material which is capable of withstanding repeated sterilization cycles. Figure 4.1. Illustrates two methods of assembling vessels for easy transfer of either inoculum or medium to the fermentor.

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in the specific growth rate (see mathematical description in section 6). Subsequently, as feeding continues, the growth rate will decline gradually as biomass increases and approaches the maximum sustainable in the vessel once more, at which point the culture may be diluted again.

batch fermentation, even considering the operating procedures for sterilization and the preventing of contamination.

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controller is the structure of the model used by the parameter estimator to analyze estimates of process dynamics. The process can be described by a set of mass balance equations, whose quantities can be measured directly or indirectly. The following figure describes schematically the concept.

Figure.4.1. Holding vessels. A. Screw-neck borosilicate glass vessel with medium/inoculum addition assembly. (a) Stainless steel rod; (b) Silicon tubing; (c) Silicon disc; (d) Hypodermic needle; (e) Air vent; (f) Screw cap; (g) Magnetic bar. B. Aspiratortype vessel for introducing an inoculum of filamentous fungi into the fermentor. (a) Cotton-wool plug; (b) Magnetic stirrer bar. What would be the next consideration to be made? Next would be the pumps required for the fermentation. There are two types of pumps which are suitable for the aseptic pumping of small volumes of culture media: the peristaltic pump and the diaphragm-dosing pump. Other pumps are unsuitable because they are difficult to sterilize and cannot be used for pumping small volumes. The peristaltic pump is typically constituted by a main body that comprises both the drive motor and electrics, and the rotating unit of rollers. This unit of rollers occludes the tube which, as it recovers to its original size passes to the nest roller until is expelled, as the unit moves round. The flow rate can be varied by either the speed setting or by changing the diameter of the tube being used. The diaphragm-dosing pump consists of a main body and a detachable heat-sterilizable head. The fluid is sucked in to the pump head. The suction inlet tube then closes and the pressure discharge tube opens and forces the fluid out. The suction and pressure forces in the pump head are generated by the reciprocating action of both the diaphragm plunger and the return spring. • That was about instrumentation. Now tell me about the control part of the fermentation. Sure. In case of fed batch fermentations, the control system adapted is called adaptive control. It is the name given to a control system in which the controller learns about the process by acquiring data from a certain process and keeps on updating a control model. A parameter estimator monitors the process and estimates the process dynamics in terms of the parameters of a previously defined mathematical model of the process. A control design algorithm is then used to generate controller coefficients from those estimates, and a controller sets up the required control signals to the devices controlling the process. An extremely important feature of an adaptive •

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Figure.5.1. Adaptive control: the controller compares the estimates from a mathematical model applied to the system to the readings obtained from the fermentation process. The controller then sends the signal to the device controlling the fermentation, for example, by increasing or decreasing a flow rate. The optimal strategy for the fed-batch fermentation of most organisms is to feed the growth-limiting substrate at the same rate that the organism utilizes the substrate; this is, to match the feed rate with demand for the substrate. This can be compared to making a boy work and feeding him such amount of food that will generate exactly the same amount of energy required for that work! Four basic approaches have been used in attempts to balance substrate feed with demand (listed in order of increasing accuracy and/or complexity): (i) open-loop control schemes in which feed is added according to historical data or predicted data; (ii) indirect control of substrate feed based on non-feed source parameters such as pH, offgas analysis, dissolved O2 or concentrations of organic products; (iii) indirect control schemes based on mass balance equations, the values of which are calculated from data obtained by sensors; and (iv) direct control schemes based on direct, on-line measurements substrates. Better and more flexible control may be obtained when there is direct measurement of substrate or an excreted metabolite in the medium, which can be used to influence feeding rates to the fermentation. This can be done off-line or semi-on-line, but on-line measurements are more useful because of •

the shorter analysis required,

•

lower personnel requirement and

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a reduced chance of fermentor contamination.

Regardless the type of control, the design is strongly influenced by both mathematical model availabilities and measurement possibilities. •

•

The feed is provided at a constant rate

•

The production of mass of biomass per mass of substrate is constant during the fermentation time and

•

A very concentrated feed is being provided to the fermentor in such a way that the change in volume is negligible.

Tell me, if the direct analysis of the control parameters is available, why do we resort to the mathematical models?

Control and optimization of bioreactors is strongly influenced by the quality of the sensors available for crucial response variables. Of primary importance is the ratio of the dynamic parameters of the sensor to those of the process. When these variables cannot be measured easily or quickly enough, a mathematical model must be used in some way in place of feedback information.

The equations that describe the system in terms of specific growth rate, biomass and product concentration (for both growth and non-growth associated products) with time are the following: Mathematical modelling of fixed volume fed-batch.

Parameter

When an exact mathematical model is at disposal, an open-loop process control can be proposed which generally proves to be insufficient. The advantage of a feedback control is that a response to unforeseen and unexpected conditions during the fermentation is achieved and the process is controlled within the desired limits. An indirect feedback control utilizes an observable parameter, such as dissolved oxygen, pH, respiratory quotient, and partial pressure of CO2, culture fluorescence or by-product formation, which is closely related to the course of microbial fermentation. As examples of fed-batch systems using this concept, one can mention the pH-stat - a system in which the feed is provided depending on the pH, - and the DO-stat - a system in which the feed is provided depending on the reading of the dissolved oxygen.

Equation

Equation #

Specific Growth Rate u = (F . Yx/s) / x

(3.6.1.1)

Biomass (as a function xt = xo + F . Y x/s . t of time)

(3.6.1.2)

Product Concentration P= Pi + qp . xo . t + qp . (non-growth F . Y x/s . t2 /2 associated)

(3.6.1.3)

Product Concentration P= Pi + rp . t (non-growth associated)

(3.6.1.4)

•

x is the biomass [mass biomass/volume]

•

xo is the biomass in the beginning of the fermentation [mass biomass/volume]

•

t is time

•

F is the substrate feed rate [mass substrate/(volume.time)] and

•

Y x/s is the yield factor [mass biomass/mass substrate]

•

u is the specific growth rate [time-1]

•

P is the product concentration {mass product/volume] and

A feedback control can be implemented accordingly to not only a single measurement, but also to obtain a finer control action in a dual-level system. Turner at al., describes a control method applied to a fed-batch culture of recombinant Escherichia coli in which a two-level control was preferred because it provided much greater flexibility and better control over the substrate concentration in the medium and the production of byproducts.

•

qp is the specific production rate of product [mass product/ (mass biomass . time)

•

rpis the product formation rate [mass product/(volume . time)]

As compared with the batch fermentation, two more parameters need to be specified to determine the operating conditions of a fed-batch fermentation: feed and initial feeding time.

(ii) the biomass increases linearly with time.

A direct feedback controller uses the concentration of limiting substrate in the culture medium as a feedback feed -related parameter for control. A direct feedback control can have the disadvantage of not being very feasible due to the difficulty associated with obtaining accurate on-line measurements of substrate concentrations or even by the absence of on-line sensors for the important compound to control. The advantage of a feedback control is that a response to unforeseen and unexpected conditions during the fermentation is achieved and the process is controlled within the desired limits.

•

All right. Now tell me how we develop mathematical formulae for fixed volume fed batch fermentation.

See, in developing the mathematical models for fixed volume fed batch fermentations, we have to assume the following:

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From equations (6.1.1) and (6.1.2), it can be observed that (i) the specific growth rate decreases with time because the biomass (in the denominator) is increasing with time and The product variation with time will depend on its being growth or non-growth associated, this is, will depend on qp (specific product formation defined as the product formation rate divided by the biomass) being dependent on the specific growth rate or not, respectively. The following figure depicts the typical behavior of a fixedvolume fed-batch culture.

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•

FERMENTATION TECHNOLOGIES

Component

Time profiles for a fixed-volume fed-batch culture. u= specific growth rate, x = biomass concentration, S(GLS) = growth limiting substrate, SN = any other substrate other than the S(GLS), P(nga) is the non-growth associated product and P(ga) is the growth associated profile for product concentration. •

And how about the variable volume fed batch fermentations? In variable volume fed-batch fermentation, an additional element should be considered: the feed. Consequently, the volume of the medium in the fermenter varies because there is an inflow and no outflow. Again, it is going to be considered that the growth of the microorganism is limited by the concentration of one substrate.

For the mathematical developments that will be presented, the assumptions are •

Specific growth rate is uniquely dependent on the concentration of the limiting substrate

•

The concentration of the limiting substrate in the feed is constant

•

The feed is sterile

•

The yields are constant during the fermentation time The following table summarizes the equations that apply to this situation. These relations are the base for all further calculations and specific cases of variable volume fed-batch fermentation.

Mass balances for the main components for a fed-batch reaction.

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Mass Balance Equation

Equatio n#

Overall

F = dV/dt

(3.6.2.1)

Biomass

dx/dt = x . (u? . V -– Kd . V -– F) / V

(3.6.2.2)

Substrate

ds/dt = F . (so -– s)/V -– u. x/ Yx/s

(3.6.2.3)

Product

dP/dt = qp . x -–P . F / V

(3.6.2.4)

•

V is the volume of the fermentor

•

t is the time

•

F is the feed rate [volume/time].

•

x is the biomass concentration [mass biomass/volume]

•

u is the specific growth rate [time-1]

•

Kd is the specific death rate [time-1]

•

s is the substrate concentration in the fermentor [mass substrate/volume]

•

so is the substrate concentration in the feed [mass substrate/ volume]

•

Y x/s is the yield factor [mass biomass/mass substrate]

•

P is the product concentration {mass product/volume] and

•

qp is the specific production rate of product [mass product/ (mass biomass . time)

•

What are the various control and analytical techniques used with fed batch fermentations?

There are many. Let’s see them one by one.

3. Calorimetry Calorimetry is an excellent tool for monitoring and controlling microbial fermentations. Its main advantage is the generality of this parameter, since microbial growth is always accompanied by heat production, and the measurements are performed continuously on-line without introducing any disturbances to the culture. Moreover, the rate of heat production is stoichiometrically related to the rate of substrate consumption and product, including biomass formation. In many cases it can be replaced by exhaust gas analysis, although this approach can not be considered in anaerobic processes which proceed without formation of gaseous products. This technique has been proved successful to indirectly determine the substrate and product concentrations continuously during aerobic batch growth of Saccharomyces cerevisiae with glucose as the carbon and energy source. In the presence of this substrate, this yeast shows diauxic growth by initially consuming the glucose with concomitant production of ethanol and then, once glucose is depleted, using the produced ethanol as an energy source. Calorimetry can then be used to control the feed rate in such a way that ethanol formation is avoided.

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•

Got it. Any other parameter that can be used?

Specific growth rate is parameter that can be successfully uses in fed batch fermentations. For the production of a growthassociated product, the production of a certain product is related with the specific growth rate of the producing microorganism. Consequently, it is of interest to feed the fermentor in such a way that the specific growth rate remains constant. Such is the case of the production of hepatitis B surface antigen by Saccharomyces cerevisiae. The yield of the antigen is ten times more than that of the fed-batch cultivation for the same volume and total substrate added. Care should be given to the value of the chosen specific growth rate, because cells may not be “activated” easily, stress proteases can be produced that may degrade the product and also there might be a threshold value of specific growth rate above which there is production of by-products. The substrate concentration, especially the carbon and nitrogen source concentration and the by product concentration can also be effectively used as a control parameter in fed batch fermentations. This approach has been used in the fermentation of Saccharomyces cerevisiae, in which acid production rate is used to provide on-line estimates of the specific growth rate. Also, in modern fed-batch processes for yeast production, the feed is under strictly control based on the measurement of traces of ethanol in the exhaust gas of the fermenter. In certain fermentations it is of interest to continuously add either an inductive or fast consumed components and not only a limiting substrate. An example is the continuous addition of an antibiotic in recombinant microorganisms bearing an antibiotic marked plasmid. Another example is given by the production of gluthathione by high-gluthathione-accumulating Saccharomyces cerevisiae, the commonly microorganism used for commercial production. Cysteine was found to be the only amino acid that enhanced gluthathione formation. However, the growth inhibition occurred and it was related to the concentration of cysteine. This problem was then resolved by an adequate addition of cysteine in exponential fed-batch culture without growth inhibition. Fed-batch proves to be an appropriate mode of fermentation in microorganisms that are producing heterologous proteins and whose elevated protein expression results in product degradation by activation of proteases. A general insight on this subject was the study of a recombinant E. coli for production of chloramphenicol acetyltransferase. A gradual induction with IPTG and phenylalanine (rate limiting precursor) addition strategies were able to reduce the physiological burden imposed on the bacterium, thereby avoiding cellular stress responses and enhancing bioreactor productivity. In this case, IPTG and phenylalanine were the driving parameters that dominated the feed. 2.521

As a final note, the addition of precursors or inducers should take into account if the product of interest is growth associated or not. For example, the use of a tyrosine-deficient strain of E. coli in the production of phenylalanine requires a balance feed of tyrosine that, if not provided in low quantities is used as carbon source with subsequent production of excessive biomass synthesis at the expense of phenylalanine synthesis. This limitation on biomass production is possible because the phenylalanine production was not growth associated. Gas analyzers, especially mass spectrophotometers are relatively fast methods used for the control of fed batch fermentations. Proton production and fluorescence are some of the other discussed methods. •

All right. Now a question about the nutrient addition. How do we find out when and how to add the additional nutrients?

This is quite an important decision to make. The times at which the feeding should start and finish, as well as the criteria to stop a fed-batch fermentation is very much dependent on the specific cultivation kinetics and the operator’s interest. For example, in substrate limited processes, the feed should start immediately after all substrate is consumed from the batch phase, otherwise the process may be difficult to control, for example, because of a lag phase due to previous starvation. The most commonly criteria to start the feed are the depletion of substrate, which can be measured by a multitude of techniques, from specific enzymatic assays, HPLC to indirect methods such as the exhaust gas analysis. Still related with the amount of substrate in the medium, the operator might not find necessary to reach the complete depletion but to be below a predetermined setpoint (eventually related with historical data, growth models and known yields). The fed-batch fermentation should be halted when the production slows down because of cell death, because the metabolic potential of the culture becomes inadequately low or because by-product excretion starts at significant levels. Some other criteria can be an increase in viscosity that implies an increased oxygen demand until the oxygen limitation is achieved, which is the case for penicillin production. •

Can we convert all the batch type fermentations into fed batch type?

Theoretically yes. Practically, though, it may not be economical and technically convenient to do so. In order to determine whether it is worthwhile to develop a fed batch process, one has to have a substantial data on that fermentation. This data includes •

Best abiotic conditions such as temperature, light, agitation, pH, growth medium, etc.

•

Specific needs of precursors, inducers or other enrichment factors The different growth phases and the consumed (substrate) and produced components (product of interest and by-product)

•

The relationship between the biomass and product formation (growth or non-growth associated product) and the oxygen uptake rates

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Another interesting description of a temperature-based controlled reactor follows a stability criterion that predicts that the range of operation is controlled by the reactant feed, being the flow rate of the cooling medium the control variable. Although the study has been performed in a chemical reactor, the concepts can be easily extended to a biotransformation process.

FERMENTATION TECHNOLOGIES

•

Limiting substrate for growth and the relationship between the specific growth rate and the limiting substrate concentration

•

Eventual inhibitions from the substrate and/or product

•

Now, the operator should define the objective functions and the best parameter to control the fermentation, considering both accuracy of data and convenience. Also, the operator should define if the control that wishes to be implemented is based on a feedback control (direct or indirect) or an openloop control based on mathematical models established for the system.

•

Ok, now tell me some of the examples of fed batch fermentations.

Notes

The use of fed-batch culture by the fermentation industry takes advantage of the fact that the concentration of the limiting substrate may be maintained at a very low level, thus •

avoiding repressive effects of high substrate concentration

•

controlling the organism’s growth rate and consequently controlling the oxygen demand of the fermentation.

Saccharomyces cerevisiae is industrially produced using the fedbatch technique so as to maintain the glucose at very low concentrations, maximizing the biomass yield and minimizing the production of ethanol, the chief by-product Hepatitis B surface antigen (HbsAg) used as a vaccine against type B hepatitis has been purified from human plasma and expressed in recombinant yeast, being now produced commercially. Again, the production of the recombinant protein is achieved using fed-batch culture techniques very similar to that developed for Saccharomyces cerevisiae. A cyclic method is used due to reports of superior productivity. Penicillin production is an example for the use of fed-batch in the production of a secondary metabolite. The fermentation is divided in two phases: the rapid-growth phase during which the culture grows at the maximum specific growth rate and the slow-growth phase in which penicillin is produced. During the rapid-growth phase, an excess of glucose causes an accumulation of acid and a biomass oxygen demand greater than the aeration capacity of the fermentor, whereas glucose starvation may result in the organic nitrogen in the medium being used as a carbon source, resulting in a high pH and inadequate biomass formation. During the production phase, the feed rates utilized should limit the growth rate and oxygen consumption such that a high rate of penicillin synthesis is achieved and sufficient dissolved oxygen is available in the medium. Some other examples are the production of thiostrepton from Streptomyces laurentii and the production of cellulase by Trichoderma reesei. The production of thiostrepton uses pH feedback control and the production of cellulase utilizes carbon dioxide production as a control factor.

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Learning Objectives

continuous fermentation is particularly applicable to those fermentations in which the growth and synthetic activities of the cells are not simultaneous; that is, synthesis is not growthrelated but occurs after the cell-multiplication rate has slowed.

In this lecture, you will learn •

Continuous fermentations – methods and controls

•

Control of continuous fermentations

•

Continuous fermentations - benefits and limitations

•

What are continuous fermentations?

Continuous fermentations are those in which fresh nutrient medium is added either continuously or intermittently to the fermentation vessel, accompanied by a corresponding continuous or intermittent withdrawal of a portion of the medium for recovery of cells or fermentation products. That is, on one hand, there is a continuous addition of nutrients and on the other; there is a continuous removal of fermented broth. This is in contrast to a batch fermentation process in which a large volume of nutrient medium is inoculated, and growth and biochemical synthesis are allowed to proceed only until maximum yields have been obtained. At this point, the batch fermentation is stopped for product recovery, the fermentor is cleaned and resterilized, and a new fermentation is started up. At first glance, the continuous fermentation would appear to be the better of the two procedures, because the fermentation equipment is in constant usage with little shutdown time and, theoretically at least, after the initial inoculation, further production of inoculum is not required. However, as we shall see, the inherent problems associated with a continuous fermentation process often do not allow the achievement of this goal. How are continuous fermentations carried out? A continuous fermentation can be conducted in various ways. It can be carried out as a “single stage” in which a single fermentor is inoculated, then kept in continuous operation by balancing the input and output of nutrient solution and harvested culture respectively. In a “recycle” continuous fermentation, a portion of the withdrawn culture, or of the residual unused substrate- ‘plus the withdrawn culture, is recycled to the fermentation vessel. For example, the immiscible hydrocarbon substrate of hydrocarbon fermentation can be recycled for further microbial attack. A portion of the organisms being produced during a continuous fermentation also can be recycled in certain instances in which the actual available substrate level in the nutrient solution for microbial growth is quite low. An example of this type of substrate is sulfite waste liquor with .its low available carbohydrate content; in this instance, the recycling of cells provides a higher population ‘of cells in the fermentor and, hence, a greater productivity. Multistage ethanol production with recycling of yeast is another such example. •

A third type of continuous fermentation, the “multiple-stage continuous fermentation. It involves two or more stages with fermentor being operated in sequence. The multiple-stage 2.521

Table Representive Chemical Products form Continuous Fermentation Growth-Associated

Not Growth Associated

Acetic acid

Acetone

Butanediol

Butanol

Ethanol

Glycogen

Gluconic acid

Subtilin

Hydrogen sulfide

Chloramphenicol

Lactic acid

PenicillinStreptomycin Vitamin B12

How do we control the microbial activity in continuous fermentations? There are several possible means by which microbial activity in continuous culture can be controlled, although only two of these approaches, the “turbidostat” and the “chemostat”. •

In the turbidostat, the total cell population is held constant by employing a device that measures the culture turbidity so as to regulate both the nutrient feed rate to the fermentor and the culture withdrawal rate from the fermentor. If the population numbers rise above a predetermined level, a greater amount of fresh medium is added to the fermentor so as to dilute the cell Concentration. Thus, there is no limiting nutrient consciously imposed with this process so that the cell growth rate should always be maximal. However, the .growth must be maintained in the logarithmic growth phase or very close to it. This factor is a disadvantage in that the fermentation must be operated at a lower maximum cell population than is possible with a chemostat, and this causes a greater residual of unused nutrient to be lost from the fermentation with the withdrawn harvested culture. Ok, and how does the chemostat work? In contrast to the turbidostat, a chemostat maintains the nutrient feed and culture withdrawal rates at constant values, but always less than that which allows a maximum growth rate. The growth rate is controlled by supplying only a limiting amount of a critical growth nutrient in the feed solution. Thus, cell multiplication cannot proceed at a rate greater than that allowed by the availability of this critical nutrient. The controlling factor for growth, however, does not necessarily have to be a limiting nutrient; it can also be a relatively high concentration of a toxic product of the fermentation, the pH value, or even temperature. The chemostat concept of continuous fermentation is employed more often then the turbidostat, because fewer mechanical problems are •

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LESSON 19: CONTINUOUS FERMENTATIONS

FERMENTATION TECHNOLOGIES

encountered, and because of the occurrence of less residual unused nutrient in the harvested culture. •

ascendancy in the culture, although certain fermentations, such as that for Torula yeast on sulfite waste liquor, provide built-in contamination control, in this instance, the presence of sulfite and a low pH. As regards the contamination problem, suggestions have been made that antibiotics or other chemicals be added to continuous fermentations to hold down the level of contaminant growth. Mutation of the fermentation organism becomes a problem only if the resulting mutant cells have a selective growth advantage during prolonged incubation and, at the same time, produce less of the desired fermentation product. It has been proposed that the answer to the question of mutation is to use multistage continuous fermentations, with the first fermentor of the sequence being periodically reinoculated. Nevertheless, the real overall solution to both contamination

Tell me, is it possible to convert all batch type fermentations into continuous ones? Again, theoretically yes. Many fermentation processes have been investigated, at least on the pilot plant scale, for their possible conversion to a continuous fermentation process. The following table presents a list of representative microorganisms investigated for their possible use in continuous fermentations.

Table Representative Genera of Organisms Grown in Continuous Culture Organisms Actinomycetes Algae Bacteria

Fungi Protozoa Yeast

Genera Streptomyces Chorella Euglena Scennedesmus Aerobacter Azotobacter Bacillus Brucella Clostridium Salmonella Ophiostoma Penicillium Tetrahymena Saccharomyces

and mutation is to reduce their rates of occurrence so that the offending cells will be flushed from the fermentors before they have a chance to multiply •

From among these potential continuous fermentation processes, only the production of beer, fodder yeast (from sulfite paper mill waste), vinegar, and baker’s yeast (from molasses) have found commercial application. However, the activated sludge system for the processing of waste waters also may be considered as a commercialized continuous fermentation, differing from the more conventional fermentations only in that it deals with mixed microbial population acting on a heterogeneous substrate and also in that the products of these fermentations are not having as much commercial importance. •

It is said that the productivity of a continuous fermentation is greater than that for batch fermentation. If this is true, then why have so few batch fermentations been successfully converted to a continuous fermentation process?

So there are some drawbacks of continuous fermentations, is it? There are. Continuous fermentations often waste nutrient substrate. Thus, the fermentation broth as it is continuously withdrawn for product recovery contains a certain amount of the residual unused nutrients of the medium as well as a portion of the fresh nutrient constituents being continuously added to the fermentation. Certain fermentation media are rather viscous and require that strong mixing activity be employed in the fermentor to equally distribute the incoming fresh medium to all parts of the broth already in the fermentor. Obtaining adequate mixing also is a problem when slow feed rates of fresh nutrient are employed, regardless of the viscosity of the medium. Antibiotic fermentations are included in this group of complex fermentation processes for which a continuous fermentation would seem to difficult to accomplish. In this regard, the feasibility of a single stage continuous fermentation for chloramphenicol and penicillin investigated by Bartlett and Gerhardt (1959) (Figure 13.2). These workers reported that, at dilution rates of 1and 0.5 volume changes of per day, respectively, they were able to obtain yields from 1/4 to 1/2 of per day, respectively, of the maximum observed in batch fermentations, and that these rates were maintained in a steady state for more than two weeks.

There are several answers. A successful continuous fermentation requires a thorough knowledge of the dynamic aspects of microbial behavior and growth, knowledge that is lacking or deficient for most industrial fermentation processes because of the complexities of the growth and synthetic patterns of the organisms. Also, contamination and mutation present a distinct problem for the development of a successful continuous fermentation process. The prolonged incubation periods associated with continuous fermentations can allow contaminating microorganisms the time that they require for gaining

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Notes

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LESSON 20: MICROBIAL BIOMASS PRODUCTION

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Learning Objectives

before being finally used to inoculate the large production fermentors of 50-350m3 capacity. Overall, this may involve up to eight scale-up stages to produce the necessary final inoculum volume.

In this lecture, you will learn Production of Microbial Biomass The Bel Process In most of the fermentation processes already discussed, the conversion of a proportion of the substrate to biomass is somewhat incidental, or for some purposes biomass formation may be actively suppressed. The main aim has been the conversion of substrate into a useful primary or secondary metabolic product, such as antibiotics, ethanol and organic acids. In such cases, once the optimal amount of target product has been achieved, the organisms produced are often merely waste materials that have to be disposed of safely and at a cost, or are simply used as a cheap source of animal feed. However, in dedicated biomass production, the cells produced during the fermentation process are the products. Consequently, the fermentation is optimized for the production of a maximum concentration of microbial cells.

Microbial Biomass is Broadly Used For Three Purposes 1 Viable microbial cells are prepared as fermentation starter cultures and inocula for food and beverage fermentations, waste treatment processes, agricultural inoculants, mineral leaching and as biopesticides 2 As a source of protein for human food, because it is often odourless and tasteless, and can therefore be formulated into a wide range of food items; and animal fodder. •

What are the common products of microbial cell origin?

Let us study some well known examples:

Baker’s Yeast A major fermentation industry has developed to manufacture the vast quantity of baker’s yeast required for making bread and associated bakery products. The skimming method’ was one of the first procedures employed for the commercial production of baking strains of Saccharomyces cerevisiae. This method used media derived from cereal grains, and was similar to brewing and distilling fermentation processes. Here the yeast floated to the top of the fermentation and was skimmed off, washed and press-dried. However, during World War I, due to shortages of cereal grains, the yeast industry sought alternate fermentation materials. In Germany a process was devised whereby molasses, ammonia and ammonium salt were used in place of cerealbased media. Baker’s yeast production commences with propagation of a starter culture, which originates from a pure freeze-dried sample or agar-medium culture. Yeast cells are initially transferred to small liquid culture flasks, then on to larger intermediate vessels

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Medium for the production fermentation normally contains molasses as the carbon and energy source, which may be pretreated with acid to remove sulphides and heated to precipitate proteins. Molasses is often deficient in certain amino acids, and supplements of biotin and pantothenic acid are usually necessary. Further nitrogen sources (ammonium salts or urea) may be added, along with orthophosphate and other mineral ions, and the pH is adjusted to 4.0-4.4. The main objective of the process is to generate a high yield of biomass that exhibits an optimal balance of properties, including a high fermenting activity and good storage properties. Aerobic fermentation favours a high biomass yield, as approximately 50% of the available carbon can be potentially converted to biomass. The maximum theoretical growth yield is 0.54 g/g, whereas under anaerobic conditions this value is reduced to 0.12 g/g. Yeast cells are separated by centrifugal separators. Harvested cells are washed, chilled and dried. The yeast blocks are generally packed in 1 Kg blocks and kept under refrigeration. What are the qualities of yeast that make it an organism of choice? There are many, actually. Some of the important ones are: •

•

high glycolytic activity

•

rapid utilization of other sugars like maltose

•

the ability to utilize a wide range of low cost carbon sources, including waste materials;

•

strain selection and further development are relatively straightforward, as these organisms are amenable to genetic modification;

•

the processes occupy little land area

•

Why and how are single cell proteins produced?

During World War I and II, interest in microbial proteins as human food and animal feed increased as conventional protein sources were in short supply. Attempts were made in some countries to use yeasts, particularly strains of S. cerevisiae and Torula yeast (Candida utilis), to supplement the shortfall of protein. In more recent times, use of microbial protein has been considered as a potential means of fulfilling the urgent need for low-cost protein in certain parts of the world, which the agriculture in those regions arguably cannot provide. The increased demand is a result of the ever-increasing populations in developing countries. This objective has not been achieved, despite the obvious need and advantages that microbial protein provides over conventional protein sources. More effort has been directed towards producing either premium products, as

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Rapid developments in microbial protein production occurred during the 1960s and 1970s. Extensive research was conducted on a wide range of microorganisms as possible alternate protein sources, motivated by large increases in the price of conventional animal feed. It was during this period that the term single cell protein (SCP) was first coined at the Massachusetts Institute of Technology. SCP is not pure protein (see table ), but refers to the whole cells of bacteria, yeasts, filamentous fungi or algae, and also contains carbohydrates, lipids, nucleic acids, mineral salts and vitamins.

microorganisms have naturally high levels and the problem is further exacerbated because fermentation conditions favouring rapid growth rates and high protein content also promote elevated RNA levels. This can be problematic as the digestion of nucleic acids by humans and animals leads to the generation of purine compounds. Their further metabolism result1 in elevated plasma levels of uric acid, which may crystallize in the joints to give gout-like symptoms or form kidney stones. Slow digestion or indigestion of some microbial cells within the gut and any sensitivity or allergy, reactions to the microbial protein must also be examined. For filamentous fungi, the possibility of aflatoxin production must be eliminated. An additional concern is the absorption of toxic or carcinogenic substances, such as polycyclic aromatic compounds, which may be derived from certain growth substrates. •

What are the various substrates used for SCP production?

Microbe

Protein percentage Nucleic acid

Bacteria

50-85

10-16%

Yeast

45-55

5-12%

As for any production, the entire economics of SCP production rests on its cost of production, which, in turn, rests on the carbon source used. The major substrates used are n alkanes, molasses, sulfite waste liquor and whey. The selected substrate will have to be cost effective, high yielding, requiring less oxygen for degradation, thermostable and requiring a minimum of downstream processing.

Filamentous fungi 30-55

3-10%

•

Algae

4-6 %

Many pilot plants have been developed over the last 30 years that utilize a range of substrates and microorganisms. However, relatively few have operated commercially, due to obstacles encountered on scale-up or for economic reasons. The physiological problems that are often encountered on scale-up include difficulties with:

Protein and nucleic acid content of microorganisms

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Fast-growing aerobic microorganisms are primarily used due to their high yields and high productivity. Bacteria generally have faster growth rates and can grow at higher temperatures than yeasts or filamentous fungi, and normally contain more protein. Yeasts grow relatively rapidly and, like bacteria, their unicellular character gives somewhat fewer fermentation problems than do filamentous organisms. However, many filamentous fungi have a capacity to degrade a wide range of materials and, like yeasts, can tolerate a low pH, which reduces the risk of microbial contamination. They are also more easily harvested at the end of fermentation than yeasts or bacteria. Selection of a suitable microbial strain for SCP production must take several characteristics into account, including: 1 performance (growth rate, productivity and yields) on the specific, preferably low-cost, substrates to be used; 2 temperature and pH tolerance; 3 oxygen requirements, heat generation during fermentation and foaming characteristics; 4 growth morphology and genetic stability in the fermentation; 5 ease of recovery of SCP and requirements for further downstream processing; and 6 structure and composition of the final product, in terms of protein •

An issue about the nucleic acid content of SCP is frequently raised. Why is it so important?

It indeed is a very important issue. All SCP products must meet the safety norms laid down for the nucleic acid content. Many

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What is the process for SCP production?

1 oxygen requirements and oxygen transfer rates; 2 nutrient and temperature gradients; 3 effects of CO2, as high levels may inhibit respiration in certain microorganisms; and 4 hydraulic pressure in deep fermentors. In some instances, the economics of production can be improved by either increasing the value of the product or reducing the production costs through: 1 use of cheaper substrates; 2 improvements in the efficiency of the organism; 3 enhanced nutritional value/composition of the microbial protein; 4 marketing the protein as a premium product for human rather than animal consumption; 5 production of other valuable byproducts, i.e. development of a multiproduct process; and 6 lowering downstream processing costs, e.g. by reducing endogenous RNA levels. The SCP production processes essentially contain the same basic stages irrespective of the carbon substrate or microorganism used. 1 Medium preparation.: The main carbon source may require physical or chemical pretreatment prior to use. Polymeric

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meat substitutes for the western diet, or animal fodder. Interest in microbial protein for animal fodder largely depends on production costs in relation to the prevailing price of the main market competitors, particularly soya protein and fish meal. The reason that more microbial protein is not currently produced for fodder is due to the present low price of these conventional protein sources. However, this may change as there have been forecasts of future shortages of soya and fish meal.

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substrates are often hydrolysed before being incorporated with sources of nitrogen, phosphorus and other essential nutrients. 2 Fermentation.: The fermentation may be aseptic or run as a ‘clean’ operation depending upon the particular objectives. Continuous fermentations are generally employed, to fully exploit the superior productivity of continuous culture. 3 Separation and downstream processing: The cells are separated from the spent medium by filtration or centrifugation and may be processed in order to reduce the level of nucleic acids. This often involves a thermal shock to inactivate cellular proteases. RNase activity is retained and degrades RNA to nucleotides that diffuse out of the cells. Depending upon the growth medium used, further purification may be required, such as a solvent wash, prior to pasteurization, dehydration and packaging. The various processes described below have been relatively successful in commercial terms, and/or involve notable technological developments.

they contain up to 3% solids and have COD values of over 20g oxygen per litre. A high proportion of the available substrate is starch, which many microbes cannot directly utilize. To overcome this problem the process was developed with two microorganisms that grow in a symbiotic association. They are the yeasts Saccharomycopsis fibuligera, which produces the hydrolytic enzymes necessary for starch degradation, and Candida utilis. The process is operated in two stages. In the first stage, S. fibuligera is grown in a small reactor on the sterilized waste, supplemented with a nitrogen source and phosphate. At this point, the starch is hydrolysed, which is the rate-limiting step of the whole process. The resulting broth is then pumped into a second larger fermentor of 300 m3 capacity where both organisms are present. However, C. utilis comes to dominate the second stage and constitutes up to 90% of the final product. The Symba process operates continuously and after 10 days the pollution load of the waste is reduced by 90%. Resultant protein-rich biomass (45% proteins concentrated by centrifugation and finally spray drum dried.

The Bel Process The worldwide dairy industry generates over 80 million tones of whey each year. This byproduct of cheese manufacture has a high pollution load with a chemical oxygen demand (COD, see Chapter 15) of 60g oxygen per litre. Consequently, it usually has to be disposed of at a high capital cost to the dairy industry. Whey contains approximately 45 g/L lactose and 10 g/L protein. It is particularly suitable for the production of SCP using lactose-utilizing yeast, although attempts have also been made to grow other organisms, including Penicillium cyc/opium. Several processes have been developed for the utilization of lactose in milk whey. Some of the more successful have been those operated by Bel Industries in France. The Bel process was developed with the aim of reducing the pollution load of dairy industry waste, while simultaneously producing a marketable protein product. A number of plants are operated using Kluyveromyces lactis or K. marxianus (formerly K. fragilis) to produce a protein, Protibel, which is used for both human and animal consumption. These processes initially involve whey pasteurization, during which 75% of whey proteins are precipitated. The lactose concentration is adjusted to 34 g/L and mineral salts are also added. This supplemented whey is introduced into a 22 m3 continuous fermentor, maintained at 38°C and pH 3.5, with an aeration rate of 1700 m3/h. The yeasts utilize the lactose and attain biomass concentrations of 25 g/L, with a biomass yield of 0.45-0.55 g/g lactose. Yeast cells are recovered by centrifugation, and then resuspended in water, recentrifuged and finally roller-dried to 95% solids. Levels of residual sugar remaining in the spent medium are less than 1g/L. •

Ok, what other processes are available for SCP production?

The SYMBA process is another famous process. It was developed in Sweden to produce SCP for animal feed from potato processing wastes. It is not economically attractive as a stand-alone operation. However, alternative routes for the purification of these waste-waters are difficult and expensive, as

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THe Symba Process • What is the next process we should see? Then there is the Pekilo process.This process began operating in 1975 and was the first commercial continuously operating process for production of a filamentous fungus. It had to overcome the special problems caused by the pseudoplastic rheological behaviour of submerged cultures of fungal mycelium, which particularly affect oxygen transfer rates. The process was developed in Finland for the utilization of spent sulphite liquor, derived ‘ from wood processing that contains monosaccharides and acetic acid. Supplements of other carbon source usually molasses, whey and hydrolysed plant was may also be added prior to inoculation with Paecilomyces variotii. This continuous process is operated aseptically and produces over 10000tonnes of SCP year from two 360 m3 fermentors. Resulting dried Pekilo protein containing up to 59% crude protein, is used in the preparation of compound animal feed. Methanol has several advantages over methane and many other carbon sources, particularly as it is completely miscible with

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Attempts to develop methanol-based processes were made in Europe, the former Soviet Union, Japan and the USA. They involved bacteria (Hyphomicrobium species, Methylococcus species and Methylophilus methylotrophus), yeasts (Candida boidinii, Pichia angusta and Pichia pastoris) and even filamentous fungi (Gliocladium deliquescens, Paecilomyces variotii and Trichoderma lignorum). A typical example of this kind of processes is the Pruteen process. Let’s find out more about the Pruteen process. This was the most technically adventurous was the process developed by ICI in the UK, which started production in 1980. This process used the methylotrophic bacterium, M. methylotrophus, to produce a feed protein for chickens, pigs and veal calves, called Pruteen. Production ceased in 1987 for economic reasons, due to the rise in price of methanol, which constituted 59% of the production costs, and a fall in the price of competing soya meal. Nevertheless, this process is worthy of examination due to the advances made in fermentation design and technology during its development. This was, apart from certain systems for wastewater treatment, the world’s largest continuous aerobic bioprocess system. It consisted of a 3000 m3 pressure cycle airlift fermentor with inner loop and a working fluid volume of 1.5 x 106 L, capable of producing up to 50000tonnes of Pruteen per annum. The fermentor weighs in excess of 600tonnes, is over 60m high, with a 5 atm pressure difference from the top to the bottom and cost US$80 million in 1979. •

Filter-sterilized compressed air was used for both oxygenation and agitation, and all streams into the fermentor were sterilized. The fermentation was performed at pH6.5-6.9 and 34-37°C with entirely inorganic commercial-grade nutrients. It was operated as a methanol-limited chemostat, the methanol being supplied through numerous distribution points within the fermentor. Bacterial cells were recovered by a novel separation technique, involving initial concentration from 3% (w/w) to 12% (w/w) by flocculation, which was promoted by acid and heat shock. This was followed by centrifugal dewatering, with recycle of water, and air drying. The dried unprocessed product contained 16% nucleic acids and over 70% crude protein. Strain development of M. methylotrophus led to several improvements in its composition and fermentation performance. Protein content of the product was increased by

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5%. The cell concentration achieved during fermentation rose from 4 g/L to 30 g/L. •

Are mushrooms SCP too?

Yes they are. Certain mushrooms and other fruiting bodies of filamentous fungi are edible and provide a good source of protein, whereas others have narcotic effects and some are highly toxic. A wide range has been traditionally used for food, but relatively few are grown commercially. In fact, of the hundreds of species that are edible, only about 10 are produced in any quantity. Mushroom production involves controlled non-axenic solid substrate fermentation. It is currently the only economically viable product from lignocellulose fermentation. Exploitation of such fruiting fungi for the generation of edible biomass has several advantages: 1 they represent examples of the most efficient conversion of plant wastes into edible food; 2 unlike many other single cell proteins, they are directly edible and many are considered to be food delicacies because of their characteristic texture and flavour; 3 harvesting of fruiting bodies is the easiest possible method of separating edible biomass from the substrate in a solidstate fermentation; and 4 compared with animal sources of protein, many have far superior protein conversion efficiency per unit of land and per unit of time.

Agaricus Bisporus In Europe and the USA, Agaricus bisporus (button mushroom) accounts for over 90% of total mushroom production value. Agarics are decomposers of cellulosic materials and are naturally found in meadows and woodlands, where they degrade plant debris. They are grown commercially in temperate regions using a substrate of com posted straw. A crop is produced within 6 weeks, whereas other mushrooms may take several months or even years to fruit. A closely related species, Agaricus bitorquis, is also grown in some areas. It is less prone to certain viruses and the bacterial blotch disease of mushrooms, caused by Pseudomonas tolaasii. The Agaricus production regime involves the following stages. 1 Inoculum preparation: growth of spawn (inoculum) on sterilized cereal grains. 2 Solid-substrate preparations: compo sting of straw, manure and fertilizers at 60-70°C for 2 weeks. 3 Substrate ‘sterilization’, so-called ‘peak heating’ of compost for 5-7 days. 4. Spawn inoculation into ‘sterilized’ compost and growth, referred to as a ‘run’ at 25°C for 2-3 weeks 5. Application of a casing (covering) layer of peat and chalk over the substrate. 6. Fruiting body production, fructification, in about four flushes (successive crops) over a period of 4-6 weeks. Specialty Mushrooms Mushroom production worldwide has increased in last 35 years from about 350 000 tonnes in 1965 to now over 500000 tonnes.

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water and is available in a very pure form. Consequently, the resultant protein does not have to undergo purification. As methane is readily converted to methanol, several oil and gas companies developed processes based on this attractive carbon source in the 1970s. However, there are some problems associated with methanol as a substrate. Only relatively low concentrations, 0.1-1.0% (v/v), are tolerated by the microorganisms that utilize it, and some methylotrophic yeasts form pseudo-mycelium while growing on methanol. During the fermentation, the oxygen requirement is high, as is the heat of fermentation. Nevertheless, the oxygen demands are somewhat lower than when using methane or other hydrocarbons.

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The majority of this increase has occurred during the last 15 years and a major occurred in the range of genera cultivated on a commercial scale.

Notes

Now, exotic mushrooms are becoming increasingly popular. China is the major producer of specialty mushrooms. The main speciality genera cultivated are Lentinula ( Shiitake), Flammulina (Enokitake), Pleurotus (Oyster mushshroom), Hypsizygus (Bunashimeji), , Volvariella (Paddy Straw mushroom) and Grifolla (Maitake). Now there is a demand for the t of improved technology to cultivate these species more efficiently, as traditional practices are not very productive. Some very valuable fungi are obtained only from wild sources and have found to be very difficult to cultivate. Cultivation of Shiitake mushroom , Lentinula edodes, started in China almost a thousand years ago and was then introduced into Japan. They are becoming popular in the west, and are now grown in Europe and the USA. Worldwide production is approaching 200,000 tonnes/annum. These mushrooms may be used fresh or dried, and apart from culinary use, several medicinal properties have been attributed to Shiitake. Components detected include antihistamines, antitumour and antiviral agents, anticholesterol substances and compounds that inhibit platelet agglutination. A problem with traditional methods of cultivation on natural logs is the time required before fruiting, which may be several years. In Japan, logs of the shii tree have been used, thus the derivation of the name Shiitake, but most production is now on species of oak. Logs of about 7-15 cm diameter are cut into lengths of about 1 m and drilled with holes spaced at one hole per 500cm2. The holes are inoculated with wood piece spawn or sawdust spawn and then usually covered with hot wax to prevent excessive drying. Spawn run, the development of fungal mycelium within the log, takes 6-9 months, after which the logs are transferred to a cooler and more moist ‘raising’ yard. This change in conditions provides an optimum environment for the growth and development of the mushrooms. The first crop is normally produced in the following year. Modern production on synthetic logs is much quicker, taking about 4 months. The synthetic logs are prepared from sawdust, straw and corn cobs, along with supplements of wheat bran, rice bran, millet, rye and corn. Water is added to raise the moisture content to around 60% (w/w). The mixture is placed into bags and autoclaved for 2 h at 121°C. After cooling they are inoculated with Shiitake spawn. The inoculum is allowed to develop mycelium for 20-25 days and then the covering bags are removed. After about 4 weeks, exposed substrate blocks begin to form fruiting body about 2mm under the surface. The stimulation of their maturation is promoted by soaking these synthetic logs in water at 12°C for 3-4h. The first crop or flush of mushrooms is ready to harvest about 10 days after soaking. •

Exercise: study and carry out the cultivation of oyster mushrooms on rice paddy and other agricultural waste products. Compare the relative yield on different substrates:

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Learning Objectives In this lecture, you will learn •

Introduction

•

General aspect of enzyme production

•

Amylase & Protease, Immobilized enzyme

•

Industrial application of microbial enzyme

Enzymes are used for a variety of purposes. They are employed in three major fields: (a) laboratory, (b) industrial and (c) clinical. In some cases, they may be used in their crude forms. But at other times, they are used in purified states (e.g. urease for urea estimation). Microbial enzymes are listed in Table Table – Microbial enzymes

of kilograms. Such a transfer is performed under aseptic conditions. Alternatively, the cultivation may be carried out in rotating drums. The fungal spores are inoculated, either in the autoclave after cooling or in trays. A series of trays are enclosed in a large vessel. Aeration is ensured by the circulation of suitably humidified air over the surface of the culture. It is necessary to; keep the temperature within narrow limits. Moreover, heat generation occurs during fermentation. Therefore, the trays should be equipped with a cooling system. It should be borne in mind that, direct air cooling is not practical, since drying of the culture takes place. Subsequently, extraction of the enzyme is performed with water. Enzymes produced by a semisolid culture or a surface culture process are:

Precautions to be Taken

(i) It is necessary to keep aseptic conditions during the fermentation, since contaminations arc considered a major problem in a semisolid culture. (ii) Large numbers of spores should be prevented from escaping into the environment. •

What are the various advantages and disadvantages of this method?

Advantages What are the various aspects of enzyme production? Enzymes arc commercially produced by two methods: (1) Semisolid culture and (2) Submerged culture. There is great competition among enzymes’ manufacturers. Therefore, manufacturers are reluctant to reveal the process details. It is not possible to undertake a survey of the production methods being used. But, it is evident that the submerged culture method has been gaining ground during the past three to four decades. These two methods have been briefly dealt with here.

Semisolid Culture The enzyme producing culture is grown on the surface of a suitable semi solid substrate. The substrate usually consists of moistened wheat or rice bra supplemented with nutrient salts. The production medium is prepared by mixing bran with a solution containing any desired nutrient salts. The desired pH for optimum growth of the mould is adjusted with acid. Then, the medium is steam-sterilized in an autoclave while stirring. This sterilized medium is spread on metal trays upto a depth of 1-10 cms. the total quantity being of the order of thousands 2.521

1. This method involves comparatively low investment. 2. It allows the use of substrates with high dry matter content. It, therefore, yields a high enzyme concentration in the crude fermented material. 3. It has to be used for cultivating some moulds which are very difficult to grow in fermentors due to wall growth. 4. This method allows the moulds to develop in their ‘natural’ state. This result in the normal differentiation in mycelium, conidophores, conidia, etc. and results in a broader variety of enzymes being formed than in submerged fermentation. Such a phenomenon is desirable in some cases (e.g. in the production of pectic enzymes).

