Biochemical Engineering

BIOCHEMICAL ENGINEERING CHP583E Course outline and objectives References 1. Biochemical Engineering, 2nd edition (1973) by Shuichi Aiba, Arthur Humhrey and Nancy Mills, Academic press 2. Principles of Fermentation Technology (1984) by Stranbury P.F. and Whitaker A., Pergamon Press 3. Biochemical engineering, unit processes in Fermentation (1958) by Steel R. (editor) Heywood and Co. Ltd 4. Biochemical Engineering (1964) by Webb F.C., Van Nostrand Company

1.0 Introduction 1.1 Historical Background About 150 years ago, Louis Pasteur pointed out the important role of living micro organisms in biochemical processes. In 1928, a major breakthrough was achieved by Alexander Fleming in discovering penicillin. The urgent need of penicillin throughout the 2nd world war led to microbiologists, biochemists and chemical engineers in a “crash” programme of developing and designing processes in areas which were hitherto unfamiliar to them. Three American companies led the way-Merck, Pfizer, and Squibb. Since then micro organisms have been known to be used in the manufacture of a host of complex chemicals, antibiotics, enzymes and vitamins. In the presidential address to the institutions of Chemical Engineers in 1952, Sir Harold Hartley made a first public reference to the term Biochemical Engineering. He recognized Biochemical Engineering as an emerging branch of chemical engineering. He further underscored the imminent contribution of biological substances and processes in industrialisation. This inevitably led to among the first series of Biochemical lectures being offered at the University of Manchester, where George Davis gave his first lectures in Chemical engineering.

However, most of the Biochemical fundamental principles had been in application since antiquity. And so, what is Biochemical Engineering? Biochemical engineering is the interaction of two disciplines-Biological sciences and chemical engineering. Biochemical engineering is concerned with conducting biological processes on an industrial scale, providing the link between Biological and Chemical engineering. Biological sciences will include microbiology, biochemistry and genetics. The heart of Biochemical engineering lies in the scale up and management of the cellular processes. Thus, therefore there is a precise need for the chemical engineer to understand the relative dynamic nature of the biological catalysts. In order to develop biological units on an industrial scale, three distinct disciplines of chemical engineering, biochemistry and microbiology are required combined with specific trade technology. The experiences gained from chemical engineering are handy at the development of this rather fast growing branch of engineering. Some interesting comparisons can be drawn between chemical engineering and Biochemical engineering: ƒ Basic concepts of material and energy transfer and fluid flow are common in both but biological processes are notoriously associated with a narrow range of temperature and nonNewtonian materials ƒ Many unit of operation are common only for the details to differ

The scope of biochemical engineering 1. Industrial fields in fermentation 2. Operations of food processing; pasteurization, sterilization and preservation 3. Industrial solvents, organic acids 4. Manufacture of sera and vaccines 5. Commercial enzymes 6. Extraction processes for insulin and other hormones 7. Processing of forests and crop products 8. Effluent disposal Among the above activities are some that may attract the prophet and visionary. However, it is important to note that a broad base of technologies must be included in Biochemical engineering, which might require additional information and knowledge. For example, vaccine manufacture requires in depth medical information while aspects of hygiene and sterilization are rather unfamiliar to a traditional chemical engineer. The awareness of Biological background is essential in

Biochemical engineering. This course assumes that the students have basic knowledge in biological sciences. The organization of this course is in such a way that it prepares students to work in the existing industries as well as applying their basic knowledge to the unborn industries of the future.

Role of a biochemical engineer ƒ ƒ ƒ ƒ ƒ

Design and operation of absolutely pure and mixed cultures Prevention of contamination – providing sterile conditions as well as “contaminant proof” environment Design of auxiliary systems of air compression, delivery systems, instrumentation and control Demonstration of pilot plant equipment and its subsequent scale-up to the production stage Separation and isolation of the product using well known techniques such as filtration, extraction, adsorption and concentrations

Primarily, the laboratory scientists, microbiologists, biochemists, genecists must continue in the discovery and advancement of desirable interactions between micro organisms and their environment. The biochemical engineer must control and translate the laboratory results to the production scale operation in an economic manner.

1.2 Exploitation of Biological processes Potential source of proteins e.g. the use of bacteria to breakdown n-alkane ƒ Co-oxidation of substrates that do not support their growth resulting into more useful ones e.g. p-xylene can be oxidized to dimethyl-cis and cis-muconic acid ƒ Production of enzymes-some of the enzymes are used in detergent manufacture, hydrolytic reactions and as analytic tools ƒ Production of polymers- Microbial cells excrete little slime outside the cell wall. For example glucan which is used as plasma substitute ƒ Pollution control-treatment of wastes with high BOD(Biological oxygen demand) Types of Biological materials ƒ

It is not easy to define a living process and it may become increasing difficult as further biochemical advances are made. This

course is designed primarily for the micro-organisms and enzymes but it is important to gain a broader sense of other forms of life-the similarities and differences between the unicellular organisms and larger animals and plants. Industrial applications of micro-organisms can be broadly classified into: Those in which great care is taken to use only one selected strain of a particular organism, typified by manufacture of antibiotics and some vaccines Those that effort is made to maintain a fairly constant mixture of strains of the same species e.g. in brewing and wine making Processes which are chiefly depend on adventitious flora of mixed species e.g. curing bacon in the manufacture of margarine, tea, coffee etc. these activities are by large dependent on tradition and experience.

1.3 MICRO-ORGANISMS Types of micro-organisms Micro organisms exist as single cells or at most in relatively unspecialized multi-cellular colonies, with no capacity to control cellular temperatures. Micro organisms may be classified into four main groups ƒ Bacteria ƒ Viruses ƒ Fungi including yeast and actinomycetes ƒ Protozoa including algae Bacteria These are single cells, in the form of cocci, rods and spirals capable of independent growth. Bacteria are ubiquitous in nature, in aerobic and anaerobic environments containing water. In the genera, synthetic abilities range from those of autotrophic species, which require only inorganic compounds for growth, to those of heterotrophic species, which may have synthetic ability and must be grown in tissue culture. Due to the diverse abilities, bacteria may be exploited industrially to accumulate both intermediate and end products of metabolism.