Disadvantages 1. This method requires more span. 2. It requires more labour. 3. It involves a greater risk of infection. 4. Modem control methods are not readily applicable to this fermentation system 5. It is difficult to introduce automation in such systems.

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LESSON 21: MICROBIAL ENZYME PRODUCTION

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•

· Ok, which is the other method of enzyme production?

Submerged Culture Nowadays submerged culture methods are widely used in the production of enzymes. The fermentation equipment used is the same as in the manufacture of antibiotics. It is a cylindrical tank of stainless steel. The tank is equipped with an agitator, an aerating device, a cooling system and various ancillary equipment (e.g. means of foam control, monitoring of p H, temperature, oxygen tension etc.) The quantity of production medium taken in the fermentation tank is in the range of 1000-30,000 gallons or more. With enzyme fermentations, the formulation of the product medium and to a lesser extent, control of fermentation conditions play major roles in the success of the process. In other words, the chemical composition of production media makes these processes tricky. The production medium should basically contain an energy source, carbon and nitrogen sources and any special grow-h requirements (e.g. essential amino acids or vitamins). However, good growth is not enough to obtain a high enzyme yield. Certain compounds present in the production medium may induce or inhibit enzyme formation. For example, the presence of lactose (inducer) in the medium induces beta -galactosidase. Other examples of inducers arc given in Table Table -Effect of various inducers on yields of selected enzymes.

As the inducers are rather expensive, it is preferable to use constitutive mutants which do not require the inducer. Such mutants have been developed for several enzymes. On the other hand certain compounds present in the medium act as represents for specific enzymes. For example, glucose represses the formation of some enzymes (e.g. a-amylases), under such conditions, the glucose concentration should be kept low. This can be achieved either by incremental feeding of glucose or by using a slowly metabolizable sugar (e.g. lactose or partly hydrolyzed starch). The presence of certain surfactants in the production medium increases the yields of certain enzymes. Non-ionic detergents (e.g. Tween 80) are frequently used. Table shows the effect of 0.1% Tween 80 on the yield of various enzymes.

Table – Effect of 0.1% Tween 80 on the yield of various enzymes

Occasionally Triton (Rohm and Haas) produces better results. But the mode of action of these surfactants is not understood. Most enzyme fermentations are carried out near neutral pH. Therefore, it is necessary to control the pH within the desired limits during fermentation. This can be achieved by adding a buffer system (e.g. phosphates or calcium carbonate to the medium). The alternative to this is to add certain com pounds, which upon metabolism, bring about a change in pH in the desired direction. This occurs due to the formation of either acid or base, as the case may be. Thus, salts of organic acids and nitrates will tend to raise the pH, while ammonium salts will tend to lower the pH. Economy is very important in a medium formulation. The raw material costs account for 60% 80% of the variable costs in a typical fermentation process. Common agricultural raw materials, which are available in a good and consistent quality, are generally preferred. For this purpose, defined or synthetic media may be used to elucidate the nutritional requirements of the culture for the formation of a desired enzyme. Once what is indispensable is understood, a search can be made for cheaper materials, giving the same result. Moreover, empirical determination of the optimum concentration of each component of the medium involves a lot of work. The enzyme recovery steps should also be taken into consideration when formulating the production medium. The composition should be such, that at the end of the fermentation the suspended solids and viscosity are as low as possible. Moreover, materials which could confuse separation of solid and liquid phases should not be present. Most production media are still steam sterilized batchwise in the fermentor. Complete sterilization of the medium is ensured at approximately 120°C. for 1-2 hours. Alternatively, the production medium can be sterilized separately by continuous sterilization. The latter method offers certain advantages: less colour formation due to Maillard reactions between proteinaceous materials and carbohydrates and superior fuel economy when heat recovery is practised. In addition, dilution of the production medium due to the condensate is avoided. After the completion of fermentation, the fermented liquor is subjected to rapid cooling to about 50C to reduce deterioration.

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Extraction of endocellular enzymes involves the disintegration of the microbial cells. This can be accomplished by a homogenizer or a bead mill. Thereafter, purification methods being employed for exoenzymes are used for purification. The different steps involved in the purification of do enzymes are schematically shown in Figure

The amylases constitute a large group of enzymes. They are characterized by their ability to hydrolyze 1, 4-glucosidic linkages in polysaccharides (e.g. Starch and Glycogen). There are two main subgroups a- and b-amylases. a- amylases are endoenzymes. They attack all the linkages between glucose units in the starch molecule. The bond hydrolyzed is that between C-1 and the oxygen atom linked to the adjacent glucose group. The process eventually results in complete degradation, through dextrins to glucose. Thus, amylose, which is a linear starch, is degraded faster than amylopectin, which is branched. aAmylases vary in their effectiveness, depending on their source. b-Amylases hydrolyze starch and other amyloses by splitting off maltose molecules until the action is blocked by the occurrence of either 1, 3 linkages or branch points. The residual molecule is then called a limit dextrin. α-Amylases are produced by the use of fungi (i.e. Aspergillus niger and A. oryzae), as well as bacteria (i.e. Bacillus amyloliquefaciens and B. licheniformis). Therefore, a-amylases are called either fungal a-amylases or bacterial a-amylases according to the nature of the microbes used for their production. Fungal a-amylases are produced by the above mentioned two species of the fungus. These fungi are grown on “heat bran (semisolid culture). It is also possible to produce fungal aamylase by submerged-culture, employing the following medium:

•

Ok, what are the advantages and disadvantages of this method?

(i) This method requires less space and labour. (ii) The method involves lower risk of infection.

There is a problem for aeration and agitation because of a very high viscosity of the medium due to the presence of mycelial body. Amylases biosynthesis is inhibited when the medium contains glucose. Bacterial a-amylases are produced by the above mentioned two bacterial species. Bacterial amylase s produced only by submerged-culture using the following medium:

(iii) Modern control methods can easily be used (iv) Automation in such methods is easier. (v) The yields are also generally higher on a dry matter basis, Disadvantage

(i) Initial investment cost is high. Let’s now study amylases, which is an extremely important class of enzymes. Amylases the most important part in food technology (e.g. bread-making, beer-making, etc). Therefore, concentrates of aand b-amylases are prepared and used in a variety of ways. These enzyme preparations must be refu1ly standardized for activity, according to the purpose for which they are to be used.

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A temperature in the range of 30 0 to 400C is satisfactory. The optimum pH for the fermentation medium is 7.0. It is necessary to maintain the pH near neutrality, since the amylase is denatured below 6. Calcium carbonate is used as the buffer to maintain neutral pH. The production of a-amylase begins when the bacterial count reaches l09 - 1010cells per milliliter after about

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Separation of micro-organisms is accomplished, either by filtration or centrifugation of refrigerated broth with adjusted pH. The colloidal particles present in the filtrate Ire eliminated with coagulating or flocculating agents (e.g. calcium phosphate). Diatomaceous earth 2%-4 may be added to the fermented broth as a body feed before filtration operation. Removal of the suspended solids is carried out by vacuum drum filtration or by a disc-type centrifuge equipped with a self-cleaning bowl. In order to obtain a higher degree of purity, the enzyme is precipitated with acetone, alcohols or inorganic salts (e.g. ammonium or sodium sulfate). Fractional precipitation gives purities higher than one-step precipitation. In the case of largescale operations, salts are referred to solvents because of explosion hazards.

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10-20 hours and continues for another 100—150 hours. Preservation of liquid preparations of bacterial a-amylase is done by 20% sodium chloride. The most active preparations contain 2 per cent active amylase protein. On the other hand, the most active solid preparations contain 5 per cent active amylase protein. Let’s Move On To The Next Class, The Proteases

Complex mixtures of true proteinases and peptidases are usually called proteases. The concentrations of the peptidases in the production medium is low, since they are endoenzymes. Proteases, like. a-Amylases, are produced by bacteria (i.e. Bacillus subtilis and B. licheniformis) as well as by Cur (i.e. Aspeigillus niger and A. oryzae). It is essential to take care during their production, because they are relatively unstable, and tend to lose their activity during dehydration. There are two types of Proteases: (a) alkaline serine Proteases and (b) acid Proteases. The production methods for both these Proteases are briefly discussed here: (a) Alkaline serine Proteases:

Subtilisin Carlsberg is the most widely used detergent protease. It is obtained from Bacillus licheniformis by submerged-culture method. The bacterium is grown on the medium with the following composition:

separate enzymes from solid substrate culture or to release enzymes fro the interior of microbial cells.

Extraction of Solid Substrate Cultures Enzymes produced by solid substrate cultivation used to be of the extracellular type. It is therefore easily conceived that extraction of mold brans is rather a washing out process. Countercurrent techniques of percolation are the most frequently used unit operation. In many cases the mold bran is dried prior to extraction. This is convenient when the utilization of the particular enzyme preparation is seasonal. The cultures can be produced in relatively small equipment all the year round, while the extraction is conducted in times of enzyme demand. On the other hand, it is easily seen that extraction from dried bran will yield solutions with higher enzyme concentrations. And last, drying avoids interference caused by the activity of living cells of fresh cultures. This argument, however may not apply in continuously operated culture plants.

Other media are available for this purpose. The temperature of fermentation in the range of 30° to 40°C has been found to be satisfactory. The pH of the production medium is kept 7.0 for satisfactory results. The production of the enzyme begins when maximum cell growth is reached after 10 to 20 hours. And this continues at an almost constant rate till the completion of fermentation. At the end of the productive fermentation, protease is only the protein present in the production medium. The reason for this is the occurrence of hydrolysis of all the proteins present in the median, by protease. The yield may be 10 per cent of the initial protein content of the medium. The enzyme is marketed primarily in the Form of dust-free granules. These granules contain 1-5 percent enzyme protein. The enzyme remains stable in liquid preparations. Liquid preparations contain about 2 per cent ct the enzyme. (b) Acid proteases

These enzymes are mostly produced by fungi. The fungi employed for producing these enzymes are: Mucor pusillus, M. miehei, Aspergillus oryzae; A. phoenicis, A. niger var. macropus. Acid Proteases may be produced by either semisolid-culture or submerged-culture, depending upon the fungal species employed. For example, Mucor pusillus is cultivated on a semisolid medium. And then we will see how this enzyme is extracted. The first step in the isolation of enzymes is their extraction. Techniques that fall into this group are employed either to

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In all cases the extractant is water which, however, may contain acids (inorganic or organic), salts, buffer, or other substances to facilitate solubilization of the enzyme or to improve its stability

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Extraction of Cells The decision on whether to employ whole cells for a biochemical process or to use isolated enzymes depends on many factors. Technical difficulties and the related cost of largescale isolation play an important role. There are a number of methods for cell disruption, as reviewed by Hughes et al. (1971). Chemical and biochemical methods, such as autolysis, treatment with solvents, detergents or lytic enzymes, have the disadvantage of being in principle batch operations. Their conduct is difficult to standardize and optimize. More recommendable are mechanical techniques. • What are the different industrial applications of enzymes? The commercial microbial enzymes are utilized in several industries. They are used to carry on certain specific biochemical reactions. Some important applications of microbial enzymes are the following: 1. Amylase a. Amylase is used in beer-making in beverage industries. b. It is used in the preparation of high-maltose syrup in the corn syrup industry. c. It is used in the extraction of jukes from fruits in food industry. d. It is used as a detergent in laundry and leather industries. e. It is used in textile industries for the preparation of paper pastes and binding substances. f. It is used in backing industry for improving the rate of fermentation of food substances. 2. Protease a. Protease is added to beer as chill-proofing agent and also to control the nitrogen level in beer. b. In backing industry it is used to improve the texture and volume the products. c. It is used in cheese-making in dairy industry. d. It is used in laundries and in the leather industry as a detergent and as a dehairing agent. e. In photography it is used to recover silver from spent films. f. It is also used in the preparation of rubber sheets from the latex.

5.Amyloglucosidase Amyloglucosidase and glucose ismerase act as catalysts in the manufacture of Corn syrup. 6.Catalase

a. It is used in dairy industry for removing waters from milk and milk products. b. It is used as a stabilizing agent in the preparation of soft drinks. It stabilizes citrus flavour in the drink. c. It is also used to control the colour of wines. 7. Glucose Isomerase

a. It is used in the removal of 02 from soft drinks. b. It stabilizes citrus flavour in soft drinks. c. It is also used to control the colour of wines.

8. Lipase It is used as a detergent in laundries and leather industry. 9. Cellulase

It is used in textile and paper industries in designing, and also for the modification of textile fibres and soft fibres. According to Fox (1974), microbial enzymes are utilized in food industries in three ways: 1. Enzymes are used in the processing of products. 2. They are involved in the improvement of the quality of products. 3. They are also used for improving the quantity of products. Besides the above uses, the microbial enzymes act as diagnostic agent in the identification of pathological conditions of an organism. Tell me about the enzyme immobilization The isolated enzymes (free enzymes) are very poor in their stability. The biological industries require enzymes which are having more reaction potential. The utility of free enzymes in industries shows some disadvantages, which are the following: •

1. The free enzymes are poor in their stability. 2. The reaction potential or turnover potential of free enzymes is very low. 3. The free enzymes go along with the product during the recovery of products. 4. The purity of the resulting product is somewhat poor. 5. There is wastage of enzymes during the harvesting of products. Because of the above disadvantages, free enzymes are protected by inserting them into a semi-solid Carrier before using them in industries. This process of arresting the free movement of enzymes is called enzyme immobilization. Such protected enzymes are often named immobilized enzymes. The immobilized enzymes have more advantages than the free enzymes.

3. Lactase It is used as a concentrating agent in dairy industry. It removes lactose from milk and whey. 4. Pectinase

a. It is used in the coffee industry for fermenting the coffee beans. b. It is used in the extraction of flavors in cosmetic industry. c. It is used in the improvement of the quality of fruit juices. d. It is also used in wine-making to catalyse the production of must.

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•

What are the various advantages of immobilized enzymes? The immobilized enzymes are used in biology- industries because of the presence of the following features in them:

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in solution, or to exclude or minimize undesired effects caused by contaminating by-products or microorganisms.

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1. The immobilized enzymes have more stability than the free enzymes. So the turn over of substrate into products is higher.

diffusion. This matrix does not allow the enzyme to escape from it. Polyacrylamide gel it commonly used for enzyme entrapment

2. The immobilized enzymes are firmly attached to the solid materials. So the recovery of the enzyme at the end of the reaction is very easy. The wastage of enzymes during the extraction of the product is avoided. 3. The reaction potential of the immobilized enzymes is more when compared with that of the free enzymes. So they catalyse the production of maximum amount of products within a unit time. 4. Only a proper species of enzyme is inserted into the solid material. So the chances of incorporation of other enzymes are very poor. Hence the enzymes produce only desired products with nearly cent percent purity. 5. The solid materials freely allow the substrate to reach the immobilized enzyme. 6. The cell-free reaction system behaves as a model system for studying the enzyme action of living cells. How is the enzymes immobilized? Generally different methods are adopted for immobilize the enzymes in solid carriers or substrates. The immobilization of enzymes depends on the specific reactions between the enzyme and the carrier. The different methods available for immobilizing the enzymes are given below: •

1. Physical adsorption It is one of the oldest methods of enzyme immobilization. In this method the enzyme molecules are adsorbed oil solid materials. The solid materials involved in the immobilization of enzymes are called carriers. Generally polymers are used for the immobilization of enzymes. Sometimes cellulose-based ion exchange resins, porous glass materials, silica gels and charcoals are also used as solid carriers for immobilizing the enzymes. The physical adsorption of enzymes on the solid carrier is due to the electrostatic forces, hydrophobic interaction. Vander Waal’s forces and hydrogen bonding between the atoms and ions of the carrier and the enzyme molecules.

3. Encapsulation T.M.S. Chang in Canada first adopted technique for immobilizing enzymes. In this method the enzymes arc entrapped in a semi-permeable membrane which acts as a bather to prevent the free movement of enzyme molecules. Thus small sacs of a definite size and porosity are formed. This process of entrapment of enzymes h a semi-permeable membrane is called encapsulation. Nylon membrane or collodion membrane is used for the encapsulation of enzymes. The sizes of the pores of these membranes are too small; the small pores do not allow the free diffusion of enzymes from the membrane sacs.

4. Liposomal entrapment Liposomes are lipid bodies consisting of two layers of lipids and enzymes. Phospholipids are generally used in the preparation liposomes. The prepared enzymes are inserted into the lipid layers or membranes. The enzyme and the livid ate mixed together in a flask or in a tube and shaken well to induce the enzymes for being inserted into the lipid bodies. As a result of proper mixing, the lipid completely surrounds the enzymes and forms liposomes.

2. Enzyme Entrapment In Polymer Matrix

In this method, polymer around the enzymes molecules is entrapped in a cross-linked polymeric matrix. Here the enzyme molecules are immobilized by the polymerization of polymer around the enzymes. During the polymerization the enzyme molecules are entrapped within the polymeric matrix. The enzyme & molecules enter the matrix by the process called

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5. Covalent Bonding Here covalent bonds are generated between the atoms of enzymes and those of polymers. The functional groups of enzymes are not usually involved in covalent bonding between the enzymes and the polymer. The groups unavailable for the enzyme reaction only take part in covalent bonding. Generally inorganic oxides and bricks and cellulose are used as carriers for

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immobilizing the enzymes. Hydrated carriers form an effective substrate for the immobilization of enzymes. Cellulose polymers are unfit for enzyme immobilization because they are susceptible attack driving industrial operations.

They also accept some non-specific enzyme proteins. So there is a chance for enzymes to produce heterogeneous products. But the synthetic polymers form effective linkers in the immobilization of enzyme. For example, acryl copolymer has aldehyde group. carboxy methyl group and hydrocyanate group; these groups are involved in the establishment of covalent bonds with the enzymes. Sometimes polymers which lack their reactive groups are used for the establishment of covalent bonding. So some small molecules are used as linkers which join the enzymes with the polymers. The linker molecule or bridge molecule must have two reactive groups; of the two reactive groups, one group reacts with the enzyme molecule while the other group reacts with the polymer. Cyanuric chloride, glutaraldehyde and cyanogen bromide are some linker molecules generally used to connect the enzymes with the polymers.

6. Copolymerintlon: Multifunctional copolymers are often used in the enzyme immobilizalion. Each polymer connects the enzyme molecules and it also connects the other polymers. That is, the polymers ate inter-connected with one another and also with the enzymes. Glutaraldehyde is one of the copolymers commonly used in enzyme immobilization. -

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Salient Features of Immobilized enzymes The Salient features of immobilized enzymes are 1. The immobilized enzymes are more stable than the free enzyme soluble enzymes; they are more stable towards heat; they are more towards urea; they are more stable towards the variability in they are more stable towards ethanol (Takata, Kayash&wa, Tos and Chibata 1982) 2. The immobilized enzymes have more reaction potential than the free enzymes. 3. They produce cent per pure products. 4. They are usually separated along with the carrier during the harvesting of products. The loss of enzymes is very much checked. 5. Immobilization methods increase the absorptive area of the enzymes. Hence immobilized enzymes show high rate of binding with the substances. 6. These enzymes easily release the products into the medium by simple diffusion 6. These enzymes do not need any modification during the catalytic reaction.

Fig 19.8 Comparison of stability of free enzyme with the stability of immobilized enzyme 7. These enzymes also strictly obey the kinetic characters of the free enzymes as explained by Michaeli and Menton.

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•

This enzyme is stable for about 14 weeks. It converts fumaric acid into malic acid (Chibata, Toss and Takata, 1983).

Are there any drawbacks of immobilized enzymes?

Yes there are. They include, 1. Some amount of enzyme may undergo denaturation during the immobilization of enzymes. 2. Sometimes denatured enzymes may be immobilized in solid carrier during the immobilization of enzymes. 3. Enzyme immobilization is an exothermic process which denatures some amount of enzymes during the immobilization. 4. Some carriers (adsorbents) are very weak in the immobilization of enzymes. Such carriers readily dissociate enzyme molecules in the solution or reaction mixture. These dissociated enzyme molecules go along with the products. Hence the quality of the enzymatic reaction becomes low. 5. The diffusion of larger enzyme molecules into the carrier is some what difficult process Hence the rate of immobilization of these molecules is very low. 6. Sometimes the entrapped enzymes are released in the reaction mixture. This leakage of enzymes causes the wastage of enzymes: 7. Sometimes immobilization reduces the reaction kinetics of the enzymes. 8. The polymers need proper chemical treatment before being used for immobilizing the enzymes. Now let’s see some example of immobilized enzymes. 1. Aminoacylase is immobilized by adsorbing it on DEAE sephadexpolymer. It converts the DL-acrylmino acid into Lamino acid 2. Amyloglucosidase is adsorbed on charcoal for immobilization.It converts dextrin into glucose. This enzyme survives for about 5 weeks, 3. Glucose isomerase is inserted into the cross-linked glutaraldehyde. Thisenzymeconvertstheglucosesyrup into frutosesyrup.This immobilized enzyme is stable for about 717 weeks, 4. Lactase is immobilized by using silica, cellulose etc. It survives lot several weeks. This enzyme converts lactose into glucose and galactose. 5. Nitrilase is entrapped in cationic acrylamide gel for immobilization. This immobilized enzyme converts acrilonitrile into acryl amide. 6. Penicillin G-acylase is covalently bonded with sephadex polymer for immobilization. This immobilized enzyme is stable for about 18 weeks. It converts penicillin-G -and Cephalosporin into 6-amino penicillianic acid. 7. Aspartase is adsorbed on vermiculite for its immobilization. This enzyme becomes stable for about 6 months It converts L-fumerate into L-aspartate . 8. Fumerase is produced by Brevibacterium flavum. The cells of B.flavum is immobilized in K-carrageenan and polyethylenirnine.

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Fumarase Fumaric acids - Malic acid. •

What are the applications of immobilized enzymes?

Immobilized enzymes have considerable practical applications in industries, in medicine and in model studies. The important applications are the following: 1. Immobilized enzymes are used as analytical agents in enzyme electrodes. The enzyme electrode is made up of a glass electrode surrounded by a thin film of immobilized enzyme. The enzyme electrode very sensitive; it is used to detect the presence of certain substances in the solution. It determines the presence of these compounds even when they are present in mild dosage. Now -a -days commercial immobilized enzymes are available for determining the presence of a small amount of drugs, pesticides and toxins in a solution. For example,. alcohol dehydrogenase enzyme is used in an enzyme electrode. This enzyme is blocked (inhibited) by morphine and also by other drugs containing barbiturate. This enzyme electrode is used to determine the presence of morphine and other drugs containing the barbiturate in the bloodstream. Similar electrodes are used in the analysis of the presence of drugs in blood and in other fluids. 2. In food industry, the immobilized enzymes play the following important roles: a. immobilized enzymes are used to convert starch into glucose. b. Milk whey contains lactase immobilized enzymes are used to convert the whey into simple sugars. c. Immobilized enzymes are used in the preparation of cottage cheese. d. Immobilize &glucose isomerase enzyme is used in the manufacture of fructose syrup. 3. In dairy industry, the immobilized enzymes are used to coagulate the milk protein during cheese-making and are also used to treat the waste whey. 4. Immobilized aminocylase enzyme is used in pharmaceutical industry. This enzyme converts DL-acyl amino acids into Lamino acids, The L-amino acids are used as ingredients in food stuffs. 5. Immobilized enzymes are used in enzyme therapy. The enzyme asparaginase is used in the treatment of leukaemias. This disease develops in human beings o to the deficiency of asparaginase enzyme. So this disease can be cured by using enzyme therapy. Insulin is treated with a small dose of zinc ions before it is marketed. The zinc ions increase the survivability of the enzyme and also increase the reaction potential of the insulin. 6. Immobilized enzymes arc used in the study of biochemical reactions of organisms under different physiological conditions. Here the immobilized enzyme reaction is used as a model system to determine the characteristic cellular metabolism in living cells.

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Review Questions 2. What are enzymes? 2: Explain the importance of microbial enzymes. 3. Write a detailed account of microbial production of enzymes 4. How do you regulate enzyme synthesis in a microbial culture? 5. Give c account of extraction of microbial enzymes. Add notes on the storage of purified enzymes. 6. Explain the various methods available for the culture of microbes for the cal-action of enzymes. 7. Explain process engineering with reference to production and purification of microbial enzymes. & Explain the various uses of microbial enzymes. 9. What are tire immobilized enzymes? Add notes on their importance in industries. 10. Write an essay on the immobilization of enzymes. 11. Write short notes on the following: a. Enzyme therapy,

d. Physical adsorption.

b. Enzyme electrode.

e. Encapsulation.

c. Protease

Notes

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LESSON 22: FERMENTED FOODS

Learning Objectives In this lecture, you will learn •

Some important fermented foods

•

Safety cabinets - design

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Safety cabinets - applications

The term ‘food’ includes all items consumed by man. Out of these, some food items are prepare by fermentation. In other words, desirable chemical changes are brought about by desirable micro-organisms (e.g. sauerkraut and yoghurt). It is also essential to preserve the food properties if the are to be consumed after a long period.

Let’s see some examples of fermented foods. Sauerkraut

Sauerkraut is a neatly shredded cabbage which has been allowed to undergo natural lactic acid fermentation in the presence of a small amount of salt. It is produced commercially in most northern and eastern European countries. It is normally consumed after cooking as a main course vegetable. At present, it ranks fifth in the order of importance amongst canned vegetables in f United States. Sauerkraut is only preceded by peas, beans, com and tomatoes. It derives its keeping quality from the acid produced during fermentation. It is a relatively stable product if it is kept cool and out of contact with air. Sauerkraut, which is properly prepared, can be quite a rich source of vitamin C. Sauerkraut also has a mild laxative action, which is attributed to the dextran content of the product. Dextran is produced during fermentation by Leuconostoc mesenteroides, dominant during the first part of the curing period. •

How is sauerkraut made? Good foods cannot be prepared from poor materials and sauerkraut is not exception to the rule. Consequently, the quality of the cabbage used has considerable bearing on the quality of the final product. Many varieties of cabbage are suitable for sauerkraut production. The heads should be solid, crisp, with a minimum of green leaf. Once the- heads have been cut, it is necessary to store them for a few days prior to shredding. They are usually, stored in well-ventilated sheds or barns. The reason for this is that a slightly wilted cabbage can be more neatly sliced, with less damage to the shreds than when it is first harvested. Another advantage of the period of wilting is hat it allows the heads to come to even temperature, and to warm up during cold weather. Just before shredding, the outer green leaves are trimmed away together with any damaged or diseased parts. The core is cut with a spiral knife without actually disconnecting the core from the lead. The heads are then sliced by a machine. The shreds should not exceed 2mm in thickness.

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Good sauerkraut can be prepared in any size of the fermentation’ vessel. The size of the fermentation Vessel, which may be used, ranges from a gallon jar to the 80 tons redwood tanks. This fermentation, vessels can be made of wood. The tiled concrete tanks are better than wooden tanks so far as sanitary aspect is concerned. All the metal equipment coming into contact with the product must be of stainless steel, or be suitably coated. The reason for this is, that iron causes discoloration, whereas copper causes certain loss of vitamin C. Copper is also responsible for off flavours. The shreds are deposited in the fermentation vessel and are mixed with 21 % by weight of salt. This is a crucial stage in the manufacture of sauerkraut. It is absolutely essential that exactly this .mount of salt should be used and that it should be incorporated evenly throughout the mass of Shreds (Pederson, 1946). This requires care and patience, and it is necessary to weigh both salt and shreds in relatively small batches. As each batch of shreds is added to the vessel, the approximate quantity of salt is scattered over it and rapidly forked in. The height of the shreds in the vessel should be maintained at an even level as it gradually rises. This is the responsibility of the operator who should be an experienced man. The shreds must also be compacted down tightly. Once the vessel is full a false head is placed in position and then weighed in some way. It is usually weighed by blocks of concrete fitted with handle. This is to compress the mass and thus, ensure that the shreds are forced under the juices extracted by the salt, and that all he air is taken out. On the other hand, such covers suffer from the disadvantage that the juices, which rise over the head, are in contact with air. This permits the growth of yeasts and moulds at the surface. These may spoil the product by causing off-odours and flavours. Various ways of overcoming this difficulty have been tried in the past, and the modern practice is of placing the wooden covers with plastic sheets weighed down with water or brine. These effectively seal off the whole surface from contact with air. Owing to the flexible nature of the plastic sheet, the gases formed during fermentation can escape freely past the seal. This method would appear to be ideal. A variety of sugars, proteins and other nutrients are present in the juices extracted from the cabbage. These support the growth of bacteria. Consequently, in a matter of a day or so fermentation sets in, this being apparent by the evolution of gas and the formation of acid. The time required for the fermentation to run its course depends on temperature, particularly filling temperature (Pederson, 1956). If this is favorable, a period of between three to six weeks will normally suffice. At the end of This time, the following changes are marked:

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other species of bacteria appear when high fermentation temperatures prevail. They are: (1) Streptococcus faecalis and (2) Pediococcus cerevisiae. These are homofermentative bacteria,

(2) All the fermentable carbohydrates will have been consumed. (3) The acid content will have risen to between 1.8 % and 2.2 %.

producingonlylacticacid.TheydonotproducetheCO2

(4) The pH will approximate to the range 3.5 to 3.7.

necessary to create anaerobic conditions. Therefore, their presence is normally associated with a lessening of product quality .

The product is now stable, provided that it is kept cool and out of contact with the air. Therefore, when fermentation is complete the vat or container must be properly sealed to exclude air.

Although, high temperatures act to the detriment of quality they do, nevertheless, favour the growth of both Lactobacillus plantarum and L. brevis. These do not grow well under cold conditions. So many early investigators advocated the use of heat during commercial sauerkraut production. Generally it is not advisable to allow the temperature to rise above 70oF. Or to drop below 45°F. The optimum range is probably 55°F. To 65°F.

If the temperature is too warm, darkening and general loss of flavour and quality will take place. The progress of fermentation should be followed by periodic checks on the development of acidity in the juice. This, not only shows whether fermentation is proceeding normally, but also enables finished vats to be recognized and sealed, and for the order in which other vats will reach completion to be predicted. A record of vat temperatures should be kept, since it is also important for predicting finishing dates •

It is extremely important that the right amount of salt should be present during fermentation. The optimum range is 2 % to 2 1/2 %. High salt contents are associated with the development of pink sauerkraut. Also, high salt content (3.5 %) favours the growth of two undesirable bacterial species (i.e. Streptococcus faecalis and Pediococcus cerevisiae), whereas the more desirable species tend to be inhibited by it . Low salt contents are detrimental to the texture of sauerkraut.

How does the fermentation take place?

Three acid-producing species of bacteria are mainly responsible for normal sauerkraut fermentation, each being dominant in succession. The first species to appear in numbers is Leuconostoc mesenteroides. This attacks the sugars leaching from the cabbage, with the formation of lactic and acetic acids, ethyl alcohol, mannitol, carbon-dioxide, etc.

•

] The pleasant flavor and odour of sauerkraut emanates from the activity of this bacterium. In addition to this, there is a copious evolution of carbon-dioxide arising from its activity. This is instrumental in quickly creating anaerobic conditions in the mass. Thus, growth of spoilage yeasts is inhibited. Since Leuconostoc mesenteroides is unable to withstand acidities approximately to 1 %, as this level is reached in the product it dies out and is replaced by Lactobacillus plantarum. This also produces lactic acid, and in turn, it is similarly destroyed by the steadily mcre2Sing acidity. Meanwhile the numbers of Lactobacillus brevis go on building up. Since this species is capable of withstanding any acidity attainable in the product by natural means (i.e. its own acidity), it takes the fermentation to completion. Both the bacteria in the first and final stages of the fermentation are producers of CO2, Lactobacillus plantarum, however, is a homofermentative species and so does not produce gas. Therefore, the first stage is indicated by the rapid evolution of gas, followed by a period of relative quiescence (second stage). Again a moderate increase in the gassing rate is an indication of the final stage of the fermentation process. The principal acids produced during fermentation are lactic and acetic acids and at the completion of the process their ratio normally approximates 4 to 1 respectively. Temperature is important in sauerkraut production in relation to quality. The grow-ill of Leuconostoc mesenteroides is not favoured at temperatures above 70oF. Since the flavour and odour are dependent on this species, high temperatures result in a poor quality product. Also, two

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Do we need to add culture to this fermentation?

The use of bacterial cultures as starters has been investigated but generally with little success and sometimes with a positive disadvantage. The reason is that the right bacteria are always present on cabbage and if given the proper conditions (i.e. salt and temperature) they will always develop and in the correct sequence. •

·How is the sauerkraut stored? Canning is best accomplished by practicing the following operations: (I)

Sauerkraut is heated to 165°F in its juice. Heating is carried out in a stainless steel or glass lined pan (steam-jacketed).

(ii)

Heated sauerkraut is immediately packed into unlacquered cans.

(iii)

Filled cans are quickly exhausted, sealed and cooled. Canned sauerkraut has a less pleasant odour than the fresh one but the disagreeable quality disappears on cooking (Cruess, 1958).

Some sauerkraut is packed in glass and vacuum capping and cool storage are advisable. Polythene bags have also been tried. Sedky et al. (1952) showed that when ascorbic acid was added to protect the colour, and the bags sealed under vacuum and kept refrigerated, a shelf-life of six weeks was attained. Recently a laminated polythene aluminum foil pouch has been tried with success (Anon., 1967). The pouches are pasteurized by heating at 170°F for 20 minutes. Freeze-dried sauerkraut has also been ‘ prepared (Anon., 1969).

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(1) The product will have acquired its typical aroma.

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The next fermented food is olives Olives are valuable pickles in the United Kingdom. Comparatively large quantities are consumed in this way and something like an average of 775 tons per year are imported. Their keeping quality depends on the absence of suitable nutrients to support microbial growth, the presence of salt, and of lactic acid and its attendant low pH. There is a difference between pickle olives and canned olives. The pH of canned olives approximates to neutral and its preservation necessitates a heat process of 240°F for 60 minutes. The olive has been cultivated by man since ancient times. There are a number of varieties of olive but only two are of major importance for pickling. These are the large Sevillano or the Gordal (Spanis Queen) and the smaller Manzanillo olive. The former is considered to have the better flavour and texture but is more difficult to pickle. Fresh olives have an insipid and intensely bitter flavour. One of the purposes of the pickling process is to remove this bitter taste. The first step in the pickling process consists of treating the fruit with a dilute alkali to remove the bitter taste. Pickled olives fall into two categories-green and black. They differ in the following two respects: (a) The time of harvesting, and (b) The manner in which they are subsequently processed. Green Olives The fruit is allowed to become full grown, but is picked before any change in the colour begins. Immediately after harvesting, the olives are placed in shallow vats. They are covered with lye which consists of water with slightly less than 2 % sodium hydroxide (i.e. caustic soda). The olives remain here for about two days until the lye has penetrated approximately two-thirds of the way to the pits. The lye penetration is followed, in practice, by the simple process of halving a fruit, washing and applying phenolphthalein to the cut surface. Lye-treated olives are washed in a number of changes of water until they are mostly free of alkali. Care must be exercised during both the lye treatment and the subsequent washing to avoid undue aeration. The reason for this is to avoid blackening of the product. Once the olives are free of alkali, they are drained and packed in large barrels. Then the barrels are headed up and 45° salometer brine (11.9 % salt) is introduced through the bung. After filling, the barrels are placed in the sun to warm the contents. Thereafter, a natural fermentation will commence, if all goes well. This is a critical period in the pickling process. If fermentation fails to start, spoilage micro-organisms may become established. Starters may be added to each barrel to ensure the rapid commencement of fermentation. Starters consist of a few pints of old brine from well fermented olives. The first bacterial species which appears during fermentation is Aerobacter aerogenes. This bacterium produces carbon dioxide and hydrogen, giving rise to ‘gassing’ of the brine. Soon lactobacilli of both the homo- and hetero-fermentative species predominate when production of gas is much reduced. As a result of this, formation of lactic acid begins to take place. More lactic acid formation can be had by the addition of small amounts of sugar to the brine. This, however, must be added

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late in the process, otherwise Aerobacter aerogenes is unduly stimulated with resultant excessive gassing. Olives grown on nutritionally poor ground may be lacking in nitrogen-containing compounds. These compounds are required by the bacteria for their growth. Samish (1955) has shown that the fermentation of such olives can be improved by the addition of a .source of nitrogen (e.g. ammonium salts) to the brine. Etchells et (1966) have investigated the use of pure cultures to induce satisfactory fermentation. After consumption of all the fermentable carbohydrates, fermentation ceases and the product is now stable (i.e. fully cured), provided it is kept out of contact with air. At this stage the lactic acid content of the brine should not be less than 0.5 % and the pH will have dropped below 4. After curing, the olives are carefully size-graded and all the blemished fruits removed by hand. Then, .they are recasked and are ready for shipment. The Manzanillo olives are pitted and their stones are removed. Thereafter, the cavities are stuffed in a variety of ways. The olives are then packed in casks with about 15 % salt brine, preferably with 0.5 % lactic acid added. The most frequently used form of stuffing is strips of pimento. These strips, before use, are preserved in strong brine. The bright red of the pimento against the green of the olive is a most attractive colour combination. Such ingenuity in the presentation of olives does much to assist in their sale. These stuffed olives are packed in jars. After filling into the jars, the olives should be rinsed with several changes of water to dislodge and remove all sediment and should then be drained. Then the jars are covered with clear brine containing between 6% to 9% salt and 0’5% lactic acid (edible grade). Capping should follow immediately, usually in vacuum, without headspace formation. Black Olives

Olives which are to be processed into the black form are not harvested until the fruit is straw 001oured on the verge of turning to red. The essential difference between the processing of the green and black form lies in the amount of aeration during the lye treatment. Aeration is reduced to a minimum during the preparation of the green olives. On the other hand, black olives are treated to a series of alternating lye and air exposures over a period of 3 to 5 days. Catechol-like substances present in the olives are readily oxidized to a dark colour. Next, we will see about the pickled cucumbers

Pickled cucumbers are made in Africa, Asia and Latin America. Cucumbers undergo typical lactic acid fermentation and change from a pale product to a darker green and more transparent product. Khalpi is a cucumber pickle popular during the summer months in Nepal. Raw Material Preparation

Fully ripe cucumbers without bruising or damage are washed in potable cold water and drained. The cucumbers can be pickled whole or sliced. With khalpi the cucumbers are washed, sliced and cut into 5-8 cm pieces. Processing

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As soon as the brine is formed, fermentation starts and bubbles of carbon dioxide appear. Fermentation takes between one and four weeks depending on the ambient temperature. Fermentation is complete when no more bubbles appear. During fermentation the brine becomes cloudy for the first few days due to the growth of bacteria. Later if the brine is not covered, a filmy yeast growth will often occur on the surface (Pedersen, 1979).

Flow diagram Packaging and Storage Cucumber pickle is usually stored in clean capped jars. They keep well if stored in a cool place. Due to the high acid level of the final product, the risk of food poisoning is low. With khalpi in Nepal, oil is added. Let’s now see an interesting fermented food, viz. Pak-GardDong (Pickled leafy vegetable) Pak-Gard-Dong is a fermented mustard leaf (Brassica juncea) product made in Thailand. The mustard leaves are washed, wilted in the sun, mixed with salt, packed into containers for 12 hours. The water is then drained and a 3% sugar solution added. They are again allowed to ferment for three to five days at room temperature. Micro-organisms associated with the fermentation include Lactobacillus brevis, Pediococcus cerevisiae and Lactobacillus plantarum. A similar product (Hum choy) is made in the South of China. This is produced by fermenting a local leafy vegetable. The leaves are washed and drained. They are then covered in salt and hung on racks to dry in the sun. The wilted leaves are placed in earthenware pots and covered with rice water, obtained after washing rice grains. The pots are sealed and the leaves allowed to ferment for four days. The product can be stored for up to two months if the seal is not broken. Tempoyak (pickled durian)

Tempoyak is the fermented pulp of a durian fruit (Durio zibethinus) from Malaysia. It has the distinctive durian smell and a creamy yellow colour. It is made by mixing durian pulp with salt and placing in a sealed container. Fermentation takes about seven days. Pickled beetroots

In Russia beetroot is pickled by cleaning, slicing and placing in a container with salt. Due to the high sucrose level, dextrans are produced giving the product a slimy texture. Lamoun Makbous (pickled lemons)

Pickled lemons are popular in Asia. In west Asia and north Africa they are known as lamoun makbous and msir. Lemons are washed in clean water,

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sliced and covered in salt. After at least 24 hours, they are drained and mixed with oil and spice. Let’s see about yoghurt or Bulgarian milk

Yoghurt is a diary product prepared by the fermentation of milk. It is consumed in large quantities as a food item by the Bulgarian people. Nowadays it is also consumed by the people of Europe and North America. According to the Russian bacteriologist, growth of Lactobacillus bulgaricus in the colony produced a high concentration of lactic acid. This highly acidic environment did not permit the growth of proteolytic flora. Therefore, disorders that were supposedly connected with adsorption of proteolytic wastes from the alimentary tract would not occur. It is believed that if a man regularly consumes 250 to 600 gms. of yoghurt daily from his childhood, he can expect to lead a healthy, buoyant and youthful life upto the age of 70 to 80 years. It decelerates the clock of aging, if not reverses or stops it. It also lowers the blood cholesterol level and, therefore, improves the functioning of the cardio-vascular system making the heart strong. Today, therapeutic, prophylactic and nutritional properties of dahi are widely accepted. How is yoghurt made? Yoghurt is commercially manufactured by the fermentation of milk with two thermophilic bacteria, viz., Streptococcus thermophilus and Lactobacillus bulgaricus . The latter rapidly grows at optimum temperature through the process of symbiosis. The final product is highly viscous, slightly sour, with its characteristicsic ‘curd’ flavour and odour. It has consistency resembling custard. The manufacturing details of the production of yoghurt have been described in some publications, although slight variations in details may exist from industry to industry. •

The essential features of all the methods of production of yoghurt are: (1)Selection of milk. (2)Standardization (fat extracted)-milk powder of concentrated milk added. (3)Warming and homogenization. (4) Heat treatment (steaming for half an hour). (5) Cooling to about 50 0C. (6) Inoculation with yoghurt cultures. (7) Packaging. (8) Incubation in a warm room (420 to 44°C.). (9) Cooling in the air. (10) Cold storage. Yoghurt contains 2 % to 3 % of fat, but even a fat-free product is acceptable to many. Usually, the SNF content is raised to 14 % to 16% to make it a more viscous product and to increase its protein content. In regard to the second aspect, the modification involves the marketing of the yoghurt as a highly viscous uniform product ‘stirred curd’ instead of the normal ‘firm-set’ or ‘junket form’. As shown above, as an alternate step of manufacture, the milk after inoculation is held in the vat at 420 to 44°C. Until it coagulates, then it is cooled by circulating

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1 kg of salt is added to every 20 kg of small cucumbers and 15 kg of large cucumbers. The brine should be formed within 24 hours by osmosis. If the brine formed by osmosis does not cover the cucumbers 40o Salometer brine is added to the desired level. A day or two after the tank is filled and closed the brine should be stirred in order to help equalize the concentration of salt throughout the mass.

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chilled water with gentle agitation and packed in suitable containers. The filled containers are then cold stored (at about 5° to 6°C.) and held overnight to develop the characteristic ‘Yoghurt’ flavour. The product so obtained will be slightly sour, but with a clean flavour. The acidity is about 0.9% but can go upto 1.1 % (pH 4.0 to 4.2). Higher acidities may be tolerated if milk fortified with extra milk solids is used. An interesting fermented product is fruit and flavoured Yoghurts

A recent innovation which has enhanced the popularity of yoghurt is the practice of fortifying it with sweetening agents, fruits and fruit flavours. Fruit and flavoured yoghurts are sweet with the characteristic flavour of the fruits or the added flavours. They retain all the nutritional and curative properties of yoghurt. They are readily accepted by children, adolescents and aged people. Common sugar is the most common form of sweetening agent added. Some people add honey which also increases the nutritive value of the product. The sugar level depends upon the consumer’s preference and is in the range of 6 % to 8 % as sucrose. Fruits and flavours can be added to yoghurt in a variety of forms. Some add synthetic flavours not to the liking of many. Quite a common practice is the addition of fruit extracts or concentrates. Fruit powders are also added in some western countries. However, the most popular practice is the addition of cut fruits or fruit pulps which retain the fresh flavour of the fruit. The types of fruits added are many and variable: The popular fruits are the berries, citrus and apples. Usually, fruits are added at the rate of 15% to 20% of the product. Fruits and sugar maybe added separately or as ‘fruits in syrup preparations. Fruit yoghurts can be preserved upto a week in a refrigerator and for longer periods at temperatures below zero. Deep freezing of the product is not desirable as it can cause ‘wheying off ’ when taken out for thawing.

Nutritive Value of Yoghurt During fermentation of milk, the composition of the minerals remains unchanged, while proteins, carbohydrates, vitamins, and to some extent, fat constituents are subjected to changes which produce special physiological effects. Dietary and therapeutic qualities of sour milk products are determined by micro-organisms and substances formed as a result of the biochemical process accompanying milk souring. These substances are lactic acids, alcohol, carbon dioxide, antibiotics and vitamins. The following processes make yoghurt more nutritive than milk: Proteolysis in Milk

Proteolysis in milk takes place by ego- or endow-peptidase of lactic acid bacteria. The biological value of protein increases significantly during yoghurt manufacture from a value of 85.4 % to 90 %. This increase is due to breakdown of protein into peptones,

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peptides and amino .acids. The contents of essential amino acids such as leucine, isoleuciae, methionine, pkenylalanine, tyrosine, threonine, tryptophane and valine increase considerably, which offer special advantages not only to healthy people but also particularly to the physically weak. Hydrolysis of lactose

Lactose in milk is hydrolysed by metabolic activity of bacteria. Approx mate 2.5 % lactose, 0.8 % to 1.3 % galactose and 0.03 % glucose are obtained from lactose hydrolysis. Lactose hydrolysis takes place due to beta—galactosidase production by lactic acid bacteria. The importance of lactose is due to the lactic acid produced from the hydrolysis of lactose, which leads to a pH change in the bowel inhibiting the growth of putrefactants. In addition to this, lactic acid is important for organoleptic properties and calcium absorption. Lipolysis

The homogenization process reduces the size of fat globules which become digestible The production of free fatty acids as a consequence of lipolytic activity increases as compared t, milk. This leads to some physiological effects. Changes in Vitamins

There is more than a two-fold increase in vitamins of B-group, especially’ thiamine, riboflavin and nicotinamide as a result of the biosynthetic process during milk fermentation Subsequently, vitamin B6, vitamin C and vitamin B1 (thiamine) decrease by approximately one half only, as they are utilized by the bacteria in milk. Antibacterial Activities

The bactericidal properties of sour products are determined by the anti biotic activity of the bacteria growing in the product. The antibiotic properties are generally associated with lactobacilli in yoghurt, and materials responsible for such antibacterial action are described a lactic acid, hydrogen peroxide and other substances such as lactobacilline, etc. Therapeutic Importance

The main advantages of regular intake of yoghurt are: 1. This product is easily absorbed and better assimilated than sweet whole milk. Assimilation of milk is 32 % in one hour, while that of yoghurt it is 91 % in the same period. (2) Yoghurt improves appetite due to its pleasant, refreshing and pungent taste. It is highly nourishing and invigorating not only to healthy persons but also to the ill and infirm (3) Gastric juice secreted by the action of yoghurt and the desirable ratio of calcium and phosphorous induced by it leads to a high digestive capability. (4) Yoghurt consists of a sufficient amount of indispensable amino acid, methionine, which removes excessive fat from the liver and enhances bile secretion. It is, therefore, an important therapeutic adjutant in gastrointestinal disturbances, hepatitis, nephritis, diarrhoea, colitis, anaemia and anorexia. It provides wonderful relief to patients of chronic diarrhoea, and ulcerative colitis. Fat-free yoghurt is necessary for those who suffer from heart disease, arteriosclerosis, hypertension and chronic inflammation of the liver.

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•

Tell me about the cheese production.

Species Propionibacterium shermanii Lactobacillus bugaricus Lactobacillus lactis Lactobacillus helveticus Lactobacillus acidophilus Streptococcus thermophilus

Yeasts, molds, and bacteria are all involved in the processes that produce different cheeses and their locations on or in the curd are what results in many cheese types. The table below shows the bacteria involved in different cheeses:

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Acid flavor

and

Acid

Acid

Streptococcus lactis Streptococcus cremoris

Acid

Acid flavor

Flavor

Product Swiss cheese family Bulgarian buttermilk, yogurt, kefir, koumiss, Swiss, Emmental, and Italian cheeses Acidophilus, buttermilk Emmental, Cheddar, and Italian cheeses, and yogurt Sour cream, ripe cream, butter, cheese, buttermilk and starter cultures. Cultured buttermilk, sour cream, cottage cheese, all types of foreign and domestic cheeses, and starter cultures.

Acid

Streptococcus diacetilactis

Streptococcus durans Streptococcus faecalis Leuconostoc citrovorum Leuconostoc dextranicum

Cheese is yet another product of milk fermentation that requires factors in addition to the traditional production of lactic acid. The coagulation of casein due to lactic acid production and the subsequent drop in pH and the addition of rennet, an enzyme derived from the lining of the stomachs of calves, form the curd of cheese Without rennet, a soft cheese such as cottage cheese or cream cheese would result

Major known function Flavor & eye formation

and

Soft Italian, cheddar, and some Swiss cheeses. Cultured buttermilk, sour cream, cottage cheese, ripened cream butter, and starter cultures.

The manufacture of cheese is a microbiological process. There are several hundred varities of cheese. The particular combination of salt, incubation, temperature, pH and culture used, determine the kind of cheese manufactured. The production of most of chesses is made from cow’s milk. The basic steps involved in cheese production are as under: 1) Curdling the milk: Curd may be prepared either by adding starter culture (pure or mixed type) or the enzyme rennin ‘to milk. The following organisms may be used as starter culture in the pure or mixed form: Streptococcus cremoris, S. lactis, S. thermophilus, Lactobacillus lactis L. bulgaricus, etc. The milk protein, casein, gets coagulated during curdling reaction. The mechanism of casein coagulation is an interesting phenomenon. The initial pH of the milk is about 6.8 at which casein does not undergo coagulation. But casein coagulation occurs when the pH reaches 4.78 to 4.64. Lactic acid produced by the starter culture lowers the pH of the milk to a sufficient extent to precipitate casein. Actually casein is dispersed in fresh milk as calcium caseinate. In some varieties of cheese production, a combination of the starter culture and rennin is used.

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(5)Yoghurt possesses potent antitumor activity according to Shahani, professor of Food Sciences at the University of Nebraska, U.S.A. It may prevent or retard the onset of diseases. Pathogenic bacteria are not able to survive in it because of its low pH and other adverse factors for their growth. Consumption of less than one liter of ‘yoghurt per day will not affect human health adversely, but more may involve the risk of acidosis. The nutritional and therapeutic effects of yoghurt do not depend on living bacteria but on their metabolites.

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the fungus has grown ‘to the desired extent. The inoculated curd is then placed at lower temperature to inhibit the fungal growth, but fungal enzymes.

Camembert cheese Like Roquefort, Camembert is said to have originated in France and it has several imitations. Named cheese varieties which are basically of the Camembert type are: Brie, Thenay, Troyes and Vend6me. The principal and obvious action of the fungus in the ripening of Camembert type cheese is that of changing the texture of the cheese, although changes in the odour and the flavour also occur. Thom was of the opinion that certain characteristic flavours are due to the action of lactic acid bacteria. Streptococcus lactis

Swiss Cheese

(2) Draining the curd to remove moisture: The strawcoloured liquid called whey is produced during curdling of milk. It is separated from curd with or without the application of pressure. The kind of cheese to be produced determines how much moisture must be removed. The production of soft varieties of cheese involves draining without pressure. On the other hand, the manufacture of hard varieties of cheese requires draining with pressure. Also, semi-hard varieties of cheese are produced, where a limited pressure is used during draining. After removing whey to the desired limit, the curd is moulded into various sizes and shapes according to the variety of cheese that is being produced. (3) Salting: Sodium chloride is practically always added at some step of production to all varieties of cheese. Sodium chloride is added in either of the following two ways: (I) It may be applied to the surface of the pressed curd by rubbing with salt. (2) Salt may be mixed with the drained curd. Salting operation has three objectives: (a) It serves as a preservative, and therefore, does not permit the development of unwanted micro-organisms. (b) It imparts flavour to the cheese. (c) It serves to control cheese moisture by withdrawal of water. (4) Ripening: Ail varieties of cheese, except a few, require ripening of curd. Cottage cheese and cream cheese are examples of unripened varieties of cheese. Usually, ripening is brought about either by bacterial or mould culture. In both cases, changes in the curd take place through enzymatic reactions. Importance of ripening operation can be understood by considering some important varieties of cheese: Roquefort Cheese

Roquefort cheese originated in southern France many years ago, and in its original form, was made only from sheep’s milk. Today most Roquefort and all of its imitations are produced from cows’ milk. The curd is inoculated with Penicillium roqueforti by mixing in bread crumbs on which the fungus has been growing. After inoculation the inoculated curd is placed in a ripening room. The temperature and humidity of the ripening room are carefully controlled. The inoculated curd is left in the ripening room until

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The ripening process in the manufacture of Swiss cheese is carried out by lactobacilli, streptococci and propinoibacteria. These bacteria are added to the milk prior to the curdling reaction. Lactose of the milk is attacked by lactobacilli and streptococci with the formation of lactic acid.). Cheddars cheese It is the most commonly sold variety of American cheeses. It is a hard curd cheese without eyes The organisms, primarily involved, are probably Streptococcus lactis and related streptococci and various lactobacilli. •

How are the food products preserved? As supermarkets were rare during most of human evolution, food was in short supply and fresh food was even more limited. Early man may not have been exactly rocket scientists, but they could tell the difference between reallyrotten and not so-rotten food. So when someone discovered a way to preserve food while it still had a reasonably decent taste and odor they were likely considered a hero. What follows is a description of some of the old and new food preservation methods.

Heat Sterilization One of the problems with war is that soldiers insist on being fed regularly (remember, they hold the weapons). In the early 1800s, Napoleon found that the joy of his life, his large army, could no longer feed itself by stealing from the local peasants and thus his plans to conquer the world were stalled. His solution was to offer a reward for anyone who could figure out how to preserve food so he could take it along with his army, thus keeping them and him happy (the only unhappy ones being those he conquered). In 1810 a man by the name of APPERT found that if he put food in a bottle, jammed a cork tightly in it and placed it in boiling water for an hour or so the contents didn’t spoil. BINGO!!, he won the prize; Napoleon got his war, and learned just how seriously cold a Russian winter could get. This procedure, known as STERILIZATION, eventually developed into the canning process. In the process of sterilization all living organisms are destroyed, including bacterial spores. As you will learn later, the most deadly biological toxin is produced by the spore-forming bacterium, Clostridium botulinum. C. botulinum is an obligate anaerobes that can grow in seal containers like cans and jars, therefore the canning process is specifically designed to destroy the C.

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In the home, sterilization is carried out using a PRESSURE COOKER. Many of you probably have seen your grandmother, or perhaps your mother, using this container to sterilize home-canned food. The pressure cooker works as follows: •

A pint or so of water is placed in the bottom of the pressure cooker.

•

The food to be sterilized is placed in the container with the lids loose.

•

The top is placed on tightly and the water is brought to a boil until all the air is vented through the outlet port.

•

Then a weight is placed on the outlet port. This weight is adjusted so that steam will only escape once the pressure has reached 15 pounds per square inch. At this pressure the temperature will reach 123oC at sea level.

Once this temperature is reached and steam begins to bleed from the port, heating is continued for a period of time necessary to bring all the food in the containers to 123oC for 15 to 20 min. •

The heat is turned off and the contents are allowed to cool.

•

Finally, the pressure cooker cover is removed, and the jar lids tightened immediately to prevent contamination from entering.

In the microbiology laboratory and commercial canning companies sterilization is achieved by using large containers that operate exactly the same as the home pressure cooker. The laboratory instrument is called an AUTOCLAVE. In commercial canning processes the sterilization containers may be as large as rooms and the food is often wheeled in on large carts. •

Can you explain the difference between Pasteurization And Sterilization. .

Cooling and Freezing As described above, except for Eskimos and other inhabitants of the far north, cooling has only emerged as a common means of preserving food since the mid 1800s when the ice-making machine was discovered. Prior to that time it was common in northern climes for people to cut large blocks of ice from local lakes and to store them in insulated warehouses for use during the summer months to cool their beer and other food items. Cooling as a food preservative is utilized at two levels, 7 to 4oC and -20oC or lower. The higher temperature is commonly used in home refrigerators. At this temperature, the growth of microbes is slowed down but not stopped. Indeed, some microbes grow optimally at these temperatures. The failure to prevent spoilage at this higher temperature is attested to by anyone who has attempted to use milk older than two weeks in a refrigerator or who has left fruits and vegetables in a ‘fridge’ for extended periods. At the lower temperature the food is frozen. As microbes are unable to grow in frozen material, freezing is one of the most successful means of preserving food with minimal change in flavor or loss of nutritional value. The major draw back to the use of cooling is that (a) it is

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expensive and (b) it also preserves many pathogens that happen to be present in the food when it was cooled. As a matter-of-fact the storage of living material at temperatures of 70oC or lower is the best way of maintaining cells in a state from which they can be subsequently cultured. Such material as sperm, ova, embryos (human and other forms of life), all types of microbes and tissue cells can be frozen and stored for years with little loss of viability providing the procedure is carried out properly.

Drying Drying as means of preserving food may very well be the oldest method of preservation known to man. Almost certainly it was an accidental discovery made by our primitive ancestors living on the hot plains of Africa. Most likely, our ancestors frequently came across carrion (a sort of road kill) that had dried in the arid conditions. Being hungry, they ripped off the dried meat and chewed down. It didn’t take them long to recognize that it wasn’t spoiled, that it was light and that it stayed unspoiled as long as it remained dry. Some budding hairy-Einstein soon realized that fresh meat could be dried by placing it in the hot sun and the human race was off to the races, so to speak. Drying is employed today as a common means of food preservation by all peoples living in warmer climates. Generally the food, such as fresh meat, is cut into small strips and placed on rocks exposed to the sun, or hung over sticks by a campfire. The pieces must be small so that the food dries fast enough to prevent spoilage. In the case of meat, one trick is to hang it high enough so the flies can’t get to it and lay their eggs in it. As the water evaporates and the food dries, the OSMOTIC PRESSURE (the result of hydrophilic molecules binding water molecules) increases to a point where microbes are unable to compete with the water-binding material in the food for the remaining water. Since microbes are unable to grow without free (available) water, the food is safe from spoilage, even though it may retain significant bound-water. In some cases (beef jerky) the food is salted prior to drying. The salt is inhibitory to many microbes and contributes to the high osmotic pressure that prevents microbial growth. Salts and other Chemicals Salt or Sodium Chloride

The use of salt as a food preservative is probably as old as drying, if not older. All mammals need salt and they will travel long distances to obtain it. Our human ancestors certainly visited the ocean or salty lakes to collect the salt that had dried on the shore. Occasionally animals or fish must have died in pools of salty water and then dried in the sun leaving their desiccated carcasses impregnated with salt. Again our hungry ancestors were unlikely to turn down a potential meal and they must have quickly recognized that the salted food was unspoiled and remained so as long as it was impregnated with salt. The salted food served a dual role as a source of nutrition and of sodium chloride, and as it dried it was easier to transport. Before canning, salted meat was the staple food on ships that traveled any significant distances away from land (hence the term “ol salt”). Nitrate (No3) and Nitrite (No2) (Saltpeter)

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botulinum spore. This is achieved by heating food to a minimum of 123oC or 253.4oF for 15 minutes.

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Nitrate and nitrite salts are used in many foods today as both a preservative and to prevent meat from browning. The bacterium Clostridium botulinum is an obligate anaerobe in that the presence of even a tiny amount of free oxygen prevents its growth. Yet, C. botulinum readily grows in prepared meats like sausage. Nitrate and nitrite are OXIDIZING AGENTS that are chemically similar to oxygen. As such they, like freeoxygen, inhibit the growth of C. botulinum in foods. In addition, they prevent certain substances in meat from becoming REDUCED, which causes them to turn the meat brown, suggesting that it may be poor quality. In recent years scientists have discovered a link between nitrate/nitrite and the formation of carcinogens. As a consequence of this the FDA has required the removal where possible of these chemicals from foods or the lowering of their concentration to the minimal level. The use of nitrate/nitrite poses a classical cost/ benefit conflict. That is, is the cost (cancer) of using these substances in our food supply balanced by the protection against death by botulism poisoning? Each of us should decide that ourselves don’t you think? Sulfite (So2) and Vitamin C

Most of you have observed the “BROWNING” of fruits and vegetables; the apple, peach or banana you eat turns brown before your very eyes, even as you chow it down. Generally, people feel that “brown” food items are spoiled or at least of lower quality. The browning results from the actions of enzymes in the fruits and vegetables that rapidly react with oxygen to produce brown-colored chemicals that protect the damaged food from microbes; i.e., the brown chemical is inhibitory to many microbes. Sulfite is a powerful “REDUCING” chemical that BLOCKS THE BROWNING RESPONSE and it is inexpensive, & effective in tiny amounts. Therefore it is common to rinse fruits and vegetables in restaurants in solutions containing SULFITE. This insures that items that were prepared several hours before will remain “fresh-looking” all day long on the customer’s plates. At the concentrations used, sulfite is not toxic, but a small percentage of people are highly allergic to sulfite and an exposure to even a tiny quantity of it on lettuce etc. may be sufficient to induce a violent allergy attack. This is why restaurants often have signs telling their customers that they are using sulfite on their foods. Another powerful reducing agent that serves the same purpose is vitamin C (ascorbic acid). This vitamin also is inexpensive, is effective in small amounts, plus it is beneficial to those who ingest it. However, because it is more expensive than sulfite and it tends to decay faster, it is not universally used.

Organic Acids As you recall, all microbes require an optimum pH or acidity in their environment to grow. If there is too much acid or base, a microbe will not grow. As the by-products of many microbial fermentations include the production of chemicals like CETIC ACID (vinegar), LACTIC ACID, and PROPIONIC ACID it is not too surprising to find that humans, and other life, can actually use these substances as nutrients. However, when they are added to foods in sufficient quantities to lower the pH below that which will support the growth of most food-spoilage microbes, they can serve as natural food 144

preservatives. Again, our ancestors recognized that “SPOILED” foods such as milk and certain vegetables retained their nutrition upon becoming acidic and remained eatable (preserved) for long periods. Thus was born choice food items like yogurt, sauerkraut, pickles, cheese and buttermilk. Artificial acids, like benzoic acid, inhibit the growth of some molds, thus it is added to breads and other bakery products that require long shelf live. In many foods, like the sauerkraut you made in lab, salt is combined with acids to preserve food.

Antibiotics Most common antibiotics are inexpensive, stable, safe and effective in small quantities. With their ability to kill or inhibit many microbes, antibiotics might seem the perfect food preservative. However, all is not what it seems. Using antibiotics for food preservation is like using 100 dollar bills for toilet paper; it gets the job done but it is not the best use for that item. As you’ve learned in, we are in grave danger from infections produced by antibiotic-resistant microbes. The use of antibiotics in preserving food and in animal feeds has been demonstrated to increase the spread of antibiotic resistance between pathogens. Although some action has been taken to limit the use of antibiotics for these purposes, it is still done in many places. Radiation Atomic radiation is becoming widely used in the preservation of food, although its use remains controversial and frightening to many people. In 1997 the FDA approved radiation as a means of preserving meats. Many of the prepared meals available on the supermarket shelves at room temperature have been sterilized by radiation. Atomic radiation is lethal to all life when used in high doses. To sterilize food by this technique, the food is placed in a protected room and exposed to a high dose, usually of gamma radiation, from radioactive wastes refined from atomic power plants. A dosage that had been determined to be lethal to all microbes, including bacterial spores, is used. Current studies indicate that increased use of irradiation to destroy contaminating microbes would slightly increase the cost, but it is suggested that the increase in cost would be offset by the reduced loss of stored foods. Use of radiation to eliminate Salmonella enteritidis contamination from eggs is under consideration. A new for of radiation involving high energy electrons has been approved and foods sterilized in this way will be on the store shelves early next year. This is NOT radiation from radioactive material, but involves the use of “fast electrons”. How well people will accept foods sterilized in this way remains to be seen. FAQ 1. Does exposure to radiation make the food radioactive? Answer: No. There is no residual radiation contaminating food exposed to sterilizing doses of radiation. 2. Does the treatment produce dangerous chemicals in the radiated food?

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Answer: Although there is still some debate over this, the vast majority of the available scientific evidence does not support this contention. There is no doubt that the high radiation does induce some chemical changes in the food, but there is no proof that any of these materials are harmful. 3. Would you eat radiation treated foods? Answer: Yes. However, I consider every new technology suspect until long use proves otherwise, so I try to keep myself informed on this and other technological matters; I would advise that you do the same. 4. How commonly is radiation used to preserve foods? Answer: At least 37 nations have approved the use of ionizing radiation as a means for decontaminating more that 50 types of food. In the US radiation is regulated as a food additive.

Notes

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LESSON 23: BEER AND WINE FERMENTATION

Learning Objectives In this lecture, you will learn How beer is produced? How wine is produced?

Beer The origin of beer brewing is lost in antiquity but archaeological evidence shows that brewing was practiced in Babylon in 6000 Bc. Beer brewing was a domestic activity until in medieval Europe large scale production was concentrated first in monasteries and then in commercial breweries. As early as 10,000 B.C. Ancient Mesopotamians and Sumarians began brewing alcoholic beverages. The first documented evidence of beer making was found in Babylonia on clay tablets dating from approximately 6,000 B.C. In Ancient Rome, beer was dedicated to the goddess of corn, Ceres. Their name for beer was cerevisia, which is the derivation for brewer’s yeast, Saccharomyces cerevisiae. During the Middle Ages, monasteries began to make beer. Around this time, hops were also introduced into the brewing process. During the 20th Century, with the exception of Prohibition, beer has been largely massproduced and automated

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Beer is produced by fermentation of an extract of malted cereals. preferably barley. Aromatic herbs were often added for additional flavouring and hops have become the standard flavouring of modern beers. Cereal grains contain little fermentable sugar; the carbohydrate is largely in the form of starch which few yeasts are able to utilize. Therefore as a preliminary to fermentation. the grain is moistened to encourage germination, during which starch and proteinhydrolyzing enzymes are formed. to provide sugars and amino acids as nutrients for the embryo plants. At an appropriate stage of development the grain is heated just sufficiently to kill the embryo plants; malt kilns generally operate at around 65-80 oC. according to type of malt required. The heating process both dries the malt, to permit storage. and improves the flavor, but does not inactivate the enzyme content of the grain. Although modern technology has improved the process of malting. it is essentially the process developed 8000 years ago. And it is interesting that the complex production of fermentable material from grain was discovered so early in human history. In modern brewing the malt is ground and extracted with hot water (‘mashed’): often ground unmalted cereal is added at this stage. Since sufficient enzyme activity remains in the malt after kilning. the starch of the ‘adjunct’ grain is hydrolysed to fermentable sugar during mashing. Mashing temperatures vary, but are generally in the range 50-80cC, and the hydrolytic enzymes of malt remain sufficiently active to completed the hydrolysis of starch.

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Beer of the ale type is traditionally fermented by ‘top’ strains of S. cerevisiae, so termed because a proportion of the yeast rises to form a thick ‘yeast head’ on the surface of the fermenter. The yeast head is collected during fermentation to provide the inoculum for a following fermentation. Beer of the lager type originated in Bavaria in medieval times and has subsequently become the dominant type world-wide. Malt for lager was kilned at a lower temperature (as we now recognize, to provide greater enzyme activity) and has a lighter colour. The yeast formerly identified as a separate species (S. carlsbergcnsis) did not form a yeast head and was harvested from the bottom of the fermentation vessels, where it settled at the end of the fermentation. ‘Bottom yeast’ grows well at lower temperatures than ‘top yeast’. The main. or primary. Fermentation was customarily at 8-10°C, followed by a prolonged secondary fermentation, of up to 3 months. At 0oC which improved the flavour and stability of the beer. The sugary extract, wort, drained from the mash tun is clarified by the husk particles functioning as a filter. The mash is sparged with either one or two further batches of hot water to ensure maximum extraction of nutrients and the collected ‘sweet wort’ is boiled with hops, primarily to extract f1avour.Boiling also sterilizes the wort and inactivates enzymic activity. Again. draining off the wort is effectively a process of clarification: the precipitate of protein. tannins and phosphate produced by boiling is filtered off by the bed of hop debris and a clear ‘hopped wort’ is obtained. After cooling, the wort is inoculated with a suitable strain of yeast. Normally air is injected immediately before inoculation to improve yeast growth.

Modern techniques have blurred the differences between the two types of beer. In particular in that the mashing systems are less distinctive than formerly, and ‘bottom yeast’ is often used for

The main products of fermentation are ethanol and CO2, but small amounts of numerous by-products of yeast growth are also formed. As important contributors to the flavour and aroma of the beer. Organic acids, alcohols and esters are especially important in this respect. During fermentation the pH falls from the initial level of 5.0-5.2 to pH 3.8-4.0. The yeast population grows approximately 8-fold during the fermentation, limited partly by falling pH and rising ethanol concentration, but mainly by its inability to grow indefinitely under anaerobic conditions. Although it is customary to aerate the wort during cooling after hop-boiling, that oxygen is rapidly consumed and aeration later in the fermentation is unacceptable because of its effect on flavour. At the end of the fermentation. with no further evolution of CO2. the yeast should settle out in the nonturbulent conditions. Clarification is accelerated by chilling. but a good yeast strain will spontaneously flocculate into clumps at the end of fermentation and settle out rapidly. Further clarification by fining agents (especially the collagen protein isinglass). filtration or centrifugation is practised if necessary. The general description given above, and illustrated in Fig. 30.1. is necessarily vague. To allow for the great variation in practice

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both ale and lager. This last change was an incidental development from the introduction of enclosed fermentation vessels. in which the space required for yeast head represents a loss of useful fermentation volume. Modern cylindroconical fermentors (Fig. 30.2) generate a vigorous natural movement of the vast through the fermenting wort rising with CO2 bubbles and sinking by cooling at the walls of the vessels. The fermentation is faster. and protected from microbial

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between breweries. In particular. the methods of preparation of the two main types of beer, ale and large, are traditionally different including different malts. Hops and yeasts). but in recent years the differences have become less obvious.

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contamination and the vessels are easily equipped with automatic cleaning and sterilizing equipment.

can + the sugar. If you are lower than 3.0 kg you will have a lower alcohol beer (like 4% to 4.5%).

Nevertheless. many breweries continue to operate open rectangular vessels of the traditional form. especially for the production of cask conditioned beers.

A home brewer can always add more malt to his/her kit to bring up the weight, or substitute wholly malt for the sugar mentioned above. As will be noted in Intermediate Brewing, malt gives a beer more colour, more body, more flavour, better head-retention and more. If you’re serious about making quality beer, you already know what malt can do for you, and what sugar can’t. 1. Onward! Place the unopened can of malt extract (and any other malt) into a sink of hot water. This will soften the contents after 10 minutes. (Remove yeast packet from under the lid). 2. Meanwhile in your clean, sterilized pail, add some hot water & then the required sugar (or malt) and stir well to dissolve. 3. Now add the can of malt extract. Stir & add cold water. If adding extra hops for added bitterness/flavour add them now. Keep adding warm & cold water until you reach the 5 gallon mark. Stir it all in and add yeast if the temperature of your wort is between 65ºF & 85ºF. 4. If using a hydrometer, original gravity should be 1.035 to 1.045. 5. Cover fermenter with a plastic sheet and tie down loosely to let gasses escape but not to let dust in. Set in a constantroom- temperature place for 4 days to ferment. Day 4 (when the foam dies down) 6. Once your specific gravity has reached 1.010 or less, you can rack (syphon) your beer into the (sterilized) carboy. Without disturbing the sediment, lift pail to a counter & place the bottom of the racking tube tube near the bottom of the pail and place the other end into the carboy. Suck on the end to start the flow. Avoid splashing the beer.

Only a small amount of beer is now conditioned in cask. Where the CO2 content of the final beer is generated by a secondary fermentation. Isinglass finings. added at filling ensures a clear beer by coagulating the yeast once the fermentation-in-cask is over. Otherwise. modern practice is to perform these processes in bulk. in the brewery. under consistent conditions. All beers require a period of conditioning after fermentation; in the case of ales only a few days chilling is required to improve the flavour of the ‘green beer’ drawn from the fermentors. At the same time CO2 is injected as required and the beer is clarified y filtration and pasteurized for longer shelf life.

Basic Beer Making The first step in making beer from kits should be to throw away the instructions that come with the kit. Generally speaking, their instructions are designed to fit on a little pamphlet, and therefore may leave out important information. Most 5 imperial gallon beer kits require 1.0 to 1.2 kilograms of dextrose (corn sugar) for the fermentation part of the process, and a further 0.2 kilograms (1½ cups) for bottling. Don’t use table sugar. A homebrewed should have a total weight of fermentable ingredients around 3.0 kilograms. This will ensure a final alcohol content of around 5%. Add the weight of the

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7. Attach airlock (half filled with water) to bung and place in opening of carboy to seal. Over the next 10 days your beer will finish fermenting and clear. Top up carboy with cooled boiled water if there is excess air space (oxygen can ruin your beer at this point). 8. Set in a dark place to finish. Day 14 (Bottling) 9. Your specific gravity should be stable for three consecutive days before you attempt to bottle. If the fermentation is not complete, you’ll have either excessively carbonated beer or bursting bottles. Gravity should be stable between 1.001 and 1.006 for light beers made with sugar or 1.004 and 1.010 for beers made with malt or for dark beers (you’ll get a feel for ending gravities as you go). 10.Have a taste...it should taste like warm, flat beer (yum). This is what the bottling sugar set aside on day 1 is for. Dissolve the sugar in 1 cup of water & put into an empty fermenter. Syphon the beer into the fermenter & stir well to ensure even carbonation in all bottles. 11.Assemble 48 x 500ml plastic bottles or 66 x 341ml glass bottles & fill up to about 1" from the top. Cap them & place in a warm place for 7 days. After that, they should be carbonated, and you can then move them to a cooler place if

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12.Put on your next batch, because running out would be a shame. In Beer the Ethanol content typically is 3.6 to 4.2%. Reinheitsgebot (Purity Laws) of Bavaria, 1516 prohibited use of brewing ingredients except malted barley, water, and hops (note that yeast was not included – the existence of yeast and its necessity for the fermentation process was not yet understood). The beer brewing process requires cool temperatures – thus restricted to autumn, winter and fall in temperate regions, and is not attainable in tropics, unless mechanical cooling is provided. A. Types of Beer

Top-fermentation – “ales” – produced by yeast (Saccharomyces cerevisiae,) which grows at the top of the fermentation vessel Bottom-fermentation – “lager beers” – produced by yeast (Saccharomyces carlsbergensis) which grows at the bottom of the fermentation vessel B. Steps in Brewing

1. Malted (germinated) grain is crushed to form a coarse flour (grist) 2. Warm water is added to grist to form the mash. Malt enzymes solubilize the endosperm of the malt. 3. Aqueous extract (wort) is separated from the solids. 4. The wort is boiled with hops (flowers of Humulus lupulus, used for flavor and aroma, and also as a preservative). 5. The wort is clarified, cooled and aerated in preparation for yeast growth. 6. The wort is fermented with yeast to convert grain carbohydrate to alcohol and carbon dioxide. 7. The beer is matured and clarified. 8. The beer is sterilized (by filtration or pasteurization) and packaged. C. Classification of beers

1. Ales – produced by top-fermentation yeasts a. pale ales – made from pale malts (heavy addition of hops) b. bitter – pale ales on draft c. brown ales – made from darker malts, usually sweeter and with less use of hops than in pale ales d. stout – dark ales; may be bitter or sweet 2. Lagers – fermented with bottom yeast a. pale (e.g. Pilsener) – made from pale malts, distinctive hop flavor b. dark – made from darker malts c. Bock, Märzen – strong beers, made at certain times of year d. e.

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Weizenbier, weissbier – made from mixture of malted barley and malted wheat, without hops African native beers – made from malted sorghum, millet; worts not boiled; no addition of hops. Served unclarified.

Intermediate Beer Making Intermediate Brewing: Enhancing Beer Kits Intermediate Brewing: Using your own malt & hops Grain Description Chart Hop List

Enhancing Beer Kits To add grains to a kit, crack them (use a grain mill if possible or we’ll crack them here for you) first. Don’t pulverize them to dust, but separate the husks. An easy way to use grains with a kit is to add the crushed grain to a Pyrex container (250 grams to 500 grams of grain is sufficient) and add 100mls of boiling water for each 100 grams of grain. Let sit 15 minutes, strain, and add the grain ‘juice’ to your primary fermenter. To add (extra) hops to a kit, boil and cool 3 gallons of water the night before and refrigerate. This will be used to reduce the temperature of your boiled wort. 1. Place a pot with 2 gallons of cold water on the stove. Add any crushed grains now (contained in a muslin bag). 2. Steep any specialty grains as your water comes to (but does not reach) a boil. Remove at about 170ºF and rinse grains with a couple cups of water over the pot to remove all the goodness. 3. Boil up the 2 gallons of water. In a sink, immerse malt extract in hot water to soften contents. When softened (3 minutes) add to boiling water. 4. Stir in malt well to dissolve. 5. Boil for up to 45 minutes. Don’t let it boil over!!! Add flavouring hops during the last 5 minutes of the boil. Use chart below for how much to use. 6. After the boil, remove hops, or let them settle out in the pot. Take care to not transfer to the primary. Carefully pour all the wort into the primary - be careful - it’s very hot! 7. Add the reserved cold water & top up to the 23L mark. Add yeast. Flavour & Aroma Amount of hops Low to Medium 1/4 oz pellets (low alpha %) Medium 1/2 oz pellets High 1/2 oz to 1 oz (higher %) Using your Own Malt & Hops Malt and hops are the only two things in a beer kit. You can essentially make your own kit by putting the two together yourself. Follow steps 1 through 5 above. When boiling, add bittering hops (as per recipe). Usually leaf hops are better (fresher) to use - pellets work fine too. Usual boil time is 45 minutes. If you can, contain all leaf hops in a muslin bag for ease of removal. 1. Add flavouring hops (as per recipe). Usual boil time is 15 minutes. 2. During last 5 minutes add aroma hops (see chart above). 3. Once boil is over, and hops removed, carefully (it’s hot!) pour liquid into primary fermenter & add reserve cold water. Add yeast if temperature is less than 80ºF

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you wish, to condition & age. Beer is usually ready to drink after 14 to 21 days in the bottle, but will improve for 6 to 9 months.

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Grain Description Chart

Most yeast strains are very viable and usually will ferment as long as the temperature of the wort/beer or wine/must is at least 70 to 75 degrees.

Choosing Malts Lager Style Lightest in colour - use 2.8 to 3.5 Kg Amber Malt Reddish (good for bitters) - use 2.8 to 3.5 Kg Dark Malt Dark (black beers; stouts etc.) - use 3.0+ Kg

•

This is the measurement of the beer or wine before fermentation starts and tells you the potential alcohol you will end up with.

Choosing Specialty Grain • Crystal Grain

Malt Reddish colour; Increases residual sweetness, mouthmost popular feel, & head retention.

Pilsner Grain Light - best used for mashing. Pale Malt Light - use for all grain beers. Lager Malt Light - use for allgrain lagers Chocolate Malt

Nutty, coffee-like flavour. Dark!

Use as base grain or up to 400g as addition to kit or malt base.

Using a Hydrometer Hydrometers measure sugar content, and can therefore be used to determine the progress of your wine or beer’s fermentation. Since the sugar ferments directly into alcohol, you will also be able to determine the alcohol content of the wine. Winemakers need to know 1. when to add the final stabilizing packages 2. (when the Specific Gravity is as low as it will go - between 0.990 and 0.999) and 3. How dry your wine is (the closer to 0.990, the drier the wine). Beermakers use the hydrometer to make sure fermentation is complete before bottling their beer (to prevent excess carbonation). Beer kits made with sugar will usually ferment down to 1.006 or lower Beer kits made with malt may only reach 1.010. Fuller bodied (and darker) beers may have a higher terminal gravity - and usually higher starting gravity too. To read a hydrometer, place it in solution and read the marking at eye level - where the liquid crosses the line. Hydrometers are accurate at 60ºF. If the mix temperature is 50º, subtract 0.001 from your reading, if 70º, add 0.001 to your reading. To determine alcohol %, subtract your Ending reading from the Original & multiply the result by 131.25 Beermaking Frequently Asked Questions

How hard is it to make beer? If you can boil one gallon of water, you can make great tasting beer. We have provided a simple to follow step by step that will show you how easy it is. •

How do I know if my yeast died?

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•

What is priming sugar? This is the sugar that is added to the wort right before bottling which will carbonate the beer in the bottle. Priming sugar can be corn sugar or dry malt extract.

Use sparingly in beers other than stouts & brown ales.

French roast coffee. In small amounts can give reddish hue.

How do I know if my fermentation stopped too soon? Unless you take a hydrometer reading at the beginning of the fermentation which is called your Starting/Specific Gravity you will not know if your beer or wine is fermenting. As long as the numbers on the hydrometer are dropping fermentation is taking place even if the airlock is not bubbling actively.

Similar to pale malt - use as grain base in all-grain pilsners. Use as grain base in ales.

Black Patent Very crisp; burnt. Great with stouts. Use on black Malt Charcoal colour beers unless dark colour desired. Roasted Barley

What is Specific or Starting gravity?

•

Five gallons of beer seems like a lot of beer. How many bottles is it? Five gallons makes approximately 2 cases (48 bottles) of beer. Believe me when your friends and family taste your homebrew they will drink it up before you do.

Wine Fermentaiton The word wine without qualification specifically means fermented grape juice, but other fruits are also used for wine production. Wine fermentations may be ‘natural’ or ‘artificial’ according to whether the natural yeast flora or an artificially grown culture yeast is used. Natural wine fermentation is now largely confined to European wine-producing areas. Particularly France. Grapes develop a microbial flora during development, and on pressing. these organisms inoculate the juice. Micro-organisms of the pressing equipment and other sources in the winery also contribute an inoculum to the juice. The juice or must. is first treated with sufficient sulphite to eliminate undesirable yeasts. moulds and bacteria, but not so much as to harm the fermentation yeasts which fortunately are more resistant to SO2or sulphite. During the fermentation a succession of yeasts develops: it is rare for only one strain of yeast to occur. Species of Saccharomyces including S. cerevisiae are involved, but also various species of other yeast genera, for example Kloeckera. Kluyveromyes.Torulaspora and Zygosaccharomyces. Each yeast contributes its own spectrum of flavour compounds. and as the yeasts involved vary from year to year. so too do the flavour and aroma (‘bouquet’) of the wine. The characteristics of the wine are due partly to the grapes. and partly to the yeast. Although the grape variety (or varieties) used is constant. the climate each year affects sugar content. acidity. etc. and so influences the flavour of each year’s production. It is possible on rare occasions that the yeast flora introduced naturally is unsuitable, in which case a specially grown culture of wine yeast is added but the wines which result are generally judged to be of poorer quality.

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Pasteurization of grape juice is used in some areas for production of cheaper wines. but is generally avoided because of its effect on flavour. Wine produced by ‘artificial’ fermentation is of consistent flavour from year to year, apart from the minor variations caused by climatic effects on the grapes themselves; Table 30 indicates the most important properties of wine yeasts for ‘artificial’ fermentations. or for rescuing ‘natural’ fermentations in difficulty. Table 30.Important properties of wine yeast The tannins and pigments of grape skins are important. 1. High ethanol production (upto 15% v/v for some wines) 2. Resistance to high sugar concentrations (upto 30% w/v) 3. Resistance to sulphite 4. Resistance to tannin (especially for red wines) 5. Resistance to ethanol (capability for growth in moderate ethanol) 6. Wide temperature range for growth (e.g. 4-32 oC) 7. Low production of volatile acidity (measured as acetic acid) 8. Good flavour compounds 9. Fermentation under pressure 10.Firm deposit in contact with the fermenting juice. Grape skin pigments are ethanol-soluble, and extracted as fermentation progresses. Also tannins are extracted. conferring an unpleasant bitterness in young red wine that mellows by chemical reaction and precipitation as the wine ages. White wines are produced from either green or black grapes. but the skins must be removed before commencement of fermentation. Therefore white wines have lower tannin or polyphenol content than red wines. Which may affect their stability in certain circumstances (for example low ethanol or sugar content) unless protected by SO2 or other presentable?

intervals until vertical. stopper downwards. and all of the yeast cells produced during the secondary fermentation has collected on the cork. The yeast is removed by freezing the neck of the bottle to provide a temporary plug of ice: the cork with attached yeast is removed and a new. (Warning: the high pressure makes this a dangerous process, NOT to be attempted by amateurs.) This is a labour-intensive and therefore expensive process. and in some areas a cheaper sparkling wine is produced by carrying out the secondary fermentation in stainless steel tanks. After the yeast has settled out, the clear wine is bottled against a counterpressure of CO2, Note that in both types of sparkling wine the CO2 is dissolved over a long period of secondary fermentation. A cheap, but inferior, sparkling wine can also be produced by injection of CO2, as in the manufacture of aerated soft drinks. Such a product is recognizable by the rapid loss of CO2 from the wine in the glass. A different type of secondary fermentation is involved in the production of sherry wines. In the Jerez region of Spain, which by mispronunciation has provided the name Sherry? the wine is matured in warehouses (bodegas) in a solera system, in effect a stack of wine casks. After drawing off half of the content of the lowest (oldest) cask for bottling. that task is topped-up from the level immediately above. Repetition of this process at higher levels leaves space in the highest (youngest) cask for new wine. The light colour and dry flavour of Fino Sherry is due to the film (flor) of surface yeast growth, Various species of filmforming yeasts may be involved, most commonly Saccharomyces cerevisae,Torulaspora fermentati and Zygoaccharomyces rouxii. rouxii.

The progress of the wine fermentation is essentially the same as the beer fermentation, but normally over a longer time and producing higher concentrations of ethanol, typically 10-12% Post fermentation treatments are widely, according to the locality and type of wine. but normally prolonged storage in cask is necessary for satisfactory clarification and maturation before bottling.

Secondary Fermentation Production of Champagne and sherry wines involves a secondary fermentation during the maturation process. In the ‘Methode Champenoise’, CO2 is dissolved under pressure by secondary fermentation in the bottles. Therefore strong bottles are required, capable of withstanding the 6 bar pressure that can develop. Bottles are filled with new wine containing 1 % fermentable sugar. Unfermented grape juice is added if necessary to provide the necessary sugar. The yeast remaining in the wine continues the fermentation in the sealed bottles. and over a period of many months the bottles are rearranged at 2.521

Saccharomyces cerevisae (Saccharomyces cerevisiae is commonly known as “bakers’ yeast” or “brewers’ yeast.” The yeast gains

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However, it is standard practice in most wine-producing areas to use a pure yeast culture or a mixture of pure cultures for all fermentations. All microbial flora of the grape juice are eliminated by addition of greater amounts of SO2 or sulphite.

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energy from the fermentation of carbohydrates. The fermentation of beer and wine was originally caused by naturally occurring yeasts present in the environment. Some wineries continue to use natural yeast strains, including Saccharomyces ellipsoideus. However, most modern brewers use highly cultured isolates. One yeast cell can ferment approximately its own weight of glucose per hour, giving rise to large volumes of CO2. Fermentation of glucose procedes by the following reaction: C6H12O6 + H2O -> CO2 + CH3CH2OH or Glucose + Water ->Carbon Dioxide + ethanol, and they produce palatable These yeasts are capable of growing in the high ethanol concentrations (over 12%) of the new wine and increasing the level to approx, 18%, producing not only ethanol but also acetaldehyde and glycerol as particularly important flavour compounds. The yeast film prevents access of air to the wine which therefore remains unoxidized. and light in colour. The darker. sweeter. sherries are also matured in a solera system. but the primary fermentation is stopped at an appropriate level of residual sugar by addition of sufficient brandy to inhibit further yeast growth. During maturation the reaction with air. both at the wine surface and through the wood of the cask. develops a darker colour and oxidized flavour.) Yet another type of secondary fermentation is of value in the wine industry to reduce the acidity of wines. However. the malolactic fermentation is not strictly a secondary fermentation. since it may occur either during the primary fermentation or during maturation. The conversion of malic (with two carboxyl groups per molecule) to lactic acid (with only one carboxyl group) electively halves the acidity. and is often a desirable byproduct of ‘contamination’by Leuconostoc oenos, the ususal bacterium involved. Deliberate inoculation of L.oenos is shunned by purists, but is done in some wine-producing areas, either late in the primary fermentation or in the early stages of maturation. Many fermentable fruit juices other than grape are also used in the production of wine. and normally the process is similar to grape wine production in all aspects. Cider and perry. from apples and pears respectively, also are produced by a similar process and both naturally and artificially inoculated products exist, as with wine production. In a few parts of the world the causative organism of natural alcohol fermentation is the bacterium Zymomonas. rather than a yeast. Although the metabolic route is different (the Entner-Doudoroff pathway in Zymomonas) the main end products are ethanol and CO2, as with yeast. However. the different range of minor byproducts of fermentation causes Zymomonas to be regarded as a troublesome source of off-flavour in the beer. cider and wine industries.

Basic Wine Making from Kits Most wine kits come complete with all the packages and instructions you need to make 30 bottles of wine. Since they’re

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all made differently, it’s best to follow the manufacturer’s instructions exactly. Below is a set of winemaking instructions from a Vintner’s Reserve (one of the more popular brands on the market) wine kit: Caution: These instructions are here for illustrative purposes & you should follow the manufacturer’s instructions for the kit that you have. 1. Empty contents of foil bag of concentrate into your sterilized primary fermenting pail. Rinse bag by adding approximately 5 liters of warm water to the bag & empty that into the pail as well. 2. If your kit contains oak chips, add them to a cup of boiling water & stir. Pour the oak solution into primary fermenter and stir. 3. The temperature should be between 65ºF & 75ºF. If it is, add yeast. 4. Cover primary fermenter and place in suitable area to maintain fermentation temperature of 65º to 75º for the next few days. Fermentation should start within the next 24 to 48 hours. Stage #2 - Secondary Fermentation

5. Once Specific Gravity has reached 1.010 or less (approx. 5-7 days) the wine is ready for transferring. Note that at lower fermentation temperatures, it may take longer to reach the target gravity. 6. Carefully syphon the wine into a clean and sanitized carboy leaving all the sediment behind. 7. Attach airlock and bung to seal the carboy. 8. Leave carboy at fermentation temperature for a further 10-12 days to finish. Stage #3 - Stabilizing

9. After the 10-12 days are over, check that specific gravity is 0.996 or less. If not, allow a few more days, and then repeat this step. 10.Dissolve Pkg #2 (Potassium Metabisulphite) and Pkg #3 (Stabilizer) in approximately 125 ml of wine extracted from the carboy. Add this back to the carboy and stir vigorously for 2 to 3 minutes 11.Shake contents of pouch 4a (Clearing Agent). Pour into carboy. Stir vigorously for 2-3 minutes. Wait 24 hours. 12.After 24 hours, shake contents of liquid pouch 4b (Chitosan - clearing agent). Pour into carboy. Stir vigorously for 2-3 minutes. Top up carboy with sterile water to within 2-5 inches from the neck. Ensure airlock is adequately filled with water and bung secured. Allow 8 days to clear.

Stage #4 - Clarification & Bottling 13.After the 8 days, your wine should be relatively clear, but it should be racked once more to polish the wine. Syphon into a clean, sanitized carboy. 14.Allow more time to clear if necessary. Top up with sterile water to eliminate airspace. If your wine is clear, you can bottle your wine at this point instead, into sterilized bottles.

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Fresh Fruit Wines You must follow a recipe for fruit wines. If you require a recipe, request it by emailing me at We sell many fruit bases, which are canned with real fruit mixed into a grape concentrate base. This may be an alternative, especially out of season. Most fruits and vegetables can easily be made into wine. You can follow the general instructions below for the process: 1. Use only clean, sound fruit. Remove any stems, leaves, pits, dirt, etc & then crush the fruit & put into your primary fermenter. 2. Instructions usually call for a “Campden Tablet, crushed”, per gallon. Adding this prior to fermentation will help sterilize the fruit. It’s a good idea to add the crushed campden tablet to the fruit (in your primary), and wait 12 hours before adding the rest of the ingredients. 3. After the 12 hours, stir the fruit-campden mixture well (to release any remaining SO2 gas that the Campden Tablet may have left behind). Add all remaining ingredients required except the yeast (consult recipe) & dissolve into the water that is needed (i.e. if making 4 liters, usually 3-4 liters of water will be required). The recipe usually lets you know how much you’ll need. 4. Add yeast & place fermenter in warm spot for 7 to 9 days. Cover with a tied-down plastic sheet or lid with airlock. Periodically, you should “punch the cap” (submerge the pulp that floats on top back into the wine). 5. Once your specific gravity has reached 1.010 or less (usually 7 days), rack the wine into a carboy (or gallon jugs) and fit with an airlock (half-filled with water). Leave to finish fermenting and clear for about 3 weeks. 6. Syphon the clearing wine after the 3 weeks into another empty carboy and add 1 to 2 crushed campden tablets per gallon to the carboy. Let sit for 4 to 6 weeks to clear further. You may add a clearing agent (such as Claro KC, or Isinglass) if you wish. 7. When clear (you may wish to filter it) bottle the wine but make sure you add 1 crushed, dissolved campden tablet (again) prior to filling your bottles. It may be easier to syphon/filter the clear wine into an empty pail first. Note: if you wish to sweeten your fruit wine POTASSIUM SORBATE (1/2 tsp per gallon) MUST be added prior to bottling, otherwise fermentation will start up again in the bottles. In fact, it’s not a bad idea to use potassium sorbate in your fruit wines, as a preventative measure.

2. Once specific gravity has reached 1.010 or lower, syphon into carboy & top up with boiled cool water if necessary. Leave in warm place to finish fermenting. 3. Once gravity has reached 0.996 or lower, you can move carboy to cool place for storage & clearing. It’s best to rack your wine into a clean fermenter every 6 weeks (add 1 crushed-dissolved campden tablet per gallon at this time for preservation). It’s recommended to take overall about 3 to 4 months to make your juice wine. 4. You may add a clearing agent 2 weeks prior to your planned bottling / filtering date. You may filter the wine prior to bottling, but remember to add the campden tablets again. 5. Age whites in bottles at least 4 months before sampling and reds over 6 months is recommended. Try the wine periodically to gain a sense for how juice wines age... it’s the fun part of the learning process.