Viruses Viruses are the smallest microbes, obligate intracellular parasites of animals, plants, insects, fungi, algae or bacteria. They contain no water and have little or no synthetic or metabolic activity themselves. Growth and multiplications take place intracellular. This frequently results in the damage and subsequent death of the host cells. The genetic material of viruses may either be ribonucleic (RNA) acid or the deoxyribonucleic acid (DNA). Myxoviruses show helical symmetry, with nucleic protein helix enclosed by a lipoprotein sheath. Bacteriophages are viruses that are parasitic on bacteria. These phages are possible contaminants of biological systems. Fungi Fungi are widely spread in environments of lower relative humidity than which favour bacteria. The metabolism of fungi is essentially anearobic, they form long filamentous, nucleated cells(Hyphae) between 4 u to 20µ wide. Generally fungi are free living saprophytes but a few are parasitic on animals and many are serious pathogens of plants. Actinomycetes are intermediate between fungi and bacteria. Industrially, this group is extremely important as a source of powerful antibiotics. Protozoa These are widely distributed in fresh and salty water, in soil and in animals. They may be unicellular or multi cellular and exhibit a wide range of morphological forms. Protozoa are either photosynthetic or non-photosythetic while algae are capable of photosynthesis. Protozoa are handy in removing bacteria from waste water in trickling filters and activated sludge plants.

1.4 Requirements for Growth and Formulation of Media Requirements for growth

Detailed investigation is prerequisite to establish the most suitable medium for individual biological processes, but certain requirements must be met by any such medium. All micro organisms require water, sources of energy, carbon, nitrogen, mineral elements and possibly vitamins plus oxygen if aerobic. It is important to appreciate that the cultural conditions that achieve maximum cell mass may not be necessarily those that give maximum yield of some products of metabolism. On a small scale, it is relatively simple to devise medium, although supporting satisfactory growth may not be suitable for use in large scale. On large scale, one must normally use resources of cheap nutrients to create medium which will meet as many as possible of the following criteria: ƒ It will produce maximum yield of products and biomass per gram of substrate used ƒ It will produce maximum concentration of the products ƒ It will permit maximum rate of product formation ƒ There will be minimum yield of undesirable products ƒ It will be cheap and of consistent quality and readily available throughout the year ƒ It will cause minimum problems in other aspects of production process particularly aeration and agitation, extraction, purification and waste management Some of the cheap sources of nutrients include cane molasses, beet molasses, cereal grains, glucose, sucrose and lactose(Carbon sources) while ammonium salts, urea, nitrates, corn steep liquor, Soya bean meal, slaughter house waste and fermentation residues(nitrogen sources).The medium selected will affect the design of equipment and processes. The best temperature for cultivation varies with species but organisms occurring in the soil naturally grow best at temperatures between 25°c and 30°c while those isolated from animals grow best at 37°c. Some organisms are actually thermophilic e.g. those used in bio-digesters (Lactobacillus), which grow best at 40-45°c. The products of microbial metabolism often cause major shifts in PH. It is important to maintain desirable PH level. Sometimes the optimum PH for product formation may not be the optimum PH for growth. When acid by-products accumulate in the medium, causing unwanted fall in the PH, ammonia is slowly fed to the culture, so supplying nitrogen for growth while maintaining PH. Calcium carbonate may also be used for the PH control in conditions where the required product is water soluble. When cell mass or some insoluble metabolite is required, acidic products are conveniently neutralised by adding sodium hydroxide.

Micro organisms vary in their need for oxygen. On one hand fungi, algae and a few bacteria are obligate anaerobes. On the other hand, a few bacteria are strict anaerobes and many bacteria and many bacteria can grow in both situations (facultative anaerobes). The physical conditions of temperature and PH will have a profound effect on microbial growth. The useful range of temperature over which the metabolic processes of a micro-organism proceed at significant rate is quite narrow, usually not more than -20°c. temperature is important in controlling the flavour balance of many fermented beverages and food. There is usually an optimum PH range, which is normally limited with complete inactivation or death on either extreme.

1.5 FORMULATION OF MEDIA This is an essential part in the design of successful laboratory experiments, pilot-scale development and the manufacturing processes. The constituent of media must satisfy the elemental requirements for the biomass and metabolite production and there must be adequate supply of energy for the biosynthesis and cell maintenance. The first step to consider is an equation based on the stoichiometry for growth and product formation. Thus,

Carbon + energy + nitrogen + other

tal requirements ⎯environmen ⎯⎯⎯ ⎯ → cell biomass + products

+ CO2 + H 2 O + Heat A quantitative treatment of the above equation is desirable in the economical design of the media if compound wastage is to be minimized. This calls for the elemental composition (K, N, O, Mg etc), whose data is not readily available. Sometimes, it is important to have excess quantity of some elements. For example, the concentration of phosphorous is deliberately raised in some culture to offer a buffering effect in addition. The carbon substrate has dual role in the biosynthesis and energy generation. The carbon requirement under aerobic conditions may be estimated from the cellular yield coefficient(Y) which is defined as

γ =

quantity of cell dry matter produced quantity of carbon substrates utilised

Analyses made to determine how the observed conversion of the carbon sources to the product compares with the theoretical maximum yield.

1.6 CONSTITUENT OF CULTURE MEDIA WATER Water is a major component. Clean water of consistent composition is therefore required in large quantities from a reliable source. Some of the factors needed to consider include PH, dissolved solids and effluent contamination. For example, the mineral content of water is important in the brewing industry; with hard waters containing CaSO4 more suitable for pilsner type lagers. It is advisable to deionise the water to make it more adaptable for the biological processes. Energy sources Energy for growth comes from either the oxidation of medium components or from light. Most industrial micro-organisms are chemoorganotrophs, therefore the commonest sources of energy will be carbon sources such as carbohydrates, lipids and proteins. Carbon sources The following are some of the examples of carbon sources: ƒ ƒ ƒ ƒ ƒ ƒ

Starch from maize grains, potatoes, cassava etc Sucrose from sugar cane or sugar beet Lactose from milk whey powder Commercial vegetable oils-both as a carbon source and anti foaming agent Corn steep liquor- a by-product of starch extraction from maize Simple organic acids and alkanes

Factors influencing the choice of carbon sources ƒ ƒ ƒ

The main products of the fermentation- determine the cost of the processes Impurities of the carbohydrate sources Government legislation e.g. in the EU, the beet sugar and molasses are encouraged compared to the cane sugar and molasses

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Local laws especially in some countries where specific acts forbid the use of some ingredients. For instance, in France, many wines may only be called by a certain name if producing vineyard is within a limited geographical area or locality. These names include vin de bordeaux, which is cultivated around the south East of France Method of media preparation e.g. it is often best to sterilise sugars separately because they may react with ammonium ions and amino acids to form black nitrogen containing compounds

The influence of carbon sources on product formation Of great significance is the rate at which the carbon source is metabolized. This will influence the formation of biomass or production of primary metabolites. Nitrogen sources Most industrially used micro organisms can utilise inorganic or organic sources of nitrogen. In organic sources may include ammonia gas, ammonium salts and nitrates. Most organic sources of nitrogen are mainly supplied as amino acids, protein or urea. Organic sources are relatively expensive. Factors influencing the sources of nitrogen sources: The nitrogen sources have been shown to influence the fermentation pattern. Antibiotic production may be inhibited by a rapidly utilized nitrogen source. For example, in the production polygene antibiotics, soybean meal is considered a good nitrogen source because of balance of nutrients. Minerals In many media magnesium, phosphorous, sulphur, calcium and chlorine are considered as essential. These are added as a distinct component. Others such as cobalt, copper, iron, manganese, molybdenum and zinc are also essential but usually present as impurities in major ingredients. Vitamins sources While many of the natural carbon and nitrogen sources contain all or some of the required vitamin, any vitamin deficiency may be eliminated by a careful blend of materials. Nutrient recycle

In cases of large scale continuous culture fermenters, there is always need for appropriate adjustment of nutrient levels. Phosphoric acid is used as a reagent for flocculating bacteria. Buffers These are added to control the PH. The buffers include calcium carbonate, phosphates, sodium hydroxide or sulphuric acid. Precursors/inhibitors/inducers Precursors help in the regulation of the product rather than support the growth of micro organisms. Examples of precursors include corn steep liquor which increases the yield of penicillin since it contains phenyl ethylamine. Inhibitors-sodium bisulphite in the production of glycerol Inducers-use of yeast in streptomycin production Oxygen This is not added in the initial media, but nevertheless an important parameter in controlling the growth of micro organisms. The medium may influence the oxygen availability in a number of ways including: 1. Fast metabolism- oxygen limited 2. Rheology-affects aeration and agitation 3. Antifoam- reduces the oxygen transfer rates ANTI FOAMS Foaming is a common problem in most Biological systems. This is caused by the proteins in the media, which denature at the air-broth interface and form a skin which does not rupture readily. Foaming may cause the removal of cells from the medium which may lead in the autolysis and further release of microbial cell protein. There are two approaches in combating foaming in the fermenters: ƒ ƒ

Partial purification of some complex nutrients and modification of some physical parameters Use of antifoam

An ideal antifoam need to have the following properties ƒ Disperse easily ƒ Active in low concentrations ƒ Long acting to prevent new foams from foaming

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Non-toxic to the micro-organisms Non toxic to animals and humans Should not cause problems in the extraction of the products Should not cause handling hazards Should be cheap Should not affect oxygen transfer Should be heat sterilizable

Examples of antifoaming agents: ƒ Alcohols, stearyl and octyl decanol ƒ Esters ƒ Fatty acids and derivatives particularly glyerides ƒ Silicones ƒ Sulphonates ƒ Propylene glycerol

1.7 Changes in the composition of cells with age and with growth rate In a batch culture, cells multiply in a closed system until some nutrient is exhausted or some product accumulate to toxic levels. The developing population passes through a number of phases, namely: 1. 2. 3. 4.

Lag phase, in which cell mass increase but no division occurs Logarithmic phase-cell numbers increase at a constant rte Stationary phase-rate of death and multiplication are equal Decline phase-rate of death is faster than the rate of multiplication

It is important to appreciate that in a batch system, the environmental conditions are not constant, even during the phase of constant growth.

Development of inocula for industrial Fermentation It is essential that a culture used to inoculate fermentation processes satisfies the following main criteria: ƒ ƒ ƒ ƒ ƒ

It must be healthy- in an active state to minimize the length of the lag phase It must be available in sufficiently large volumes to provide an inoculum of optimum size It must be in a suitable morphological form It must be free of contamination It must retain its product forming capabilities

The quantity of the inoculum normally used is between 3 and 10% of the medium volume. This implies that starting from a stock-culture; the inoculum must be built up in a number of stages to produce sufficient biomass to inoculate the production stage fermenter. The master culture is reconstituted and plated on a solid medium; to form colonies of sub master culture. The Isolation, preservation and improvement of the industrial micro organisms The first stage is screening for micro organisms of potential industrial application in their isolation.

Isolation involves obtaining of either pure or mixed cultures followed by their assessment to determine which can carry out the desired reactions or produce the desired products. The criterion for the choice of organisms is a follows: 1. The nutritional characteristics of the organisms-so that the process may be carried out using very cheap medium 2. The optimum temperature of the organism- the choice of an organism having an optimum temperature of above 40°c reduces on the cooling costs of a large scale fermentation 3. The reaction of the organism with the equipment to be employed and the suitability of the organism to the type of process to be used 4. The productivity of the organism, in terms of ability to convert substrate into the product and to give high yield of product per a unit time 5. The stability of the organism and its sensibility to genetic manipulation 6. The ease of product recovery from the culture Before the process may be put into commercial operations, the toxicity of the product and the organism must be checked and assessed. The above account implies that cultures must be isolated from the natural environments. However, the industrial microbiologists may also isolate micro organisms from culture collections. It is probably cheaper to buy a culture than isolate from nature. The ideal isolation procedure starts with an environmental source (frequently soil) and incorporates a simple test to distinguish the most desirable types. Selective pressure may be used in the isolation of the micro organisms which grow on particular substrates, in the presence of certain compounds or under cultural conditions adverse for these types. In other cases, it may be possible to design a procedure to select microbial strain which is known to show certain characteristics at a relatively high frequency e.g. the production of antibiotics by the Streptomycetes. Another method may apply random isolation or isolation by taxa. Isolation methods utilizing selection of the desired characteristics Enrichment of liquid culture The process involves taking a mixed population and providing conditions either suitable for the growth of the desired type or unsuitable for the growth of the other types e.g. providing a particular substrates and inclusion of inhibitors. The enriched culture is inoculated

in a fresh medium and subsequently the procedure is repeated several times before the dominant organism is isolated by spreading a small inoculum of the enriched culture in a solid medium. This is normally hampered by the time of transfer and selection on the basis of specific growth rate. This may be overcome by the use of continuous process. Preservation of industrially important organisms The isolation of a suitable organism for commercial process may be long and very expensive procedure and it is therefore essential that the isolated species retains the desired characteristics that led to its selection. Also the culture must be viable and free from contamination. Industrial cultures must be stored in a such away as to 1. Eliminate genetic change 2. Protect against contamination 3. Retain viability Methods of Micro organism preservation 1. Storage at reduced temperature Some of the common mean include ƒ Storage on agar slopes, which are refrigerated at 5°c or frozen at -20°c and sub cultured at intervals of about 6 months ƒ Storages of spores in water in which it’s suspended in a sterile distilled water and stored at 5°c. this technique is limited in application ƒ Storage under liquid nitrogen- the metabolic activities of the micro organisms may be reduced considerably by storage at very low temperatures(-150°c to -196°c), which is achieved by liquid nitrogen refrigeration 2. Storage at dehydrated form ƒ Soil culture-inoculated before being allowed to dry for a period of about 2 weeks, thereafter refrigerated ƒ Lyophilization- the culture is frozen followed by its drying under vacuum which results in the sublimation of the cell water. This culture may remain stable for upto 10 years.