Winemaking Faqs • Is wine made with juice or concentrate as “real” as wine made with grapes? Yes! During September and October, when grapes are available, fresh-pressed juice is also available. These high quality varietal grape juices make excellent wine with a plus being that they are already balanced for acid and sugar and a serious amount of work has been removed by not having to crush, destem and press the grapes. Which also means it’s not necessary to have such large equipment such as a grape crusher and grape press. Concentrates are available 365 days per year and come in many varietal styles. The most common and easy to use types of concentrates come as 28 day or 4 week wine kits. Most of these wine kits produce 6 gallons each, are very easy to use and produce an excellent wine. Many of our new winemaking customers are shocked and pleasantly surprised to find out what a good wine these kits can produce.

Wines From Fresh Grape Juice Basic Procedure

1. Empty contents of juice pail into your primary fermenter. Add yeast & cover with plastic sheet tied down, or with lid & airlock. Let sit for 7 days in a warm place to ferment.

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I know these instructions may be a bit vague, but that’s because most kits are that easy to make and are made differently from each other. If you would like me to expand on any particular part of the process, email me here:

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LESSON 24: ETHANOL FERMENTATION

Learning Objectives

Brazilians nave been using sugarcane molasses and cane, juice as raw materials for the production of ethanol.

In this lecture, you will learn •

What is ethanol fermentation? Conventional and nonconventional methods of ethanol fermentation.

The French companies have been producing ethanol by using sugar beets as raw materials.

•

Cellulose based production of ethanol

•

Countercurrent analysis method of ethanol fermentations

Japan has been producing ethanol by using cassava, yams and other agricultural products.

•

What is ethanol and what are its uses?

India, Australia and the USA use sugarcane molasses or sugar beet molasses for the production of ethanol.

Ethanol -otherwise known as ethyl alcohol, alcohol, grain spirit, or neutral spirit -is a clear, colorless, flammable oxygenated fuel.

Grains of cereals like maize, com and sweet sorghum are also used as raw materials for the production of ethanol. •

What are the different microorganisms useful for the production of ethanol? Zymomonas mobilis has a strong tendency to produce ethanol by consuming carbohydrates (simple sugars). The fermentation potential of Z. mobilis is two times more than that of yeast. Thermoaerobacter ethanolicus also produces enough amount of ethanol during fermentation.The species of Monilia and Fusarium produce ethanol directly from carbohydrate wastes.Clostridium thermocellum, C. thermohydrosulphuricum and C. thermosaccharolyticum degrade the complex polysaccharides into simple sugars. Even though they do not convert them into Ethanol, they play an important role, because they produce simple sugars. These simple sugars form a substrate for the action of other organisms which convert the simple sugars into ethanol.

Uses It is mixed with gasoline to create ethanol/gasoline blends at volume levels of 5.7%, 7.7%, and 10%. Ethanol not only is used to increase octane and improve the emissions quality of gasoline but also is used as an alternative fuel to replace gasoline.

The simple sugars produced by the action of Clostridium are fermented by Zygomonas mobilis and Thermoanerobacter this fermentation results in the formation of ethanol.

It is used in the manufacture of ethyl tertiary butyl ether (ETBE), an environmentally friendly substitute of the hazardous methyl tertiary butyl ether (MTBE) derivative. ETBE and MTBE are fuel additives developed primarily to reduce tailpipe pollution. Unfortunately, MTBE has been found to pollute waterways.

Status Most of the world’s ethanol is produced by fermentation of crop biomass (93%) with synthetic ethanol production from crude oil and natural gas at 7%. The UK is the world’s largest producer of synthetic ethanol. Recent advances in biotechnology enable genetically engineered bacteria to produce ethanol from waste biomass. • How is ethanol produced? Ethanol is produced by using a wide variety of substrates or raw materials. The utilization of correct raw materials depends on the availability of the materials and the cost of the materials. But the raw materials must contain a high proportion of carbohydrates for the production of ethanol. Ethanol is also known as gasohol. 154

Genetically engineered yeast play an important role in the’ production of ethanol; they directly convert the wastes into ethanol. •

How is ethanol produced on an industrial scale?

The major steps involved in the industrial production of ethanol are:

1. Formulation of Medium The preparation of the medium is the first step of ethanol production. For this purpose, the sugar concentration of cane molasses and of other carbohydrates in the medium is diluted to 10 - 18%. This sugar concentration favours the growth of the microorganisms. Ammonium sulphate or ammonium phosphate is added to the diluted medium. The pH of the medium is adjusted to 4 – 5 by using dilute sulphuric acid. Sometimes lactic acid bacteria are inoculated into the medium when the medium has high pH value. The lactic acid bacteria grow well and, initiate the production of alcohol. Other microbial contaminants’ should be avoided during the

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2. Structure of a Fermentation System: The fermentation system consists of three molasses tanks, three seed tanks, a fermentor and a wash chamber on of the three molasses tanks, one is larger in volume and is loaded with ample molasses; it named molasses storage tank. The stored molasses enter another tank where the, molasses are diluted properly with-water. The diluted molasses then enter the sterilization tank where the diluted molasses are sterilized clearly for the production of ethanol. The sterilized medium then enters both the fermentor and the seed tanks through pipelines. The seed tanks, are rather smaller vessels which are interconnected by pipeline through which the microbes and the medium flow from one tank to the others. These tanks participate in the production of enough amount of microbial inoculum for the production of ethanol. A fermentor is a large vessel where sugar is converted into ethanol by the action of microorganisms. The fermentor is connected with 5 pipelines: 1. The first pipeline is concerned with the supply of enough amount of sterilized medium into the fermentor.2. The second pipeline is concerned with the supply of enough amount of inoculum into the fermentor. . 3. The third pipeline participates in the addition of certain chemicals into the fermentor 4. The fourth pipeline is concerned with the supply of cooling water to the fermentor for helping to maintain proper temperature inside the fermentor. 5. The fifth pipeline is used to harvest the spent medium from the fermentor. The spent medium is then transferred to a wash chamber where it is processed for extract ethanol from it. The actual process of ethanol production can be briefly summerised as follows: The ethanol-producing microbes are inoculated into the fermentor containing full of nutrient medium and chemicals. These microbes convert the carbohydrates present in the medium into ethanol and carbon dioxide which is release from the fermentor. They require 2 - 3 days to produce 80% ethanol in the medium. After a sufficient period of incubation, the spent medium is transferred to a wash chamber where it is processed (distilled) to extract 96% of ethanol. It must be borne in mind that that this is the conventional method of ethanol production. Modern production techniques are based on advanced continuous fermentation. • ·hat are the drawbacks of ethanol production? Ethanol is highly toxic to the microorganisms like bacteria; it sterilizes the bacteria and reduces their biomass. This is true, 2.521

but some organisms resisting ethanol are used for the extraction of ethanol. Carbohydrates are very costly and so the produced ethanol must fetch a high price. This problem can be solved by using wastes as substrate for the production of ethanol. The isolation and purification of ethanol is mainly effected by distillation which needs some amount of energy. So there is wastage of energy during the manufacture of ethanol. •

What are the various applications of ethanol? Ethanol is an active solvent of dyes, lubricants, adhesives, a few detergents, some pesticides, paints, explosives and resins. It is also used as an organic solvent for the extraction of some organic compounds from living things. Ethanol is used in the manufacture of synthetic rubber.It is used in the manufacture of synthetic fibres like rayon, polyester, etc.It is used in the extraction of certain pharmaceutical products.It is used in the manufacture of acetaldehyde.It is used in the manufacture of perfumes.Ethanol is used as fuel in internal combustion engines either, in the form of anhydrous ethanol (98.S% of ethanol) or mixed with Petrol or in the form of hydrated ethanol. The yield of energy will be very high when it is used along with petrol. In most cases, 15% or 10-20% of ethanol is mixed with petrol for fuel purpose in engines and in chemical industries. The energy contents of ethanol are 19 MJ/t.

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In Brazil nearly one-third of cars are running by consuming ethanol as fuel. Brazil produces above 66 million hectoliter of ethanol per year for its need.

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Americans use ethanol along with petrol for running their cars. They are marketing petrol along with ethanol. This country produces 55 billion liters of ethanol per year.

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India produces nearly 10 million hectoliter’s of ethanol per year.

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Can we produce ethanol from carbohydrate based waste? We certainly can. Let us study the following case.In order to produce ethanol from biomass and waste, two stages are required:

1. Hydrolysis to break the material down into simple sugar molecules 2. Fermentation to produce ethanol

Feedstock Composition Feedstock may include purpose-grown crops (including maize and corn), crop waste, paper mill sludge, forest residues and household waste (including sewage). These are mostly lignocellulosic materials containing cellulose, hemicellulose and lignin. Cellulose and hemicellulose are long chain polymers that make up the bulk of plant material, and lignin is the chemical “glue” that holds them together.

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industrial production of ethanol. The starchy media like corn, rye and barley are hydrolysed with dilute acids before they are pumped into the fermentor. The hydrolysis of starch yield simple sugars which are directly converted into ethanol. Sometimes starchy feed stock is treated with amylase enzyme, extracted from Aspergillus and Rhizopus. Amylase converts the starch into 80% maltose and 20% dextrine.

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Hydrolysis This process breaks the long cellulose and hemicellulose chains into simple sugars. Cellulose yields primarily glucose (a sixcarbon sugar) whereas hemicellulose, in the region of 20% of the material, gives a mixture including several five-carbon sugars. Methods of hydrolysis include using enzymes and using dilute or concentrated acids. Whereas in the past hydrochloric or hydrofluoric acid may have been used, sulphuric acid is found in newer processes.

Sugar Separation After hydrolysis, the sugar for fermentation must be separated from the acid. A new process developed by the company Arkenol in the United States, which is still at the pilot stage, makes use of ion exchange to improve the separation, allowing a greater proportion of the acid to be concentrated and re-used. Final traces of acid are precipitated as “gypsum” (calcium sulphate) by addition of lime.

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Fermentation Fermentation is a complex series of reactions, which convert carbohydrates, mainly sugars and starches, into ethanol and carbon dioxide. Several enzymes, such as zymase in yeasts, catalyse these reactions. Yeast is a living organism, and these are the products of anaerobic respiration.

Conditions Fermentation with yeast works best at temperatures in the range 25 - 37°C, in the absence of oxygen (anaerobic) and will produce aqueous solutions of up to 14% ethanol. Below 25°C the reaction rate is too slow, but at higher temperatures the enzymes start to denature and lose efficiency. If oxygen is present, aerobic respiration will occur producing carboxylic acids, in this case acetic acid (vinegar). The toxicity of ethanol to the organisms used limits the ethanol concentration possible. • What are the benefits of Arkenol process? Conventional yeasts cannot make use of five-carbon sugars that arise from the hydrolysis of hemicellulose. Conventional methods for ethanol fermentation do not utilize this resource, which may count as 20% of the feedstock. The process developed by Arkenol uses specially bred yeast (not genetically engineered) that feeds preferentially on C5 sugars, as well as on C6 sugars. In this way, a greater proportion of the feed is utilized. Another approach is to use genetically engineered bacteria.

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Can ethanol be produced from cellulose based feedstock ? How? The production of ethanol from corn is a mature technology that is not likely to see significant reductions in production costs. Substantial cost reductions may be possible, however, if cellulose-based feedstocks are used instead of corn. Producers are experimenting with units equipped to convert cellulose-based feedstocks, using sulfuric acid to break down cellulose and hemicellulose into fermentable sugar. Although the process is expensive at present, advances in biotechnology could decrease conversion costs substantially. The cost of producing ethanol could be reduced by as much as 60 cents per gallon by 2015.

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The two most common methods to increase the oxygen level of gasoline are blending with MTBE and blending with ethanol. Because ethanol has higher oxygen content than MTBE, only about half the volume is required to produce the same oxygen level in gasoline.

Interesting. Tell me more about this. Yeast is very good at converting glucose, and other sixcarbon sugars into ethanol. Unfortunately, a significant proportion of waste biomass consists of complex natural polymers made from sugars that are not “digested” readily by yeast enzymes.These include hemicellulose, which on hydrolysis produces a range of sugars including: mannose, xylose, arabinose and galactose, depending on the original source.

Genetically engineered Bacteria A genetically modified bacterium, developed by the microbiologist Lonnie Ingram in 1987, has enabled these sugars to be converted to ethanol. The bacterium, referred to as KO11, would normally produce acids, but the modification means ethanol is produced instead. The advantage over yeast is that a wider range of sugars can be processed, enabling the utilization of biomass waste such as wood waste, corn stalks, rice hulls, and other organic waste, which would otherwise require disposal by some other method, or which could only be partially utilized by conventional fermentation methods, making them uneconomic.

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What is the process like? Ethanol is produced from the fermentation of sugar by enzymes produced from specific varieties of yeast. The five major sugars are the five-carbon xylose and arabinose and the six-carbon glucose, galactose, and mannose. Traditional fermentation processes rely on yeasts that convert six-carbon sugars to ethanol. Glucose, the preferred form of sugar for fermentation, is contained in both carbohydrates and cellulose. Because carbohydrates are easier than cellulose to convert to glucose, the majority of ethanol currently produced in the United States is made from corn, which produces large quantities of carbohydrates. Also, the organisms and enzymes for carbohydrate conversion and glucose fermentation on a commercial scale are readily available.

The conversion of cellulosic biomass to ethanol parallels the corn conversion process. The cellulose must first be converted to sugars by hydrolysis and then fermented to produce ethanol. Cellulosic feedstocks (composed of cellulose and hemicellulose) are more difficult to convert to sugar than are carbohydrates. Two common methods for converting cellulose to sugar are: 1. Dilute acid hydrolysis and 2. Concentrated acid hydrolysis, Both these processes include use sulfuric acid. Dilute acid hydrolysis occurs in two stages to take advantage of the differences between hemicellulose and cellulose: The first stage is performed at low temperature to maximize the yield from the hemicellulose, and The second, higher temperature stage is optimized for hydrolysis of the cellulose portion of the feedstock. Concentrated acid hydrolysis uses a dilute acid pretreatment to separate the hemicellulose and cellulose. The biomass is then dried before the addition of the concentrated sulfuric acid. Water is added to dilute the acid and then heated to release the sugars, producing a gel that can be separated from residual solids. Column chromatographic is used to separate the acid from the sugars.

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What are the drawbacks of this process? Dilute acid hydrolysis of cellulose tends to yield a large amount of byproducts. Concentrated acid hydrolysis forms fewer byproducts, but for economic reasons the acid must be recycled.The separation and reconcentration of the sulfuric acid adds more complexity to the process. In addition, sulfuric acid is highly corrosive and difficult to handle. The concentrated and dilute sulfuric acid processes are performed at high temperatures (100 and 220oC) which can degrade the sugars, reducing the carbon source and ultimately lowering the ethanol yield. Thus, the concentrated acid process has a smaller potential for cost reductions from process improvements. What is the alternative process then?

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It is called countercurrent hydrolysis. This is a two stage process: In the first stage, cellulose feedstock is introduced to a horizontal co-current reactor with a conveyor. Steam is added to raise the temperature to 180oC (no acid is added at this point). After a residence time of about 8 minutes, during which some 60 percent of the hemicellulose is hydrolyzed, the feed exits the reactor. It then enters the second stage through a vertical reactor operated at 225oC. Very dilute sulfuric acid is added to the feed at this stage, where virtually all of the remaining hemicellulose and, depending on the residence time, anywhere from 60 percent to all of the cellulose is hydrolyzed. The countercurrent hydrolysis process offers more potential for cost reductions than the dilute sulfuric acid process. Its is estimated that this process may allow an increase in glucose yields to 84 percent, an increase in fermentation temperature to 55oC, and an increase in fermentation yield of ethanol to 95 percent, with potential cumulative production cost savings of about 33 cents per gallon. The use of cellulosic biomass in the production of ethanol also has environmental benefits. Converting cellulose to ethanol increases the net energy balance of ethanol compared to converting corn to ethanol. The net energy balance is calculated by subtracting the energy required to produce a gallon of ethanol from the energy contained in a gallon of ethanol (approximately 76,000 Btu). Corn-based ethanol has a net energy balance of 20,000 to 25,000 Btu per gallon, whereas cellulosic ethanol has a net energy balance of more than 60,000 Btu per gallon. In addition, cellulosic ethanol use can reduce greenhouse gas emissions. Argonne National Laboratory estimates that a 2-percent reduction in greenhouse gas emissions per vehicle mile traveled is achieved when corn-based ethanol is used in gasohol (E10), and that a 24- to 26-percent reduction is achieved when it is used in E85. Cellulosic ethanol can produce an 8- to 10percent reduction in greenhouse gas emissions when used in E10 and a 68- to 91-percent reduction when used in E85. •

In American context, ethanol has enjoyed some success as a renewable fuel, primarily as a gasoline volume extender and also as an oxygenate for high-oxygen fuels, an oxygenate in RFG in some markets, and potentially as a fuel in flexiblefuel vehicles. A large part of its success has been the Federal ethanol subsidy. The future of ethanol may depend on whether it can compete with crude oil on its own merits. Ethanol costs could be reduced dramatically if efforts to produce ethanol from biomass are successful. Biomass feedstocks, including forest residue, agricultural residue, and energy crops, are abundant and relatively inexpensive, and they are expected to lower the cost of producing ethanol and provide stability to supply and price. In addition, the use of corn stover would lend continued support to the U.S. corn industry. Analysis of NREL technological goals for cellulose ethanol conversion suggests that ethanol could compete favorably with other gasoline additives without the benefit of a Federal subsidy if the goals were achieved. Enzymatic hydrolysis of cellulose appears to have the most potential for achieving the goals, but substantial reductions in the cost of producing cellulase enzymes and improvements in the fermentation of nonglucose sugars to ethanol still are needed. Significant barriers to the success of cellulose-derived ethanol remain. For example, it may be difficult to create strains of genetically engineered yeast that are hardy enough to be used for ethanol production on a commercial scale. In addition, genetically modified organisms may have to be strictly contained. Other issues include the cost and mechanical difficulties associated with processing large amounts of wet solids. Proponents of biomass ethanol remain confident, however, that the process will succeed and low-cost ethanol will become a reality. Ethanol production is a multifaceted operation. From the processing and storage of incoming raw materials through to the storage and shipment of the final products, many industrial processes come into play. Ethanol production combines aspects of both the grain handling and chemical production industries. Despite the best available technology and strictest attention to safety procedures, human error and mechanical failure must be taken into account when considering potential hazards to employees and the surrounding community.

Notes

So what do we conclude from all this?

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Learning Objectives

microorganisms against which it is most effective and which is called its “inhibition spectrum,”

In this lecture, you will learn •

Antibiotics & alkaloid products

Antibiotic Fermentations- a brief history

•

Penicillin fermentation

The industrial fermentation industry received its greatest impetus for expansion and profits with the advent and exploitation of antibiotics as chemotherapeutic agents. The demand for penicillin during World War II, and later for streptomycin and other antibiotics, brought on the undertaking of intensive research programs designed to find organisms capable of producing good antibiotics, and oriented towards the development of means for producing antibiotics on a large scale. New cultural procedures were devised, and the technique of submerged-agitated aerated fermentation in deep-tank fermentors came into being. As a result, much of the knowledge gained during the development of antibiotic fermentation processes then became available for the commercial development of other new nonantibiotic fermentation processes not previously possible on a large-scale production basis.

Antibiotics are probably the most important group of compounds synthesized by industrial microorganisms. They are not produced in the greatest quantity, nor are they the most economically valuable. Nevertheless, over the last 60 years their influence in improving human health has been immense. ‘The other major health-care products derived from microbial fermentations and/or biotransformation are alkaloids, steroids, toxins and vaccines; along with vitamins, certain enzymes, and viable microbial cell preparations used as probiotics. In addition, genetic engineering techniques have made it possible for microorganisms to produce a wide variety of mammalian proteins and peptides that have various therapeutic properties. Those of considerable medical importance and with established markets include insulin, interferons, human growth hormone and monoclonal antibodies. Apart from these therapeutic agents, which cure or reduce the incidence of disease, many diagnostic products are also derived from microorganisms. These are extensively used to test for the presence of various health and disease states. Let us first see about the most well known product group, viz. antibiotics. Before we start with the study of antibiotic fermentations as such, let us go back a little and start with the definition of an antibiotic. So, what is an antibiotic and how does it differ from other antimicrobial substances? If antibiotic is a substance that kills microorganisms or inhibits their growth, can common disinfectants like phenyl, chlorine, iodine etc. can be called antibiotics? No? Why not? If antibiotic is a substance that kills microorganisms or inhibits their growth at very very low concentrations, then can we call cyanide or peracetic acid an antibiotic? No? Why not? If antibiotic is a substance that is produced by one organism and kills or inhibits their growth of other organism, can we call snake venom an antibiotic?

No? Why not? The answers of all the three above questions are obviously negative. This is because the definition of an antibiotic is actually a combination of all that is mentioned above. Thus, an antibiotic is an organic compound produced by one organism and which is capable of inhibiting the growth of other organism at very very low concentration. Though we use the term ‘organism’ here, it is generally implied to microorganisms. For any one antibiotic there is a specific group of

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Antibiotics–the Producers Well over 4000 antibiotics have been isolated from various organisms, but only about 50 are used regularly in antimicrobial chemotherapy. The best known and probably the most medically important antibiotics are the blactams, penicillins and cephalosporins; along with aminoglycosides, such as streptomycin, and the broad-spectrum tetracyclines. Antibiotics are produced primarily by bacteria, Streptomyces, Nocardia, and fungi, although several other classes of microorganisms have at least limited abilities in this area. However, antibiotics produced by Streptomyces species have found greatest commercial application. Many bacteria produce antibiotics, and this is particularly true for bacteria of the genus Bacillus. However, many of the bacterial antibiotics are polypeptides which have proven generally to be somewhat unstable, toxic, and difficult to purify. Antibiotics produced by fungi, with a few notable exceptions, also generally have been found too toxic for medical use. One obvious exception is the penicillin group of antibiotics produced by various molds. The fact that an antibiotic possesses toxicity, which usually rules out its internal administration to the animal or human body, does not necessarily, prevent its medical application, since in some instances the antibiotic can still be used in topical applications, such as for the treatment of burns or skin infections.

Manyantibioticsfailto fulfillcertain importantcriteria, particularlytheirlackofselectivity,exhibitingtoxicityto humans oranimals,ortheirhigh production costs.Some antibiotics haveapplicationsotherthan in antimicrobialchemotherapy.For example,actinomycin and mitomycin,produced byStreptomyces peucetius and S. caepitosus, respectively roles as antitumour

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LESSON 25 : MICROBIAL PRODUCTION OF HEALTH-CARE PRODUCTS-I

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agents; and other antibiotics are used for controlling microbial diseases of crops or as tools in biochemistry and molecular

biology. Several antibiotics are also added to animal feed as growth promoters. However, worries about the development of resistance has meant that some antibiotics used or intended for human use, may be withdrawn from use in animal feed. For example, the EU commission voted to ban the application of bactericin, spiromycin, tylomycin and virginiamycin as growth promoters after January 1999. •

·How are antibiotics produced by the microorganisms? The metabolic reactions leading to antibiotic formation usually do not seem to be components of the normal metabolic systems responsible for growth and reproduction of microorganisms. In fact, antibiotic biosynthesis might be regarded as “a series of inborn errors of metabolism superimposed on the normal metabolism of the organism.

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Industrial antibiotic fermentation processes have further exaggerated these errors by subjecting the microorganisms to mutagenic agents and to highly nutritious media and growth conditions quite different from those encountered by the organism in its natural habitat. The peculiar metabolic reactions involved in antibiotic formation are reflected in the unusual chemical structures that occur in some antibiotics. For example, antibiotics are known to contain fused rings, rare sugars, and unnatural isomers of amino acids. However, in at least one instance, there seems to be a rationale for the production of an antibiotic by a microorganism, and this occurs with the antibiotic bacitracin, a polypeptide antibiotic containing both D- and Lamino acids, as produced by Bacillus licheniformis. This antibiotic is formed by the cells only under conditions that support spore formation and, as such, the antibiotic appears to serve a structural function as a chemical component of the spore coat. These unusual chemical structures observed in many antibiotics have been a boon to the industrial fermentation industry in that, with one exception, these antibiotics have proven difficult, expensive, or even impossible to prepare by chemical synthesis. The notable exception is the antibiotic chloramphenicol produced by Streptomyces venezuelae. This antibiotic was first produced commercially by a fermentation process, but later it was found that its chemical structure was relatively simple and amenable to chemical synthesis and, thus, present-day production of this antibiotic is by chemical synthesis. Let us now see one classical antibiotic fermentations.

Penicillin Fermentation Penicillin, because of the impetus of World War II, was the first antibiotic to be produced on a large scale, and it still is one of the best antibiotics available. It is active against many Grain positive bacteria, Nocardia, and Actinomyces, but not against most Gram negative forms except at higher dosage levels. It interferes with cell-wall synthesis of sensitive organisms and is active only against growing cells. In addition, it presents the favorable characteristic of being almost nontoxic to mammals, except for certain allergic reactions that develop with a small percentage of individuals.

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Several different fungi are able to produce one or more of the penicillins, although this activity resides chiefly with the aspergilli and penicillia. Today, however, the principal organisms for commercial penicillin production are highly mutated strains of Penicillium chrysogenum.

Biochemistry of Penicillin molecule and precursors Whereas in present day commercial terms penicillin is regarded as penicillin G, penicillin also is a generic name applied to a group of compounds having the same nucleus and approximately similar antibiotic activity characteristics against sensitive microorganisms. The various penicillins differ primarily in the nature of their “R” side chains, which are attached by an amido linkage to the chemical nucleus of the molecule. Study of the R side chain in relation to the use of precursors has been highly profitable to the development of high-yielding penicillin fermentations. Fleming’s original Penicillium notatum strain, when grown on his medium, produced largely penicillin F, also known as 2-pentenyl penicillin, in part because he did not utilize precursors in his studies. However, the particular type or types of penicillin produced without added precursors are, to some extent, also a function of the particular mold strain being employed. Thus, descendants of Penicillium chrysogenum Q-176 in the absence of precursors produced largely penicillin K with smaller amounts of dihydro penicillin F. As is evident to this point, the medium constituents have a profound effect on penicillin yields. The corn steep liquor provides peptides, amino acids, and amines which are deaminated to provide the ammonia required in the early stages of the fermentation, as well as some of the carbon nutrients. The glucose is rapidly utilized to provide mycelial growth but allows very little penicillin production. The lactose, however, is only slowly degraded to glucose plus galactose, and it is this slow glucose availability from the lactose that allows the starvation conditions required for penicillin production. In fact, a series of publications by Johnson and his coworkers have shown that penicillin yields equivalent to those with lactose can be obtained from glucose alone, if the glucose is added only slowly to the fermentation as required by the mold. In this regard, it is probable that with the relatively lower cost of commercial glucose today, a carbohydrate regimen somewhat similar to that described above, in fact, may be commercially employed in penicillin production.

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Lipid nutrients also are utilized by the fungus during penicillin production, and fatty oils, such as lard oil, soybean oil, and linseed oil, and fatty acids of greater than 14 carbon chain lengths and their esters are especially effective. Some of the oil is added as antifoam, and the rest is purposely added directly to the medium. These nutrients increase both the amounts of mycelium and yields, but high levels can be deleterious in both early and late stages of the fermentation. Also, these nutrients can provide too great an acidity, but this is usually neutralized by the calcium carbonate of the medium. These oils probably are degraded by the fungus to the two-carbon acetate or similar compound level before being used in formation of mycelium and penicillin. Various synthetic media have been developed for penicillin production, and it has been claimed that these media provide penicillin yields equivalent to those from a medium containing cornsteep liquor. Obviously, such media are far too expensive for their industrial use in the production of penicillin, but they have been of value in studies on the mechanisms and factors involved in penicillin production. •

How is the penicillin recovered from the fermented broth?

Penicillin in the acid (anion) form is solvent extractable, and the antibiotic, as dissolved in an organic solvent, can be backextracted as a salt into aqueous solution. These considerations, in general, are made use of for the recovery and purification of penicillin from harvested culture broths, although the exact procedures to be used depend somewhat on the particular production medium employed and on the final penicillin yields in this medium. Obviously, high yields in conjunction with a medium that does not interfere with recovery and purification greatly simplify the procedures required to obtain a pure product. A general flow sheet for the commercial recovery of antibiotics presented in Figure 17.12 although, as will become apparent, the recovery of Penicillin does not require all of the steps presented. Figure 17.13 is an over all view of commercial antibiotics recovery plant. To be more specific, at harvest, the completed penicillin fermentation culture is filtered on rotary vacuum filter to remove the mycelium and other solids although, under the right conditions, this may not even be required. Phosphoric or sulfuric acids are added to lower the pH to 2 to 2.5 in order to convert the penicillin to the anionic form, and the broth is immediately extracted in a Podbielniak countercurrent solvent extractor, with an organic solvent such as amyl acetate, methyl isobutyl ketone, or butyl acetate. The penicillin is then backextracted into water from the organic solvent by adding enough potassium or sodium hydroxide to form a salt of the penicillin, and the resulting aqueous solution is again acidified and reextracted with methyl isobutyl ketone. These shifts between water and solvent aid in purification of the penicillin. The solvent extract finally is carefully back-extracted with aqueous potassium or sodium hydroxide, but more often with sodium hydroxide, and from this aqueous solution various procedures are utilized to convert the penicillin to crystallize as sodium or potassium penicillin. The resulting crystalline penicillin salt then is washed and dried, and the final product must pass rigorous

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There actually are several penicillins, all closely relate in structure and in activity against sensitive microorganisms. These penicillins have a common chemical nucleus and differ principally in the chemical structure of a side chain attached to this nucleus. The various penicillin fermentations also are unusual in that various compounds resembling the side chains can be added as precursors to the fermentation medium, and these compounds, through microbial action, are directly incorporated into the penicillin molecule. Also, the side chain can be enzymatically removed, liberating the penicillin nucleus so that unnatural side chains can be chemically added to the nucleus in order to create new penicillins.

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government standards. Spent solvents resulting from the above procedures are recovered for reuse.

followed by its conversion to the preferred precursor, 7amino deacetoxycephalosporic acid (7-ADCA), by ring expansion. A suitable side chain can then be readily attached. •

Exercise: find out details of one fungal and one bacterial fermentation. Use the space provided to write your findings.

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Tell me more about antibiotic resistance. The success of penicillin in the mid-1940s led to the search for other antibiotic-producing microorganisms. One of the most notable early successes was the discovery of streptomycin from a soil actinomycete, Streptomyces griseus. Subsequently, actinomycetes, especially Streptomyces species, have yielded the majority of the antibiotics used in clinical medicine today . However, the increasing development of bacterial strains that exhibit resistance to antibiotics demands the continued search for new antibiotics and alternative agents for treating microbial diseases. Antibiotic resistance is not a recent phenomenon, it was recognized soon after the natural penicillins were introduced. The use of antibiotics creates selection pressure favouring the growth of antibiotic-resistant mutants, which is promoted by the misuse and overuse of these drugs. Over the last 10 years the situation has become alarming, due to the emergence of pathogenic bacterial strains that show multiple resistance to a broad range of antibiotics. One of the most important examples is the development of multipleresistant strains of S. aureus. Certain strains, particularly methicillin-resistant S. aureus (MRSA) cause serious nosocomial (hospital-acquired) infections. They are resistant to virtually all antibiotics used in antimicrobial chemotherapy, including methicillin, cephalosporins and other b lactams, the macrolide erythromycin, and the aminoglycoside antibiotics streptomycin and neomycin. The only compound that can be used effectively against these staphylococci is an older and potentially more toxic antibiotic, vancomycin. Resistance even to this antibiotic has been detected in some strains.

Production of semisynthetic penicillins and Cephalosporins As we have seen, the objective in semisynthetic penicillin production is to generate compounds with improved properties, e.g. acid stability, resistance to enzymatic degradation, broader spectrum of activity, etc.. It involves removal of the side chain of the base penicillin to form 6-APA. This is achieved by passage through a column of immobilized penicillin acylase, usually obtained from Escherichia coli, at neutral pH. Penicillin G, for example, is converted to 6-APA and phenylacetic acid. The 6-APA is then chemically acylated with an appropriate side chain to produce semisynthetic penicillin. Yields of cephalosporins from direct fermentations are much lower than those for penicillins. Consequently, as 6-APA can also serve as a precursor of cephalosporins, it is often used as the starting material for their semisynthetic production. A base natural penicillin is converted to 6-APA, as described above,

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Many of the antibiotic-resistance genes of staphylococci are carried on plasmids that can be exchanged with species of Bacillus and Streptococcus, providing the means for acquiring additional genes and gene combinations. Some resistance genes are carried on transposons-segments of DNA that can exist either in the chromosome or within plasmids. •

What is the future of antibiotic Fermentation? Antibiotics have found use in medical and veterinary applications, treatment of plant diseases, as an aid in animal nutrition when mixed with feeds or water, and in the preservation of food and other materials. In recent years, however, relatively few new antibiotics have come into commercial production and, in fact, the search for new antibiotic products has been somewhat curtailed. This is due largely to the fact that extensive screening programs have turned up only a few commercially usable antibiotics over those discovered in the 1940’s and 1950’s, and because any new antibiotic must be better than those already in

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•

produced commercially. The second group, based on lysergic acid (LSA) and containing a tripeptide or an amino alcohol are found only in Claviceps species. Examples include ergometrine, ergocristine, ergosine and ergotamine. Ergotamine, for example, is a structural analogue of serotonin (a neurotransmitter) and is formed from LSA by the action of a peptide synthetase that adds alanine, proline and phenylalanine.

All right. What are the other important health care products we should study?

Look at the following table.

These LSA-based alkaloids have medical roles as analgesics in migraine therapy, as hallucinogens and for treating circulation problems. Others have particular applications in obstetrics, for inducing the smooth muscle of the uterus to contract during labour and after childbirth.

First, let’s see about the ergot alkaloids. Alkaloids are a diverse group of small nitrogen containing organic compounds produced by certain plants and microorganisms. Many are toxic, but some have various therapeutic properties. Species of the filamentous fungus Claviceps, which are pathogens of grasses, produce a range of alkaloids. Some of the best known are the ergot alkaloids. These compounds are produced within the sclerotia (fruiting bodies) of Claviceps purpurea that develop naturally when this organism infects developing cereal grains. Infected grains become black and are referred to as ergots. These structures contain indole alkaloids, derived from a tetra cyclic ergo line ring system, which are classified into two groups. Members of the first group are based on clavin and contain no peptide component. Clavin-based alkaloids are also produced by other groups of fungi, including species of Aspergillus and Penicillium. Some possess antibiotic and antitumour activity, but few are 2.521

Previously, the alkaloids were extracted from ergots that developed within infected cereal crops, usually rye, or by chemical synthesis. Most are now produced by fermentation of Claviceps fusiformis, C. paspali or C. purpurea in surface, submerged or immobilized cell culture. Inoculum for the production fermentor may be developed by mycelium or conidiospores. The production medium contains an organic acid of the tricarboxylic acid (TCA) cycle and a carbohydrate, such as citrate and sucrose, the specific combination depending upon the target alkaloid. In later stages, the organic acid stimulates the necessary metabolic change from the TCA cycle to the glyoxylate cycle. Alkaloid production, like that of many secondary metabolites, exhibits phosphate regulation. The synthesis is delayed until the medium phosphate has been utilized during the trophophase and the culture enters the idiophase. However, phosphate inhibition can be overcome by addition of tryptophan or a tryptophan analogue, which act as inducers and precursors. •

Exercise: Find out more about ergot alkaloids and their medicinal applications. Search the internet for companies and organizations involved in the commercial production of alkaloids.

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commercial usage. This is not to say, however, that new antibiotics of great value will not be discovered in the future, although it may be that the approach to discovering valuable new antibiotics may lie in a slightly different direction. Thus, there are many potentially good antibiotics already known which, because of moderate toxicity or some other feature, are not presently usable, and manipulation of the genetic characteristics of the organism, changes in fermentation conditions, or even the use of chemical reactions to alter the structures of the antibiotic molecules might provide a change in the antibiotic that would allow its commercial acceptability. Such altered chemical structures conceivably could provide an additional valuable feature in that sensitive organisms might be less likely to acquire the antibiotic resistance that is characteristic of some antibiotics after long exposure of the cells.

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LESSON 26: STRAIN IMPROVEMENT

Learning Objectives

product, or used directly in a further bioconversion step if it is an intermediate compound.

In this lecture, you will learn •

Steroid biotransformation

Microbial production of health care products Steroids & other products We have seen the production of antibiotics and alkaloids in the last session. In this lesson, we will see some more products related to human health that can be produced by fermentations. Steroid Biotransformation The term steroid is probably famous for all the wrong reasons. Actually, they are quite useful substances especially for the treatment of allergies, inflammation, skin diseases and as oral contraceptives. These are called therapeutic steroids. Initially, they were prepared by extraction from animal tissues or via complex chemical synthesis, both of which were extremely costly. Many steroids are now manufactured using a combination of chemical and microbial transformation steps. These processes employ relatively cheap sterols as the starting materials, often diosgenin extracted from e Mexican yam (Dioscorea composita), or stigmasterol, a byproduct of soybean oil manufacture. The microorganisms involved are mostly filamentous fungi Rhizopus, Curvularia, Fusarium and Aspergillus species) and mycobacteria, in the form of suspensions immobilized growing cells, resting cells, spores and cell free extracts. They perform key reactions to modify the basic steroid structure, a cyclopentanoperhyophenanthrene , including hydroxylations at positions 11 and 17; various side-chain cleavages, hydrogenations and dehydrogenations; and ring expansion , from a five-membered to a six-membered ring. When live vegetative cells are used for steroid biotransformations, the medium is kept as simple as possible in order to make later purification less problematical. Even simpler media can be formulated for use the spore preparations and there is less need for undesirable antifoam, which may otherwise affect product extraction. For vegetative cells, the culture is grown through exponential phase to obtain maximum biomass before the substrate is added, or the biomass may be harvested to set up immobilized column systems. The steroid precursors are insoluble in water and must be dissolved in solvents, e.g. methanol, ethanol or acetone. These solvents and some substrates are toxic. Consequently, substrate concentrations rarely exceed 2-5 g/L, but their conversion approaches 100%. Processing of the product depends on whether it is accumulated within the cells or excreted into the medium. Water immiscible solvents, usually methylene chloride, ethylene chloride or chloroform, are used for extraction of the product from clarified medium or cell extracts. The product may be further purified if it is the end-

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Having seen about the steroids, let’s see about the bacterial vaccines. We know that the administration of vaccines induces immunity against that organism. Early bacterial vaccines consisted of whole cultures of bacteria that had been inactivated by heat or formaldehyde, but now they can be divided into two categories, living vaccines and inactivated vaccines. Living vaccines are composed of live attenuated (weakened) strains of the parent virulent strain. Inactivated forms are composed of whole bacterial cells, or a cell component or metabolic product (cell wall antigen, capsular antigens, toxin, etc.), which now may be products of recombinant DNA technology. Microbial protein toxins can serve as vaccines following their inactivation with formaldehyde or heat to form toxoids. Vaccination with an antigenic toxoid vaccine leads to the generation of antitoxin that neutralizes the pharmacological effect of active toxin. These vaccines have been successful against

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Vaccine production requires highly controlled operating conditions and strict adherence to good manufacturing practices.1t normally involves the growth of bacterial cultures in sophisticated high-grade fermentors of usually no greater than 1000 L capacity. The fermentations are designed for optimized yield of antigen (cells or cell components) and containment, involving the very strict adherence to protocols that prevent bacterial release into the environment. Internal pressures never exceed atmospheric pressure, to reduce the risks of leakage, and exhaust gases from fermentors must pass through sterilizing filters, incinerators or both. Fermentations for the production of vaccines based or whole cells aim to maximize biomass production for inactivated whole cell vaccines, downstream processing usually follows cell inactivation by heat treatment or the addition of formaldehyde. The microbial cells, inactivated or live; are then separated from the medium by centrifugation. All harvesting equipment incorporates absolute microbial containment and is situated within a room maintained under negative pressure, so that any escape is contained. Live attenuate; vaccines are usually prepared as freeze-dried products. For the production of vaccines based on toxins or surface antigens (cell wall or capsular components), the growth conditions are aimed at producing maximum levels of these specific cellular antigens. Excreted toxin and loosely bound surface antigens that are shed into the medium are purified from the clarified culture broth and the harvested cells are safely discarded. As mentioned above, toxins are usually inactivated by treatment with heat or formaldehyde to become bacterial toxoids that have no toxicity, but retain their antigenicity. However, in some cases, notably Clostridium botulinum toxin, the active neurotoxin is also prepared for other therapeutic uses.

Viral vaccines were previously available only via culture in live animals or from animal tissue and cell cultures. However, genetic engineering has allowed the production of recombinant viral vaccines through the cloning of viral antigens into an appropriate host microorganism. For example, the virulence factor of hepatitis B and viral protein of foot-and-mouth disease virus can be expressed in E. coli for the production of valuable vaccines; recombinant hepatitis B vaccine alone has worldwide sales worth over US$ 1000 million. Also, the safe production of recombinant vaccines for dangerous bacterial pathogens is now possible, using benign host organisms well suited to large-scale fermentation. This has the added advantage that the host can be manipulated to amplify antigen production. Some lactic acid bacteria are suitable hosts and are being evaluated for use in oral immunization. These bacteria have generally recognized as safe (GRAS) status and low immunogenicity, and can be used to express antigens, such as fragments of tetanus and diphtheria toxins.

Recombinant therapeutic Peptides and Proteins Previously, mammalian therapeutic proteins and peptides could be prepared only from animal or human tissues and body fluids, and were available in very limited quantities. These preparations were extremely costly to produce, some had unwanted side-effects, and in certain cases there were unfortunate problems with virus and prion contamination. Recombinant DNA technology has allowed the production of many recombinant therapeutic proteins from various sources, including over 400 human proteins and peptides with potential medical applications. At present, only about 10% have received approval for use by the US Food and Drug Administration (FDA). Overall, the worldwide market for recombinant pharmaceuticals is worth around US$20 billion and is rising at a rate of over 10% per annum. However, the fermentation volume for industrial-scale production of human therapeutic proteins is usually no greater than 2000-5000 L. Unlike recombinant vaccines, where it is essential to retain or even enhance antigenicity, these recombinant therapeutic products must be free from antigenicity.

Vaccine antigens for human immunization are highly purified. Purification procedures may include conventional ammonium sulphate precipitation techniques and various chromatographic steps. Affinity chromatography is usually incorporated, utilizing specific antibodies as ligands, preferably monoclonal antibodies. For maximum effectiveness as a vaccine, the purified antigens are adsorbed onto an adjuvant that increases the immunizing power. Adjuvants include mineral salts, such as aluminium hydroxide and potassium aluminium sulphate, or water-in-oil emulsions. For some diseases with a number of serotypes, blends of antigens are used in the final vaccine. Vaccine preparations can be injected parenterally or administered orally. Inactivated forms are usually injected and living vaccines are mostly taken orally, particularly those for enteric diseases. Injected vaccines stimulate antibody production in the bloodstream, whereas oral vaccines stimulate local production of antibody at the mucosal surface of the intestine.

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Gram-positive bacteria responsible for diphtheria, tetanus and several other diseases caused by Clostridium species. Similar toxoid vaccines employed to counter some Gram-negative bacterial diseases have proved to be less effective. However, their surface antigens, some of which are involved in their adhesion to epithelial tissues, have been used to develop effective vaccines.

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of other diseases, particularly those associated with ageing, and in wound healing. This hormone is a single protein chain that is synthesized in the body as a precursor, prehormone, composed of 217 amino acids. The prehormone contains a signal sequence of 26 amino acids that is enzymatically cleaved to give the biologically active protein. Active hGH has a molecular weight of 21500Da and consists of 191 amino acids with two disulphide bonds linking cysteines at positions 53 and 165, and 182 and 189. The search for sources of hGH started with animal-derived products, but bovine and porcine growth hormones were found to be ineffective. Greater quantities of a safer supply are now provided through recombinant hGH, which began development in the late 1970s. Two companies, Kabivitrum in Sweden and Genentech in the USA, formed a joint venture to develop a recombinant DNA-derived hGH. However, at that lime production of recombinant products was not permitted in Sweden, so the work was carried out in the USA.

DNase Cystic fibrosis is a fatal genetic disorder involving a malfunction in epithelial tissue. This condition is characterized by the presence of a thick mucus which is produced in a number of organs, particularly the lungs, where it impairs breathing and increases the risk of microbial infection. As a consequence of infection, part of the immune response involves phagocytes attacking the microorganisms, which often include Pseudomonas aeruginosa, Burkholderia (formerly Pseudomonas) cepacia, Staphylococcus aureus and Haemophilus influenzae. This results in the release of free DNA from both bacteria and phagocytes into the lungs. The DNA is very viscous and further thickens the mucus. Genetically engineered DNase preparations are now available that can help clear these secretions by breaking up the long DNA strands into smaller sections to reduce the viscosity of the mucus. Pulmozyme, a genetically engineered DNase developed by Genentech, received approval from the US FDA in 1996. This 37000 Da human glycoprotein, consisting of 260 amino acid residues, is produced in cell lines from Chinese hamster ovary (CHO) and can be administered in an aerosol. The annual sales of this product are now in excess of 110 million dollars. Human Growth Hormone (somatotrophin) Human growth hormone (hGH) is a protein hormone that is synthesized in the pituitary gland at the base of the brain. This hormone is involved in controlling both growth and stature. Preparations of hGH are used to treat children with ‘hypopituitary dwarfism’, a congenital disease in which the pituitary fails to secrete sufficient hGH for normal growth. This hormone cannot be administered orally, but must be injected. The standard dose is 0.5-0.9 ill/kg body weight per week. In addition, hGH has therapeutic value in the treatment of a range

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Insulin Insulin is a hormone produced by the pancreas and is responsible for the metabolism of carbohydrates. The inability of pancreas to secrete adequate quantity of insulin or the

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In case of type II diabetes, the patient is almost completely dependant in external supply of insulin. Dead bodies of human beings and animals have long been looked upon as a source of insulin. Due to the insufficient supply of insulin through these methods, microbial fermentations have emerged as a promising field for its production. Recombinant microorganisms cloned with A and B chain producing genes of insulin have been successfully producing insulin identical to that produced by the human body. Recombinant insulin is now available from either E. coli or Saccharomyces cerevisiae.

fragments, respectively, and may be safe future sources of this protein for medical and surgical use.

Notes

Interferon Interferon (IFN) is a member of the cytokines, a large family of small signaling proteins involved in regulation of cell-mediated immunity, which also includes interleukins (see below), tumour necrosis factor (TNF), colony-stimulating factor (CSF), erythropoietin (see above) and thrombopoietin. All vertebrates produce a variety of interferons, and mammals, including humans, produce three types; a, 13 and y. IFN-a forms are primarily produced by leucocytes and they consist of a single polypeptide chain of 165-166 amino acid residues. Some are glycosylated with varying amounts of carbohydrate moieties and their molecular mass is in the range of 16000-26000 Ad. The carbohydrate portion does not appear to confer any functionality on IFN- and may be removed without affecting their activity. This property allows recombinant IFN- to be produced in prokaryotic systems, such as E. coli, which are not capable of the post-translational modifications necessary to form glycosylated polypeptides. Recombinant IFN-a received approval from the FDA in 1991 for use in the treatment of hepatitis C. Subsequently, it has been approved for the treatment of a number of conditions, including hairy cell leukemia, chronic myeloid leukemia, renal cancer, melanoma, multiple myelomas and genital warts, and in a nasal spray to provide protection against colds caused by rhinoviruses. Recombinant IFN-a is now manufactured by a number of companies Interleukins The interleukins are also a subclass of the cytokines. They are usually single-chain glycosylated proteins with molecular masses of 8000-30 000 Ad. There are at least 15 different members of the interleukin family. In 1992 the Chiron Corporation received approval from the FDA for a recombinant IL2 preparation, marketed as Proleukin, which is used in the treatment of metastatic renal cell carcinoma. Several other interleukins exhibit therapeutic potential. Collagen Collagen is the most abundant protein in the human body and is used by surgeons for suturing and repair. It is currently obtained for this purpose from cattle or human cadavers. Consequently, there are concerns about its safety with regard to potential contamination with viruses and poisons. The yeasts Piehia augusta and Pichia pastoris have now been genetically engineered to produce human type I and III collagen

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inability of the body to utilize the insulin results in inadequate carbohydrate metabolism. Clinically, this syndrome is called diabetes.

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LESSON 27: INDUSTRIAL WASTEWATER TREATMENT

Learning Objectives In this lecture, you will learn •

Different methods of wastewater treatment

•

The activated sludge process

•

Anaerobic wastewater treatment

Introduction Many of us think that the study of fermentation technology ends with the harvesting of the fermentation product. Well, they are in for a surprise. A sector as important as the proper fermentation is the treatment of the waste water generated. •

·Why do we need to study the waste water treatment? Fermentation generates all three types of waste products, viz. solid, liquid and gases. But the liquid effluent is highest both in terms of quantity and the pollution load as reflected by the biochemical oxygen demand. Some industrial wastewaters resemble domestic wastewater to a remarkable degree whereas others may contain high concentration of toxic or non-biodegradable materials. Clearly, biological processes would be considerable potential in treatment of wastes of the former type, but would be of little use in the case of waste of the latter type. Historically, the options available for the treatment of industrial wastewater have been broadly similar to those used for the domestic wastewater. These are direct discharge to surface water, direct discharge to sea through long outfalls or treatment prior to discharge of an effluent of acceptable quality. Treatment can be conducted at the industrial site in which case the plant will often have been designed specifically to cope with certain waste composition. Alternatively, industrial wastewater can be discharged to sewer with or without pretreatment and treated in admixture with domestic wastewater at a municipal treatment works employing conventional processes. Discharge to sewers has been practiced for as long as sewers have been in extensive. Clearly, not all wastewater are suitable for discharge. This was recognized at an early stage, and led to the important of trade effluent controls by the authorities responsible for wastewater treatment. The development of industrial wastewater treatment processes has been due largely to changes in trade effluent policy, which have imposed economic pressure upon the dischargers. What is wastewater treatment? Wastewater is the liquid waste released from the community. It includes worthless materials discharged from industries, kitchens, food industries, health sanitaries, municipalities, etc. The wastewater has been treated for reducing the pollution hazards. The wastewater treatment process includes three main steps. They are primary treatment, secondary treatment and tertiary treatment. The primary

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treatment consists of purely physical operations; it includes the screening and sedimentation of floating and sedimentable solids from the wastewater water. The secondary treatment includes biological and chemical methods employed for the removal of organic matter from the wastewater water. The tertiary treatment includes the addition of some substances to the wastewater water in order to remove nitrogen and phosphorus from it.

I. Primary Treatment It includes the removal of solid wastes floating on the wastewater and the other sedimentable wastes from the wastewater. They are removed by screening and sedimentation methods. 1. Screening: It is the first step of the wastewater treatment. It is used to remove the solid particles from the wastewater, The wastewater is passed through a screen fitted in an opening. Usually screens of uniform size are used for the screening. The screening of wastewater protects the operation unit against the excessive dumping of solid wastes. The screens are classified into three groups, namely coarse screens, medium screens and fine screens. They are explained below: a. Coarse Screens: Coarse screens are used to remove the floating materials like rags, paper, wood etc. from the wastewater. The screens are made of steel bars with a space of 50mm between the two adjacent bars. These types of screens are also called racks. b. Medium Screens: Medium screens are used for removing some amount of organic materials from the wastewater. Here the steel bars are arranged properly with a space of 40 mm between each of two adjacent bars. c. Fine Screens: Fine screens have perforations of 1.5 mm to 3mm size. These screens help in the removal of some suspended particles from the wastewater. 2.Sedimentation: The separation of sedimentable wastes in the wastewater is carried out by allowing the wastewater to remain stagnant in large tank. The wastes get sedimented in the bottom of the tank. The process of separation of solid wastes (suspended wastes) from the wastewater is called sedimentation and the tank is often referred to as sedimentation tank or settling tank. Sedimentation is carried out by adding some coagulants to the wastewater. Such a sedimentation process is called chemical precipitation. Sometimes sedimentable wastes are removed by using the physical forces such as gravitational force, flocculation of particles and so on. The removal of particles by using the physical forces is called plain sedimentation. Alum, ferric chloride, ferric sulphate and chlorinated copper are generally used as coagulants in the

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1. It reduces the total sedimentable solid contents in the wastewater. 2. It prevents the formation of sludge in the wastewater treatment unit. 3. It reduces the Biological oxygen demand (BOD) in the wastewater. 4. It is used to prepare wastewater water for further treatment. 5. It is used to prepare wastewater water to be used in irrigation

II. Secondary Treatment During the secondary treatment, the non-sedimentable wastes are removed from the wastewater. So the level of organic chemicals and of nutrients like nitrogen and phosphorous become lowered in the wastewater water. The secondary treatment is carried out either by the chemical method or by the biological method. The biological method of wastewater treatment is explained below:

The Activated Sludge Process The activated sludge process surely is the most widely used biological process for the treatment of municipal and industrial wastewaters. Normally, the activated sludge process is strictly aerobic, although anoxi1 variations are coming into use for denitrification,. In simple terms, the activated sludge process consists of a reactor called the aeration tank, a settling tank, solids recycle from the settle to the aeration tank, and a sludge wasting line. The aeration tank is a suspended-growth reactor containing) microbial aggregates, or flocs, of microorganisms termed the activated sludge. The microorganisms consume and oxidize input organic electron donors collectively called the BOD. The activated sludge is maintained in suspension in the reactor through mixing by aeration or other mechanical means. When the slurry of treated wastewater and microbial flocs pass to the settling tank, the flocs are removed from the treated wastewater by settling and returned to the aeration tank or wasted to control the solids retention time (SRT). The clear effluent is discharged to the environment or sent for further treatment. Capturing the flocs in the settler an recycling them back to the reactor are the keys to the activated sludge process, because they lead to a high concentration of microorganisms in the reactor. Thus, the sludge is “activated” in the sense that it builds u to a much higher concentration than could be achieved without the settler and recycle. The high biomass concentration allows the liquid detention time to be small, generally measured in hours, which makes the process much more cost effective. Wasting the sludge through the separate sludge-wasting line makes the solids retention time (SRT ) separate from and much larger than the hydraulic detention time. The basic principle of aerobic treatment is that the waste-water is brought into contact with a mixed microbial population of aerobic organisms and oxygen. Soluble, suspended and colloidal biodegradable materials that contribute to the BOD are then metabolized: Aerobic microbes + BOD +O2

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New cells (biomass) + CO2 + residual BOD + H2O During the process, part of the biodegraded material is converted into CO2 (mineralization) and a proportion becomes new biomass (assimilation). Under ‘starvation’ conditions, some of the microbial biomass (intracellular storage compounds) may also be metabolized; this is referred to as endogenous respiration. A major problem associated with aerobic treatment is the disposal of excess biomass produced during the degradation of the pollutants. Approximately 30-70% of the biodegraded carbon is transformed into new cells, and the remainder is converted to CO2, the specific values being process dependent. Although the efficiency of the systems relies on the production of new active cells, this simultaneously produces a new form of pollution, the excess waste biomass, which must be safely disposed of. Therefore, aerobic treatment can be classed as only 30-70% efficient, depending upon the specific process. The fermentor, with a depth of 100m, is sunk in the ground to reduce noise, odour and land usage, and has significantly lower biomass yields. Neverertheless, the most commonly used aerobic processes are still the conventional activated sludge processes and trickle filters described below.

An outline of conventional wastewater treatment. An activated sludge plant Homogeneous activated sludge Processes

The activated sludge process was originally developed in 1914 by Arnold and Locket. The basic principles of the process are that the waste-water is brought into contact with a mixed microbial population, in the form of a flocculated suspension, within a continuously aerated and agitated tank. These processes are routinely used to treat domestic wastewater and industrial waste-waters that have usually undergone primary treatment. Typically, primary treated wastewater entering the system contains150-200mg/L TSS, 150-200mg/L BOD, 20-40mg/L ammoniacal nitrogen and 6-10 mg/L phosphorus. However,

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sedimentation tanks, The important uses of sedimentation processes are:

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these values vary depending on the location and the nature of the wastes deposited into the system treatment This is a two-stage process, involving biological treatment and secondary settlement. Biological treatment is performed in an aerated basin containing a diverse range of flocculated microorganisms, the mixed liquor suspended solids (MLSS), that biodegrade the polluting material present. As the microorganisms grow in the aeration basin they clump (flocculate) together to form stable flocs of activated sludge. The formation of stable flocs of 2-3 mm diameter is essential for the efficient operation of the plant with respect to BOD removal and rapid settlement in the secondary sedimentation stage. The microorganisms present include a range of bacteria, e.g. carbon oxidizers, filamentous carbon oxidizers, nitrifies, denitrifies, etc., along with fungi, protozoan, withers, nematodes and algae. Despite being widely used, the microbiology and community structure of activated sludge processes is not well characterized. However, bacteria such as species of Acinetobacter and Zoogloea ramigera are considered to playa key role in floc formation by the synthesis and secretion of polysaccharide gels. Protozoa act as bacterial scavengers, ensuring low turbidity in the final treated effluent. Some 200 protozoan species have been isolated, but the sludge recycle most important are the ciliated forms, e.g. Vorticella apercu/aria. Overall, activated sludge must contain a microflora capable of producing all enzyme systems required for the biodegradation of both soluble and insoluble pollutants. These microorganisms should form flocs with good absorbing properties that are stable and settle rapidly. Secondary settlement occurs after one hydraulic retention time, when the treated effluent from the aeration basin passes into a secondary settlement tank. This is similar in design to primary sedimentation, i.e. SLR, 15-30 m3/m2/day; SLR, 50-100kg/ m2/day; HRT, 2-4h. Here the flocculated microorganisms rapidly settle to form a secondary sludge, normally containing 13% (w/v) total solids, and a clarified supernatant. Often the supernatant is suitable for final disposal, but where necessary it can be subjected to a tertiary treatment to remove inorganic nutrients. Depending on the design sludge loading rate (SLR), a proportion of the settled flocculated MLSS (secondary sludge) is returned to the aeration basin to maintain the required operating MLSS (microbial biomass) concentration. This allows a high concentration of biomass to be maintained in the aeration basin independent of the growth rate of the microorganisms, thus preventing microbial wash-out. These systems are comparable to stirred tank bioreactors with biomass recycle. Numerous designs and configurations of activated sludge plants exist, but they vary in only four key aspects: sludge loading rate (see below), MLSS concentration (kg/m3), configuration and method of oxygen supply. The main parameters that have to be taken into account when designing activated sludge systems are:

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•

Hydraulic loading rate (HLR, kg BOD/m3/day) raw BOD (kg/m3) x flow rate (m3/day) Aeration tank volume (m3)

• • • • • •

Hydraulic retention time (HRT, h) Reactor volume (m3) Flow (m3/h) Sludge loading rate (SLR, kg BOD/kg MLSS\day) Raw BOD (kg/m3) x flow rate (m3/day) MLSS (kg/ m3) x aeration tank volume (m3)

The operating SLR affects the level of treatment achieved and is basically the food to biomass ratio, which is the mass (kg) of BOD provided per kilogram of biomass (MLSS) per day. Therefore, as a rule and up to a limit, the more food (BOD) that is added to each kilogram of MLSS, the faster the microorganisms grow (see Chapter 2, Microbial growth). However, for maximum purification (percentage BOD removal) the food to biomass ratio should be low. This maintains the cells in a partially starved state, thereby ‘encouraged’ to actively metabolize any biodegradable pollutants present, to produce a low residual BOD (i.e. the substrate is limiting). Conversely, with increasing food to biomass ratios, the food availability increases, allowing higher microbial growth rates and greater biomass yields. Also, as substrate is no longer limiting its residual concentration in the final effluent increases. What are the different modes of operation of activated sludge plants? There are three main modes of operation for activated sludge plants: conventional, extended aeration and high rate treatment. The major difference is the operating SLR .However, percentage BOD removal, HRT, biomass yield and sludge age (residence time) vary depending on the nature of the waste being treated. This is particularly true for industrial waste-waters whose concentration and composition vary considerably. As a rule, the more concentrated the influent BO D, the longer the required operating HRT to achieve the necessary degree of treatment and the lower the sludge age.

•

Conventional processing is used for complete treatment of waste-waters such as domestic wastewater. Here the lower the SLR operated, the greater the level of purification obtained. Over 95% removal of BOD can often be achieved at the lower end of the SLR range (0.25kg BOD/m3/day), falling to 85% removal at the higher end of the range (0.5 kg BOD/m3/day). Extended aeration operates at a lower SLR than conventional plants and achieves approximately the same degree of purification, but the operating HRT is significantly longer. First impressions suggest that this system has no advantages over conventional treatment, particularly as this system is often more costly to construct and operate. Higher costs are due to increased HRTs that require a reactor with a greater volume and, consequently, more energy for aeration. Nevertheless, the main advantage of this system is the significantly reduced biomass yield (0.2-0.3 kg biomass per kilogram of BOD removed). This is approximately 50% of that found in conventional plants and substantially reduces the costs of its disposal. The reduced biomass yield is a function of the lower SLR, which maintains

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High rate treatment is mostly used for the partial processing of strong industrial waste-waters and is designed to remove only 60-80% of BOD. Consequently, the treated waste-waters normally remain too polluted for direct disposal into the environment. The high food to biomass ratio (kg BOD: kg MLSS) favours the faster-growing- microorganisms and results in an increased biomass yield (0.5-0.7kg biomass per kilogram of BOD). However, the high SLR produces short HRTs. Generally, for any given waste-water, the lower the SLR, the larger the aeration basin volume required. This results in longer operating HRTs, reduced biomass yields and improved percentage BOD removal. The opposite occurs as the SLR increases. A smaller aeration basin volume is required, which gives reduced HRTs, increased biomass yields and lower BOD removal rates. Any specific SLR value chosen is therefore a function of the degree of treatment required, land availability, running costs and the cost of disposal of the excess sludge generated.

Dissolved oxygen in activated sludge plants The operating dissolved oxygen (DO) level is a function of the value chosen in the design requirements. If the objective is full nitrification, a dissolved oxygen concentration of at least 2 mg/ L is necessary. However, where only carbon oxidation and denitrification is required, a lower DO will suffice. The DO concentration required within the aeration basin can be determined by either mathematically modeling the system to predict oxygen demand at different times of the day, or by use of feedback mechanisms incorporating oxygen electrodes. Oxygen requirements may be supplied by mechanical aerators installed in the aeration basin, bubble diffusers (spargers), or a combination of the two. • All right. So this is how we treat the liquid part. And what to do about the sludge and the solid waste generated? Disposal of sludge has to be done to meet the local regulations laid down by the governing bodies. Methods routinely used for the disposal of final sludges and other solid wastes are as follows: 1. Landfilling: which is also used for other agricultural, industrial and urban wastes. However, there are potential pollution problems as materials can leach into adjacent water courses when unsuitable or ill-prepared sites are used. Also, it is becoming increasingly expensive due to a lack of suitable landfill sites. Nevertheless, landfilling has potential as a means of methane production. This may be provided that problems associated with the establishment of suitable microbial populations can be overcome, possibly by inoculation with appropriate methanogens. 2. Incineration: is routinely used for solids and well dewatered sludges with solids contents in excess of 30% (w/v). For sludges, the system operates with limited energy input due to their high calorific value, leading to self combustion.

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3. Biologically stabilized dewatered sludge: may be used as a low-cost fertilizer and soil conditioner on agricultural land, often incorporated with composted solid organic agricultural and household wastes. This mode of disposal is becoming increasingly popular, but the regulations regarding disposal are very stringent, particularly regarding nitrogen, phosphorus and heavy metal concentrations, and pathogen content. The fine colloidal particles present in the wastewater water ,are removed in the form of gelatinous precipitate. This precipitate is named floc. The precipitation is carried out by adding some chemical coagulants like alum, ferric chloride, ferric sulphate, etc. to the wastewater water. The heavy metals present in the wastewater water are treated before the disposal of the. wastewater water. Thus the wastewater water becomes more or less pure, which is fit for agricultural purpose.

•

·Well, now what is the next type of treatment?

It is called trickling filters. The basic principle of aerobic trickle filters is that a microbial population is allowed to develop as a biofilm on an inert support material within a biological reactor. Polluted wastewater is continuously sprayed over the surface of the support material and percolates (trickles) through the filter bed, where it is biodegraded by the microbial population. Aeration is achieved by exploiting the difference in temperature between the inside and outside of the reactor, resulting in a counter current of air. High microbial activity within the reactor causes a rise in temperature, and the warm air rises and allows fresh air to enter at the bottom of the reactor. As treatment proceeds, the biofilm grows and increases in depth until a critical thickness is achieved, at which point oxygen becomes limiting at the surface of the support material. This results in the biomass falling off, called sloughing, after which the biofilm starts to redevelop. Microbial populations vary considerably depending on the position within the filter. At the top, a range of microorganisms develops, including bacteria, fungi, protozoan and algae; along with microorganisms, especially insects and their larvae. Below the surface, carbon-oxidizing microorganisms predominate, whereas nitrifies are mostly found at the bottom of the filter. Overall, a highly complex food chain is created within these filters. The three most important features of the packing material (inert support material) are as follows. 1 The specific surface area to volume ratio for biological attachment: the larger the surface area, the greater the biomass concentration per unit volume of the reactor and therefore the faster the rate of biodegradation. 2 The voidage volume: a high void space is required to prevent clogging and short-circuiting of the waste-water as it passes through the filter bed. Also, a high voidage volume aids oxygen transfer. 3 The density of the support material: the more dense the support material, the stronger the construction has to be to support and contain the total weight.

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the cells in starvation conditions, so that a proportion of cells respire endogenously.

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•

·What are the different types of trickling filters? Trickle filters can be designed to operate under two modes of operation. Low rate filters almost invariably have stone or some other dense mineral medium with a low surface area and high density, whereas high rate filters use plastic media with high voidage and high surface areas. As these systems are non-homogeneous and have complex ecology, it is impossible to quantify the total biomass concentration attached to the inert support material. When designing such filters it is not practical to use the sludge loading rate (kilogram of BOD per kilogram of MLSS per day) as in the activated sludge process, because the biomass concentration is unknown. Therefore, it is normal to use the organic loading rate (OLR, kilogram of BOD/m3/day), which is the mass (kg) of BOD added to each m3 working volume of the reactor per day. This does not take into account the microbial biomass concentration within the bioreactor. Low rate filters as used in wastewater works are usually designed to produce effluents of high quality. They employ mineral support material (e.g. slag and granite), which develop a mature biological film within 424 weeks. These mineral low rate filters are normally circular or rectangular. Their depth is often restricted to 1.5-2.5 m, due to the dense nature of the support material and the associated construction costs. Most circular filters do not have diameters greater than 40m and rectangular filters are less than 75 m long and 45 m wide. Mineral support media usually have surface areas in the region of 80-110m2/m3 and a voidage of 45-55%. This relatively low void age can result in filter blockages, known as ponding. When operating such filters it is important that the biomass should not be allowed to dry out as it affects their overall efficiency. Therefore, recycling of clarified supernatant from secondary sedimentation is often required. Low rate filters operate with an OLR of 0.06-0.12kg BOD/ m3/day, a wetting rate of 0.5-4.0m3/m2/day, and an HRT in the region of 20-60 min. They remove 90-95% of the BOD, resulting in high-quality effluent. High rate trickle filters are often used for treating concentrated industrial waste-waters, acting as a ‘roughing’ process rather than a complete treatment, comparable with the high rate activated sludge process. They remove 50-80% of the BOD and their OLR is about la-fold higher than with low rate filters. To overcome problems associated with low rate filters (dense support material, low surface area for attachment, potential ponding problems, and limited depth), plastic support materials have been developed. These plastic materials are chemically stable, but are gradually degraded by light. Their low density reduces associated civil engineering costs and permits filter depths of 6-9 m, which minimize land requirements. In operation, a high voidage volume, normally greater then 95%, reduces ponding. The very large surface area for microbial attachment, normally in the range 100-300 m2/m3, also results in high biomass concentrations. This allows greater OLRs to be used, while maintaining good levels of BOD removal. However, the large surface area of the support material necessitates higher

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wetting rates, in order to keep the biomass moist. Recycling of clarified supernatant from the secondary sedimentation tank satisfies this requirement. Overall, high rate filters are defined as those where the operating OLRs are in excess of 0.6 kg BOD/m3/day, but this may reach as high as 10 kg BOD/m3/day. However, the higher the operating OLR, the lower the degree of purification attained. For example, depending on the nature of the waste, at an OLR of 1 kg BOD/m3/day, BOD removal efficiencies of 80-90% can be expected, falling to approximately 50% with OLRs of 3-6kg BOD/m3/day. Ok, now what happens after the secondary process? After the secondary process, the tertiary treatment is carried out.

•

Tertiary Treatment

Tertiary treatment is any additional treatment process designed to achieve higher standards of water quality. Disinfection systems such as ultra-violet or micro filtration remove any remaining viruses after secondary treatment and can remove up to 99.9% of faecal bacteria. Other tertiary treatments, called nutrient stripping, concentrate on nitrogen and phosphorus removal. Several techniques are available to remove dissolved salts from wastewater effluent, but all are quite expensive.

Ultra-Violet (UV) UV light systems appear to present no threat to the marine environment, since this treatment is non-additive (i.e. does not involve use of chemicals). Most water companies have a number of UV tertiary treatment schemes included within the current improvement programme. Physical (micro-filtration) Wessex Water opened Europe’s first ultra-filtration membrane technology plant in April 1998. Since then, a few more have been built around the country. Like UV, this treatment is nonadditive and removes the majority of harmful pathogens. Chemical This is an additional disinfection treatment that can be applied to both secondary and primary treated wastewater. The chemicals used may include sodium hypochlorite, per-acetic acid or ozone. None of these have been adequately tested to ensure their safety with regard to marine life and human health when used in the disinfection of wastewater. • Ok, can we now learn about the anaerobic treatment of effluents? The treatment of wastewater and wastewater creates a problem; that of the disposal of the by-products of this process. For an engineer, this problem may create the single most complex and costly process of the whole wastewater treatment process. Municipal wastewater treatment plants generate sludges as a by-product of the physical, chemical and biological processes used in the treatment of wastewater. Generally, this sludge must be subject to some form of treatment in order to alter its character. It may then be disposed of without creating health problems or further hindrance. This treatment has many objectives. First, to reduce the volume of excess material by eliminating the

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Background The first step in the wastewater treatment process is pretreatment. The purpose of this step is to remove large solids and grits through screening. The screenings by-products are disposed of separately from the other wastewater sludges. The next step in the wastewater treatment process is primary treatment, which involves the use of clarifiers and sedimentation tanks to settle particulates in the wastewater. Primary treatment removes approximately 50 to 65 percent of the suspended solids and 30 to 40 percent of the biochemical oxygen demand (BOD) from the wastewater. The sludge removed from this process contains mostly organic matter that is highly putrescible. Following primary treatment, the wastewater is subject to secondary treatment. In nearly all municipal wastewater treatment facilities aerobic microorganisms are used to biologically remove the remaining BOD and suspended solids. This effluent then passes through a secondary clarifier, producing a sludge consisting of nearly 90 percent organic matter. Overall, this sludge is composed of approximately 2 to 4 percent solids and if not treated correctly, it becomes highly odiferous. In most cases, the sludges from primary and secondary treatment are combined and undergo another form of treatment before their disposal. First, the sludge is thickened, by gravity or floatation, removing as much water as possible. Thickening may reduce the amount of sludge to as little as half of the original volume. The liquid effluent from this process is recycled back to the beginning of the treatment process. Once this has been accomplished, the sludge is subject to some form of stabilization. This process converts the organic solids to more inert forms so that they may be disposed of without causing health problems or further difficulties. The Anaerobic Digestion Process An anaerobic treatment system is a complex three-step process that produces methane gas (in addition to other products) from the biological digestion of wastewater waste. The first stage is the hydrolysis of lipids, cellulose, and protein. Extracellular enzymes produced by the inhabiting bacteria breakdown these macromolecules into smaller and more digestible forms. Next, these molecules are decomposed into fatty acids such as propionic, acetic, and butyric acid. This decomposition is performed by several facultative and anaerobic bacteria such as Clostridium, Bifidobacterium, Desulphovibrio, Actinomyces, 2.521

and Staphylococcus. Finally, methanogenic bacteria such as Methanobacterium, Methanobacillus, Methanococcus, and Methanosarcina digest these fatty acids, resulting in the formation of methane gas (Metcalf & Eddy, 457). The production of methane gas is the slowest and most sensitive step of the anaerobic digestion process because it requires specific environmental conditions for the growth of methanogenic bacteria. These bacteria can only digest effectively at a pH of 6.6-7.6, and if the growth of the acid forming bacteria is excessive, there will be an overproduction of acid leading to a decrease in the pH causing many problems. (Metcalf & Eddy, 457). Also, the methanogenic bacteria have a limited temperature range for optimum performance, usually in the mesophilic range (90 - 105 °F). Often this requires preheating of the waste before entering the digester (Owen, 203).

Anaerobic Digesters Utilization of Methane

Once the methane gas has been collected from the reactor, it must be cleaned and separated from other biogas constituents such as carbon dioxide, hydrogen sulfide, and excess moisture. Hydrogen sulfide is corrosive to metal piping and may damage gas engines and therefore must be removed by scrubbing the gas with an iron oxide sponge or a gas scrubber. Metal ions added to the sludge before anaerobic treatment can also reduce the hydrogen sulfide content, forming insoluble salts which are removed during digestion (ASD, 31). Although most carbon dioxide is stripped during the removal of hydrogen sulfide, additional carbon dioxide may be removed to reduce the total volume and increase the gas value. Removal of carbon dioxide is expensive and is only economically feasible when the gas is to be sold commercially. The most common method of carbon dioxide removal is absorption through a chemical or aqueous solution, as in a scrubber (Owen, 262). After cleaning and purifying the methane gas, it can either be stored for later use or used immediately. It can either be burned by direct firing or within a gas engine (internal combustion). Digester gas can be used as fuel for hot water boilers, water pump engines, blowers, and electric generators. It can also be used to fire incinerators or burned to heat the influent sludge during pretreatment. The benefits of this process are optimized when the gas is used on site; most commonly to heat the digester influent. Any excess gas that can not be used by the treatment plan can be sold commercially (ASD, 31). The wastewater is treated by using anaerobic microorganisms. These microorganisms digest the solid wastes into simple inorganic chemicals. The essential steps of anaerobic wastewater treatment are summarised below: 1. The wastewater coming from the drainage channels and other sources are collected in a large primary settlement tank. The soild and semi-soild waste materials present in the wastewater are allowed to settle at the bottom of the sedimentation tank. 2. After the sedimentation of solid and semi-solid wastes, the wastewater sludge is separated from the supernatant. The wastewater is then allowed to pass into an aeration tank. An air-compressor is attached to the aeration tank; it gives

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liquid portion of the sludge. The second goal is to decompose the highly putrescible organic matter into relatively stable or inert organic and inorganic compounds; thus allowing water to separate more easily. By using anaerobic digestion in the treatment of wastewater sludge, the overall cost of wastewater treatment is reduced and it also furnishes a considerable power supply. Although many sludge stabilization methods exist, anaerobic digestion is unique for it has the ability to produce a net energy gain in the form of methane gas, it optimizes cost effectiveness and minimizes the amount of final sludge disposal, thus decreasing the hazards of wastewater and wastewater treatment by-products.

FERMENTATION TECHNOLOGIES

proper aeration to the aeration tanks the aerobic bacteria thrive well and consume more and more organic matter present in the supernatant wastewater water. 3. After the processing of wastewater water, the water is properly tested to determine the amount of organic matter and ammonia found in the water and also to determine the BOD. The wastewater water has a low BOD, which indicates the purity of the water. 4. The purified wastewater water is then either discharged into a river or directly used for irrigation. 5. The sedimented coming from the primary settlement tank is allowed to pass into primary digester. The digester is concrete, insulated tank having the capacity ranging from 103 to 104 M3. The sludge is then allowed to remain as such for 20-30 days completing the anaerobic digestion. For this purpose, the digester is heated to 35 oC by using a heat exchanger. 6. During anaerobic digestion, biogas is also being released from the fermenter(digester). The gas is stored in gas cylinders and used to run engines, and as a source of electricity to work the heat exchanger and the air-compressor. 7. The digested sludge is then allowed to pass into the secondary digester. Here the sludge is allowed to digest itself under the cold condition for about 25 days. The supernatant solution released from digestion tanks is then allow to pass into the primary settling tank, while the solid waste is thickened and dried properly. The dried solid waste is used as manure for agricultural and horticultural crops.

Notes

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LESSON 28: BIOENERGY FROM WASTE

Learning Objectives In this lecture, you will learn

• • •

Biomass for energy production Biogas production Hydrogen gas production

Introduction Energy is the source of economic growth. Energy consumption reflects the state of development of the country. Energy requirements of man on the whole are believed to be doubling every 14 years. Energy sources fall into renewable and nonrenewable categories. Energy has been derived from various conventional and non-conventional sources like coal, oil, natural gas, nuclear, solar, wind power, tidal, biogas etc. The sources which are exploited for obtaining energy differ in different countries. In India 40% of total energy requirements are supposed to be met by non-commercial sources like firewood, agricultural waste, cow dung, animal power and manual labour, Fossil fuels like coal, gas and oil are exhaustible and nuclear energy has its own limitations. So it will not be surprising if we are forced to shift our attention to solar energy, biomass, biogas and the similar. Biotechnology can make a lot of contribution to increase the acceptability of biomass, biogas, and fuel alcohol as feasible, viable energy options for the future. Advantages of bioenergy generation will be ecofriendly, less polluting, cheap, plenty etc. If bioenergy generation is coupled with the tapping of unutilized biomass, wasteland utilization for biomass or treatment of solid liquid wastes pollution abatement, resources utilization will simultaneously be achieved.

Wood Fuel While3 x 1024 J/a solar energy is received by earth, the total estimated proven reserves of oil, coal, natural gas, uranium are only equivalent to 2.5 x 1022 J. Thus, in one week, the earth receives an amount of energy equivalent to total reserves of non-renewable energy. In other words, energy in one day’s sunlight is equivalent of 1/5 of the known reserves of fossil fuels.

Development in reactor designs, gene manipulation in microorganisms has made the task easier and bioenergy from wastes has become a reality when we see the number of applications in the field.

Biomass for Energy Production Although there is enough of energy in our surroundings to meet all conceivable needs, we do not have the right technologies to tap all of them. Most of our energy today is derived from sun, including wood, coal, oil, natural gas and even wind and electric power.

Wood Renewable Energy Cycle By photosynthesis, solar energy can be converted into biomass which in turn can be stored and used as fuel in various forms. So far, plant material was mainly used as fuel wood for burning. 2.521

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Currently a number of thermal conversion systems are in various stages of development. These include pyrolysis, gassification and hydrogenation.

(7) Low conversion efficiency (% solar energy trapped).

For direct burning, moisture contents should be less than 30% Advanced fluidized bed incinerators can take biomass of even 55% water. wood and straw is normally used for combustion. Animal wastes sewage sludges, compost sludges which contain upto 75% water can also be used. pyrolysis, liquification and gassification are upgrading processes converting biomass into stable, transportable fuel, solid, liquid, gaseous forms thus produced have similar properties as coal, oil and natural gas. All these processes require feedstock of relatively low water content and operate at a higher temperature. Biological conversion processes, however, can handle feedstock of high water content and operate at a temperature range 25-65°C.

Sources of Biomass Sources of biomass for fuel conversion included: 1) Land and crops - lignocellulosic material from trees of Eucalyptus, like maize, cassava and sugar crops like cane and beet. (2) Aquatic plants - Unicellular algae, multicultural algae, aquatic weeds like water hyacinth, Hydrol etc (3) Wastes like manure, domestic rubbish, and municipal waste/sewage (4) Agro-industrial wastes - wood and crop residues like straw, husks, citrus peels, bagasse molasses, willow dust, press mud, paper sludge etc. Biomass as a source of energy has its advantages and disadvantage which decide whether solar energy - biomass utilizable energy route can be exploited or not. But as said above, biological conversions are easier than other technologies and if environmental pollution control is simultaneously achieved, then the utilization of biomass for energy has a heavier side in balance. Thus, various agro-industrial wastes as biomass definitely suits the bioconversion processes for energy and chemicals. Advantages of biomass as a source of energy: (1) Storage is possible; (2) Transportation possible; (3) It is renewable; (4) High energy fuels can be obtained; (5) Low capital input required; (6) Can be developed with present man and material abilities; (7) It is ecologically safe and is inoffensive; (8) It does not increase CO2 content of the atmosphere. Disadvantages to be listed are: (1) Land and water use competition; (2) Solar energy a source of biomass is diffuse and intermittent; (3) Collecting and storing it is bulky and costly; (4) Supply uncertainty initially; (5) Costs uncertain; (6) Fertilizer, soil, water required;

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Reactors Using Immobilized Cells for Biomethanisation

Immobilized cell anaerobic reactors are now used in more numbers for treating industrial waste-water. This is due to their capacity to retain biomass and better performance. Following systems are available in immobilized cell technology with anaerobic conditions: (a) Up flow anaerobic sludge blanket reactor (UASB). (b) Hybrid reactors. . (c) Up flow fixed film anaerobic filter. (d) Down now fixed film anaerobic filter. (e) Expanded bed reactor. (f) Fluidized bed reactor. It is difficult to compare the above systems as far as superiority is concerned. Low-loading rates or low biogas production do not necessarily reflect poor reactor design. The amount of active biomass decides the loading capacity and subsequent biogas production and also waste-water depollution. Immobilized cell anaerobic reactors are only able to treat wastewaters with low concentrations of particulate material. Agro-industrial waste-waters and to a lesser extent, diluted and filtered animal manures can be used as substrates. The development of stable associations of micro-organisms is required for methanogenesis, Concentration of biomass in the reactor and minimum hydraulic retention time are achieved which result in a smaller reactor volume and reduced investments. Biogas Production from Food Processing Industries

Effluents from food processing industry are most suitable for biogas production. These effluents have a high BOD due to easily biodegradable organic matter that they contain and hence are of immediate concern while releasing into the water bodies. The overall trend of industries to conserve energy and if possible, to generate it from wastes has increased anaerobic digestion of effluents in general. Milk processing unit’s wastes could be processed to cause 99% reduction in BOD and gas production 0.85m3 kg.l BOD in 6 days’ retention time. Disposal of whey is the most serious problem for the cheese manufacturing units. One tonne of cheese gives rise to 10 tonnes of whey. Each cubic meter of whey produces approximately 38 m3 of biogas. One m3 of whey is equivalent to

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For vegetable canning wastes (carrot peelings) with a two stage reactor (liquefaction separated from methanogenesis) 2.4 m3 methane/m3 digester/ day is produced while with a one stage system, only 1.35 m3 methane/ m3 digester/day can be produced. Waste-waters from manufacture of wheat gluten, starch from flour contain proteins, carbohydrates, mixture of amino acids, hemicellulose, pentose gums, suspended starch granules etc., and COD is 10000-25000 mg I-I with a loading rate of 2.6 kg COD/m2/day. Methane yield is 0.33 m3 per kg of COD. Methane content in the biogas is 65% and BOD reduction achieved is 95%. Size of the plant is 1000 m3. In citric acid production from molasses using Aspergillus niger for fermentation 18-20 tonnes of wastes-water is produced for every 1 tonne of citric acid produced. COD of this waste-water is 30000 - 50000 mg per liter. Biogas can be produced from it and COD reduction achieved in the treatment.

Substrate Organic fraction municipal solid waste Vegetable processing waste Brewer’s and grains Distillery wastes(fruits) Slaughter house waste Pressed grape skin Mown grass Fats from skimmined tanks

Biogas l/Kg 450 600 500 550 450 400 600-700 1000

In India M/S Ashok organics treat their distillery wastes to generate biogas. With a residence time of 8-10 days pH of digester 7.2 and temperature around 48-52°C, 40 to 50 M3 biogas is generated for M3 effluent that is treated. BOD reduction achieved is 80-85%. The Sakthi Sugars Ltd. uses French and Italian technology and carries on in fixed fIlm reactor biomethanisation to achieve 90% BOD reduction or 6570% COD reduction and biogas production. Each tonne of COD reduced produces 530 m3 biogas. Upflow anaerobic filter and gas collection system can be used for soluble carbohydrate wastes for example for soft drink bottling industry, as a pre-treatment process. COD can be substantially reduced, and methane is produced. Filter operating at 41 hour HRT can remove 85-90% of soluble COD.

From the Fruit pulp and apple juice the united states produces 1.5 million tones of pomance every year and that requires 10 million for the disposal. The Cornell University, N.Y., U.S. has developed an anaerobic process for the treatment of pomance and returns in the form of gas expected are 10-30 dollars per wet tonne of pomace. In citrus processing industries peels can be used for anaerobic fermentation. But first oil is removed from them since it is inhibitory to microbes 0.5 m3 biogas can be produced per 1kg total solids. Wastes from bean leaching, pear and potato peeling can be used for biogas production with anaerobic contact process or UASB as the bioreactor. Table: Industrial Anaerobic Treatment Plants Waste Molasses Starch Sugar beet Potato Dairy Yeast production Papemill

Anaerobic fluidized bed biofilm reactor has been used for acid whey, soft drink bottling waste-water, whey permeate molasses, municipal wastes etc. COD removal efficiency is >90%, load rate is 35 kg/m3/day and 332I methane produced per kg COD removed.

Reactor Type CSTR CSTR/USAB CSTR UASB UASB Filter UASB

Table : Biogas Potential of Food Processing Wastes

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daily pollution by 600 people. The strength of whey is 3200060000 mg BOD 1.1.35% of the costs of cheese manufacturing unit could be recovered from biogas generated from effluents. Biogas produced is 1500 m3/day to 4000 m3/day depending upon whey production. Methane contents of biogas produced are 62%. Biogas produced can be used for boilers or for generating electricity (generators using biogas for electricity generation are available) or after treatment and compression, it can be used for vehicle propulsion or can be sold to other users.

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Jute caddis, the unspinnable short fibres deposited by jute mill looms can generate biogas when fermented. Jute caddis is lignocellulosic waste. India produces 0.28 million quintals of this material and is used as boiler fuel or wasted and causes pollution. The Calcutta based Jute Technological Research Laboratory (ITRL) has produced biogas using 2.5% of this material, in 20 days. If alkali-treated caddis are used instead of raw caddis, the same is possible in 15 days. The remaining slurry after biogas production is rich in N,P,K nutrients and is comparable to the farm yard manure. Alkali treatment of caddis helps solubilization of lignocellulosic material. hence time of fermentation is reduced.

Biogas production from poultry manure of large farms is ecologically and economically effective technology. 40% COD reduction takes place with 1m3 biogas produced for every 1 kg of degraded organic matter with 15-40 days retention time. It removes aggressive odour, reduces number of pathogens, and converts organic nitrogen to ammonia. Agricultural firm ‘Ogre’ in the USSR uses l00m3 bioreactor with pig slurry for fermentation. Biogas production is 0.5 m3/kg dry organic matter/day. It contains 65% methane. Yields are 2.6 m3/mJ sludge/day There is another 50 m3 bioreactor at agrofirm ‘Uzvara’ in the USSR which produces 15-20 m3 biogas/m3 brown juice with 70-80% COD reduction. Biogas production is a convenient way of agricultural wastes disposal for more than one reasons. Substrate detoxification, deodorization, inactivation of pathogens, dehelminthization Occur along with biogas production and fertilizer or humus forming substance as a byproduct. Rapid production’ of methane from chicken manure by microbes immobilized on ceramic and placed in continuous plug flow reactor is feasible and has been operated for 9 months continuous. A carrier with porosity between 2-35.!J. pore diameter is better. Piggery wastes produce maximum biogas when compared with other animal wastes and it is observed that 60% of organic substances could be converted into gas from the pig manure. Sheep manure, silkworm waste produced less gas, may be due to more nitrogen content of these wastes. Cattle dung Sheep manure Piggery wastes Poultry wastes Water hyacinth Soyabean waste

%Dry Matter 17 34.5 57 57 13.4 17

Biogas Produced 14,615 cc/21/6 month 125700cc/21/6 months

In such examples. the question is not as to how widely such sources can be used for biogas production but the suggestion is wastes (causing pollution) from whatever source can be disposed of for generating energy while simultaneously reducing environmental hazards. . Similarly, the textile industry in India generates willow dust which is one of the solid cellulosic waste material produced during the processing operations. 30000-33000 tonnes of willow dust is generated per year by the textile industries in India. Composting, direct burning and anaerobic digestion for biogas production are the .three alternatives available for the disposal of willow dust. Biogas production from willow dust was first demonstrated by Cotton Technological Research Laboratory (CTRL), Bombay. Willow dust contains celluloses, hemicelluloses and C:N ratio is 25:1. Plant producing 17m3 biogas from 100kg willow dust in 30 days is operating satisfactorily. A large scale trial was taken by the Apollo Mills, Bombay. With the help of 6 digestors, 12 tonnes of willow dust were digested per month. 350 m3 biogas was obtained in each digester handling 2 tonnes of willow dust with 90 days retention period. Slurry obtained after digestion serves as good manure With a modified process requiring less water (H2OP to substrate ratio 1.5:1), 250 m3 biogas can be obtained from 1 tonne of willow dust in 60 day. Anaerobic digestion by the BiotimR System in Malaysia is the 1st full-scale application for rubber factory effluents. These effluents have high sulfates and ammonical nitrogen which are inhibitory to digestion. Smell problem in the ponding system and high operation cost in oxidation ditch hence alternate anaerobic system is set up. Loading rate is 18.5 kg COD/m3 day. Hydraulic Residence Time(HR1) is I and 1.85 days respectively in BCR (Biological Conditioning Reactor) and MOR (Methane Upflow Reactor). 70% COD is removed and biogas yield is 580 m3/day.

184 72 cc/21/6/months 45 lit/kg dry matter/2month

Earlier agricultural wastes, animal wastes, food industry wastes were primarily thought suitable for biogas production. But the range of possible wastes for biogas generation is continuously increasing.

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H2 gas has 3 times calorific value per unit weight of petroleum and it does not generate CO2 (a green house effect gas) or other air pollutants. Energy conversion can be increased almost 20%.

Table H 2 Production from Waste-water Source of waste water Alcohol factory Refinery Straw paper mill W.W. containing organic acid Icecrem and butter factories

Organisms Cl. butyricum Rhodopsedomonas palustris Rhodospirillum molischianum Rhodopseudomonas rubrum

1. Exercise: AARTI ,an institute involved in the development of sustainable technology for rural development has developed a mini bio gas plant that can be operated on domestic waste. Find out more about it and write a report. 2. Exercise: what do you know about biodiesel? Write a report.

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Hydrogen Gas Production Hydrogen gas is a perfect renewable fuel. It is produced by sun. Raw materials used for the production can be water which is abundant. Hydrogen when burnt does not cause any pollution but regenerates water. Hydrogen production process operates at a normal temperature. No toxic materials are produced in the process. It is the cost factor which has prevented hydrogen from becoming a common fuel. Coupling of solar energy for H2 production using stabilized photo synthetic components and enzyme hydrogenase is the system. The enzyme responsible for making hydrogen gas (joining two hydrogen ions and two electrons to form a molecule of gas) is hydrogenase. This enzymes so far has been extracted from 15 species of bacteria and algae. Attempts to commercialize it are on. Clostridium butyricum when supplied with sugars produces hydrogen but the system is unstable. After a while, bacteria stop making hydrogen. Japanese biotechnologists have immobilized Cl. butyricum and these cells produce gas for a month instead of a few hours when fed with wastewater containing sugar from a alcohol factory. Anaerobic packed bed reactor and agar entrapment is used for the purpose. Rhodopseudomonas palustris has been grown in anaerobic bioreactor. Plat~ of agar immobilized organisms are used. The system is easy to operate and build 0.78 lit of production from sugar refinery wastes and 2.2 l/lit H2 from straw paper mill effluent is reported. Current 13-15 mA was generated for 20 days. Rhodopseudomonas rubrum gives H, production from waste. water containing organic acids (acetic, propionic, butyric). Organisms can be irnmobilised with alginate. . Rhodospirillum molischianum gives 2.6 1/lit of H, production when. straw papermill waste-water is used. H2 produced can’ be used for fuel cell and current generation of 0.5 - 0.6 A or 0.16 0.18 V for 6 hours could be produced. Scientists at NEERI have identified the photosynthetic bacteria that use solar energy to generate H2 from waste-waters from ice creams and butter factories. With this technology, H2 production is coupled with removal of pollutants. The gas can be used along with others to form enriched fuel or in edible oil industry for hydrogenation of vegetable and animal fats. The gas has immense potential for use as a chemical feedstock in the production of NH3, methanol or other chemicals. NEERI proposes to set up a plant to process 20m3 of whey waste to produce to m3. Rhodopseudomonas gelatinosa produced more from organic acid than from sugars like glucose, sucrose, lactose 50 ml of H2 is produced per gram of total organic carbon used. Technology comprises initial pretreatment of Whey waste which involves neutralisation pasteurisation. Preheated waste is then transferred to bioreactor containing anaerobic photosynthetic bacteria Japan’s Fermentation Research Institute and Agency of Industrial Science and Technology have jointly developed a system to efficiency produce H2 gas. Bacteria used for the system is Rhodobacter sphyroid (photosynthetic bacteria).

UNIT-5 BIOSAFETY & FUTURE OF FERMENTATION TECHNOLOGY

LESSON 29: BIOHAZARDS IN FERMENTATIONS

FERMENTATION TECHNOLOGIES

Learning Objectives

workers. Similarly, the size of the aerosol particles decides the portal of entry.

In this lecture, you will learn

• • •

Product & worker safety Aerosol generation & management

Introduction Biotechnology, and especially fermentation technology appears to be a very safe science compared to its other sister branches in terms of the hazards and risks associated. Unfortunately, it is not always so. As a matter of fact, the hazards posed during fermentation technology could actually be more dangerous than most other branches of science.