Quality control of preserved stock cultures Each batch of newly preserved stock culture should be routinely checked to ensure their quality in terms of viability, purity and productivity. The viability of a micro organism is the ability to reproduce. Reasons for controlling Micro organisms

It is interesting to consider a few reasons for wishing to reduce adventitious contamination: 1. Avoid spoilage of a product after a period of storage 2. Avoid pathogenic organisms 3. Avoid unwanted end products 4. Maintain the yield of the desired products Sources of contaminants ƒ Present in raw materials ƒ Airborne contamination ƒ packing supplies ƒ personnel The multiplication of the contaminants is prevented by: ƒ ƒ ƒ ƒ

design of buildings to prevent cross-infection and avoid crevices and ledges design of equipment to maintain hygienic standards control of storage and holding conditions at all stages from raw materials to the ultimate consumer choice of operating procedures and conditions

The use of advance methods for prevention and control of contaminants will be discussed under sterilisation.

Variations of micro-organisms When a micro-organism is sub cultured so as to give a number of generations of progeny, the final culture obtained contains many individuals differing from the first parents. This is a result of mutations or the relatively permanent change in the genetic apparatus of the cell. This is mainly associated with genetic changes. Though most changes in genes are disadvantageous or even lethal to the organisms, enough survive with higher incidences of variants after a few generations. Thus, every culture of micro-organisms loses its original character by subcultivation. To avoid this, a few generations should be allowed to intervene between the tested master- culture (obtained from single colony isolation) and the production stage. In addition, a number of arbitrary treatments such as heat- and coldshock, pasteurisation and sub-cultivation on special selective media may be employed. Besides its deleterious effect on the yield of product, mutation makes itself manifest in a number of ways:ƒ Alter the degree of pigmentation e.g. the pale-green normal penicillin Chrysogenum throws white, dark green, yellow orange and other coloured mutants ƒ The power to sporulate may disappear

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The new organism may lose the power to synthesis essential amino acids or vitamins The colony form may change as well as growth rate

However, mutations have a beneficial side also, and to this end may be produced artificially. A suspension of organisms is subjected to treatment and desirable properties may be sought by examination of individuals in the resultant population. The principal methods of artificially induced mutations include:1. Exposure to ultra violet light Ultra violet light is absorbed by the nucleoprotein material, which forms the genetic material, resulting in the resonance and leads to destructive changes. This method is not highly selective and is accompanied by a good deal of damage to the rest of the cell. 2. Mustard gas Mustard gas is a more drastic agent reacting with generic centres and disrupts chromosomes. Once the generic centres are changes, information on heredity is interrupted. 3. X-rays and Gamma rays X-rays act almost the same way as the mustard gas, giving rise to free radicals in the ambient medium. The free radicals are responsible for further changes in genetic information. Gamma rays are the more recent. 4. Use of radioactive substances The use of radioactive materials is known to give higher mutants rates with a lower killer rate. The classical isotope used is the sulphur-35 which decays in 87 days to give chlorine-35. The major advantage of this method is that mutations may occur without appreciable cell trauma. It can also be simple to control. It also enjoys higher rates of survival, almost about 100%. From the mutated cultures, conventional testing techniques are used to obtain a higher yield of antibiotic or other desired quality. For example, the yield of penicillin has been raised from 10units/ml in 1942 to over 2000units/ml in 1960. As an example of the commercial use of mutant strain, Escherichia Coli has been mutated in lysine fermentation. Hybridization The selective sexual breeding is finding limited application in microorganisms.

STERILIZATION OF AIR, MEDIA AND EQUIPMENT Definition: Sterilization is the destruction of all forms of life in a medium and environment. In the laboratory, this is achieved by the use of heat; the bacteriological medium is held at temperatures of about 120°c for 20 minutes. Dry heating in an oven is yet another method commonly used to sterilize equipment in the laboratory. Besides, the use of heat, there are other methods which can be used to sterilize the equipment, air and media. These methods include:a) Use of ultra violet light b) Irradiation by chemical regents or high frequency c) Filtration especially for the sterile air STERILIZATION OF EQUIPMENT AND MEDIA The sterilization of equipment and media together is achieved by the use of steam. This is a simple operation in which a jacket or a coil is fitted to the bioreactor is supplied with pressure steam at a temperature and duration suitable for the destruction of life. This is followed by subsequent cooling to the fermentation temperature. A vent is provided through which air is expelled from the bioreactor and the space above the medium is filled with steam. A supply of sterile air must be connected to the fermentor to prevent formation of a vacuum when the system is cooled. STERILIZATION OF THE BIOREACTOR AND MEDIUM SEPARATELY The procedure of sterilizing an empty bioreactor is exactly as described in the last section. There are however, several methods of sterilizing the medium separately:1) Batch cooker A vessel fitted with coils or a jacket for heating and cooling. An agitator may be fitted to aid heat exchange. The interconnecting piping between the cooker and the main bioreactor must be sterilized at the same time so that sterile medium may be transferred. This method reduces the time in which the main reactor is unoccupied between fermentations; against this is the higher cost of the extra equipment involved, and the increased steam usage. 2) Continuous sterilization The medium is sterilized as it is pumped from the medium make-up vessel to a fermentor which has been sterilized empty. This method offers flexibility in choice of time temperature conditions in which the medium is exposed and advantage taken of lower sterilizing temperatures or shorter holding periods if the medium has low PH.