•

How? Let’s see what makes an aerosol dangerous. First is the duration for which the aerosol can remain airborne. Lighter aerosols can remain freely suspended in the air for longer durations of time, and hence, are more hazardous. Similarly smaller aerosols can penetrate deep into the respiratory tract and are able to cause more serious infections. The size of aerosol also decides the ease with which the aerosol can be removed and the survival and infectivity of the organism involved. In a nutshell, smaller and lighter aerosols are more potent in causing infections and allergic symptoms amongst the workers.

•

What are biohazards?

Why fermentation technology is hazardous and what are the hazards associated with it?

There are three distinct types of hazards associated with fermentation technology. 1. Safety to the workers 2. Safety to the product and 3. Safety to the environment

How are aerosol generated? Handling of microbial suspensions, fermented broth in this case, results in the release of aerosol in the air. Large aerosols are generated when low energy operations are performed. Smaller and hence more dangerous aerosols are generated during high energy operations like cell disruption, centrifugation, lyophilization etc. which are common during downstream processing. Spray factor is an indicator of aerosol generation by a particular operation.

•

What is spray factor? The spray factor is defined as the number of viable organisms released in the air per minute divided by the number of viable organisms being handled per minute. The spray factor is an indicator of aerial contamination levels by common industrial operations.

We will see these hazards one by one. Probably the most important safety hazard by fermentations is posed to the workers who work in the fermentation industry. The various aerosols that are released during the fermentation operations pose the major hazard to the workers. These aerosols affect the workers by three portal of entry.

•

1. Inhalation

•

2. Ingestion

Four major operations associated with industrial fermentation have been identified as potent aerosol formers.

3. Skin contact Of these three, inhalation is most dangerous. Why? An average industrial worker, who works for eight hours in a shift, inhales about ten cubic meter of air. Considering that the level of aerosols is only one part in one hundred million, then the worker inhales about 0.1 mg of aerosol. This is a very large dose and is more than sufficient to cause airborne infection and/or allergic reactions in the workers. Interestingly, it is the portal of entry that decides the extent of hazard posed by the aerosol.

•

How? Let’s take the example of an organism Francisella tularensis. This organism, if inhaled causes pulmonary disorders. If ingested, the same organism causes typhoidal diseases and if it comes in contact with the skin, it causes infection of bubonic form. Thus, the route of entry of an aerosol, generated during the fermentation process, decides its degree of hazard to the

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What are the major industrial operations that generate maximum number of aerosols?

1. Breakdown of bacteriological factors. 2. Failure in antifoam system. 3. Failure in culture transfer pipe work. 4. Explosive breakage of fermentor. Of these four reasons failure in antifoam systems is considered to be the most dangerous.

•

Why do you think it would be so? Use the space below to justify your answer.

•

·How do we take care of these aerosols? Aerosols generated during industrial fermentations can be taken care of by two basic techniques.

Aerosol Removal This technique involves the physical removal of aerosols in the defined area. The settling of aerosols by inertial separation, filtration, scrubbing and electrostatic precipitation are some of the techniques applied here.

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1. Hazards involved during sterilization of the media, handling of heat, steam etc. 2. Hazards due to solvents inflammable chemicals and other toxic/allergic chemicals. 3. Hazards due to the fermentation media ingredients, especially in causing allergic reactions. 4. Hazards due to the handling of laboratory chemicals with reference to their toxicity and carcinogenicity.

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5. Hazards due to the exposure to microorganisms. – this is probably the most important occupational hazard and is further classified into five major types.

•

Infection by a pathogen: Pike in 1979 reported 4079 cases of infection and 168 fatalities due to infection by a pathogen. Medical laboratory staff in United Kingdom has been shown to have seven times more incidences of tuberculosis and three times more incidences of unspecified diarrhea and hepatitis. Aspergillus and Pseudomonas have been shown to cause lung infection Aspergillosis and cystic fibrosis especially among immunocompromised industrial staff.

•

Generalized allergic reactions: inhaled allergens have been found to bind with IgE resulting into the release of histamines and other vasodilators. This causes allergic reactions inflammation of skin and nose irritation. Allergic reactions also include constriction of airways, difficulty in breathing and other asthma like symptoms. As we are aware, this allergy can be immediate or delayed type. While it is easy to find out the causative agent of immediate type of allergy, it is not so in case of delayed hypersensitivity due to the time elapsed between the contact of the organism and the appearance of symptoms. Sometimes, the same allergen can cause both immediate and delayed types of reactions, further complicating the diagnosis.

•

Allergy to biological products: Pollens, wood dust, soya bean flour are common allergens. Bacterial proteases commonly used in washing powders are a cause of respiratory disorders among workers. These disorders include wheezing and breathlessness, cough, chest pains, fever and malaise. Continued exposure to an allergen can result into reduction in lung capacity. Regular monitoring of lung capacity with the help of a simple spirometer can help in early detection of these symptoms.

•

Allergic reactions to microorganisms: There are several microorganisms that can dangerous allergic reactions among industrial workers. Common among them are Actinomycetes used in composting, Aspergillus spp. Used in various fermentations, Penicillium spp used in citric acid production and cheese making, Candida spp. used in the production of proteins. It has been observed that 4.9% workers from citric acid producing units are suffering from asthmatic symptoms. The recovery of fungal mat at the end of the fermentation generates a large number of aerosols especially consisting of spores of Aspergillus. These spores have been recovered from the lungs of the workers, although no colonization was found. The level of microorganisms required to cause sensitization depends on the length of exposure, particle size of the allergen and the concentration of other pollutants in the air.

•

Endotoxin reactions: Lipopolysaccardides from the cell wall of Gram-negative bacteria are potent endotoxins. E.coli cell wall accounts for 3-4% of the cellular dry weight. These endotoxins are extremely heat stable and may require a treatment above 180 0C for over 3 hrs for inactivation. These conditions do not exist either during the fermentation or the downstream processing. Bacterial endotoxins can cause pyrogenic reactions when injected into the blood. Severe

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Aerosol inactivation Aerosols released in the air, can be inactivated by application of heat, irradiation and various disinfectant chemicals. But the inactivated aerosols can still act as allergens amongst the workers causing immediate or delayed hypersensitivity. • OK. Then what are the various factors that affect the survival of microorganisms in the aerosol? There are several factors. Let’s study them one by one. Stability of the organism. - Microorganisms from stationary growth phase have low metabolic activity and hence are better suited for survival in aerosols. The method of culture and the composition of growth medium also affects the survival of organisms in the aerosol. Whether the culture is grown in batch type or continuous type also makes a difference. Particle size - particles of 1 um size are 800 times more potent in causing infection compared to those of 12 um size. However, survival is better in larger particles. Relative Humidity/Temperature- Bacteriophages survive better at high relative humidity, whereas, the same causes surface damage, leakage of ions and decreased DNA, RNA and protein synthesis in bacteria. The survival of Bacteriophages at different relative humidity depends on the composition of the humidifying liquid. Oxygen – Bacteria and algae survive poorly at higher oxygen concentration, especially if they are in their logarithmic growth phase. This is supposedly because of the inactivation of cell division process at higher oxygen concentration. Bacterial spores, Bacteriophages and viruses are not affected by the oxygen concentration in the air. Sunlight and Ultraviolet light – many organisms, especially bacteria are sensitive to thermal and UV light inactivation. Ultraviolet light causes severe damage to the nucleic acid of many microorganisms, especially bacteria. Some bacteria though can resist this damage by what is known as “dark repair mechanism.” Protecting factors- Several media ingredients have been found to offer protection against inactivation to the organisms. These ingredients include spent culture media, di and tri sachrides, raffinose, dextran, glucose, glycerol, sorbitol, polyhydric alcohols, inositol and sodium glutamate. Presence of these ingredients in the aerosols render better survival to the organisms involved. • What are the major health hazards involved in fermentation biotechnology? These hazards can be classified into five major classes.

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allergic reactions are found among workers who were exposed to the inhalation of these endotoxins. There is also a linear relationship between the reduction in lung capacity and the concentration of airborne endotoxins. Following organism are found to be involved in the production of endotoxins: Enterobacter agglomerans - cotton milling

the blood. Provision of airline hoods and gloves was found to reduce this problem. Workers in fermentation units manufacturing penicillin, streptomycin and tetracycline have been found to suffer from increased incidences of candidiasis and general gynecological problems.

•

What could be the occupational hazards a worker in a distillery is exposed to? Use the space below to justify your answer.

•

What are the hazards posed by genetically modified microorganisms (GMMOs) to the safety of workers?

Flavobacterium spp- humidifiers Methylophilus methylotrophus- SCP production Methylomonas methanolica- SCP production Pseudomonas aeruginosa- downstream processing Serratia marcescans- Military research The endotoxins are found to give rise to different types of clinical symptoms in Gram-positive and Gram-negative organisms. In Gram-negative organisms, exposure to endotoxins results in kidney and stomach pains, conjunctivitis and aching limbs. It is also seen that previous exposure to the endotoxins is not necessary for the appearance of these symptoms. In Gram-positive organisms, the symptoms are more localizes and include rhinitis, asthma, dermatitis etc. in this case, pre-sensitization is necessary for exciting this kind of allergic response. Case study: A Gram-negative organism, Methylophilus methylotrophus was used for SCP production in the ICI plant in the United Kingdom. Workers complaints included headache, aching limbs, chest tightness and shivering. Sore eyes with discharge were also seen. Similar examples were found elsewhere in the world. The reason for these complaints was narrowed down to the endotoxins produced by Methylophilus methylotrophus. Prevention of particle formation, providing protected clothing and eye protection was found to reduce these complaints considerably. Gram-negative organisms are often involved in the production of intracellular enzymes. For the harvesting of these enzymes cell rupturing is needed. This is a high-energy operation and releases fine aerosols in the air. It has been found that a concentration as low as 30 nanogram of endotoxins per cubic meter of air can give rise to allergic reactions.

•

All right. Are there any other toxic reactions caused by the fermentation products or byproducts?

Yes, there are. Interferons and hormones produced by genetically modified microorganisms (GMMOs), when inhaled even in very very small amounts can have disastrous effects on human health. In an example of a company manufacturing oral contraceptive hormones, workers were found to suffer from increasing menstrual problems, loss of libido, and gynaecomastia (growth of mammary glands in male workers.) The solution offered by this company is the shifting of these workers to another department and the recruitment of postmenopausal women for this job. Obviously this is not practical in all such industrial units. In another example, where barbiturate being produced by fermentation, the concentration of the product in the blood of workers was found to be half the therapeutic dose. This was due to inhalation of barbiturate aerosols and their entry into

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There is a fear of worldwide pandemics and ecological imbalance caused by genetically modified microorganisms (GMMOs) especially in the developed countries. Discharge of organisms through effluents and aerosols is particularly objectionable. It is however, noteworthy that enough precautions are taken to avoid such catastrophic consequences even if such organisms are discharged in the environment in exceedingly high quantities. All GMMOs are fitted with a genetic switch, which makes them impossible to survive for longer periods of time outside a laboratory. It is therefore not surprising that more and more acceptance is being gained to GMMOs. In the baking industry, UK has approved the use of modified yeast. The genetically modified animal cell culture however, presents a very potential hazard to the safety of the workers.

•

How?

Firstly, most if not all, products produced by animal cell cultures, are proteins, used for therapeutic use. Exposure to these products, as we have seen, can be extremely dangerous. Secondly, genetic modification is carried out in animal cells for producing these products in higher quantities, making them even more unsafe. Thirdly, despite the strict screening offered during the initial stages, it is possible that the animal cells, especially from primates, may carry infectious viruses and oncogines. The importance of these can not be overemphasized as many primates share their sensitivity spectrum with human beings. Last but not the least, most animal cell culture media employ the use of serum from animal origin. This serum can carry micoplasmas and other infectious agents that can cause infections in human being. Strict screening of all cells used for culture can minimize these hazards. Since the products are highly bioreactive extreme care should be taken during the downstream processing. The use of mechanical barriers or air curtains is strongly recommended. The hazards posed by plant cell culture are similar and product specific.

•

What are the various hazards of bioprocessing equipments?

All the bioprocessing operations involved during fermentation, downstream processing and product recovery are capable of posing serious health hazards to the workers. Fermentation–large volumes of fluids, containing potentially allergic microorganisms and biochemicals are handled during fermentation. The air inflow and outflow, agitation, impeller

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Centrifugation–If sealed buckets and rotors are used for centrifugation, no aerosols are generated. However, the manual removal of cell paste and the poorly functioning seals in the centrifuge are seemed to generate high quantities of aerosols. It must be emphasized that industrial centrifugation is highenergy operation and hence, as seen before generates aerosols of smaller size. We have already seen that these are more dangerous as compared to their larger counterparts.

7. To formulate and implement local codes of practice for the safety of the personnel.

Notes

Cell disruption–Like centrifugation these are also high-energy operations and hence generate fine aerosols. Again, poor quality seals are found to be the reason for aerosol generation. The cell disruption and homogenization of gram negative bacteria presents an additional hazard due to the endotoxins present in the bacterial cell wall. Filtration–Gravity filtration is a low energy operation and hence is less hazardous. Use of rotary vacuum filters with violent removal of biomass and removal of microbial mats from the filter generates extremely potent aerosols. Product handling–It is often assumed that this is the safe area as compared to the production of downstream processing of bio products. Conversely, this is the most hazardous area. First, because the product is in its most concentrated and bioreactive form when it reaches this area. Second, because mainly the product is in the form of dusty solid, rapid aerosolisation takes place. The spray dryers and freeze dryers often generate heavy aerosol dose.

•

How do we prevent the various hazards in fermentation biotechnology?

This can be done on many levels. It can start with the selection of least hazardous products for manufacturing. Because of commercial reasons this may not be always possible. The second line of defense comes from proper training of the staff and good housekeeping. Regular health surveillance of the workers including skin test, lung function test etc. is not only legally binding but also is a good idea to reduce the health hazards. Ager and Nourish in 1988, laid down principles of occupational hygiene, which are relevant to all industrial operations, especially involving biotechnology. These principles run as follows:1. To keep workplace and environmental exposure to any physical, chemical or biological agent to the lowest practicable level. 2. To exercise engineering control measures at source – to support this with appropriate personnel protective clothing and equipment. 3. To test adequately and maintain control measures of equipments. 4. To test, when necessary, for the presence of viable process organisms outside the primary physical containment. 5. To provide training of personnel. 6. To establish biological safety committee or subcommittee as required.

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operation, sampling valves, preparation and mixing of media ingredients are some of the critical areas. Filtration and the clogging of filters often present questionable safety practices.

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LESSON 30: CONTAINMENT IN FERMENTATION

Learning Objectives In this lecture, you will learn

• • •

Types of containment Safety cabinets - design Safety cabinets - applications

In the last lesson, we have seen what are the various hazards posed by the biotechnological operations, especially fermentations. In this lesson, we will study the processes and equipments used to control these hazards. This control of biological agents and their products is called containment. It is, in the first place, important to appreciate that it is IMPOSSIBLE to avoid the hazards altogether. Attempts can, however, be made to keep these hazards under acceptable limits. We must also understand that in case of living microorganisms, due to their ability of multiplication, it is necessary to limit and prevent their release in the atmosphere. In case of microbial products, however, it would be sufficient to limit their release within acceptable norms. One must also understand that the containment is needed right from the generation of seed culture to the effluent treatment.

•

What are the various levels of containment needed in fermentation?

Imagine a prison in which some highly dangerous prisoners are kept. Obviously, no stones are left unturned to prevent these prisoners from breaking the prison and escaping into the society. What are the different methods adapted for this? First and foremost, the prisoners are kept locked in individual cells. This would prevent most of them from even making an attempt to get out. Secondly, there would be a tall and barbed fence around the entire prison. Then probably, there would be armed guards patrolling around the prison offering additional security.

Containment in biotechnology can be compared with this example. There are three levels on which biological containment is offered. 2. Primary containment : This is the first line of defense against the proliferation of microorganisms and their products. This is also the immediate physical barrier to their uncontrolled release. The filters, caps and mechanical seals present at various levels offer the primary containment. Failure or breakage of this primary containment measures can directly release the organisms and their products in the surroundings. 3. Secondary containment: These are the back up designs to work in case of failure of primary containment. Secondary containment is offered by the physical enclosures and includes the use of working hoods, tables etc. 4. Tertiary containments: this includes the use of defined operational facility like directional air flows and air filters.

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In addition to these three levels of containment, there are specific measures to prevent the proliferation of hazardous microbes and their products. Personal physical protection equipment like gas masks, gloves, shoes, goggles etc. is one such example. The genetic switch incorporated in the genome of GMMOs which renders them incapable of growth outside specified laboratory conditions is another example. Further, temporal elements for a specified situation like fumigation, disinfection and use of antimicrobial agents offer additional containment. It is important to note that the purpose of containment in fermentation operations is to offer protection not only to the workers, but also to the product. No doubt, the health and the safety of industrial workers is of paramount importance and must come before anything else. The safety of the product being manufactured is also important. We have seen that many products produced today by fermentation are of high commercial value. Their contamination by other products has serious commercial implications. All attempts therefore should be made to keep the purity of the final product as unaffected as possible.

•

Tell me more about the safety cabinets. In case the primary containment fails, secondary containment is offered by the safety cabinets. All these units are compact metal or plastic boxes. Depending on their level of sophistication, British Standard (BS 5726, 1979) has classified them as class I, class II and class III cabinets. Let’s see what these cabinets are one by one.

Class I cabinets – These are open-fronted cabinets that operate with negative pressure ventilation and have a minimum inward air velocity at the front opening of 0.75 m/s. Exhaust air passes through a high efficiency particulate air (HEPA) filter before being exhausted to the outside. Class I cabinets are intended to protect workers carrying out simple routine microbiological operations and to prevent dissemination of possibly hazardous materials from the immediate work area.

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•

What are the drawbacks of Class I cabinets? We must remember that Class I cabinets are designed for basic biotechnological operations only. They do not protect materials within it from possible external airborne contamination. Also Class I cabinets do not provide total containment. A protection factor, defined as the ration of exposure to airborne contamination generated in the open to the exposure from the same dispersal generated within the cabinet, is required to set a minimum standard for containment. For BS 5726, this factor should not be less than 1.5 multiplied by 105 . Where high-energy processes, such as centrifugation, are involved, particles of hazardous materials might be projected out of the cabinet against the airflow. Hazardous materials might also escape on removal of the gloved hand of an operator or as a result of spillage. Care must also be taken to prevent undue perturbation of the airflow into the cabinet by inappropriate positioning of equipment within the cabinet or by external air movement caused by personnel movement, opening doors etc. Release of airborne material through the work opening is almost inevitable should the exhaust air system fail. For these various reasons, Class I cabinets are not suited to contain many biotechnology process operations.

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Ok, now, how are Class II cabinets different from Class I cabinets?

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It is not always appreciated that Class II cabinets may not act as effective safety cabinets in so far as worker safety is concerned; therefore in the UK their use for category 3 and 4 pathogens is strictly forbidden. As with Class I cabinets, the internal air flows are subject to perturbation by cross draughts, the formation of air bulges at the working opening and movement within or adjacent to the cabinet. Hence these cabinets are considered as inappropriate for containing biotechnology process operations involving hazardous materials.

•

So it is the Class III cabinets that are totally safe, right?

Correct These are totally enclosed, ventilated cabinets of gas tight construction and are designed to separate the worker from the cabinet interior at all times (Figure no. 3). The cabinets have

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Class II CabinetsThese are open fronted cabinets that not only have inward air movement (75 ft/min) at the work opening but also provide HEPA filtered laminar airflow within the cabinetwork space ( see the figure below). The cabinets are intended to provide operator protection, as for Class I cabinets, whilst protecting materials within the cabinet from external airborne contamination. In these cabinets there is a downward airflow of HEPA – filtered air over the work surface to which is added air from the work place that enters the cabinet but is diverted by the descending air stream through the front of the cabinet floor. Subsequently all air is HEPA-filtered; a portion of the filtered air is discharged and the remainder is recirculated downward over the work area.

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flexible gauntlets attached mechanically to the cabinet by means of which the operators may carry out work within the cabinet. In use the cabinets operate at a negative pressure with air drawn into the cabinet through a single HEPA filter. Exhaust air is drawn through a HEPA filter (or filters) before being exhausted. The use of an air inlet filter offers the advantage of preventing release of hazardous materials in the event of a fan failure. In addition, it prevents external airborne contamination of materials being handled within the cabinet. This pharmaceutical manufacturing processes involving hazardous microorganisms or products. BS 5726 requires there to be airflow of 0.75 m/s into the cabinet when gauntlets are detached and at least 3 m3 /min through the inlet filter when the gauntlets are attached. In addition the dimensions of the inlet and exhaust filters should be such as to achieve a minimum negative pressure of 200 Pa within the cabinet under operating conditions. BS 5726 requires there to be airflow of 0.75 m/s into the cabinet when gauntlets are detached and at least 3 m3/min through the inlet filter when the gauntlets are attached. In addition the dimensions of the inlet and exhaust filters should be such as to achieve a minimum negative pressure of 200 Pa within the cabinet under operating conditions. Have a look at the following diagram and you will know how the Class III cabinets look.

Unfortunately, no. laminar air flow units employ the HEPA filters. It should be remembered that HEPA filtered laminar flow work stations designed to provide horizontal or vertical clean air flows are suitable for aseptic purposes only and offer no protection to the worker. Hazardous biotechnology processes must not be carried out in such areas.

•

All right. Now how are these safety cabinets applied in fermentation technology?

The Porton Mobile Enclosed Chemostat (POMEC) described by Evans and Harris-Smith was the first fermentation system designed and built to enable stirred batch (20 l) and continuous (2.5 l vessel) culture of pathogenic bacteria to be carried out without risk of escape of any aerosols released from the fermentor vessel. A specially designed and constructed culture apparatus was contained within a purpose-built Class III type cabinet constituted of glass reinforced polyester resin. The various controls and measurement indicators were panelmounted and accessible on the exterior of the cabinet. An inclined airlock with two UV lights and a liquid disinfectant lock (dunk tank) were set into the cabinet wall to allow safe ingress and egress of material. The safe transfer of culture into a transfer was achieved by passing a tube from the culture vessel to a receiver bottle through the dunk tank. The cabinet had additional safety features, including an accident well, to contain gross spillage in the event of culture vessel rupture and means to decontaminate either by formaldehyde vapour and/or drenching in formaldehyde. A system to separate and recover pathogenic bacteria grown in POMEC using a continuous flow centrifuge is described by Evans et al. Continuous flow centrifuges, particularly those of the vertical rotating cylinder type, are notorious generators of aerosols and their containment for harvesting of pathogens is essential. Evans et a/ described a novel arrangement whereby a continuous flow centrifuge contained within a Class III type cabinet was connected to the POMEC. For this, the two cabinets were connected via two ports fitted to the outside of the disinfectant lock of the two cabinets. Connecting tubes were passed between the fermentor, centrifuge and effluent receiving vessel. The principles established with the POMEC are still used today although cabinet design and fabrication have been modified to meet current operating standards. Typically fermentors are steam sterilizable in situ and require steam and water for temperature control. In addition they are equipped with sophisticated electronic control and monitoring systems. A feature of modern cabinets is the need to provide interfaces at the cabinet wall that allows physical and electronic services to pass into and out of the cabinet without affecting the biosafety integrity of the cabinet. These interfaces should be capable of being disconnected while maintaining cabinet integrity.

•

But most laboratories have only laminar flow units. Aren’t they sufficient to offer protection to the worker and the product?

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Although the use of disinfectant fluid locks is still an effective means for safe entry and exit of materials from cabinets, alternative methods are being increasingly used because of safety regulations for the use of large volumes of hazardous chemicals such as formaldehyde and the unsuitability of such chemicals for use in pharmaceutical manufacturing areas. Items can be safety passed into and out of cabinets through double-

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•

Tell me, is it possible to enclose all the industrial scale giant fermentation units into these safety cabinets that we have seen?

Obviously not. These cabinets are designed mainly for laboratory-scale fermentors of up to 50 liter working volume. Even the pilot plant fermentors cannot be completely enclosed into these cabinets. Therefore, the design engineering of both the pilot plant and production scale fermentors has to take care of the containment procedure. The following diagram shows a highly simplified diagram of a fermentor highlighting the various containment points.

Fluids can be passed into and out of cabinets using sterilizable male/ female connectors. With such devices, it is possible to make and break fluid lines aseptically and to allow sterilization of all surfaces exposed to fluids both before and after use. Exercise: find out more about the various disinfectants commonly used for industrial applications. Discuss their merits and demerits. Use the following space to express your views.

•

Fine- Where else cane we use the Class III cabinets?

Class III cabinets incorporating such features have been designed and constructed to contain a variety of modern downstream processing equipment including continuous flow centrifuges, cross-flow filtration units for concentration steps, bead mill homogenizers for disrupting cells and fast protein liquid chromatography (FPLC) systems for protein purification. These have been used for the production of hazardous biological substances such as neurotoxins of Clostridium botulinum. Because of the relatively compact size and large operating capacity of most modern downstream processing equipment these cabinets can readily be used for large-scale (up to and possibly beyond 500 l initial fermentation volume) processes.

•

All these cabinets appear to be bulky and non – portable. Is there a lightweight and portable arrangement for offering containment?

This is a very good point. It has been shown that rigid cabinets offer many advantages; they are robust, can support heavy items of equipment and peripherals such as pass boxes and interface panels. However, it is practical to consider the use of plastic film isolator technology as an alternative approach to providing physical encapsulation. The use of flexible film isolators is a well established means of providing barriers between patients or animals whilst allowing essential support duties, such as nursing or animal husbandry respectively, to be carried out safely. The technology also offers an effective means of creating aseptic environments within relatively ‘dirty’ environments. The isolators can be operated to the same containment standards as rigid construction Class III type cabinets and may provide the most effective solution to containment problems. They offer advantages including good visibility, improved worker comfort and cost over rigid cabinets but are perhaps best suited to operations that do not involve heavy, bulky, complex equipment.

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The main biosafety features of the fermentation system include the use of steam barriers on double O-ring seals, supply lines and mechanical seals on stirrer shafts, multiple O-ring seals, piping of condensate lines and pressure relief systems to a ‘killtank’, double (in series) filtration of inlet and off-gases, elimination of unnecessary piping joins and use of welded piping and hermetically sealed steam condensate traps. A mobile flexible isolator unit can also be used to allow localized containment of sample valve and probe entry ports. The safe operation of such a complex plant requires effective validation and integrity testing. Inevitably, it is not practical to engineer secondary containment features on all primary containment barriers without incurring considerable expense and so planned preventive maintenance (PPM) is an important aspect of the safe operation of such a system.

•

Exercise: Imagine you are in charge of a fermentation unit. This unit produces antibiotic. What different containment processes you would adapt? Justify your answer.

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ended pass boxes after being sanitized by spraying with disinfectants (e.g. 70% v/v isopropanol). Transfer port systems were developed for the safe transfer of radioactive materials between contained handling areas. The lid of the transfer container is designed to fit to a special port fixed into the cabinet wall such that once locked to the port the container can be opened directly to the cabinet interior without exposing the exterior of the container or its lid to the cabinet interior environment. While such systems offer high safety they have disadvantages, being expensive and not convenient to use, they impose limitations on the size and shape of items to be transferred and, most importantly, all cabinets are required to be equipped with compatible ports.

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LESSON 31: CONTAINMENT IN DOWNSTREAM PROCESSING Learning Objectives

aerosols of potentially hazardous material and process equipment design should minimize such risks.

In this lecture, you will learn

• • •

What is containment in downstream processing?

•

Containment in cell separation Containment in cell disruption

Recent advances in molecular biology and recombinant DNA (rDNA) technology have enabled products of animal, plant or microbial origin to be produced in large quantities by culturing bacteria, yeast, plant or mammalian cells. A typical bioprocess will consist of growing cells in a suitable nutrient medium, followed by the recovery and purification of the product: downstream processing. If the desired product is extra-cellular then the first stage in processing will be the removal of large solids and cells by centrifugation or filtration. The broth is then fractionated or extracted into major fractions; this can be done using processes such as chromatography, liquid-liquid extraction or precipitation. The fraction containing the product may then be purified further, often with more specialized chromatographic techniques. However, the majority of products remain intracellular, enclosed in a soluble or insoluble form within the cell. Some of these products are cytoplasmic, others are associated with cell membranes, cell wall components or the periplasm (where present). In this case, the cells must first be harvested to form a concentrated slurry or paste, then disrupted to release their products into solution for subsequent extraction and purification. We have seen that the chances of microorganisms coming in contact with the workers are more in case of downstream processing as compared to the fermentation as such. Downstream processing operations are high energy operations and hence generate fine sized aerosols, which, as we have seen, more potent in terms of causing infections and allergic reactions. Therefore, containment of downstream processing is probably more important than containment of fermentation. Of all process equipment, centrifuges, in addition to fermentors themselves of course, are most likely to release microorganisms. It is possible to kill process micro-organisms after the fermentation is complete so there may be no need for containment in further processing steps to eliminate the infectious risk. However, even dead micro-organisms could present an allergenic risk. Most reported health problems have been associated with downstream processing. The greatest demands on biosafety occurred from the time the broth leaves the bioreactor to the final processing steps, as this involves dealing with large amounts of cell debris. Downstream processing frequently involves the use of machinery that rotates at high speeds (centrifuges) or exerts increased pressure (liquid extrusion homogenizers, cross-flow microfiltration and ultrafiltration units). Such energetic processes may generate

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Right. But why do we need to study containment of different downstream operations? I mean, why can’t a singe containment protocol hold good for all downstream processes?

I will tell you that. Let’s take the example of various types of seals used for containment. A single seal system is the basic arrangement to provide a barrier between microorganisms and the workplace environment. If the equipment is operating above ambient pressure, any failure of the seal would result in flow into the workplace environment. A double seal arrangement offers extra security although it could be argued that both seals are likely to wear at the same rate. Failure of the primary seal would be checked by the secondary seal against emissions into the workplace. Failure of the secondary seal would not result in any obvious problem unless the primary seal failed. In both cases, it would be difficult to know if one of the seals failed. It could also be argued whether the gap between the two seals could be adequately sterilized. If not, migration by microbial growth could give rise to contamination problems. Regular maintenance and/or seal replacement would be the obvious recommendation for the double and single seal arrangement before seal failure occurred. A higher security system can be designed by employing a barrier fluid between the two seals. Steam is often used as the barrier fluid (steam tracing), so that any micro-organisms breaching the primary seal are killed and removed from the system. Commonly, the condensate will be directed to a kill tank rather than to the steam boiler for heat recovery. Other barrier fluids used are sterile water, biocides and glycerol, usually at higher pressure than that in the contained device. Now, this containment practice cannot be generalized. The types of the main and back up seals, the material of construction, the engineering design, the method of sterilization, even the barrier fluid will vary from one downstream operation to another. That’s why we need to know about the containment of all types of downstream operations.

•

Ok, what different operations are important from the containment point of view?

For the purposes of this account downstream processing has been split into two main areas: cell separation and cell disruption. First, let’s see about the cell separation.

Cell Separation Filtration is one of the commonest processes used, at all scales of operation, to separate suspended particles from a liquid, using a porous medium which retains the particles but allows the liquid to pass through. There is potentially a wide variety of

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What about the membrane filtration?

In order to concentrate and purify the product more, it is possible to use membrane filtration. Here, some form of semipermeable membrane is used to separate the components of a liquid stream. In most of the commercially important processes the driving force is pressure, the solvent (usually water) is driven through the membrane while the solute(s) are retained. This type of process includes reverse osmosis, ultrafiltration and microfiltration. Cross flow membrane filtration has attracted attention in recent years as an alternative to high g force centrifugation. Scaling up from laboratory or pilot scale is relatively easy, as additional modules/units can be added to increase the surface area for filtration; this can, however, be costly. The major disadvantage of these techniques is the detrimental effect of membrane fouling on filtration rates and subsequent product recovery. Generally, membranes are considered to have less potential for the emission of aerosols or breach of containment, compared with centrifuges. Difficulties may be encountered when cleaning membranes in situ. It may only be achieved adequately through the dismantling of the filter units. This process could be hazardous in terms of aerosol production, so adequate precautions should be taken, i.e. the use of secondary containment. Traditionally, most membranes have been fabricated from plastics such as polysulphones and cellulose acetate. In recent years, inorganic membranes, made from materials such as ceramics and metals, have been introduced and these have found application in cell recovery. The robustness of inorganic membranes are generally higher than plastic membranes, offering higher temperatures (suitable for sterilization) and higher operating pressures. Ceramic membranes, however, are

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vulnerable to heat shock and mechanical shock, i.e. they are brittle and can be broken. A wide range of membrane equipment designs are available for cell recovery and other applications at both pilot and production scale. The inherent containment features vary widely. Plate and frame membrane filters rely on seals on each plate and the clamps on the assembly for containment. Hollow fibre systems are pressure limited and are often fitted with a pressure switch in order to prevent the recirculation pump reaching the bursting pressure of the fibres. Tubular membrane systems appear to offer the best containment features because they are usually constructed with hard piping and require fewer seals to the outside environment. The collection shrouds on the low pressure side would provide a convenient shield should the membranes or the filter seals fail. Metal membranes can be constructed using welding and this negates the need for seals.

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The centrifugation operations would also require strict containment. Won’t they?

Most certainly. The separation of biomass from growth media is a difficult operation, as cells have almost the same density as their surrounding medium, are small, are able to form stable colloids and are cohesive. Sedimentation of cell debris presents an even more difficult problem for biotechnologists and the choice of separation technique is limited. Solid bowl and tubular bowl centrifuges are relatively inexpensive and have in the past been chosen for use in the biotechnology industry. They are useful for small batches, but are labour intensive because the solids have to be dug out by hand. The scroll decanter centrifuge has limited use in the biotechnology industry because of the low g forces generated. It should, however, be better contained than the traditional solid or tubular bowl type of centrifuge. In reality, only the higher g force devices such as disc stack and tubular bowl centrifuges are used at large scale. Decanter and solid bowl centrifuges are however used for separating bigger particles, such as yeast or flocculated bacteria.

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Could we study such an example of commercially available centrifuge?

Why not? Let me tell you about the Centritech Cell Separator. This is relatively recent development. The Centritech Cell Separator is designed for aseptic separation of mammalian cells in a completely closed system without any rotating seals. It contains a spinning disposable bladder which lies within the rotor that spins at speeds up to 1200 rpm. The centrifugal force created within the bladder separates the culture into cell concentrate and fluid. A system of tubing and pumps enables the cell culture to enter the bladder directly from the fermentation vessel. The tubing is connected to the rotating bowl in a way that allows one end of the tube to rotate while the other end is standing still. Thus the separation system is totally enclosed. Further primary containment is provided by a sealed lid on the rotor chamber and an external hood which acts as built on secondary containment. The separation insert is delivered as a pre-sterilized disposable plastic bladder. The novel design of the Centritech Cell Separator, with no openings to atmosphere and no rotating seal, means that it is unlikely to

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filtration devices available for initial cell separation. However, the choice is restricted in biotechnology due to the limitations imposed by the nature of fermentation broths. The filters used for initial solids recovery (e.g. recovery of biomass from fermentor broths) are of two main types: the rotary vacuum drum, a continuous filter, and the filter press, a batch filter. Generally, filter presses are slow and labour intensive and are usually only used at small scales. They are often found in the older style biotechnology processes such as brewing and distilling. Rotary vacuum drum filters can be used for larger scale continuous operations and they are more often found in the pharmaceutical and food industries. It is easier to contain a rotary vacuum drum filter, e.g. using local exhaust ventilation, than a filter press but it is not possible to operate a rotary vacuum drum in an aseptic manner. Filter presses usually operate at pressures between 5 and 7 bar. Rotary vacuum drum filters operate such that the vacuum pressure is applied internally so the filtrate is drawn through the filter, into the drum and finally into a collecting vessel. Considering the low pressures and low rotational speeds used in such devices, their operation should not present a problem in terms of containment and aerosol formation. However, when the cake is removed from the filter there is potential for considerable release of biological material.

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produce biological aerosols during normal operation. The containment of this device can be tested by simulating rupture of the bladder. Micro-organisms are detected outside the primary containment of the sealed lid; however, none are detected outside the secondary containment. If the interior becomes heavily contaminated, decontamination may be difficult. The Centritech Cell Separator has a very low separating capacity (100 1/ hour) and therefore cannot compete with disc-stack separators.

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What are disc-stack separators?

Disc-stack centrifuges predominate at production scale in biotechnology. They consist of a solid bowl containing a series of hollow truncated cones (‘discs’) stacked one upon another. Feed suspension enters the centrifuge through a central feed pipe, passes out of the edge of the bowl then upwards and inwards through the stack of discs. Solids settle onto the lower surface of each cone and clarified liquid moves inward and upwards to reach an annular overflow channel, emerging at the neck of the bowl around the feed pipe. The sedimented solids slide off the disc and collect in the space between the stack of discs and the bowl wall. The different types of disc-stack centrifuge are distinguished by the method in which they discharge solids from the space between the discs and the wall. In solid-bowl or solids-retaining disc-stack centrifuges, the machine has to be stopped for solids to be removed manually. In nozzle discharge disc-stack centrifuges solids are discharged continuously. Opening bowl, solids-ejecting or intermittent discharge disc-stack centrifuges discharge solids either at preset time intervals or discharge is automatically triggered by the load on the bowl.

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What are nozzle- discharge disc – stack centrifuges?

Solids discharge from nozzle discharge disk-stack centrifuges is normally continuous. Two different types exist. In the Alfa Laval BTUX 510, the solids are collected in conical storage spaces, with concentrate tubes located around the largest diameter of the bowl in the apex of the cones. Solids pass through the concentrate tubes and the vortex nozzles into the paring tube chamber. The concentrate is skimmed off by the paring tube and discharged under pressure. The clarified liquid phase is displaced towards the centre through the disc-stack. The centrate is then discharged under pressure via a paring disc pump at the top of the frame hood. In the BTUX 510, the unique vortex nozzles automatically compensate for variations in feed flow rate or feed solids concentration to ensure a constant concentration of the discharged solids phase. (See diagram) In the second type of nozzle-discharge disc-stack centrifuge the solids are collected in a triangular storage space with nozzles located around the largest diameter of the bowl. The size and number of nozzles can be optimized for each application, so that too dilute slurry is not discharged, but it is sufficiently fluid to flow through the nozzles. A further development of the nozzle-discharge disc-stack centrifuge incorporates an additional annular valve at the periphery of the bowl. This centrifuge therefore has the same

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type of solids discharge as a solids ejecting disc-stack centrifuge. This hybrid is also equipped with extra nozzles around the bottom of the bowl. As well as giving the centrifuge a CIP facility, the additional feature means that blocked discs can be cleared by initiating a full desludging. Another type of disc stack centrifuges are opening bowl disc stack centrifuges. What are they? Opening-bowl, or solids-ejecting disc-stack centrifuges are very common in large and pilot scale biotechnology plants. They have been the most widely researched in terms of sterile or contained operation and are similar in design to solid bowl and nozzle-discharge disc-stack centrifuges, but here peripheral ports in the solids collection area are held closed by water or air pressure to retain sedimented cells during separation. At a predetermined time interval, the feed-stream ceases and the ports open to allow the solids to eject (termed ‘desludging’). They are often the only type of centrifuge capable of continuous separation of cells and cell debris because the frequency of solids discharge can be set to maximize the sedimented solids concentration.

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Most devices have cyclone receivers to contain the discharge of sludge. However, a considerable shock wave is generated by the centrifuge and the air which is then displaced from the cyclone may contain aerosols of cells or debris unless suitable vent filters are fitted. Lawrence and Barry report shock waves during discharge from an Alfa-Laval AX 213 Separator, thought to be sufficient to allow aerosol to escape from cartridge housing air vents. Walker et at. describe modifications to a Westfalia CSA 19-47476 centrifuge. The vent filter was blocking due to massive aerosol formation during desludging, so it was removed and attached to the main frame drain, thus increasing the distance between the solids receiver and the filter. This alleviated the vent filter blockage problem. There are several such types of centrifuges. But we will see about just one more.

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Which is that?

That is solid bowl centrifuges. Solid bowl centrifuges have the feed stream entering from the bottom of the bowl and moving upwards. Solids are sedimented in the bowl and centrate flows out over a weir. Single chamber, triple bowl and multichamber devices are available, each with a larger surface area and hence greater efficiency. Sedimented solids can be removed intermittently manually or automatically using a plough with the bowl rotating slowly. Normal operational speeds lie between 450 and 3500 rpm, developing centrifugal forces in the range of 500 to 1200 g force. A novel design, the Alfa Laval-Sharples SP-725 Superhelix, is shown in the figure. This is a vertical solid bowl centrifuge. The product stream is fed through a stationary feed nozzle at the bottom of the bowl and gently accelerated to bowl speed in the conical feed zone. Under the action of centrifugal force, the solid phase moves to the bowl wall where the helical conveyor forces it downwards to the beach. Here, the solid phase is further concentrated. Solids are finally discharged into the solids chute at the bottom of the centrifuge, and to prevent escape of

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material the method of solid collection must be contained. The manufacturers state that they can supply suitable equipment for solids handling. The centrate is discharged by a centripetal pump at the top of the bowl. The automatic solids discharge of the Alfa Laval-Sharples SP-725 represents an improvement in solid bowl centrifuge design.

The SP-725 Superhelix Exercise: Find out what are the different types of centrifuges, their manufacturing companies and their brand names. Feel free to use internet and make a list in the space provided below: The Btux 510 Nozzle Discharge Disc Stack Centrifuge

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After we have separated the cells we need to break them up. How do we do that? We can do that by a number of cell disruption techniques. Let us see more about cell disruption first.

Cell disruption

Disruption may involve physical, mechanical or chemical steps to allow intracellular products (usually proteins) to be extracted from cells. Alternatively, it may consist of merely removing certain components from the cell wall or membrane, to permit product leakage. There are many methods of disrupting cells. The suitability of each method depends on the scale of production, the protein to be isolated, the individual cell suspension, and the disruption techniques available. The performance of each technique is dependent on cell type, culture conditions, pretreatment, and the device used. Physical or mechanical methods of cell disruption are the most widely researched in terms of containment. The underlying principle is either by breakage of the cell wall by mechanical

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contact, the application of liquid or hydrodynamic shear forces, or the application of solid shear forces. Cell disruption by nonphysical methods generally involves simple operations which may be carried out in large tanks or vessels, which mayor may not require agitation.

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What are the various physical methods of cell disruption? Again, there are several methods. We will see some of them.

Agitation with abrasives Micro-organisms in dry or frozen solid form can be disrupted by conventional ball or vibratory mills used in the chemical process industries. Whilst the method of dry milling may be efficient, it raises a number of problems, including caking of fine powders (at around 1 mm most bacteria are smaller than powders that are generally milled in the chemical process industries), erosion of the mill surfaces including liners and balls (which leads to contamination of the disruptate), and the generation of heat energy which can denature the desired product. In the biotechnology industry, it is much more common to employ wet milling where disruption is caused by a mixture of hydrodynamic shear forces and mechanical crushing. Bead mills are generally operated at near ambient pressure. When disrupting very thick cell pastes, there may be a slight build up of pressure in the vessel, but it is unlikely to exceed 0.2 bar, so bead mills are unlikely to cause aerosols to be released during operation. In the event of seal failure or a leak, however, even this low pressure is likely to lead to aerosol formation. Design of a typical continuous bead mill is given below:

The earliest devices to employ this principle were the French Press and the Chaikoff Press. Both these devices are relatively crude and simple which can only disrupt small batches of cell suspensions. The next stage in the development was the introduction of dairy industry homogenizers. This consists of a ram pump which forces product through a one way valve into a homogenizing valve. The feed enters this valve at high pressure, typically 533 bar. As the feed passes between the homogenizing valve and its valve seat, there is a rapid increase in velocity with a corresponding decrease in pressure. This results in cavitation, which, coupled with impaction of the cells against an impact ring, causes cell disruption. The gap between the valve and the valve seat is adjusted by a spring-loaded hand wheel. The important design features are the suction valve, discharge valve, pump plunger and plunger packing. On the suction stroke of the plunger, the suction valve opens, allowing product to enter the pump chamber. On the compression stroke of the plunger, the discharge valve opens, the suction valve closes and product is forced along a bore to the homogenizing valve.

Ultrasonic Techniques Ultrasonics are sound waves of greater than 16 kHz frequency; when these are applied to solutions, they cause ‘gaseous cavitation’, areas of rarefaction and compression which rapidly interchange. As the gas bubbles collapse, shock waves are formed. Sonication in batch or continuous processing has been employed successfully for the disruption of many types of microbial cells. A number of commercial laboratory-scale and pilot-scale ultrasonic disruption systems are available. Devices such as the Soniprep 150 (MSE, Crawley) use glass tubes with rubber sealing caps. This should provide an efficient seal between probe and tube, preventing the escape of aerosol. The filling and emptying the tubes will lead to a breach of containment so this operation should take place within secondary containment, exhausted through a HEPA filter if high-risk micro-organisms are being disrupted. Life Science Laboratories Ltd, Luton, UK supply the FLOCELL, a continuous flow cell manufactured by Heat System Inc, Farmingdale, New York, USA. The device consists of a chamber which screws onto an ultrasonic probe. Cell suspension is fed into the chamber under pressure at the bottom and flows out of a port after disruption above the orifice. An overflow port is provided for recycling cell suspension if necessary. The flow cell is sealed with single Orings. Operating pressure is up to approximately 7 bars.

Liquid Extrusion This method has been widely studied and relies on the principle that forcing a cell suspension at high pressure through a narrow orifice will provide a rapid pressure drop. This is a very powerful means of disrupting cells. It is a relatively simple matter to design equipment to subject the cell suspension to shear forces before releasing the pressure. By varying the pressure applied, cells may be completely or only partly disrupted (the latter usually being sufficient for the release of periplasmic enzymes).

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LESSON 32: PATENT AND SECRET PROCESSES

Learning Objectives In this lecture, you will learn

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What is a patent? Composition of a patent Rights of an inventor and their protection

The economic and competitive position of a fermentation process depends on several factors, some of which are recoverable yields, research costs, size of the market, profit potential, and patent or secret process position of the fermentation process or product. The latter consideration is particularly important, because patents and secret processes provide a degree of protection to the competitive position of a commercial fermentation. But which approach should be taken by an industrial Concern? Should a fermentation process or product be patented, assuming that they are patentable, to provide 17 years of monopoly, or should the fermentation process and know-how be kept a secret so as to possibly allow many additional years of protection for the process? A yet more basic question, however, concerns what can and cannot be patented. The answers to these and other questions relating to the patenting of inventions will become clearer if the basic concepts behind the granting of patents by the Patent Office of the Federal Government are examined. Patents are granted to inventors in return for a public disclosure of their inventions. These disclosures add to the knowledge of the respective art and help to advance the state of that art. The patent, in turn, gives the inventor the right to exclude others from making, using, or selling his particular invention as disclosed in the “claims” of the patent. Most inventors will settle for the 17 years of protection rendered to them by a patent and will apply for a patent on their invention instead of attempting to maintain secrecy during the use of their invention. Obviously, secrecy about a fermentation process is difficult to maintain. Special contracts must be negotiated with trusted employees so that they will not reveal their knowledge of the process and, in addition, other problems arise which will not be discussed here. Regardless of these considerations, however, in a few instances, secrecy concerning a fermentation process has allowed the maintenance of a good competitive position for a commercial fermentation for many years in excess of the 17-year monopoly of a patent. A notable example is the commercial production of citric acid by Aspergillus niger as the fermentation process is conducted by the Charles Pfizer Company, Incorporated. A fermentation process or product protected by a patent, however, also may have secrets associated with it. Special fermentation and recovery techniques associated with obtaining high yields and good product recoveries are often secret information known only to the inventor and his associates and not disclosed in the patent itself, since such data are generally

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developed after the application for the patent has been filed. Also, the patent describes a workable process and a product without necessarily describing minor variations in process technique and product recovery, considerations that may be, however, of extreme importance in maintaining the competitive position of the fermentation. There is some protection available from state or federal courts for this type of information. Thus, an individual or individuals can be prosecuted for stealing microbial cultures and technical data such as secret process information. The individual working in an industrial research laboratory or any laboratory in which fermentation processes of potential economic value are under study should know how to read a patent in order to be able to determine the points of the invention which are actually protected by the patent. This knowledge should prevent his infringing on the rights of other inventors. He should also understand the types of information that are required for filing a patent application so that research can be directed towards obtaining this information. He should be able to decide the extent of process variations or ranges in variations in chemical structure of a product which should be claimed in a patent application. As we shall see, claiming too little or too much about the process or product can be disastrous. Guidance in these problem areas frequently can be obtained by consulting a qualified patent attorney.

History of the Patent Concept It will be easier to understand patents if we first consider how the concept of patents has developed during the past few hundred years. Patents in one form or another have been in existence at least since 1332, when Bartolomeo Verde of Venice received a revocable 12-year patent on a windmill. A patent statute enacted in Venice in 1474 allowed the granting of exclusive rights for 10 years to “inventors of new arts and machines.” In 1501, Aldus in Venice obtained a patent on italic type. Galileo, in 1594, received a patent for a machine to be used for raising water and irrigating land. This patent was for a 20year term, and within one year of granting of the patent, he had to construct his new form of machine. This patent also provided for the punishment of infringers, and it was based on the assumption that the machine had not previously been invented or thought of by others, and that it had never been the subject of a previous patent or grant. Thus, Galileo’s patent included several principles found in modern patent law. It provided a reward to the inventor and to the first inventor only, it provided a requirement for compulsory working of the invention, and it provided the right to exclude others from practicing the invention. Monopolies of a different sort were commonplace in England during the reign of Queen Elizabeth. These monopolies were granted by the Crown on a favoritism basis to various individuals, although this practice was illegal, based on English

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The early American colonists were well aware of the injustices that had occurred in England through the granting of monopolies. Therefore, a Massachusetts statute of 1641 stated that monopolies should not be granted or allowed, although such new inventions that might be profitable to the colony might receive a monopoly for a short period of time. In succeeding years, patents were granted by the various states and, in fact, an inventor had to obtain a separate patent from each state. The constitution of the United States, however, provided for patents, and the first Federal patent act enacted in 1790 allowed the patenting of “any useful art, manufacture, engine, machine, or device or improvement therein not before known nr used.” There have been many changes in the patent laws of the United States since this first Federal patent act, and further changes are under consideration at the present time. What does the patent contain? A patent consists of three parts: the grant, specifications, and claims. The grant is filed at the patent office and is not published. It is a signed document and is the agreement that grants patent rights to the inventor. The specifications and claims are published as a single document (Figure 15.1) which is available to the public at a minimal charge from the Patent Office. Thus, this is the part of a patent that one normally sees. The specifications section is a. narrative description of the subject matter of the invention and of how the invention is carried out. Therefore, anyone skilled in the particular branch of learning relating to the patent should be able to reproduce the invention on reading the specifications section. The claims section specifically defines the scope of the invention to be protected by the patent. That which others may not practice is defined here and, if the patent relates to industrial fermentations, it should be clearly understood from the claims just how the invention differs from known products and processes. The inventor may claim a part or all of that which is described in the specifications. In fact, the exact wording of the claims is important, because it states exactly what is to be protected and what is not. Thus, a patent stands or falls depending on the statements included in the claims section. It is obvious, then, that the inventor must decide as best he can that which he should and should not claim. If he does not

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claim certain variations in the process or product, it may be possible for others utilizing these variations to carry out the process or make the product without infringing on the rights of the inventor. In contrast, if in the claims the inventor attempts to protect all possible variations in the process or product without experimentally establishing the validity of each of these variables, he may find that the patent is in jeopardy because of nonworkability of some of the claims. Thus, the inventor should claim those variables of which he is sure and, if possible, he should experimentally test all other variables which, if not claimed, might allow others to circumvent the patent.

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·What are the characteristics of a patent?

Inventions are divided into various categories or “classes” of subject matter. Several of these classes do not apply to microbial processes or products, however, and therefore are not considered in the present discussion. Microbial processes or products usually fall under one of two classes: an “art or process” or a “composition of matter.” The second class pertains to new chemical compounds or novel compositions or mixtures, and a microorganism or its enzymes may be used to aid the accomplishment of a chemical synthesis or in producing such compositions. The art or process class of patents pertains to methods, including microbial fermentations, for bringing about useful chemical or physical results. Implicit in the patentability of a microbial process is the concept that man has adjusted the environment of the microorganism to such an extent that the organism will carry out, in the laboratory or commercial production plant, a process that it could not carry out, to any extent, under the conditions occurring in nature. Thus, Clostridium acetobutylicum), in nature, possibly might produce small amounts of acetic and butyric acid, but it is considered to be highly unlikely. Those natural environmental and nutritional conditions would be favorable for the further formation of acetone and butanol. Likewise, many investigations designed to demonstrate antibiotic production by microorganisms in soil, for the most part, have yielded negative results. If this phenomenon does occur in nonsterilized soil, it is probably on a micro scale in the immediate vicinity of the individual microorganism or on the surface of a particle of readily decomposable organic matter. The microorganism utilized in a microbial process cannot be patented, and this statement also is true for mutants. However, the utilization of a microorganism not previously described or of a newly derived mutant does lend “novelty” or newness to an invention. This consideration has caused considerable difficulty for the classification of industrial microorganisms, because a large number of supposedly new microorganisms have been given species names in patent applications, although these organisms often differed only slightly from recognized species. In fact, extensive studies of certain groups of these organisms are underway to establish their taxonomic relationships so as to reduce the numbers of species described in the patent literature to those that really differ taxonomically. It is possible that the inventor of a microbial process may have the alternatives of patenting either the process or the product, or of patenting both. Obviously, if the product is a compound

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common law. Such products as salt, starch, glass, and paper were included in these monopolies~ The power to enforce the monopoly was also granted to the holders of the monopolies and, obviously, this led to high prices and poor quality. Regardless of these monopolies, however, a patent was granted by the Crown in 1565 for a furnace, and this probably was the first example in England of a reward to an inventor. In contrast to monopolies, the granting of a patent such as this was legal under English common law. This question of the legality of patents versus monopolies was decided when the Statute of Monopolies was enacted in 1623 during the reign of King James, since this statute protected inventions by the granting to inventors of monopolies (patents) by the Crown, but it prohibited the Crown from granting other forms of monopolies. These early English patents did not include a written description of the invention, nor were drawings included. In fact, it was not until the middle of the eighteenth century that these became a part of a patent.

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already well known (for example, the amino acid L-lysine), a product patent cannot be obtained, and the patent application must cover only the process for manufacture. In contrast, a newly discovered product not previously described, such as certain new antibiotics, may allow application for a product patent. This product patent has distinct advantages over a process patent in that there may be more than one microbiological route or even a chemical route to formation of the product. A specific example is the Pfizer product patent, U.S. 2,699,054, on the antibiotic tetracycline, since there are at least two processes involving microorganisms yielding this antibiotic. Thus, tetracycline can be produced by a direct fermentation, or chlorotetracycline can be first accumulated through fermentation followed by its chemical conversion to tetracycline. The United States has an “examination” patent system in contrast to the “registration” system in use in some other countries. The examination system requires that a patent application be studied for “novelty” in the light of “prior art” and for usefulness or “utility.” The prior art consists of ail printed material, including patents, that was available previous to the time of patent application. This material may be from any country and in any language. The invention must be new in respect to use in this country, so that any unpatented process, even if secret, already in use cannot be patented. For example, Pfizer’s secret citric acid process cannot now be patented. Prior experimental use, an abandoned experiment, or lost art does not affect novelty. Based on this, a research worker should not abandon experiments in his research book, and he should not state in writing that he has given up on the experimental approach. A research worker also must be careful that his own scientific publications do not constitute a bar to obtaining a patent. Thus, the Patent Office considers that a patent for which the application was filed within one year after a pertinent publication or public use by the inventor’ still may be granted, but that longer time periods forfeit novelty. Publication or prior uses by others than the inventor can be antedated. Too many patent applications are presented to the U.S. Patent Office for a rapid determination of novelty. This backlog results in a prolonged period between the time of patent application and patent issue-as much as three years or more. The requirement for usefulness or utility means that the invention must perform some beneficial function. Thus, it is considered that utility is absent for inventions that are inoperative, frivolous, or in jurious to the health, morals, and good order of society. No particular degree of utility is required, however, and there need not be a presently existing practical usefulness. For example, a compound may be useful under the patent statutes if it can be employed for research purposes, as in ‘the case of intermediates that may be used in the synthesis of other compounds of a useful class. The basic criteria for patentability are set forth in the patent statutes. For example, Section 101 of Title 35 U.S.C. states: Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement there of, may obtain a patent therefore, subject to the conditions and requirements of this title. 196

Thus, apart from utility, the basic requirements for patentability are new and nonobvious subject matter and adequacy of disclosure. In regard to the latter point, in microbiological applications, the drafting of a proper disclosure is quite complex and involves problems that require competent professional assistance. Also, where a new organism is involved, in addition to a proper description in the specification, a culture should be deposited with a recognized depository culture collection prior to filing, although public access to the culture may be restricted until the patent issues. Based on novelty and utility, the Patent Office decides whether a patent should be granted for a particular invention. If this decision should be unfavorable as it relates to a particular patent application, an appeal can be made to a Board of Appeals within the Patent Office, and one can even appeal to various federal courts. Also, amendments can be made to the original patent application in order to make the invention more acceptable in the eyes of the Patent Office. There is no requirement that a patent granted in the United States must be put to actual use. In fact, there are many “defensive” patents and patents on small improvements that are never sold or used commercially. Despite such nonuse, these patents seldom impede economic development, since they provide valuable information and encourage invention of modifications or alternatives. Also, in many cases, a patent of this type may be licensed to “unblock” another invention. A study by the Patent, Trademark, and Copyright Foundation of George Washington University (Washington, D.C.) showed that 55 to 65 percent of assigned United States patents and 40 to 50 percent of unassigned United States patents were ‘actually produced for sale or used for making commercial articles at some time during the life of the patent, which indicates a rather high rate of commercial utilization of patents. The patent concepts discussed thus far apply not only to “basic” patents but also to “improvement” patents that follow and are closely related to the basic invention. Thus, if an inventor obtains a basic patent for an invention, but later finds an improved way of carrying out the invention, he may obtain an improvement patent. The situation is more complex, however, if an inventor other than the original inventor obtains the improvement patent, since it is likely that the second inventor cannot utilize the improvement without permission or license from the holder of the basic patent. Thus, the solution usually calls for negotiation and sale of rights so as to allow utilization of the improvement patent. A specific example of basic and improvement patents is associated with a fermentation process for the manufacture of L-lysine (see Chapter 20). The basic patent (U.S. 2,771,396) pertains to a fermentation process for utilizing an Escherichia coli mutant to produce 2,6diaminopimelic acid (DAP), with this compound then being decarboxylated to L-lysine by a wild-type strain of Aerobacter aerogenes. This basic patent was followed two years later by an improvement patent (U.S. 2,841,532), which was secured to protect the decarboxylation of (DAP) by Escherichia coli back mutants that accumulated during inoculum growth and production, a situation in which the DAP decarboxylase of the Aerobacter aerogenes was not needed. In this instance,

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Who holds the right of an invention?

In the United States, a valid patent is granted only to the first inventor, regardless of filing dates for patent applications. It is not always clear, however, just who really is the first inventor and, therefore, the patent laws provide for “interference proceedings” to determine priority of invention. These interference proceedings may be initiated by the Patent Office before issue of the patent, or they may be initiated by another applicant who has a copending application or who files within one year after the patent issues. The inventor is considered to be that individual who first conceived the idea of the invention, regardless of whether the idea had at that time been reduced to practice. But, to be considered as the inventor, one also must have demonstrated reasonable diligence in carrying out the invention (reducing it to practice), or be able to prove reduction to practice prior to the other party’s conception. As can be seen, it is of utmost importance for the inventor to establish the actual date on which he conceived the idea of the invention. To establish this date, the inventor should immediately get the inventive idea down in writing, sign and date the paper on which it is written, and have witnesses sign and date the document who can verify the date and content of the description of the invention. This recording of the invention is often done in a research book, and it should include what is considered to be the results and means for obtaining the results of the Invention. In addition to recording the invention, every page in the research book on which further experimental work has been recorded should also be signed and dated by the inventor and by one or two witnesses who understand the invention. These witnesses possibly may be called on at a later date to identify the writing in the research book and to establish its date and content. The procedures outlined above establish. both the date of conception of the invention and the progress in its reduction to practice but, to prove reduction to practice, the inventor must be able to produce corroboration that the invention was actually carried out, generally through witnesses who actually observed the experimental work. Alternatively, however, a patent application itself can serve as a “constructive” reduction to practice without further proof being required, but by so doing, the invention is placed in a less favorable position as regards considerations for patentability. Implicit in the concept of granting of patents is the premise that a “spark of genius” or “inventive ingenuity” is involved. In other words, individuals other than the inventor, even with the knowledge at hand which was available to the inventor, could not have conceived the invention. At times, however, questions arise as to whom among several individuals working in a single laboratory or for an industrial concern is the actual inventor. In other words, who actually possessed the inventive ingenuity? If a supervisor presents an idea and the experimental approach for reducing it to practice to a laboratory technician, then the invention is the property of the supervisor and not of the technician who merely carried out instructions to reduce it to practice. However, the supervisor who presents the idea without a solution may possibly not be the sole inventor. At 2.521

times, the actual inventor may be difficult to decide, and the names of more than one inventor may appear on the patent application as joint inventors. Usually, however, an invention is thought to be conceived by a single individual. Aside from determining who the inventor is, there is the question of who owns the patent. This is a particularly pertinent question, because patents can be of great economic value in addition to the monopoly that they afford, since they can be sold, licensed for a return of royalties, or assigned to an industrial concern or to the federal government. Also, patents are heritable property and can be part of an estate. Research workers employed by an industrial concern often sign contracts stating that they will assign their inventions to the concern so that the resulting patents are actually owned by the concern. In fact, it is often a condition of employment that such agreements be signed. Problems arise at times when an individual with such a contract invents something “in hi.; basement” which may he directly related to the interests of the company, or when the individual utilizes company equipment and facilities to make an invention, whether or not the invention is directly related to the interests of the company. Although there are some ground rules applying to these situations, it still may be difficult to make a fair decision as to actual ownership of such a patent. A published patent states at the top of its first page the type of ownership that is to be associated with the patent. There are three categories. The first category, in which most patents occur, is that in which commercial rights are retained by the inventor or the concern to which the patent has been assigned, although these rights may be further licensed, assigned, or sold (see Figure 15.1). In the second category are those patents that are assigned to the federal government, for instance, those assigned to the U.S. Department of Agriculture. The assigning of patents to the federal government allows it some control in the licensing of the use of these patents. The last category includes those patents “dedicated to the public.” Such patents do not provide a monopoly for any individual or any industrial concern but make the inventions available for all In regard to this category, the absence of exclusivity for these patents may not provide adequate incentives to invest in the development or marketing of products • How do we protect the rights of an invention? A patent grant to an inventor confers on that inventor the right to exclude others from making, using, or selling his invention for a period of 17 years from the date of patent issue. In case of “infringement,” that is, a violation of these patent rights, the inventor can call on the federal courts for help. In the courts, the inventor may sue the infringer for up to triple damages plus court costs, and the damages include profits that the infringer has accumulated in the practice of the invention. Thus, the infringer has a great deal to lost: when he violates the patent rights of others. However, the potential infringer may feel that he has not infringed the subject matter specifically stated in.t4e claims of the patent. Also, he may be able to show that certain of the claims, or all of the claims, are seriously defective so that the patent grant is not valid. If, as a result of these proceedings, certain of the patent claims are declared invalid by the courts, the patent may still be preserved by filing a “disclaimer” with the

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negotiations were not required, since both the basic and improvement patents were assigned to the same Fermentation Company.

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Patent Office for the faulty claims. Also a “reissue” patent may be granted to correct errors in the patent as originally granted. In certain instances, there are apparent infringements of inventor’s rights in which United States courts generally do not enjoin or penalize the infringer. This occurs when someone uses information from the patent claims on an experimental basis, or for private, noncommercial, use. For example, a patent covering a new process for the manufacture of wine might be employed by an individual in his basement to make a small batch of wine for home consumption. While this is technically an infringement, it is difficult to conceive of expensive infringement proceedings being brought against such an individual. Drugs, pharmaceuticals, and other fermentation products manufactured in a foreign country and imported for use or sale in the United States present special problems. If such products are protected in the United States by a product patent, then importation and use or sale of the product constitute infringement. A specific example can be cited for the antibiotic tetracycline as produced by fermentation in Italy where patent coverage cannot be obtained for pharmaceutical products or processes of manufacture. In this instance, importation and use or sale of tetracycline by American concerns not licensed by the holder of the tetracycline product patent are considered to constitute infringement, although the Italian companies themselves cannot be sued.

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What are the costs involved in a patent?

The costs for patent application and issuance have recently been greatly increased. Formerly, a fee of $30 was paid to the Patent Office at application and an additional $30 at patent issue. At present, these fees are $65 on filing plus certain extra charges for claims, and $100 on issuance plus $10 for each printed page of specifications. Also, there are additional fees for drawings and appeals, and patent attorney fees may be considerably more. In regard to this point, in rare instances patent attorneys will accept a percentage of royalties from the monies accruing from use of the patent instead of a specific fee. Research personnel for an industrial concern do not pay fees since these are absorbed by the employer as a part of the agreement for assignment of patents. Obviously, the real expenses associated with a patent occur if an infringer is challenged. Nevertheless, usually the high potential monetary returns from a useful patent outweigh all these considerable.

Possible Changes In United States Patent Law As previously stated, over a period of years, various changes have occurred in United States patent law, and further changes are still being considered. Fur instance, under consideration is a change to grant the patent to the first-to-file rather than to the actual first inventor, a procedure that would totally eliminate patent interferences. Another change would permit the filing of one or more preliminary patent applications prior to the filing of a complete application within one year. In addition, all applications would automatically be published 18 to 24 months from the earliest filing date. Finally, the life of the patent would be for 20 years from the date of filing of the patent application, rather than for the present 17 years from the date of granting of the patent. 198

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What is the scenario in other countries?

To protect his invention, an inventor may well wish to apply for patents in countries other than the United States. However, this is more complicated than it would seem, because the procedures for application, the structures of patents, and the protection afforded by a patent vary markedly from one country to another, so that separate and differing patent applications must be prepared for each country. Several countries use a “registration” system for filing patent applications instead of the “examination” system employed in the United States. In the registration system, the proper filing of the patent application and payment of government fees automatically result in the granting of a patent. This system is operative in France, Switzerland, Italy, Belgium, Luxemburg, and some other countries. Also, the small and less industrially developed countries utilize this system, because it is simpler and less costly. Some of the countries utilizing the registration system do not require a definite listing of claims, but merely a short resume. Other countries require that the claims be stated, but this is only to help in deciding whether more than one invention is involved. With the registration system, the scope of the patent is not limited by the exact wording of the claims and, if infringement litigation ensues, the entire disclosure is studied in light of prior art. Thus, the registration system for patents makes no decision on scope and validity before granting of the patent, so that these considerations are left to the decision of the courts. Many countries, excluding the United States and Canada, consider the inventor to be the individual who first files a patent application, and not necessarily the one who first conceives the invention and reduces it to practice. Also, in many countries, the determination of novelty and prior art includes all publication and public use of the invention prior to the date of filing of a patent application. This is in contrast to the one-year grace period allowed to an inventor in the United States between the time of published description or use of an invention and the filing of a patent application. At present, pharmaceuticals and the microbiological processes for making them cannot be patented in Italy. In contrast, Germany and the Netherlands provide patent grants for processes for making chemicals and pharmaceuticals, but the products themselves cannot be patented. Most countries other than the United States require the payment of periodic maintenance fees to keep a patent in force. Also, many countries require that a patent actually be used within the country granting the patent if the patent is to stay in force. In fact, if the government feels that the public would benefit from the exploitation of a patent not being used, it may request the granting of a compulsory license to others who are willing to use the invention. In a few countries, a patent may be revoked if not used. The many variations in patents and patent application procedures make it difficult for an inventor to know how to proceed in obtaining patents in countries other than the United States. However, a summary guide for the inventor may be found in a special report entitled “Common Patents for the Common Market” published in Chemical and Engineering

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News (June 16, 1964, pp. 96-97). In this guide, the following patent considerations are compared for 22 countries: (1) what is considered as prior art, (2) what cannot be patented, (3) language patent written in, (4) type of examination, (5) opposition, (6) life of patent, (7) maintenance fees, and (8) working requirements.

Notes

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LESSON 33: FERMENTATION ECONOMICS

Learning Objectives

demands of subsequent purification stages for high product concentration and high product purity which may in turn be overridden by safety considerations. In other words, economics of fermentation will have to give way to product safety and purity which also will come second to the overall safety of the operation.

In this lecture, you will learn Ways to economize a fermentation To cut the corners- where and how? Economizing the downstream operations Well, we have seen how different parameters affect the overall process of fermentation. This leads us to a basic question. Why is it necessary to take all the pains and try to optimize various process parameters? The answer is easy to fathom. The objective of any industrial process is to generate maximum profits. Now when we say that we have to increase the profitability, what is the first thing that comes to your mind? In other words, how can we increase the profitability of any industrial operation?

Either by reducing the cost of that operation, the cost of fermentation in this case or by increasing the selling cost of the product or preferably both. The selling cost of the fermented product is dependant on several factors like competition, demand – supply gap etc. and is not really under the control of the manufacturer. So the best a manufacturer could do to increase the profitability of fermentation is to cut down the cost of production without any compromise on the quality of the product. This is where fermentation economics comes into picture. • How do we make a fermentation process commercially viable? A number of basic objectives are commonly used in developing a successful process which will be economically viable. 1. Minimum capital investment in the fermentor and ancillary equipment. 2. Cheap, easily available and efficiently utilizable raw material. 3. Searching for possible alternative materials. 4. Use of the highest-yielding strain of micro-organism or animal cell culture. 5. Saving in labour whenever possible and use of maximum possible automation. 6. Employment of shortest possible growth cycle in a batch process to obtain the highest yield of product and allow for maximum utilization of equipment. 7. Adapting the simplest possible recovery and purification procedures. 8. Minimum effluent discharge. 9. Efficient use of heat, power and other resources. 10.Strict compliance of safety guidelines and regulations. Three key objectives emerge in economizing the fermentation: maximum product yield, process productivity and substrate utilization. However, these criteria may be overridden by the

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Ok, which of the above are the major areas where maximum expenses incur?

Four basic components contribute to the process cost in the following decreasing order: raw materials, fixed costs, utilities, labour. The cost of the various components of a production medium can have a profound effect on the overall cost of a fermentation process, since this account for 38 to 73% of the total production cost. A particular material may be selected because it is cheap locally, rather than the best substrate. When raw materials are a major part of the total cost of fermentation, maximum thrust must be given on the hunt for a cheaper, easily available or high product yielding substrate. For example, cane molasses is the commonest substrate used for alcohol fermentation in India. But due to the recent shortage of cane molasses in India, a continuous search for an alternative substrate has been on. Starch found in food grains appears to be a natural substitute. Similarly, Saccharomyces cerevisiae is conventionally used for alcohol fermentation. A shift from molasses based fermentation to say, starch based fermentation will also mean a shift from single stage molasses fermentation to two stage duel fermentation. Here, saccarolytic organisms will convert starch to simple sugars in the first stage and conventional yeast will convert the sugars to alcohol in the second stage. Many fermentations employ industrial wasters as their raw materials. Examples of these are sulfite waste liquor, corn steep liquor etc. The availability and chemical composition of these raw materials is subject to considerable fluctuations. In many cases, it is advisable to prefer a substrate of inferior quality if it is available locally so that the freight expenses are minimum. Indirect costs due to change in the substrate will also need consideration. For example, with the use of an alternative carbon source, the medium may become more viscous, thus requiring increased aeration and/or agitation. The cost of this extra provision must therefore be less than the savings from the change of substrate if the process is to be feasible. It is therefore worthwhile to develop a series of cost optimized media formulations so that the most cost effective growth medium can always be used when necessary. During the 1970s there was considerable interest in using petrochemicals as substrates for SCP production as protein animal feed by a number of major chemical and petroleum companies. A number of processes were developed using methane or methanol as the main carbon substrate, None are

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Mineral components normally constitute a smaller part of the cost of media, e.g. they account for 4 to 14% of the manufacturing cost of single-cell protein. Although feed grades of phosphates are more expensive than fertilizer grades, they do not contain impurities such as iron, arsenic and fluoride. This is an important consideration in the production of foods and drugs. The hydroxides and sulphates of potassium, magnesium, manganese, zinc and iron are preferred to the chlorides to minimize corrosion of stainless steel. The source of basic materials can cause considerable variation in product yield. Corbett (1980) compared six samples of calcium carbonate, and found that five of them reduced the titre of penicillin G in a production medium. Problems concerned with the storage, handling and mixing of media should not be neglected. Powders must be kept in dry conditions because of the possibility of substances becoming rock-like or glutinous. Some bulk liquids with a high solids content need to be kept warm to prevent them solidifying, e.g. glucose and corn-steep liquor. If storage temperatures are too high there could be degradation. It is also vital for workers to follow instructions for media preparation very carefully to prevent ‘lumpy’ media, etc. (Corbett, 1980). Monoclonal antibodies are produced using hybridomas between T-Lymphocytes and cancer cells. The growth of hybridomas takes place on media containing bovine calf serum. This serum forms the major part of the media cost. When this growth of hybridomas is achieved on serum free media, the cost of production is considerably reduced. Can you think of another such example? Use the space below to briefly write your thoughts.

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Clear. Now what about the fixed costs, utilities and labour?

Government aid or taxation can determine the viability of many fermentation processes. Some fermentations, in themselves, could be cost effective, but ultimately become uneconomical due to the heavy taxes and duties levied on the products produced. On other hand, some fermentations become economical due to the situational support offered by the Government even though the absolute cost of such fermentations does not do justice to the cost of the product. For example, concessions are offered in the taxes and duties levied on the production of pharmaceutical products like antibiotics during wartime. Agricultural-aid programmes in the United States of America made available low-cost supplies of grain and potatoes and enabled fermentations to be operated when they would not have been economically viable in a free market. Coming back to the example cited above, the worldwide low prices of molasses permitted the production of ethanol by fermentation in India till a couple of years back. A sudden rise in the price of molasses made its export economically more attractive. The simultaneous drop in global price of ethanol further made its import commercially sustainable, drastically reducing the alcohol production in India.

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The cost of utilities and labour also vary considerably all over the world making a particular fermentation operation economical or uneconomical in that geographic area. The fact that labour is available at a very low cast in India is well known. This has attracted many multinational companies for establishing their production units in India. However, the cost and reliability of utility services has been a cause of concern to many of them.

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The economics of a fermentation process will also depend on the efficiency of the organism employed, won’t it?

It certainly would. That is the reason most industries are continuously spending time and money on the improvement of strains of organisms used for fermentation. The most appropriate micro-organism for a potential process is usually found by isolation from a variety of sources, most commonly soil. The classical method of screening to obtain a suitable organism tended to be very time consuming, expensive and often without a very clear objective. It has been speculated that the screening of 100,000 soil micro-organisms may lead to the isolation of 5 to 50 new compounds, but there is no guarantee after evaluation that a useful new drug or other product will be found. The isolation may begin with pretreatment of samples which favour the survival of the preferred organism. This is followed by growth on selective or non-selective media and often associated with batch or continuous enrichment. Important factors which will be of economic significance which might be selected in a well planned screen could include: 1. Growth on a simple cheap medium. Why? The simpler (and cheaper) medium the organism uses, the lower will be the cost of production. 2. Growth at a higher temperature. Why? To reduce the cooling costs. 3. Better resistance to contamination. Why? To ensure better survival of the desired organism during the fermentation process. However, the synthesis of other microbial products (e.g. antibiotics) does not give the producing organism any selective advantage which might be used in an isolation procedure. Therefore a collection of these organisms must be made before testing for the desired characteristic. Because many isolation procedures will lead to the rediscovery of known organisms with known activities it is important to use well planned, efficient isolation procedures which can prove very productive. There are many way in which a desired organism can be isolated. We have seen the details of this in the screening part. It is however, significant to note that more interest has now been shown in isolation of strains of the less common genera of organisms as compared to the conventional ones. For example, species of Actinomadura, Actinopianes, Kirosarospona, Streptoalloteichus, etc. which are producing other novel bioactive compounds are getting more attention compared to the conventional genera like Streptomyces. Attention is also being paid to isolate organisms from unusual habitats, which may include extreme environments, to ensure that the greatest microbial diversity.

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now being operated, Major factors which contributed to making these processes uncompetitive included the increased cost of the substrate and the availability of cheaper alternative.

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So, when we have isolated a desired organism, what next? Next step in economizing a fermentation process is to improve upon the selected strain. Strain improvement using a mutation/selection programme for improving an organism is effectively used for reducing the cost of fermentation. Historically, mutation/selection programmes to improve strains of Penicillium chrysogenum were time consuming, labour intensive and very random because of the lack of knowledge about penicillin biosynthesis. These mutation programmes did, however, contribute significantly to increases in penicillin yields from less than 100 units cm -3 in the 1940s to over 51,000 units cm -3 by 1976 and a four-fold increase in yields between 1970 and 1985 at Gist Brocades. Improvements for streptomycin, chlortetracycline and erythromycin are reported in Table

•

It is however, a matter of considerable debate whether the mutation / selection programme for improvement of the economics of the fermentation process is worth its while.

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Why?

Because first, the mutation is a matter of pure chance and there is no guarantee whatsoever that a certain number of mutation experiments will produce the desired mutation. More often than not, an extremely large number of mutations will be needed before a single desired mutation is detected. Secondly, the actual increase in yield because of a mutation has to justify the cost incurred in carrying out all the strain improvement studies. It would be, in some cases, more prudent to continue with the fermentation using the wild type of organism rather than keep on experimenting with the hope to find a high yielding mutant.

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But can’t we carry out a more directed attempt to change the desired genetic character?

Sure we can. A better understanding of cell metabolism and its regulation has enabled the development of more logical targeted’ methods to” be introduced to select for mutants with desirable ‘blue prints’ where there may be a need to block undesirable enzyme activities or eliminate negatively acting control mechanisms. This approach is much more efficient and economic in terms of resources and time. It was first employed extensively in the preparation of mutant strains used in amino acid fermentations. Although the main targets in strain improvement are normally to increase the product yield or specific production rates, it is also important to consider strain stability, resistance to phage infection, response to dissolved oxygen, tolerance to medium components, production of foam and the morphological form of the organism. These are very important in helping to achieve targets in a research and development programme as they can have a significant impact on the process and/or product. In short, the output of the strain improvement experiments should justify the cost incurred on them. And then, of course, we have to consider the ultimate market price of the product which affects the economics of the fermentation process.

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How the price of the product will affect the fermentation economics?

Four categories of microbial product can be recognized economically and it is important to consider to which category a compound belongs: The first category is that of low price bulk chemicals. e.g. solvents, biomass, high fructose syrups. The next category is that of mid price chemicals. e.g. organic acids, amino acids, biopolymers etc. The third category is of high price microbial and animal-cell products e.g. enzymes, vitamins, antibiotics, corticosteroids, vaccines etc. And the last category is that of very high-price animal-cell products, e.g. monoclonal antibodies, tissue plasminogen activator etc. The third and fourth groups can normally be produced only by a microbial or animal-cell based process and therefore do not have to compete with an alternative chemical process which is usually much cheaper. This includes compounds which have complicated structures, are chemically or thermally unstable or for which a multi-stage chemical synthesis would be expensive. Many microbial products are not exploited because cheaper synthetic processes are available. Again coming back to the example of ethanol production, the fermentation would only be competitive with synthetic ethanol from crude oil if the fermentation plant was in an area where cheap supplies of carbohydrate were available. In addition to that, the fermentation would be commercially viable only if the prices of petroleum products consistently remain above a threshold level. Many times, in order to safeguard the interests of local industries, the government levies heavy duties on the import of petroleum products, thus MAKING the fermentation commercially viable. Another example in this connection would be that of products produced using genetically modified microorganisms (GMMOs). Generally these products are expensive by themselves. Further, the cost of production increases because of the additional containment procedures that must be adapted. The level of containment increases directly with the degree of hazard associated with the product. The cost of production, too, increases accordingly.

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Next, is it possible to economize the fermentation process itself ?

Sure. It is technically possible to cut down on the costs of all major operations involved in the fermentation. This includes sterilization of the air used for fermentation. The problems associated with producing large volumes of sterile air for aerobic fermentations are unique. Although sterilization by heating is technically possible, it has generally been regarded as too costly for full-scale operation, although it might be used in the treatment of exhaust gases. Absolute fixed-pore membrane systems using pleated membranes of PTFE are now widely used in the fermentation industry and have proved to be very reliable. This is very important when considering the costs associated with loss of fermentation batches due to contamination and production downtime due to filter failure. A contamination probability of 1 in 1000 is economically acceptable for microbial batch fermentations; while in large scale

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Operating costs will be based on the estimated life of the filters. Factors to consider include the cost of replacement filters or filter materials, servicing and labour. Even if the filters could be cleaned there must be an, allowance for depreciation due to normal wear and tear. Savings may also be made by introducing series filtration whereby the major part of the foreign matter from the air stream is taken out by varying degrees of coarse filtration, thus reducing renewals of the more expensive highefficiency filter media such as membrane filters. The treatment of fermentor exhaust gases to satisfy containment requirements is also important. Treatment is normally by filtration with 0.2-ll-m hydro filters, but in-line incinerators may be an alternative approach. Filtration is usually cheaper but it may be necessary to supplement filtration with incineration depending on the process and scale.

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Fermentations could be exothermic. In that case, it would be necessary to cool the fermented broths. On the other hand, organisms may require higher temperature for their growth. There you would need to heat the fermentation medium. Can these processes be economized too?

Very good question. Of course, you can cut down on the costs incurred on the heating and cooling operations. Ideally there should be no heating or cooling at any stage in a fermentation process, but because this is virtually impossible, heat should be conserved and cooling minimized by careful process design. Where all do we need to undertake heating and cooling operations? 1. Sterilization or boiling of the medium to 100° or above followed by cooling to 35° or below. 2. Heating the fermentor and ancillary equipment to sterilize it, followed by cooling. 3. Heat may be generated during the fermentation. This heat output has to be removed by cooling to maintain the growth temperature of the microorganism within prescribed limits. Cooling requirements will be influenced by the size and type of an individual process. 4. After harvesting, heat may be required to remove water from the product.

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Now, how can we minimize the energy inputs required for the heating and cooling operations?

To reduce cooling requirements, the specific energy input may be minimized through the use of air-lift fermentor. Cooling equipment has .been estimated at 10 to 15 % of the investment cost for single-cell protein. Another way to minimize cooling costs is to use micro-organisms with higher optimum growth temperatures, if it is feasible. The selection and use of thermophiles and thermotolerant organisms would have obvious advantages to reduce cooling demands. Can you think of any other methods which would help in reducing the energy required for heating and cooling? Use the space below to explain your thoughts.

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•

·Ok, where else can we save and how?

In addition to the above, considerable savings could be achieved by carefully controlling the energy requirements for aeration and agitation. Nearly all fermentations require some form of mixing to maintain a constant environment, and many also need aerating. Fermentations may be broadly classified into: 1. Fermentations which are anaerobic where oxygen is undesirable, e.g. acetone-butanol. Obviously, no aeration is required here. 2. Fermentations which have a minimal oxygen demand, e.g. ethanol. 3. Fermentations which have a high oxygen demand, e.g. antibiotics, acetic acid, single-cell protein. In categories 1 and 2, aeration is not generally regarded as a major economic consideration. During acetone-butanol fermentation carbon dioxide and hydrogen are evolved. Once this gas production starts it will help to maintain anaerobic conditions and stir the mass of broth without the need for mechanical agitation. Anaerobic conditions are achieved initially in a production fermentor by maintaining a positive pressure of filtered carbon dioxide and hydrogen obtained from another established fermentation. For ethanol production, the yeast inoculum in the vessel is initially dispersed in the medium by compressed air or by mechanical stirring. Aeration or agitation is stopped once the biomass concentration reaches a predetermined level. A vigorous anaerobic fermentation commences, and the evolution of carbon dioxide bubbles stirs the contents of the vessel and disperses the cells in the medium so that mechanical agitation is unnecessary. In this process aeration and agitation are considered to be a minor component of the total production costs. Fermentations having a high oxygen demand must be agitated with sufficient power to maintain a uniform environment and to disperse the stream of air introduced by aeration. It has been mentioned that the cost of energy necessary to compress air for yeast production proved that a considerable amount (10 to 20%) of the total production expenses was due to aeration. The mixing costs in penicillin fermentation for example, are over 15% of the total production costs. In single-cell protein processes, the carbon substrate yield coefficient is the most critical physiological factor. It is also well documented that much higher carbon-substrate yield coefficients are obtained with methane or n-alkanes instead of carbohydrates. Unfortunately, cells grown on hydrocarbons have greater oxygen requirements. The oxygen requirements of a hydrocarbon yeast fermentation is almost triple that of a yeast fermentation grown on a carbohydrate substrate and producing an equal quantity of cells. Therefore, if there is to be effective utilization of a hydrocarbon substrate, which can account for over 50% of total production costs, the production fermentor must have a high oxygen-transfer capacity. The demands on fermentor design are further complicated by the hydrocarbon fermentation being highly exothermic, which necessitates the provision of good cooling facilities if a constant temperature is to be maintained in the fermentor.

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animal-cell culture processes contamination rates as low as 2% are now achievable.

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A few companies developed SCP processes using mechanically stirred Fermentors with sparged air. BP Ltd. constructed vessels of up to 1000 m3 capacity for their n-alkane process in their Sardinian Ital protein project. In the Swedish Norprotein process it has been estimated that the total utilities costs, which included aeration and agitation for 100,000 tons/ year of SCP would only be 16% of total production costs. A number of companies, including ICI plc and Hoechst decided to develop Fermentors based on the air-lift principle. The main advantages of these Fermentors are simpler design and reduced energy and cooling water costs. Since the energy supplied to an air-lift fermentor is only supplied with the air, it is crucial to obtain a fermentor design which minimizes the energy requirement for biomass production yet creates high oxygen-transfer facilities to ensure efficient substrate utilization. In the ICI process, the estimated manufacturing costs for all utilities were 14%, with aeration accounting for 70% of fermentation utility costs.

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All right. Now that the fermentation is over, where else can we cut the corners and make a saving?

After the process of fermentation is over, the desired metabolites must be recovered out of the fermented broth. Earlier, the costs of product recovery were very low compared to the cost of production. Now a days, the products produced by fermentation are low volume high value ones that demand extremely complicated and expensive recovery processes. Therefore the recovery costs are routinely several times more the production costs. It is therefore of paramount importance that the recovery techniques are appropriate and involve lowest possible expenses. For example, the separation of microbial cells from the broth can be done either by filtration or centrifugation. However, it is considered that removing cells by filtration is less energy consuming than by centrifuging. If filter aids are to be used in the most economical way this cost can be further reduced.

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Why are the recovery costs so high?

1. Yield losses, even if only modest, are certain to occur at each stage of the recovery process. This also emphasizes on having the minimum number of stages in the recovery of the product. The more number of stages you have in the recovery system more are the losses incurred. 2. High energy and maintenance costs associated with running filtration and centrifugation and equipment. 3. High costs of solvents and other raw materials used in recovery and refining of products.8% for citric acid and 4% for penicillin G used to get lost in the recovery and purification stages before conversion to the potassium salt in production processes. It is thought that depreciation, return on capital and maintenance can account for over 80% of the’ overall cost for a large-scale rotary filtration or centrifugation plant. One of the main factors affecting centrifuge economics is the size of the particle to be separated. Filtration costs are less dependent on particle size. At a particle size greater than 1 to 2 um, centrifugation is more economical. Below this size ultrafiltration becomes more economical.

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There are some extraction procedures which are economically attractive but cannot be approved by the local governing authorities because of reasons of pollution, safety etc. In such cases, we have to forego the economic benefits and abide by the regulations. Well, a lot is heard these days about water recycling and waste water management in industries. Will this have anything to do with the fermentation economics? Most certainly. Ideally an industry will be optimally utilizing its water resources if its discharge of liquid effluent is zero and all the liquid effluent is recycled after treatment. Indeed, this can be done with the employment of modern techniques in water and waste water management. But again, the capital investment made for such operations and the recurring costs involved have to justify the savings offered. In such an example, the water from the mash cooler in a bacitracin plant was collected and reused to charge the mashing vessels and wash the Fermentors. Water from the cooler coils was used to wash down the discharge cake from the filter presses. Recycling of water was an integral stage of large scale SCP processes developed during the 1970s to minimize water consumption, reduce effluent treatment costs and reduce media costs by recycling of spent media. When ICI pic’s SCP Pruteen plant was operating, it was designed to recycle most of the fermentor medium water. Under optimum conditions they claimed that the water loss could be reduced to 3% of the flow through the fermentor using water recovery systems. Well, if we want to economize on the water and waste water expenses, we need to know about the water volume, the organic and solids loading, range of pH variation, nutrient level, temperature fluctuation, and the presence of any toxic compounds. It will also be necessary to consider company finance policies, the site location and government legislation for waste disposal. How? Think about it and write your thought in the space provided below.

Exercise 1. Visit a distillery in your area. Find out the areas where economization is possible. Write a report. 2. Make groups of two students. Write an imaginary case of a fermentation. Give it to your partner. Ask him / her to suggest ways to economize the fermentation. Do the same to the case designed by him / her. An example is given below: Activity of the unit: SCP production Location of the unit: Gorakhpur, India Organism used: Saccharomyces cerevisiae Medium used: A mixture of beet and cane molasses and various salts including ammonium and phosphate salts. Cornsteep liquor may be added to supply organic nitrogen compounds. Fermentation procedure: To prepare the fermentation medium, the molasses and cornsteep liquor (if used) are adjusted to pH

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4.5 to 5, heated, filtered (or sedimented and decanted), and diluted to provide a concentration of 0.5 to 1.5 percent sugar. Aqueous ammonia also is added during the fermentation as needed. The fermentation temperature is maintained at 30°C or less, and the culture broth pH is maintained between 4 and 5 to aid in controlling the growth of bacterial contaminants. At initiation of the fermentation, the culture is aerated, with the aeration rates then increasing during the next 8 to 12 hours as the fermentation progresses. This aeration requires a considerable volume of air, and the costs attributable to aeration can be as high as 20 to 30 percent of the total cost of yeast production. At the end of the 8- to 12-hour fermentation period, the aeration rate, decreased, and the sugar and ammonia additions are stopped. The cells are allowed to mature for an hour. At harvest, the culture broth is cooled, and the cells are removed from the fermentor by centrifugation. The yeast cells are then washed in water and centrifugation to remove the residual impurities. This washing may be repeated several times. The cells finally are separated on a filter press from the aqueous phase, mixed with plasticizer (small amounts of vegetable oils,), and extruded in block form. This block then is cut into portions of commercial size and weight, wrapped, and stored under refrigeration. Now where can we cut the cost of production cost?

See where the plant is located. It is an area where cane molasses is abundantly available. Even if the price of molasses is same all over, due to the geographic location of the plant, we would be able to save considerably on the freight if we eliminate all other carbon sources and carry out the fermentation exclusively based on cane molasses. We will have to search for a suitable cheaper nitrogen source (urea?). Beet molasses is out of question because of the cost. Now see the temperature profile of the fermentation process. It involves both heating and cooling. Can these two operations be coupled using a heat exchanger and the energy inputs be reduced? After the cells are separated from the broth, the liquid effluent is discharged for treatment. Can this be recycled? For what? One possibility is for the dilution of molasses. Another is for cooling purposes. Can you think of anything else? Think on similar lines.

Notes

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LESSON 34: FUTURE OF FERMENTATION

the future if changes should occur in the economic and demand pictures for their respective fermentation products.