Increased yield have been reported in cases where continuous sterilization is employed. There are two principle that may be applied in continuous sterilization a) Heat Exchange Principle This is a three section plate type heat exchange. This methods realizes some important steam saving economy. It is necessary to avoid any leakage or short-circuiting between the non-sterile medium entering and the sterile medium leaving the system. b) Continuous Retention Tube In this system, medium is made up in a mixing vessel and is pumped from this into one end of a long retention tub. At this end, steam is injected to heat the medium to the desired sterilizing temperatures for the requisite time, before being passed to a cooling section and thereafter a bioreactor; in the design of the retention tube, it is important to ensure that there is no end-to-end mixing. This would result in the non-sterile medium by-passing sterilizing temperatures zone and infecting the rest of the sterile system. In sterilization, there are principles of securing and maintaining sterile and pure culture conditions. These include:1. No direct connection should be made permanently between non-sterile and sterile parts of the system 2. Welded construction should be used where possible and convenient 3. Where joints have to be used they should be of high quality finish, employing rubber or any other impervious material as the seal 4. The type of valve used should be easily sterilizable and serviced when necessary 5. After sterilization, all parts of the system which are to be kept sterile should be kept under a positive pressure, either with sterile air or sterile liquid 6. Each part of the system should be capable of independent sterilisation, without interfering with operation of the rest of the plant 7. Isolated danger spots, which are difficult to sterilize at the star of a batch, should be provided with steam connections for continuous or intermittent use. STERILIZING OF AIR

Since the time of Pasteur, air has been recognized as a source of microbial contamination. At this time, it was discovered that a plug of cotton wool would allow air to reach the culture but would prevent other micro-organisms from contaminating it. Over the years, the demand for pure air for industrial operation has been on the increase. This calls for the sterilizing of air in terms of thousands of cubic feet of air per hour. The most surely effective method is heating the air, but the cost is prohibitive when large volumes of air have to be sterilized. Therefore, other methods of sterilizing the air have been applied:1. Electrostatic precipitation 2. Exposure to UV light 3. Filtration through columns packed with glass wool, slug wool, cotton wool, activated carbon or other filtering media coupled or without counter-current scrubbing through phenols, caustic soda, acids or other germicidal agents. Direct chemical treatment is not possible because of the danger of carry-over into the culture. Most antibiotic plants use filters packed with fibrous media such as glass or slug wool or granular material such as activated carbon. The efficiency of filtration is calculated from 100( N 1 − N 2 ) N2 Where N1 is the number of micro-organisms per unit volume of air before filtration and N2 is the number of micro-organism per unit volume of air after the filtration. effeciency % =

The mechanism of filtration is not a simple sieving action but is due to retention of airborne particles on the fibres or granules of the filter media. Therefore, the efficiency of the filter media, and the efficiency of filtration would therefore depend on a number of factors like ƒ Diffusion ƒ Inertial impingement ƒ Electrostatic attraction A mathematical evaluation on the dimensioning of filters for sterilisation of air a addressed as assignment and will be dealt during tutorial session. CHEMICAL STERILIZATION Sterilization can sometimes be achieved by the use of chemicals. There are numerous terms used in connection with the control of contaminants. These terms include antiseptic, disinfectant, prophylactic, sanitizer, germicide, bactericide, fungicide.

Due to the large spectrum of sterilizing agents, different methods of testing sterilising agnts have been developed. 1. Rideal_walker test This depends on a comparison between the inhibitory effect of phenol and the other agent on the growth of selected micro-organism, under closely monitored condition 2. Chick-Martin test Due to the inadequacy of the comparison test (Rideal-Walker) in terms of variations dependent on the organic nutrient present, dried yeast is used in a more standardises procedure. 3. Reddish and other official tests A number of detail modifications have been introduced into the chickMartin test to make results of wider applicability. In spite of these modifications, no method has been found which consistently reflects the behaviour of sterilising agent under actual working conditions. There is a growing tendency for each user to devise tests to suit their own requirement. Factors affecting chemical sterilisation I. Presence of other matter- the presence of some other matter may interfere with the sterilising action e.g. the presence of grease can offer a large measure of physical protection to the organisms unless the agent can penetrate it. In addition, grease can offer nutrients for the organisms. This has dual effect; allows undesirable faster multiplication and bringing organisms into an actively mitosing condition in which there are vulnerable to attack. II. Rate of destruction of micro-organisms: the use of a single sterilising chemical agent on a single strain follows approximately logarithmic course in respect with time N 2.303 log o t Nt where No, Nt are the concentration of organisms initially and at a time t respectively, k is reaction velocity constant and t is time usually in minutes the hourly basis for air change has been calculated by N 138 KA = log o t Nt III. the level of initial contamination- assuming the logarithmic relationship, then it follows without doubt that absolute sterility can only be approached asymptotically and arbitrary final level N t = N o e −κt or κ =

must be quoted. The higher the initial contamination, the higher the probability of including pathogenic or resistant microorganisms in th sterile environment. IV. Concentration of the sterilizing agent- a general relationship between the concentration of the sterilizing agent and the time to reach a low count can be expressed C1n t1 = C 2n t 2 Where n is considered as the concentration coefficient V. Decay rates of the disinfectants-sterilising agent can progressively loose their effectiveness as a result of chemical change, adsorption and various other factors. For instance hypochlorite (jik) is quite unstable or highly reactive compound whose KA value approaches 20. VI. Physiological effect of sterilising agents-unless there is clear evidence to the contrary, it should be assumed that any compound capable of disorganising a unicellular organism as to cause its death, will also have a significant physiological effect on larger creatures including man. These effects include allergy, cumulative effect of ingestion in small amounts over a long period of period. Caution needs to be exercised at all times! Detergents Detergents in a broad sense are the foundation of industrial cleaning procedures and may be included in the final products either deliberately or fortuitously as residues. The chosen detergent may occasionally be relatively pure compound, but is usually a mixture, either blended on site or purchased from a specialized company There are important aspects of detergency, which must be considered in the choice of the most efficacy detergent:a. Surface and interfacial tensions are reduced, allowing emulsification of grease. But this poses of a negative side effect of increasing wettability of the surface b. Dirt and grease should be held in suspension c. Calcium and magnesium are held in control ƒ Form soluble Ca and Mg salts ƒ Form true soluble complexes with Ca++ and Mg++ ƒ Phosphates and other builders may combine with these ions to form insoluble but finely divided precipitates d. A high PH level, increasing detergency Uses of sterilising agents ƒ The use can be considered under the following headings ƒ Sterilising water ƒ Cleaning equipment ƒ Preservation of food and cosmetics