Learning Objectives In this lecture, you will learn

• • •

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The future of fermentation industry Biotechnology in future Opportunities & challenges of tomorrow’s biotechnology

It is difficult to say just what place in our society the future holds for industrial microbiology. Many different factors are involved, and it is the interaction of these factors which will control its future. It is apparent that many different industrial fermentations are presently available for commercial usage, but that relatively few are actually practiced. Some of these fermentations were practiced in the past but, because of changes in the competitive positions of the fermentation products, they are no longer able to compete on the open market. This state of affairs was brought on by increased costs of substrate materials and labor and equipment, by competition from chemical synthesis routes, and by the discovery of other compounds or products more suitable for the particular applications over which the fermentation products had held rein. Another group of available industrial fermentation processes which are not practiced at present include those for which a fermentation process has been developed, but for which a public demand has not been created. Included in this group are fermentation-derived products that are not quite as good as or do not perform quite as efficiently for specific uses as do other compounds already in commercial usage. Also included are fermentation products which, because of high production costs, including low yields and recoveries, have not been in a position to compete on the open market. A third group of fermentations is concerned with a vast array of microbial metabolic by-products for which workable fermentation processes, other than small-scale laboratory studies, have not yet been developed. These metabolic byproducts are often unusual compounds not yet producible by chemical synthesis, but frequently no commercial Usage can be found for them. Thus, Raistrick (l950) has described many unusual and often chemically reactive compounds produced by fungi, but these compounds have not been studied in enough detail or considered thoroughly enough from the commercial standpoint for their present commercial exploitation. Of course, the development of fermentation processes for the production of these compounds also is deterred because of a poor patent position. Without a defined demonstration of utility (which of course strengthens the case for the granting of a patent), the economic drive for process development is lacking. These various groups of presently unused industrial fermentation processes all could find commercial exploitation in

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But what about those industrial fermentation processes being practiced? What will be their future status in the open market? It has already been observed that chemical synthesis routes to products similar to those produced by fermentation have made deep inroads into the fermentation market. A notable example, of course, is that of petroleum-derived chemicals. These petrochemicals will probably become a yet greater threat to industrial fermentations, particularly as the chemical synthesis technology advances and the costs of fermentation substrates increases. Thus, the production of the simpler chemical molecules will probably be relegated to the chemical synthesis industries. However, microbial processes for producing the more complex organic molecules, the molecules for which biologically active isomers are required, and the molecules requiring microbial enzymatic transformation steps should retain their competitive position on the market. This can also be said for biologically active microbial products such as various enzyme preparations. Certain fermentation products have enjoyed, in the past, and still do enjoy an unusual economic position. In particular, beverage alcohol has had no problem in competing with petrochemical alcohol, while fermentation-derived industrial alcohol has not been in such a fortunate position. These two types of alcohol are in actuality the same compound, the difference being that government regulations protect the fermentation production of beverage alcohol. So, the question may be raised, however, as to whether government protection will continue. For the most part, this is not a bothersome question, because the flavor, aroma, and other complex characteristics of the various beverage alcohol products are difficult if not impossible to reproduce by purely chemical means, so that fermentations of this type should continue to be able to compete on their own merit. Nevertheless, the question might be raised as to whether other industrial fermentation processes should be protected by government regulations against the competitive inroads by chemical synthesis processes. This question can be answered by stating that there probably is little need for this coddling of the fermentation industry. There are enough possibilities, both explored and unexplored, for economically sound industrial fermentations that such regulations should not be required. Government regulations of fermentation processes and products presently play a large role in another area of the fermentation industry that of the safety of pharmacological compounds used in medical practice. Obviously, such

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Tremendous strides have already been made in discovering and developing processes for fermentation-derived products for the treatment of disease, and it is hoped that government regulation of these products in the future will be such that the economic reward accruing to those who search for new products of this type will be great enough to entice them to continue their search. Government regulation in the form of patent grants is a boon to the industrial fermentation industries. The cost of research is so great that the gamble is considered worthwhile only if it is known that strong patent protection for the product or process can be obtained. Thus, the future statement and workings of our patent laws will have a profound effect on industrial microbiology. In this regard, however, there are distinct indications of impending changes in the patent laws, although most of these changes may take place in countries other than the United States. There have been discussions concerning the possibility that, at least in portions of Western Europe, a single patent might be granted that would provide protection in several countries to the inventor-a multination patent. There is also the possibility that countries such as Italy may change their outlook on the issuing of pharmaceutical patents. The question of patent protection in the underdeveloped nations, obviously, is unanswered at the present time. The patent laws of these nations are bound to change, particularly as developments occur in the availability of fermentation substrates, requirements for fermentation products, and economic climates for fermentation industries. Although not presently included in tile protection provided by patent grants, it is likely that courts in the United States and in other countries will deem it necessary to provide greater protection than is presently available for company secrets, microbial cultures, and so forth, since these are vital factors in the ability of a fermentation process or product to maintain a competitive position in the market.

•

What changes are likely to take place in the processes of existing fermentations? Those industrial fermentation processes that are presently practiced will require continued research and development. In particular, many of these fermentations will require increased yield capacity if they are to maintain their economic positions. An important aspect of the yield capacities for these fermentations involves continuous research on strain selection, both from natural sources, and through induced changes such as by mutation or hybridization in strains already at hand. Alternatively, other organisms (even organisms from quite different genera or families) with the capacity to produce a particular fermentation product should be evaluated as a substitute for the organism normally utilized as the fermentation agent.

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How the screening processes will be changed in future?

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To obtain and evaluate organisms for these processes, as well as to obtain organisms for new and different fermentation processes, will require the development of better and more efficient primary and secondary screening techniques, including those techniques for the detection and assay of fermentation products. The latter also includes the ability to detect the presence in fermentation broths of new fermentation by-products, whether or not these compounds have ever been previously observed from any source. Some of these techniques should allow the screening of groups of microorganisms from soil and marine environments which, as yet, have not been investigated to any extent, or which, although existing in these environments, have rarely or possibly never been cultured successfully in the laboratory. For instance, a group of microorganisms which occurs in soil in large numbers, but which is difficult to grow in the laboratory by conventional techniques, has as yet not been evaluated for its possible ability to produce fermentation products of value. Also, another potential group of industrial organisms, the nonspore-forming obligate anaerobes, is known to be present in soil, but little is known of their numbers and types in this environment. Thus, the vast numbers of possible microorganisms and strains of these organisms existing in natural microbial sources, compounded with the mutational and metabolic control possibilities for each individual strain, should allow virtually unlimited source material for investigation. The mutational and other genetic approaches to obtaining industrially important microbial strains offer fascinating possibilities. Included among these approaches are the use of mutagenic agents, hybridization of fungi, and various transfers of genetic material in bacteria. In a sense, the fermentation industry is waiting for the microbial geneticist to determine for it the finite points of the genetic make-up of the cell, and to develop better means for its manipulation. With this knowledge at hand, greater use could be made of genetic blocks to unmask useful but minor metabolic sequences of the cell, and of genetic transfers to introduce new genetic material into cells. New mutagenic agents are continuously being discovered that enhance the frequency of specific mutation types. We hope that new mutagenic agents also will be found that will broaden, through mutation, the total phenotypic spectrum of individual microorganisms so that new commercially valuable strains of microorganisms with undreamed-of synthetic capabilities will result. Greater use also should be made of nongenetic manipulations of the regulatory mechanisms of the cell. Specific enzyme poisons and various substrate and product inhibitions of specific metabolic pathways should be employed in order to bring into play minor but valuable metabolic sequences from the industrial standpoint. A somewhat similar approach would be to make use of the faulty metabolism of aging microbial cultures; that is, cultures which have been incubated for considerable periods of time.

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regulations are of benefit to the public as well as to the fermentation industry. This is true, however, only so long as these regulations are wisely defined and administered.

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However, Raistrick’s approach was laborious in that he often had to employ difficult and involved chemical fractionation procedures to detect and characterize these products. In contrast, however, modern-day procedures will detect and often characterize products in one or a few operations, and the use of these procedures should markedly simplify the detection and characterization of products such as those studied by Raistrick. The application of new screening techniques, as well as of these presently available, should engender success in the search for alternate products present in fermentation broths of commonly practiced industrial fermentations, products which, thus far, have been overlooked. These fermentations were developed, with one product in mind, because that product could be easily identified and possessed commercial value.

•

The search for new products and from new and existing fermentations will continue, isn’t it?

Of course, it will. For example, the presence of 6aminopenicillanic acid in penicillin fermentation broths was overlooked for many years, although this compound at the present time has considerable economic’ value for the production of new penicillins. It is difficult to predict what other compounds might be present in various fermentation broths without first performing a thorough search for such compounds, assuming of course the availability of the methods for their detection. Thus, it is entirely possible that the culture broths of some fermentations may contain compounds with pharmacodynamic action if we could but devise methods for the detection of these compounds. The possibilities for the existence of valuable by-products in fermentation broths are multiplied when mutation and other regulatory controls of the metabolism of the organism are employed since, even though these metabolic controls are directed towards increasing the yield of a specific and known product of economic value, the alterations in unconsidered metabolic pathways may well yield other valuable products during the prime fermentation which remain only to be detected. In fact, it is always possible that such alternate products may be of more economic value than the primary fermentation product so that the fermentation could be redesigned to make such a product the primary product of the fermentation. It is obvious from the foregoing discussion that vast screening programs may be required to attain these ends. But screening programs are becoming increasingly more expensive in labor, time, and materials. Obviously, the better designed a screening program is, the more quickly will it yield the desired organisms and with the least cost. Nevertheless, even the best of designed screening programs are still, expensive, and methods for decreasing screening costs obviously will need to be worked out. One approach to reducing screening costs, now being employed to a limited extent, is to have the screening carried out in nations that have low labor and overhead costs. While this solution may be attractive at present, it certainly is not a longterm answer.

•

How would be the effluent treatment looked upon in future?

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Spent fermentation medium should be studied not only for its potential content of valuable by-products, but also to find better uses for it than to be sewered to become a problem of waste disposal. The same applies to spent microbial cells at the termination of fermentation. The spent media and cells still contain a lot in the way of fermentation nutrients and, if properly handled, could well be used to supply at least some of the nutrients for fresh fermentation media. This is analogous to the slopping-back procedure employed in the acetone-butanol fermentation. The use of spent microbial cells may require that the cells first be lysed or broken up in some manner, thus liberating their store of potential fermentation nutrients. Obviously, such reuse of spent medium and microbial cells would contribute greatly to the economic position of a fermentation, since this procedure would help hold down the high cost of fresh fermentation nutrients. Spent media and cells also could find further use as animal feeds or feed supplements. In addition, with an improvement in their palatability and acceptability, they might find use as food for man. To date, most industrial fermentation processes have been developed to comply with the existing designs of fermentation equipment. This equipment mayor may not be ideal for individual fermentations and, in fact, there are fermentations (for instance, those utilizing hydrocarbons as substrates) which probably would provide greater yields in a shorter period of time if the fermentation tanks and auxiliary equipment were redesigned specifically for these fermentations. Thus, at least for some fermentation, the fermentation equipment should be designed for the fermentation instead of vice versa. The design of fermentation tanks and auxiliary equipment should take several factors into account. There may well be a greater future use bf continuous fermentations, multistep fermentations, and even fermentations employing mixed cultures or unusual microorganisms.

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How would the fermentor design and the instrumentation and control techniques change in future?

Thus, specific equipment will need to be designed to provide a high level of efficiency for such fermentations. Special fermentation tanks also will be needed for highly oxygensensitive anaerobic microorganisms, for fermentations employing gaseous substrates, and for other fermentations employing unusual substrates or microorganisms having unusual or sensitive growth requirements. Fermentation tanks should also be designed to provide better aeration and agitation with less power input and, in addition, the design should consider means for more adequate foam control and prevention of contamination. More refined electronic controls for the monitoring and control of pH, temperature, nutrient addition, and so forth, and for the programming of these variables in a defined manner, should be made an integral part of the fermentation equipment. Computers should more frequently be associated with production fermentors, especially in order to make immediate decisions between alternative steps to be taken if contamination or the biological variability of the fermentation microorganism should gain the upper hand in the

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•

The fermentation media composition and sterilization technology will also undergo some changes in future, won’t they?

Yes, of course. As has been pointed out, the expense of media sterilization is great enough that certain industrial fermentations employ unsterile media so as to maintain economic feasibility for the process. Also, it has been noted that the media employed in some industrial fermentations are difficult to sterilize because of their crude organic substrate components, and that the heat requirement for sterilization of these media components can mean that other components of the media become overcooked. Thus, new, more efficient, and cheaper means of media sterilization are needed. Gaseous sterilization, as with ethylene oxide, is a possibility, but this procedure is probably far from any semblance of economic feasibility for large-scale fermentation processes. The availability of cheap organic compounds as fermentation medium carbon sources today is a critical problem of fermentation economics, and it will become more serious as time progresses. In fact, it is possible that carbohydrate and protein fermentation nutrient sources in the temperate areas of the world in the future will become so expensive that they cannot be employed as fermentation nutrients. Because of the rise in population density, these nutrients will have to be channeled for use as human food and animal feed. However, carbohydrate sources for fermentation media are presently available in high quantity and low price in the tropics, and it is likely that, in the near future, various fermentations (including those yielding microbial cells as food and feed as well as those yielding fuel alcohol) will be practiced more extensively in this part of the world. Other fermentations also may gain prominence as some of the underdeveloped nations in these areas achieve a higher technological level. Obviously, new sources of fermentation nutrients, particularly carbon nutrients, will have to become available. Hydrocarbons as fermentation substrates have already been discussed, and it is probable that hydrocarbons will be employed much more extensively as the carbon substrate for the fermentative production of chemical compounds as well as for the production of microbial cells for food and feed. Also, hydrocarbons may find extensive utilization as substrates for microbial conversions, such that in a one- or two-step transformation a reactive chemical group or groups becomes incorporated into the hydrocarbon molecule to make it acceptable for various commercial uses and as a starting point for further chemical alterations or syntheses. Cellulosic agricultural by-products present a possible source of carbohydrate fermentation nutrient but for most fermentations the cellulose will need to be hydrolysed by some means. Acid hydrolysis is possible, as is the uses of micro-organisms (or their enzymes) which have strong cellulolytic activity.

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Spent fermentation media and the exhausted cells from harvested fermentations, as previously stated, also are possible sources of fermentation nutrients. Another possible source rich in available nutrients is domestic sewage. For most fermentation processes, the sewage waters would need to be sterilized before they could be used, but the expense of sterilization, as contrasted to the expense of a source of readily available fermentation nutrients, might be minimal. A similar Source of fermentation nutrients exists in the high BOD wastes from food-processing plants.

•

What would tomorrow’s agriculture and food industry look like?

The potential for industrial microbiology applications to agriculture and food production is vast and certainly has been only nominally explored. Various fermentation products already find extensive use in agriculture (for instance, the use of antibiotics in the prevention of plant and animal disease, as an animal-growth stimulant when incorporated into feeds, and as an aid in food preservation.) Vitamins, gibberellins, auxins, and related substances also should find continued and possibly expanded use as plant- and animal-growth stimulants. However, microorganisms may well find other extensive applications in agriculture. Thus, batches of microorganisms grown in fermentors could be incorporated into the soil to fight soil-borne plant diseases although, admittedly, more information on the ecology of soil microorganisms will be required to increase the feasibility of this application. Microorganisms also could be added to soil to release plant nutrients that are bound in soil in forms unavailable to growing plants. Non symbiotic nitrogen fixation in soil has received considerable study in the past, but without resulting in the discovery of any decisive means for increasing the Content of the fixed nitrogen of the soil. Russian investigators on In any occasions have reported increased crop yields on incorporation of Azotobacter strains into soil, but this may have been more a growth-stimulation phenomenon for the plants than due to for adding nitrogen-fixing strains of Azotobacter or Clostridium species to soil, or for incorporating various chemicals into soil to adjust the soil ecology in favor of the nitrogen-fixing microorganisms, are still areas for exploration. Rhizobium inoculant has been and still is employed extensively for the inoculation of legume seed. A somewhat similar symbiotic association that of the mycorrhizal fungi with the roots of trees and other plants, should receive extensive study for possible commercial exploitation. Although the exact reasons why the mycorrhizal fungi stimulate plant growth are not yet clear, these fungi obviously are beneficial to the plant, and possibly could be grown in large volumes in fermentors for incorporation into soil with seeds or seedlings of the proper plant species. Microorganisms such as Bacillus thuringiensis which destroy insects have present agricultural applications, and other

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fermentation. It is assumed, of course, that these refinements or changes in the design of fermentation equipment will not only provide fermentations with higher yields in a shorter period of time, but also will greatly reduce the total expense associated with the fermentation.

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microorganisms with this capability doubtlessly will be evaluated and find use in agriculture.

residual hydrocarbon. The protein from several yeasts is low in one or more of the essential amino acids, and this has caused hesitation in the development of fermentation processes for growing these yeasts as protein sources. However, the critical amino acids can be produced by specific bacterial fermentations, so that the yeast protein could be fortified with the deficient amino acid(s) as obtained from the bacterial fermentation sources.

Bacillus thuringiensis viewed by phase contrast Microscopy Microorganisms that destroy lower animal forms also may well find application in .agriculture, for instance, the nematodetrapping fungi that entrap and kill these tiny wormlike parasites of the roots of growing plants. A word of warning is in order, however. The above and other similar uses of microorganisms in agriculture can cause limited to extensive alterations in the ecological balance of the microorganisms that occur in soil, on plants, and in the intestinal tracts of animals, birds, and so forth, so that a better understanding of microbial ecology again would be of distinct value, This also is true for those situations in which antibiotics and nonmicrobially produced chemicals such as herbicides, fungicides, bactericides, and insecticides are incorporated into the soil, onto plants, and into other natural environments. • With the increase in population, is it possible that more thrust would be given on the microbial production of proteins? The production of microbial cells for food and feed probably will come under increased pressure from the population explosion in all parts of the world. Investigations already are underway to evaluate Torula yeast, as produced by sulfite-waste liquor fermentations, and Candida yeast, from hydrocarbon fermentations, for this purpose. Cellulose agricultural byproducts are potential carbohydrate sources for growing yeasts of this type, but these substrates will require preliminary acid hydrolysis of the cellulose, or the use of cellulolytic microorganisms or their enzymes for the hydrolysis. Present fermentation procedures for producing yeast protein from hydrocarbons utilize liquid hydrocarbons; the yeast removes most of the paraffinic portion of the hydrocarbon, leaving behind the aromatic fraction and some of the more highly branched paraffins. This aromatic fraction is difficult to separate completely from the cells at harvest-a procedure, however, which must be accomplished because of the potential carcinogenic activity of certain of the components of the

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Microorganisms other than yeasts are also known to produce protein in good yields. Various bacteria produce considerable protein during growth, including some that do so during growth on hydrocarbons, and these organisms may well be useful as a protein source if their cells can be made palatable and acceptable as a food or feed. Studies are already underway to utilize methane and other small molecular weight gaseous hydrocarbons as the carbon substrate, so that no residual hydrocarbon will be associated with the bacterial protein. Algae and, possibly, photosynthetic bacteria are particularly attractive sources of protein, because their photosynthetic capabilities practically negate the requirement for substrate organic carbon nutrient. Again, however, palatability, digestibility, and acceptability of these organisms as food or feed must be evaluated. The fat-producing yeast Rhodotorula gracilis is a particularly intriguing source for food and feed. This organism undergoes normal cellular growth during its logarithmic growth phase in a medium containing adequate nitrogen. However, at the onset of nitrogen starvation, this organism accumulates large amounts of lipid within its cells. It has been pointed out, however, that the protein-lipid ratio occurring in this organism can be varied at will by the way in which its nitrogen nutrition is handled, while at the same time allowing it to produce other products, such as B vitamins, ergosterol, and pro-vitamin A. The discovery and development of microbially mediated means for producing new foods and flavoring agents for human consumption, and of better methods for the production of similar presently available foods and flavoring agents, are distinct possibilities. For instance, the mushroom of commerce, the Agaricus mushroom, presently is grown in a commercially acceptable form only on compost beds, and it would be hoped that submerged, aerated fermentations to yield this organism in an acceptable form will be accomplished in the near future. The somewhat exotic foods such as soy sauce, various cheeses, and fermented milks have been microbially produced for centuries. Monosodium glutamate is presently produced by fermentation, and the nucleotide flavoring agents are under study. There is no reason to believe that similar or even other types of foods and flavouring agents will not soon be discovered and developed as products of microbial action. The rumen of the cow is known to be a vital component of the metabolism of this highly productive animal. The rumen allows the functioning of an anaerobic balanced ecology designed for the natural. fermentative breakdown of cellulose and for the conversion and degradation of other natural compounds. Since the rumen exists within the living animal, the temperature and certain other incubation conditions are constant, so that the rumen actually is an ideal fermentation vat.

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•

What shape the pharmaceutical industry take in future?

Many good antibiotics have been discovered in recent years, and new antibiotics are still being discovered. New uses for antibiotics may well be found so that additional impetus will be applied to the search for organisms producing new antibiotics, and for ways of chemically modifying those antibiotics that presently are of little commercial value because of toxicity or other problems. Antibiotics are needed which are active against common viruses, against L-form bacterial states (which apparently are induced in the body by continuous antibiotic therapy), and against various carcinomas. Already there is some evidence that antibiotics are effective for these treatments, and that there is sufficient difference in the toxicity level for the disease agent as contrasted to the healthy tissue. The build-up of antibiotic resistance in certain pathogenic microorganisms, such as Staphylococcus, has caused alarm in the past, and the search will continue for new antibiotics to use in instances in which this has occurred. Chemical or microbiological modifications of the structures of existing antibiotics have already yielded a certain amount of success, for instance, in the use of microbial enzymes to remove the side chain (R group) of penicillin to yield 6-aminopenicillanic acid so that new penicillins can be synthesized.

deterioration prevention, and other uses for their biological activity. Antibiotics such as tetracycline are presently being employed for the preservation of fish and poultry. It is possible that a similar application of antibiotics for the preservation of meat and other foods will gain acceptance, and it is also possible that, if the particular economic situation will allow it, antibiotics will be used for protection against microbial deterioration of many other forms of natural and man-made materials. • What other areas of biotechnology, and particularly, fermentation technology will undergo a change in future? Microbial enzymes enjoy extensive present-day usage, and there is no reason why this usage should not be expanded. It is hoped that enzymes from microbial sources will gradually replace those obtained from the tissues of higher plants and animals. In favor of this is the high rate of production of microbial enzymes, the relatively lower production and purification costs, and the possibility that enzymes of commercial value may be found which are unique to microorganisms. Enzymes from microbial sources may well find further medical uses; particularly if a means concrete, and so forth. Also, means for preventing the fouling of submerged objects in marine environments will require extensive study. The fact that microorganisms can be employed as scavengers is only beginning to be realized. Thus, microorganisms should find use in the scavenging of phosphate and other ions from waste waters, and from phosphate and other mining waters. In addition, they should find use in scavenging heavy and rare metals from mining process waters, sea water, and waters resulting from nonminingrelated industrial processes. There also is some evidence that microorganisms can be employed to scavenge radioactive materials from water, food, soil, and so forth. Fermentation processes employing unique microorganisms, or unique means for making use of the activities of microorganisms, may well be commonplace in the years to come. Thus, fermentative agents for producing commercially valuable products could include protozoa, lichens, microbial spheroplasts or protoplasts, stable bacterial L-forms (or unstable L-forms purposely maintained in this state), and animal or plant cells maintained as reproducing tissue cultures. As a specific example, the tissue-culture approach might yield alkaloids, hormones, and other biologically active compounds.

•

Structural model of penicillin G

There is no reason why success should not be achieved in the modification of other antibiotic structures in order to reduce toxicity; broaden the microbial inhibition spectrum range, or introduce specific activities into the antibiotic molecules. Antibiotics previously discarded because of high toxicity, or for other reasons, may soon find pharmacological, agricultural,

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What would be the role of microorganisms in energy production in future?

Microorganisms may be useful as agents to partially degrade highly viscous petroleum so as to reduce its viscosity. This would allow the oil to be more easily pumped through long pipelines. This property of microorganisms also could be employed to release oil from underground reservoirs; the reduced viscosity would allow the petroleum to become detached from the sand and rock to which it is bound so that it could flow underground toward the location of oil wells. This would require that the organisms be introduced into the ground along with the waters used in secondary oil recovery.

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This raises the possibility that the rumen of the living animal, or even an artificially established rumen in the laboratory, could be employed. to produce defined chemicals of commercial value simultaneously with its normal metabolic function. Obviously, the microbial ecology of the rumen would have to be well understood, and the specific chemical fermentation products would have to be nontoxic to the animal and not absorbed by it. The latter, of course, would not be pertinent if the artificial rumen were employed.

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Microbial enzymes, either cell-free or retained within the organisms that produce them, will find greater application in the mediation of specific steps of chemical syntheses and of transformations difficult by purely chemical means. This is already being accomplished in the modification of specific sites on steroid molecules; such transformations should also be feasible on antibiotic and other complex molecules. Thus, in specific instances, it should be possible to change the molecular configuration of a complex molecule such that specific chemical properties are added, retained, or deleted from the molecule. Increased knowledge of the treatment of municipal and industrial wastes doubtlessly will provide means for treating these wastes in a manner such that the effluent waters, which contain oxidized inorganic salts promoting aquatic weed and algal growth, and toxic and obnoxious inorganic or organic materials that survive present procedures of waste treatment, do not find their way into natural bodies of water. The modifications in waste-water treatment procedures required for obtaining these ends at least to some extent will necessitate a better under standing of the natural microbial ecology that occurs during the various treatment steps of the process1ng of the waste waters. Industrial and municipal wastes undergoing treatment are a gigantic and continuous reservoir of highly nutritive materials for microbial growth and, as such, it is possible that byproducts can be recovered from the treated waste waters. Vitamin B 12 occurs naturally in these waters and, in certain instances; it presently is being recovered commercially. It should also be possible to direct the microbial activities of waste-water treatment microorganisms such that other products of commercial value can be recovered, either from the waters or from the settled sludge. Another possibility, although obviously more remote, is the utilization of waste-water treatment facilities as a giant microbial fuel cell for the production of electricity. Microbial deterioration has always been a problem, and it will continue to be so. Much study will be required to find means for preventing the deterioration of paper, fabrics, food and feed, wood, metals, concrete, and so forth. Also, means for preventing the fouling of submerged objects in marine environments will require extensive study. Chemosynthetic or photosynthetic autotrophs also could serve as fermentation microorganisms, particularly if mutated to expose components of their complete biosynthetic metabolisms. Unless introduced during mutation, a carbohydrate nutritive requirement would not be involved; hence these microorganisms would by-pass this costly ingredient of fermentation media make-up. Separate from or complimentary to this use of these organisms is the ability of some of them to oxidize inorganic ions; perhaps, more application will be found for the ability of the thiobacilli to oxidize hydrogen sulfide, mineral sulfides and sulfur to sulfuric acid. Microorganisms may be useful as agents to partially degrade highly viscous petroleum so as to reduce its viscosity. This would allow the oil to be more easily pumped through long 212

pipelines. This property of microorganisms also could be employed to release oil from underground reservoirs; the reduced viscosity would allow the petroleum to become detached from the sand and rock to which it is bound so that it could flow underground toward the location of oil wells. This would require that the organisms be introduced into the ground along with the waters used in secondary oil recovery. Microorganisms also might be employed for the releasing and removal of the contaminating metals, sulfides, and so forth which occur in crude oil fractions. Another potential microbial application as regards hydrocarbons relates to the fact that hydrocarbons are completely reduced compounds and, as such, they contain considerable quantities of hydrogen in their molecules. Therefore, microorganisms might be employed to release this hydrogen as gaseous hydrogen for use in chemical catalytic reductions. Microbial spores as fermentative agents for mediating chemical transformations of various compounds probably will find greater exploitation. These spore fermentations would be analogous to the present use of spores in methyl ketone fermentations and steroid transformations. The nonsporeforming, highly oxygen-sensitive anaerobe has never really been investigated for its fermentation potential. Thus, it is not known just what products of commercial value these organisms might produce, if means could be found for their growth under mass culture conditions. Microbial dextrans and other polymers are known to be synthesized in high yield by microorganisms; more and better uses should be found for these polymers. Poly-,B-hydroxybutyrate is a specific example of a polymer produced in large amounts by many organisms. This compound could serve as a basal material for chemical modification, or it could be partially or totally hydrolyzed. Nucleic acids and their partial degradation products are already being recovered from cultures of microorganisms and these compounds should find increased applications in medicine, as food-flavoring agents, and in other ways. The advent of space travel has generated several microbial problems that need solving. Leaving and returning spacecraft require some means for decontamination. Sampling devices and auxiliary equipment are needed for detecting and, possibly, identifying the microbial life on other planets. Algal and other balanced ecosystems for space travel are under study, as a means for supplying oxygen and removing carbon dioxide from the air, and as a means for decomposing and utilizing human wastes. Biochemical fuel cells also might be employed for utilizing human wastes and, at the same time, for producing electricity for space travel. Microbial fuel cells, or as they are more often known, biochemical fuel cells. are of various types, but at least one type could be described as a unit made up of electropositive and electronegative half-cells joined by a potassium chloride agar bridge with each half-cell containing an electrolyte and an electrode. Reducing conditions are maintained in the electronegative cell by microbial activity on a nutrient substrate, and the electropositive side is maintained by oxidizing conditions such as by air, oxygen, hydrogen peroxide, and so forth (Figure below). Apparently, the microorganisms can

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tolerate a high electric charge, but other problems remain to be solved for these fuel cells (Sisler,1964). For instance; they rapidly discharge under load, because the media are not sufficiently poised for accumulation of a sustained charge. Also, unfavorable reaction kinetics is encountered which lead to a, sluggish electromotive response. It is theoretically possible that, if the problem of these fuel cells could be solved, electricity could be generated for municipalities. Thus, the activated sludge system or other microbial processes employed by municipal sewage-treatment plants could be used as the electronegative half-cell. It should now be obvious that industrial microbiology presents many possibilities for-the future, and many directions in which to go. Industrial microbiology does not stagnate. The thinking of man may stagnate at times, but usually someone manages to make the critical observation or discovery, design the proper equipment, and evaluate the market so that financial rewards accrue. Advances in the understanding and practice of the industrial applications of microorganisms will occur in university, government, and industrial laboratories, and it is only through working together and understanding each other’s goals and purposes that the apparently purely theoretical discoveries made in these laboratories will have industrial significance. By the same token, research carried out in industrial microbiology laboratories would do well to include more of the basic or theoretical approaches that may seem to have little if any potential for economic return in the foreseeable future. Thus, what appears at first glance to be a purely theoretical discovery may, when considered from the proper viewpoint, be the key to a profitable industrial fermentation process.

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FINAL EXCERSICE

Well, you have just completed a comprehensive study on various aspects of the fermentation technology. Now try the following self test exercise and see how well you have done! Here it goes…The production of an amylase from starch by Aspergillus niger is often performed in a fed batch fermenter. This is because

j a. A fed-batch reactor can be used to minimize starch k l m n concentrations and prevent A. niger from fermenting. j k l m n

b.. A fed-batch reactor can be used to minimize starch concentrations and thus minimize the inhibitory effects of starch.

j c. A fed-batch reactor can be used to minimize starch k l m n concentrations and thus reduce the viscosity of the medium. n d. All of the above are correct. j k l m 2. Vinegar is typically produced in fed batch reactors because j a. a fed batch reactor can be used to maintain low acetic k l m n acid concentrations j k l m n

b.. a fed batch reactor can be used to maintain low ethanol concentrations

j c. acetic acid bacteria tend to ferment at high ethanol k l m n concentrations n d. All of the above are correct. j k l m 3. Yeast biomass is typically produced in fed batch reactors because j yeast cells respire at low glucose concentrations k l m n j yeast cells ferment at low glucose concentrations k l m n j yeast cells produce ethanol at low glucose concentrations k l m n j All of the above are correct k l m n 4 Antibiotics are typically produced in fed batch reactors because

j a. antibiotic yields are generally higher when cells enter the k l m n stationary phase j k l m n

b .the precursors are often toxic to the cells

j c. antibiotic yields are generally higher when cell growth k l m n slows j d. All of the above are correct. k l m n 5 Monoclonal antibodies are typically produced in fed batch reactors because j a. hybridoma cells respire at high glucose concentrations k l m n j b.. hybridoma cells ferment at low glucose concentrations k l m n j c. hybridoma cells produce higher lactate yields at high k l m n glucose concentrations j k l m n

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d. All of the above are correct

6 Which of the following reactors would have mixing profiles which are closest to plug flow?

j a.A continuous air lift bioreactor k l m n j b.A continuous fluidized bed bioreactor k l m n j c.A continuous packed bed reactor k l m n j d.A continuous stirred tank reactor with biomass recycle. k l m n 7 In which of the following would mixing per unit volume be poorest?

j a. Continuous air lift bioreactor k l m n j b.Continuous fluidized bed bioreactor k l m n j c.Continuous packed bed reactor k l m n j d.Continuous stirred tank reactor with biomass recycle. k l m n 8 In which of the following would heat transfer rates (per unit volume) be poorest?

j a. Continuous air lift bioreactor k l m n j b.. Continuous fluidized bed bioreactor k l m n j c. Continuous packed bed reactor k l m n j d. Continuous stirred tank bioreactor with biomass k l m n recycle. 9 Mass transfer rates in fluidized bed bioreactors are higher than in acked bed bioreactors because

j a.the size of the immobilization particles are smaller in the k l m n fluidized bed bioreactors j b.the level of mixing is higher in fludized bed bioreactors k l m n j c.the particles move with the fluid in a fluidized bed k l m n bioreactor j d.All of the above are correct k l m n 10 Mixing in an anaerobic sludge blanket reactor is due to j a. the microbes swimming through the reactor k l m n j b. the water temperatures changing rapidly throughout the k l m n reactor j c.the release of gases by the microbial populations k l m n j d.All of the above are correct k l m n 11 When modelling a fed batch bioreactor, the rate of change in the bioreactor volume is assumed to be equal to

j a. the initial volume k l m n j b.the final volume k l m n j c.the flow rate k l m n j d.the solids content of the reactor k l m n 12 The mass of substrate utilized during a fed-batch fermentation is calculated from which of the following equations?

j a.Initial mass of substrate in the reactor - final mass of k l m n substate in the reactor + flow rate x [substrate] in the feed © Copy Right: Rai University

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b.. Iinitial mass of substrate in the reactor + final mass of substate in the reactor + flow rate x [substrate] in the feed

j c. Initial mass of substrate in the reactor - final mass of k l m n substate in the reactor - flow rate x [substrate] in the feed . j k l m n

d. Initial mass of substrate in the reactor + final mass of substate in the reactor - flow rate x [substrate] in the feed

13 The mass of biomass produced during a fed-batch fermentation is calculated from which of the following equations?

j a.Initial mass of biomass in the reactor - final mass of k l m n biomass in the reactor j b.Initial mass of biomass in the reactor + final mass of k l m n biomass in the reactor j c.Final mass of biomass in the reactor - initial mass of k l m n biomass in the reactor j d.Final mass of biomass in the reactor + flow rate x k l m n [biomass] in the reactor 14 The mass of substrate in the reactor is calculated from which of the following equations?

j a.[Substrate] in reactor x flow rate k l m n j b.[Substrate] in reactor x volume of reactor k l m n j c.[Substrate] in reactor x mass of reactor k l m n j d.Flow rate x volume of reactor k l m n 15 Mixing in a fluidized bed reactor is facilitated by

j a. the upward movement of the incoming feed only k l m n j b.gravity k l m n j c.both upward movement of the incoming feed and k l m n gravity n d.diffusion j k l m 16 In an activated sludge process, the biomass is recycled to

j a.They are more stable as the microbial population willnot k l m n be washed out should a slug of an inhibitor enter the system. j b.A higher concentration of cells can be maintained in the k l m n reactor. j c.A higher dilution rate can be used before the cells k l m n washout. j d.All of the above k l m n 19 A fed-batch reactor initially contained 2 litre of medium. It was fed at 1 litre per hour. After 10 hours, the volume of the reactor was n a.12 l n j k l m j b.6 l n k l m j c.3 l n k l m j d.2 l k l m 20 A fed-batch reactor initially contained 2 litre of medium and 1 g.l-1 of substrate. It was fed at 1 litre per hour with a medium containing 1 g.l-1 of substrate. After 10 hours, the concentration of substrate in the reactor was 0.5 g.l-1. The mass of substrate that was used by the culture in the reactor was j a.12 g n k l m n j b.6 g n k l m j c.3 g n k l m j d.1 g k l m 21 A fed-batch reactor initially contained 2 litre of medium and 0.1 g.l-1 of biomass. It was fed at 1 litre per hour with a medium containing 1 g.l-1 of substrate. After 10 hours, the concentration of biomass in the reactor was 0.2 g.l-1. The mass of biomass that was produced during the 10 hour period was n a.5.5 g n j k l m j b.3.3 gn k l m j c.2.2 g n k l m j d.1.1 g k l m 22 A fed-batch reactor initially contained 2 litre of medium. The concentration of substrate and biomass in the reactor was 1 g.l-1 and 0.1 g.l-1 respectively. It was fed at 1 litre per hour with a medium containing 1 g.s l-1 of substrate. After 10 hours, the concentration of substrate and biomass in the reactor was 0.5 g.l-1 and 0.2 g.l-1. The biomass yield during this 10 hour period was j a.0.57 g.g-1 n k l m n j k l m g.g-1

b.0.47 g.g .g-1 n j k l m

c.0.37 g.g-1 n j k l m

d.0.27

j a.increase the concentation of cells in the reactor k l m n j b.increase the efficiency of the process k l m n j c.reduce sludge volumes k l m n j d.All of the above k l m n 17 A few years ago, a process involving the production of citric acid from alkanes was developed. The fermentation is performed by an aerobic yeast using a fed-batch culture in which alkanes were slowly fed to the yeast. The reason a fed batch culture is used is that

j a.citric acid is toxic to the cells k l m n j b.alkanes will cause foaming k l m n j c.high concentrations of alkanes will inhibit the cells and k l m n reduce oxygen transfer rates j d.high concentrations of alkanes will cause the cells to k l m n grow too quickly 18 Which of the following describes an advantage of using immobilized cell reactors for wastewater treatment?

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j k l m n

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GLOSSARY FOR THE FERMENTATION

absorption Removing a particular antibody or antigen from a sample (from serum, for example) by adding the corresponding antigen or antibody to that sample. adsorption Nonspecific adherence of substances in solution or suspension to cells or other particulate matter. adventitious agents Acquired, sporadic, accidental contaminants. aerobe An aerobic organism is one that grows in the presence of oxygen. A strict aerobe grows only under such a condition. aggregate A clustered mass of individual cells — solid, fluffy, or pelletized — that can clog the pores of filters or other fermentation apparatus. —amino acids A class of 20 hydrocarbon molecules that combine to form proteins in living things. anaerobe An anaerobic organism grows in the absence of air or oxygen. Some anaerobic organisms are killed by brief exposure to oxygen, whereas oxygen may just retard or stop the growth of others. antifoam agent A chemical added to the fermentation broth to reduce surface tension and counteract the foaming (bubbles) that can be caused by mixing, sparging, or stirring. aseptic Sterile, free from bacteria, viruses, and contaminants such as foreign DNA. bacteriophage A virus that infects bacteria, sometimes used as a vector. batch culture Large-scale cell culture in which cell inoculum is cultured to a maximum density in a tank or airlift fermentor, harvested, and processed as a batch. bioactivity A protein’s ability to function correctly after it has been delivered to the active site of the body (in vivo). bioavailability Measure of the true rate and the total amount of drug that reaches the target tissue after administration. biologic A therapeutic agent derived from living things. biopharmaceutical A therapeutic product created through the genetic manipulation of living things, including (but not limited to) proteins and monoclonal antibodies, peptides, and other molecules that are not chemically synthesized, along with gene therapies, cell therapies, and engineered tissues. bioprocessing Using organisms or biologically derived macromolecules to carry out enzymatic reactions or to manufacture products.

catabolites Waste products of catabolism, by which organisms convert substances into excreted compounds. chemostat A growth chamber that keeps a bacterial culture at a specific volume and rate of growth by limiting nutrient medium and removing spent culture. CIP (clean in place) A way to clean large vessels (tanks, piping, and associated equipment) without moving them or taking them apart, using a highpressure rinsing treatment, sometimes followed by steam-in-place (SIP) sanitization. clearance Demonstrated removal according to specified parameters. cryopreservation Maintenance of frozen cells, usually in liquid nitrogen. cytokine A protein that acts as a chemical messenger to stimulate cell migration, usually toward where the protein was released. Interleukins, lymphokines, and interferons are the most common. dalton The unit of molecular weight, equal to the weight of a hydrogen atom. downstream processing Bioprocessing steps following fermentation and/or cell culture, a sequence of separation and purification activities needed to obtain the required drug product at the necessary level of purity. DNA (deoxyribonucleic acid) The nucleic acid based on deoxyribose (a sugar) and the nucleotides G, A, T, and C. Occurring in a corkscrew-ladder shape, it is the primary component of chromosomes, which thus carry inheritable characteristics of life. DNA fingerprinting Sequences of nucleic acids in specific areas (loci) on a DNA molecule are polymorphic, meaning that the genes in those locations may differ from person to person. DNA fragments can be cut from those sequences using restriction enzymes. Fragments from various samples can be analyzed to determine whether they are from the same person. The technique of analyzing restriction fragment length polymorphism (RFLP) is called DNA typing or DNA fingerprinting. efficacy The ability of a substance (such as a protein therapeutic) to produce a desired clinical effect; its strength and effectiveness. endogenous Growing or developing from a cell or organism, or arising from causes within the organism.

bioreactor A vessel capable of supporting a cell culture in which a biological transformation takes place (also called a fermentor or reactor).

enzymes Proteins that catalyze biochemical reactions by causing or speeding up reactions without being changed in the process themselves.

broth The contents of a microbial bioreactor: cells, nutrients, waste, and so on.

fermentor A bioreactor used to grow bacteria or yeasts in liquid culture.

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fusion partner When making a small protein or peptide in E. coli, it is often necessary to produce the protein fused to a larger protein to get high levels of stable expression. The resulting fusion protein must be cleaved (chemically or enzymatically broken) to yield the desired protein or peptide. The nonproduct fusion partner is left over and usually thrown away. gene The unit of inheritance consisting of a sequence of DNA, occupying a specific position within the genome. Three types of genes have been identified: structural genes encoding particular proteins; regulatory genes controlling the expression of the other genes; and genes for transfer RNA or ribosomal RNA instead of proteins. genetic engineering Altering the genetic structure of an organism (adding foreign genes, removing native genes, or both) through technological means rather than traditional breeding. HPLC High-performance liquid chromatography or highpressure liquid chromatography, a commonly used method for separating liquid mixtures. Immortalize To alter cells (either chemically or genetically) so that they can reproduce indefinitely. inoculate To introduce cells into a culture medium. inoculum Material (usually cells) introduced into a culture medium. interferon A cytokine that inhibits virus reproduction. Interferons also affect growth and development (differentiation) in certain normal and tumor cells.

PCR Polymerase chain reaction, a method of duplicating genes exponentially. peptides Proteins consisting of fewer than 40 amino acids. pilot plant A medium-scale bioprocessing facility used as an intermediate in scaling up processes from the laboratory to commercial production. plasmid Hereditary material that is not part of a chromosome. Plasmids are circular and self-replicating and found in the cytoplasm of cells (naturally in bacteria and some yeasts). They can be used as vectors for introducing up to 10,000 base-pairs of foreign DNA into recipient cells. polymerase An enzyme that catalyzes the production of nucleic acid molecules. posttranslational modifications Protein processing done by the Golgi bodies after proteins have been constructed by ribosomes.

in vitro Performed in the laboratory rather than in a living organism (in vivo).

protein Macromolecules whose structures are coded in an organism’s DNA.

ligase An enzyme that causes fragments of DNA or RNA to link together; used with restriction enzymes to create recombinant DNA.

Each is a chain of more than 40 amino acids folded back upon itself in a particular

media A (usually sterile) preparation made for the growth, storage, maintenance, or transport of microorganisms or other cells. microbiology The study of microscopic life such as bacteria, viruses, and yeast. microorganism A microbe; a living thing too small to be seen by the naked eye. mutagen An agent (chemicals, radiation) that causes mutations in DNA. mutation A permanent change in DNA sequence or chromosomal structure. mycoplasma parasitic microorganisms that infect mammals, possessing some characteristics of both bacteria and viruses. nucleic acids DNA or RNA: long, chainlike molecules composed of nucleotides. organism A single, autonomous living thing. Bacteria and yeasts are organisms; mammalian and insect cells used in culture are not.

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way. proteolytic Capable of lysing (denaturing, or breaking down) proteins. recombinant Containing genetic material from another organism. Genetically altered microorganisms are usually referred to as recombinant, whereas plants and animals so modified are called transgenic (see transgenics). restriction enzyme An bacterial enzyme that cuts DNA molecules at the location of particular sequences of base pairs. ribosome Cell organelles that translate RNA to build proteins. RNA Ribonucleic acid; similar to DNA but based on ribose, and with the

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floc A fluffy aggregate that resembles a woolly cloud.

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base uracil (U) in place of thymine (T). Various forms of RNA are found: mRNA (messenger RNA); tRNA (transfer RNA); and rRNA (ribosomal RNA). Most RNA molecules are single-stranded, although they can form double-stranded units. roller bottle A container with large growth surfaces in which cells can be grown in a confluent monolayer. The bottles are rotated or agitated to keep cells in suspension, but they require extensive handling, labor, and media. In large-scale vaccine production, roller bottles have been replaced by microcarrier culture systems that offer the advantage of scale-up. scale-up To take a biopharmaceutical manufacturing process from the laboratory scale to a scale at which it is commercially feasible. seed stock The initial inoculum, or the cells placed in growth medium from which other cells will grow. sequence The precise order of bases in a nucleic acid or amino acids in a protein. serum The watery portion of an animal or plant fluid (such as blood) remaining after coagulation. When cheese is made, whey is the milk serum that’s left. SIP Steam in place or sterilize in place (see CIP). somatic cell In higher organisms, a cell that (unlike germ cells) carries the full genetic make-up of an organism. sparge To spray. A sparger is the component of a fermentor that sprays air into the broth. strain A population of cells all descended from a single cell. substrate Reactive material, the substance on which an enzyme acts. substratum The solid surface of which a cell moves or on which cells grow. supernatant Material floating on the surface of a liquid mixture (often the liquid component that has the lowest density). surfactant Any substance that changes the nature of a surface, such as lowering the surface tension of water.

or to improve livestock strains. Transgenic plants have been created for increased resistance to disease and insects as well as to make biopharmaceuticals. ]translation The DCprocess by which information transferred from DNA by RNA specifies the sequence of amino acids in a polypeptide (protein) chain. trypsin, tryptic digestion Trypsin allows the growth of cells as independent microorganisms distinct from tissue culture by causing cell disaggregation. Excised tissue is softened and treated with a proteolytic enzyme, normally trypsin, then washed and suspended in a growth medium to produce a primary culture. Subculturing from the primary culture usually involves treatment with an antitrypsin (such as serum) to produce a secondary culture. Cell lines are established by repeated culture through cycles of growth, trypsinization, and subculture. Trypsin is also used to remove anchorage-dependet cells from their attached substratum. turbidostat A variation on a chemostat. Whereas a chemostat is designed for constant input of medium, a turbidostat is designed to keep the organisms at a constant concentration. A turbidity sensor measures the concentration of organisms in the culture and adds additional medium when a preset value is exceeded. turbulent flow field The state that results from mixing the contents of a fermentor or bioreactor to provide oxygen to the cells. That must be balanced against the shear that causes cell damage and death. unicellular Composed of only a single cell. vaccines Preparations of antigens from killed or modified organisms that elicit immune response (production of antibodies) to protect a person or animal from the diseasecausing agent. vacuolation In cell and tissue culture, excess fluid, debris (aggregates), or gas (from sparging) can form inside a cell vacuole. A vacuole is a cavity within the cell that can be relatively clear and fluid filled, gas filled (as in a number of blue-green algae), or food filled (as in protozoa).

suspension Particles floating in (not necessarily on) a liquid medium, or the mix of particles and liquid itself.

vector The plasmid, virus, or other vehicle used to carry a DNA sequence into the cell of another species.

symbiotic Living together for mutual benefit.

vessel jacket A temperature control method consisting of a double wall outside the main vessel wall. Liquid or steam flows through the jacket to heat (or cool) the fluid in the vessel. Because biopharmaceutical products are so sensitive

synthesis Creating products through chemical and enzymatic reactions. titer A measured sample. (To draw a measured, representative sample from a larger amount is to titrate.) transgenics The alteration of plant or animal DNA so that it contains a gene from another organism. There are two types of cells in animals and plants, germ line cells (the sperm and egg in animals, pollen and ovule in plants) and somatic cells (all of the other cells). It is the germ-line DNA that is altered in transgenic animals and plants, so those alterations are passed on to offspring. Transgenic animals are used to produce therapeutics, to study disease,

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and vessel jackets can cause uneven heating (hot or cold spots), shell-andtube or plate-and-frame heat exchangers are more common in biopharmaceutical production systems. viability Life and health, ability to grow and reproduce; a measure of the proportion of live cells in a population. virus The simplest form of life: RNA or DNA wrapped in a shell of protein, sometimes with a means of injecting that genetic material into a host organism (infection). Viruses cannot reproduce on their own, but require the aid of a host.

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viscosity Thickness of a liquid; determines its internal resistance to shear forces. yeast A single-celled fungus.

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