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Pharmaceutical preparations

FERMENTATION PROCESSES ANAEROBIC FERMENTATION PROCESSES: ACETONE-BUTANOL Historically, this type of fermentation was developed as a source of nbutanol which could be used in the preparation of butadiene, a major ingredient in the manufacture of synthetic rubber. After the world I, this process was used as a source of acetone. The acetone was used in the manufacture of cordite. After some years, the demand for acetone reduced and the pressure reverted to be a source of butanol. This time, the butanol was the main raw material for the production of butyl esters, particular solvents of nitro-cellulose lacquers. The organism used for this fermentation is the Clostridium Acetobutylicum, which by definition is an anaerobic spore forming bacterium. The name acetobutylicum implies that it produces acetone and butanol. It does this in a medium of molasses, made up of about 6% sugars (calculated as sucrose). From a good fermentation it produces a solution mixed solvents composed of about 65% butanol, 30% acetone and 5% ethanol. This solution is distilled and fractionated to obtain particular products. There is also considerable gas produced, a mixture of CO2 and hydrogen gas. Animal feed may be prepared from the residue, largely composed of the bacteria. AEROBIC FERMENTATION PROCESSES: PENECILIN The principal advances in the technology of aerobic fermentation were made in the course of development of penicillin fermentation. Technically, penicillin is a generic name applied to a group of compounds. Therefore, a large number of penicillin occur naturally or could be artificially prepared. For example there exist penicillin G and Penecillin V, which are distinguished by the side chain-benzyl and phenoxymethyl group respectively. However, most penicillin are labile in the acid form and they are normally prepared as much stable salts or esters e.g. sodium benzyl penicillin. Penicillin are formed by several different organisms; chiefly aspergi and penicillia. Penicillium Chrysogenum group is known to give the highest yields. At the industrial scale, the penicillium spores are developed in a flask of sterile corn in sufficient quantity to provide inoculum for the seed mash. After inoculation the seed mash is maintained at temperatures optimal for the germination of the spores and the growth of the organisms. It is necessary to aerate and agitate vigorously to promote metabolism and growth. This may take between 24 and 48 hours depending on the

size of the vessel and the inoculum. The termination is usually based on some empirical criterion such as ƒ Age of the culture ƒ The exhaustion of the sugars ƒ Tendency of the PH to rise The seed is then transferred along the sterile connection into the fermentor charged with mash. Aeration, agitation and temperature control of the mash commences at least as soon as the seed is received into a fermentor and continuous until the mash is harvested between 3 and 5 days later. The first step in harvesting consists of separating the mycelium from the medium by the use of rotary vacuum filter in which the mycelium is continuously stripped as felt. YEASTS Yeasts have uniquitous distribution throughout the plant and the animal kingdom and also in the soils. From the industrial viewpoint, there re two main genera of interest, the broad range of saccharomycetes and the more limited candida or torulopsis yeasts. Industrial yeasts can be classified broadly into six groups a. Beer or ale yeasts-these are referred as top fermentation. They rise to the surface of the wort as the head of foam, which can be skimmed off for re-use b. Lager yeasts- brewing lager is carried out at a much lower temperature than beer and for longer periods. This yeast must withstand the conditions and also settle out to the bottom of the vessel towards the end of the fermentation c. Distiller’s yeasts- they are selected strains of the beer/ale yeast adapted for high sugar and alcohol tolerance and capable of giving high conversion ratio. d. Baker’s yeast-while its selected from beer/ale yeast, the baker’s yeast is bland in flavour, lacking hop bitterness from beer, is more rapid in action and has extended shelf life e. Wine’s yeast f. Food and fodder yeast BREWING Large scale brewing has been developed empirically in various parts of the world. There are differences also in raw materials and the methods evolved to deal with them. Barley malting-clean and uniform is selected and stored for a resting period before it can be germinated. For malting, it is steeped in several

changes of water for about 2days at 50°f, taking care to avoid germination. The final water is then drained off and the soaked barley is spread on the malting floor for about a week to sprout. During this period, various enzymes are activated and potentially able to convert up to 75% of the solid contents into soluble sugars, mainly maltose, dextrins and some polypeptides. The next stage is kilning, which stops further activity by reducing water availability. This is then milled. Preparation of wort- All the ingredients are broken down to an intermediate size by a roller mill. The mixture is fed with hot water through a mashing machine. Mashing carries out several functions namely:ƒ The soluble fractions of malted grains is extracted ƒ The amylotic enzymes present have an opportunity to hdyrolyse unattacked strch still in malt ƒ The smaller proportion of proteolytic enzymes must be allowed to break down proteins into polypeptides ƒ The balance of ingredients and technique are arranged so that the final wort has the correct specific gravity Fermentation- The temperature of fermentation depends on the type of beer required and the yeast strains in use. The cooled wort is pitched in a cream made from yeast taken from previous brews and the fermentation allowed to proceed for about 3 days. During this time, the temperature is maintained by passing cold or hot water through immersed pipes, known as attemperators. Industrial Alcohol Although a large proportion of industrial alcohol in the world is made from petrochemicals, considerable amounts of fermentation spirits are still made through the world the raw materials include grain, beet/cane molasses etc. After mashing the final wort is adjusted to conditions to suit the chosen yeast. Temperatures are in the range of 23°c to 30°c and the initial PH is adjusted to 4.5 to 6.0. The balance of nutrients and total concentration are adjusted t provide only a limited growth but maximum conversion of carbohydrates to ethanol. Utilisation of by-products Carbon dioxide-about ¾ is recovered for sale as compressed liquid or dry ice. Since pure grade is required, the CO2 is scrubbed through water, from which ethanol is recovered, then sulphuric acid and finally deoderized an activated charcoal. Fermentation residues- these are insoluble material filtered off and dried to be used as animal feed. Alcohol distillation: The filtered liquor after fermentation contains not only ethanol and water but also small amounts of volatile components (acetahyde, esters, fusel oil). After distillation, the ethanol obtained is

good enough for certain sales outlets. However, some dehydrated form of ethanol may be required. The methods used including 1) absorb it as water of crystallisation on a mixture of sodium and potassium acetates 2) azeotropic distillation – use of a solvent (benzene) to form a ternary azeotrope with the water and a small part of the ethanol. In the production of industrial alcohol, the alcohol is denatured for purposes of excise duty control; to avoid reaching the public in potable forms. Depending on the intended final use, it is normally denatured by methanol. Methanol is highly neurotic. CONTINUOUS FERMENTATION Applied relatively fast chemical reactions or physical processes, continuous fermentation methods offer very great advantages compared to batch methods, provided the demand of the products is high enough. These advantages are:ƒ Non-productive time i.e. time spent cleaning, filling, heating, cooling and emptying are drastically reduced ƒ This affects the useful loading of the plant, so that smaller equipment and buildings can be used ƒ Labour can significantly reduced, partly because it is easier to mechanize some operations ƒ Automatic controls and warning systems are easier to install and they in turn can reduce the requirements of skilled supervision ƒ The product can be held at optimum reaction conditions, unaffected by heating and cooling, producing better yields ƒ Product uniformity should be improved However against these advantages may be listed certain possible disadvantages:ƒ Although the plant may be smaller, instrumentation and mechanical handling devices may make it equally expensive ƒ The design and operation of such equipment requires the services of highly educated staff ƒ The shift work necessary may increase some costs, highly hourly rates, provision of steam, laboratory services etc ƒ It is rare for manual efficiency of the night workers to be as high as that of day workers In practice, a careful balance is struck between these factors, often a compromise is decided on e.g. large scale operations are carried out continuously while minor operations e.g. cleaning of the equipment is carried out during the day shift. Experience with continuous fermentation indicates that there are quite severe limitations to the time an operation may be run. These factors that limit include:-

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Adventitious contamination can become serious after even a few days There is a tendency for fast growing strains of organisms to become dominant Some organisms develop the morphological and biochemical mutation under continuous culture Physical problems such as removal of fungal mycelium limit to the operation The yield of the product and/or the concentration significantly reduces

THEORY OF CONTINUOUS FERMENTATION Interest has been focussed on the continuous stirred-tank reactors (CSTR), used singly or a few in sequence. The essential feature of this form of reactor is that stirring is sufficiently vigorous so that the composition of the efficient is identical with the bulk of the tank contents; the danger of short-circuiting of vital nutrients from feed to the effluent must be taken into account and conditions have to be selected to keep this minimum. Mathematical analysis of such continuous fermentor is much easier if the following assumptions are made:The growth rate of the organism is held constant Only a single strain of organism is present which maintains its growth characteristics Therefore, the density of the organism within a single vessel can be described by the following equation F F dN = Nf + κN − N . V V dt Rate of change of the fermentor = Increase due to organism in feed + increase by growth- loss of effluent N= Concentration of organisms, Nf= organisms of feed, F= flow into and out of the fermentor, V= volume of fermentor, k= specific growth The dimensions of the equipment may be eliminated by referring the dilution rate, D= F/V SINGLE VESSEL STERILE FEED Under steady-state conditions dN = 0 and the first term, NfD disappears dt F OR κ = D therefore, κN = N = ND V

for the population to remain steady, the holding time 1/D, must be equal to the generation time or reciprocal of growth rate. The specific growth factor, k may be identical to the value found on the logarithms part of the batch growth curve. In practice it may be well below this value because. The feed rate of limiting nutrients is insufficient to maintain maximum growth The organism may be subject to environmental factors more nearly corresponding to early stationary phase If avalue of kn is taken as logarithmic value, then by definition the maximum achievable, then D=km, thus the process may be treated as batch. Thus, F = Vκ m If this flow rate is exceeded i.e. F is greater than km, then clearly the organisms are carried out in the efficient than they can grow in the fermentor. The classical work of Monod, showed that the growth rate of many organisms can be related to the concentration of the limiting component by formula similar to Michaeli’s enzyme equation.

S ) K+S Where k= saturation constant, numerically equal to Km/2

κ = km (

The dilution rate ⎛ S ⎞ D = κm⎜ ⎟ where ⎝K+S⎠

⎛ D ⎞ ⎟⎟ S = κ ⎜⎜ ⎝κm − D ⎠

MICROBIAL GROWTH KINETICS Microbial growth is defined as the orderly increase of all chemical constituents of an organism. It may result from the synthesis and accumulation of a cellular material. As dictated by the type of the product being produced, fermentation may be carried out as batch, continuous and fed-batch processes. Batch Culture- This is a cloud culture which contains an initial, limited amount of nutrients. In this process, the lag phase must be limited as much as possible, while the exponential phase is encouraged. The exponential phase is described by the equation dX = µX ………………………………………………………………1 dt X=concentration of microbial biomass, t= time(hrs) and µ is the specific growth rate, hrs-1 Equation 1 is intergrated as follows ƒ X t =Xoeµt Xo is the original biomass concentration On taking the natural logarithms, ln X t = ln X o + µt

During exponential growth, the micro-organism is growing at its maximum growth, µmax for the prevailing conditions. However, the nature of limitation of the growth ay be exposed by growing the micro-organisms in the presence of a range of substrates concentrations. The situation may be described as X = Y (S R − S ) Where X IS THE BIOMASS produced, Y is the yield factor, SR is the original substrate concentration and S is the residual substrate concentration. CONTINUOUS CULTURE

A media displaces an equal volume of culture from the vessel to achieve continuous production. The flow of medium into the vessel is related by the term dilution rate, D, defined as F D= V The net change in cell concentration over a time period may be expressed as

dX = growth − output dt dX = µX − DX dt Under steady state conditions, the cell concentration remains constant, thus dX/dt =0, Then µX + DX and

µ=D Thus under steady state condition, the specific growth is controlled by the dilution rate. For the substrate X⎛ S ⎞ dS ⎟ = DS R − DS − µmax ⎜⎜ Y ⎝ K S + S ⎟⎠ dt Fed-batch culture- there are batch system which are fed continuously.

THE RECOVERY AND PURIFICATION OF FERMENTATION PRODUCTS The recovery and fermentation products may be difficult and costly to ensure good recovery or purification, speed of operation may be the overriding factor because of the labile nature of the product. The choice of the recovery process is based on the following criteria:a. The intracellular or extra-cellular location of the products b. The concentration of the product in the broth c. The physical and chemical properties of the desired products d. The intended use of the product e. The minimum acceptable standard of purity f. The impurities of the fermentor broth g. The marketable price of the product The main objective of the first stage for the recovery of an extra-cellular product is the removal of large solid particles and the microbial cells b centrifugation or filtration. In the next stage the broth is fractionated or extracted into major fractions using adsorptions or ion-exchange chromatography liquid-liquid solvent extraction or precipitation, further more precise chromatographic and crystallisation. It may be possible to modify the handling characteristic of the broth so that it can be handled much faster with simpler equipment making use o a number of techniques ƒ Selection of the micro-organisms which does not produce pigments or undesirable metabolites ƒ Modification of the fermentation conditions to reduce the production of undesirable metabolites ƒ Precise timing of harvesting ƒ PH control ƒ Temperature treatment after harvesting ƒ Addition of flocculating agents ƒ Use of enzymes to attack cell walls Removal of microbial cells and other solid matter. Due to the small size of microbial cells, filter aids are applied to improve filtration rates while heat and flocculation are employed as techniques for increasing sedimentation rates in centrifugation. a. Foam separation- this is done by exploiting the differences in surface activity of materials. The material is selectively adsorbed or attached to the surface of gas bubbles, concentrated and finally removed by skimming b. Precipitation- It is possible to obtain some products from the broth, either by adding a compound which leads to the formation of insoluble complexes or salts or by adding a suitable organic solvent.

c. Filtration- this is the commonest method of removing suspended particles from a liquid or gas, using a porous medium which retains the particles but allows the liquid or gas to pass. The following factors influence the choice of the most suitable filtration equipment to be used:ƒ Properties of the filtrate, viscosity and density ƒ Nature of solid particles especially size, shape and size distribution ƒ Solids:liquid ratio ƒ The need for recovery of the solid or liquid fraction or both ƒ The scale of operation ƒ The need of batch or continuous operations ƒ The need for aseptic conditions ƒ The need for pressure/vacuum suction to ensure an adequate flow rate of the liquid d. Centrifugation-micro-organisms and other similar sized prticles can be removed from broth by using centrifuge when filtration is not satisfactory separation method. Although a centrifuge may be expensive when compared with a filter it may be essential when:a. Filtration is slow and difficult b. The cells or other suspended matter must be obtained free of filter aids c. Continuous separation to a high standard of hygiene is required e. Liquid-liquid extraction- this is the separation of a component from liquid mixture by treatment with a solvent in which the desired component is preferentially soluble. The solvent is then recovered by distillation. f. Chromatography- this is used to isolate and purify relatively low concentrations of metabolic products. This will involve the passage and separation of different solutes as the liquid is passed through a column. Depending on the mechanism by which solutes may be differentially held in a column, the technique can be grouped as ƒ Adsorption chromatography-binding of the solute to the solid phase primarily by weak van der waal forces ƒ Ion-exchange-reversible exchange of ions between liquid phase and solid phase which is not accompanied by radical change in the solid structure ƒ Gel-filtration-separates on the basis of size in which the small particles diffuse through the gel rapidly ƒ Affinity chromatography-depends on the interactions between pairs of biological materials such as enzymessubstrate

g. Ultra-filtration- the process in which solutes of high molecular weights are retained when the solvent and the low molecular weight solutes are forced under hydraulic pressure through a membrane of very fine pore size. h. Drying- this is often the last stage, which involves the removal of water from the heat sensitive material ensuring that there is minimal losses of viability, activity or nutritional value. Drying is undertaken because:ƒ The cost of transport can be reduced ƒ The material is easier to handle ƒ The material can be more conveniently stored in dry state A spray drier is the most widely used for drying biological materials when starting materials is in the form of a liquid or paste which can be initially atomized into small droplets through a nozzle or by contact with a rotating disc. i. Crystallisation- This is best applied in the initial recovery of organic acids and amino acids

FERMENTATION ECONOMICS If a fermentation process is to yield a product at a competitive price, the chosen micro-organism should give the desired end products is predictable, and economically commonly used in developing a successful process:a) The capital investment should be confined to minimum b) Raw materials should be a cheap as possible and utilized efficiently c) The highest yielding strain of micro-organism should be used d) There should be saving in labour whenever possible e) Growth cycle must be short as possible for batch fermentors f) Recovery and purification procedures should be simple and rapid as possible g) The effluent discharge should be efficiently h) Space requirement should be kept minimum There a number of steps in a biological process, which need to be carefully analysed in terms of cost. The major steps are as follows:-. 1. Isolation of micro-organisms-Isolation programmes are unfortunately time consuming, expensive and may be something of a gamble e.g. Pfizer spent about £1430000 on screening programme for a broad spectrum antibiotic producer during which terramycin was detected in 1952. In search for suitable micro-organisms for use for single-cell protein production, many objectives will have an economic basis. 2. Strain improvement- The strain improvement by a mutation selection for improving an established process can be very cost effective. Obviously time and money worth spending on mutation/selection programme will depend on the size of the manufacturing process. 3. Media-The cost of the various components of a production medium can have a profound effect on the overall cost of fermentation. The carbon source is usually the most expensive contributing to the cost of the process. In addition, the price of natural material may fluctuate due to other competing demands annual variations in quantities harvested. Problems concerned with storage, handling and mixing of media should not be neglected. 4. Air Sterilisation- although air sterilization by heating is technically possible, it has generally been regarded as too costly for full-scale operation of a plant. Filtration of air

through deep bed of fibrous or granular material is preferred. While the capital costs are dependent on the size of the plant, the operating costs will be estimated on the life of the filters. 5. Heating and cooling-A biochemical process may include heating and cooling in the following major areas:ƒ Sterilization or boiling of medium to 120°c followed by cooling to 35°c ƒ Heating of fermenter and other ancillary equipment ƒ Removal of water from products The cooling costs of micro-organisms can be minimized with the use of micro-organisms with a higher optimal growth temperature. 6. Aeration and agitation-Fermentation having high oxygen demand must be agitated with sufficient power to maintain uniform environment and to disperse the stream of air introduced by aeration. Some companies have introduced air-lift principle, with benefits of simple design and reduced energy costs. 7. Batch-process cycle times- For shorter growth cycles such as baker’s yeast (14 to24 hrs), the turn around time will be as important as the time between inoculation and harvesting while when production cycle is long e.g. penicillin(6 to 7 days),a few extra hours of turn around will be insignificant on the total cost. 8. Recovery cost-A number of factors contribute to the recovery costs:ƒ Yield losses ƒ High energy and maintenance associated with the recovery and purification e.g. depreciation accounts for 80% of the overall cost of a large-scale rotary filtration ƒ High costs of solvents and other raw materials used in the recovery and refining of the products 9. Water usage and recycling- As the charges for water increase, many of the biological process will become vulnerable to cost escalation because of relatively large volumes of water required per unit volume of product. There is a wide spread interest in reducing the overall consumption. 10. Effluent treatment-In the majority of fermentation processes it is impossible to dispose of effluents at zero

cost. The various alternative disposal procedures may be compared using economic considerations. Before deciding on the most economic form of treatment, the water column, the organic and solids loading, range of PH variation, nutrient level, temperature fluctuaction, company finance policy, the site location and government legislation for waste disposal must be known. 11. Plant and equipment- It is most logical to build equipment as large as possible because of the economy of scale. However, there are a number of restraints which have to be considered before deciding on scale of operation such as restriction include:ƒ Cooling and aeration requirements ƒ Methods of fermentation vessel construction Due to the capital investment and operational costs there is now a trend of fermentor design to consider unconventional designs of simple construction with every efficiently oxygen transfer to be used for specific purposes e.g. single protein cell. A more detailed guide is prepared by the Institution of Chemical Engineers. MARKET POTENTIAL It is necessary to estimate the size of the present and potential market and increase in the demand for a compound. The life expectancy of the compound will have to be predicted when covered by a patent.