Lect6(Inoculum Preparation and Development).pdf

Inoculum Preparation and Development

Strain of microorganism - most important part of a fermentation process. (pure and produce the desired product at optimal level). Two most important types of microorganism bacteria and fungi. More plant and animal cells are also being grown in bioreactors for production of highly specialized product.

General course of fermentation in the production of primary and secondary metabolites

Stage Course of the fermentation I

Inoculum preservation

II

Inoculum build-up

a. 1-2 Shake flask cultures b. Spore formation of solid medium

III

Prefermenter culture

1-3 Preculture fermentations

IV

Production fermenter

a. Batch fermentation b. Continuous fermentation

DEVELOPMENT OF INOCULUM FOR INDUSTRIAL FERMENTATION

Culture used to inoculate a fermentation satisfies the following criteria:

1. must be in a healthy, active state thus minimizing the length of the lag phase in the subsequent fermentation. 2. must be available in sufficiently large volumes to provide an inoculum of optimum size. 3. must be in a suitable morphological form. 4. must be free of contamination. 5. must retain its product-forming capabilities.

The process adopted to produce an inoculum meeting these criteria is called inoculum development. Design of a production medium is determined by: nutritional requirements of the organism and requirements for maximum product formation. Formation of product in the seed culture is not an objective during inoculum development - seed medium may be different composition from the production medium.

However, the lag time in fermentation is minimized by growing the culture in the 'final-type' medium. Inoculum development medium should be sufficiently similar to the production medium to minimize period of adaptation of the culture to the production medium reducing the lag phase and the fermentation time. Quantity of inoculum normally used is between 3 to 10% of the medium volume.

Inoculum built up in a number of stages (two or three stages in shake flasks and one to three stages in fermenters, depending on the size of the vessel) to produce sufficient biomass to inoculate to the production stage fermenter.

Throughout this procedure there is a risk of contamination and strain degeneration and necessitating stringent quality-control procedures. Compromise may be reached regarding the size of the inoculum to be used and the risk of contamination and strain degeneration.

Culture purity checks are carried out at each stage to detect contamination as early as possible. The procedure for the development of inoculum for bacterial fermentations, which with minor modifications, is applicable to any type of culture. The procedure involved the use of one sub-master culture to develop a bulk inoculum, which was subdivided, stored in a frozen state and used as inocula for several months.

A single colony, derived from a sub-master culture, was inoculated into liquid medium and grown to maximum log phase. This culture was then transferred into nineteen time (19) its volume of medium and incubated again to the maximum log phase, at which point it was dispensed in 20-cm3 volumes, plug frozen and stored at below -20°. At least 3% of the samples were tested for purity and productivity in subsequent fermentation and, provided these were suitable, the remaining samples could be used as initial inocula for sub- sequent fermentations.

One of the thawed samples was used as a 5% inoculum for a seed culture, which in turn, was used as a 5% inoculum for the next stage in the programme.

This procedure ensured that a proven inoculum was used for the penultimate stage in inoculum development. Yeasts, bacteria, fungi and Streptomycetes have different requirements for inoculum development. Table 1– inoculum development. (MSword)

Development of inocula for yeast FERMENTATION

brewing of beer and the production of biomass (bakers’ yeast - longest established)

Brewing It is common practice in British brewing industry to use the yeast from the previous fermentation to inoculate pitch, (in brewing terms) a fresh batch of wort (liquid extract of barley malt). High risk of contaminants and the degeneration of the strain, the most common degenerations being a change in the degree of flocculence and attenuating abilities of the yeast.

In breweries employing top fermentations these dangers are minimized by collecting yeast to be used for future pitching from ‘middle skimming’.

During fermentation, yeast cells flocculate and float to the surface, the first cells to do this being the most flocculent and the last cells the least flocculent.

As head of yeast develops, surface layer (the most flocculent and highly contaminated yeasts) is removed and discarded and underlying cells (‘middle skimming) are harvested and used for subsequent pitching.

Therefore, ‘middle skimming will contain cells which have desired flocculence and which have been protected from contamination by surface layer of the yeast head.

The pitching yeast is treated to reduce the level of contaminating bacteria and remove protein and dead yeast cells by treatments such as reducing the pH of the slurry to 2.5 to 3, washing with water, washing with ammonium persulphate and treatment with antibiotics such as polymixing, penicillin and neomycin. Despite these precautions yeasts are rarely used for more than five to ten consecutive fermentations necessitating the periodical production of a pure inoculum.

This would involve developing sufficient biomass from a single colony to pitch a fermentation at a level of approximately 2 grams of pressed yeast per litre.

Pure inocula and devised a yeast propagation scheme utilizing a 10% inoculum volume at each stage in the programme and employing conditions similar to those used during brewing has been used.

However, modern propagation schemes use inoculum volumes of l% or even lower and may use conditions different from those used during brewing. Therefore, continuous aeration may be used during the propagation stage, which seems to have little effect on the beer produced in the subsequent fermentation.

Bakers yeast

The commercial production of bakers’ yeast involves the development of an inoculum through a large number of stages.

Although production stages of the process may not be operated under strictly aseptic conditions, a pure culture is used for the initial inoculum thereby keeping contamination to a minimum in the early stages of growth.

Development of inoculum for production of bakers’ yeast and quoted a process involving five stages, the first two being aseptic while the remaining stages were carried out in open vessels. The first two stages were carried out in closed vessels without aeration or nutrient feeds.

Development of inocula for bacterial Fermentation

Main objective: produce an active inoculum with short lag phase in subsequent culture.

Long lag phase: time wasted and medium is consumed in maintaining a viable culture prior to growth.

Length of lag phase is affected by size of the inoculum and its physiological condition. Bacterial inocula should be transferred in logarithmic phase of growth, when cells are still metabolically active.

Age of inoculum is important in growth of sporulating bacteria, for sporulation is induced at the end of the logarithmic phase and use of an inoculum containing a high percentage of spores would result in a long lag phase in a subsequent fermentation.

5% inoculum of a logarithmically growing culture of a thermophilic Bacillus has been used for production of proteases. Two-stage inoculum-development programme has also been used in production of proteases by Bacillus subtilis:

Inoculum for a seed fermenter was grown for 1 to 2 days on a solid or liquid medium and then transferred to a seed vessel where the organism was allowed to grow for a further ten generations before transfer to production stage.

The inoculum development programme for the production of bacitracin by Bacillus subtilis Stage 1.

2. 3.

4.

Cultural conditions

Incubation time

4-dm3shake flask inoculated with a stock culture Stage 1 culture inoculated into 750-dm3 fermenter 750-dm3 culture inoculated into point of 6000-dm3 fermenter

18 to 24 hours

6000-dm3 culture inoculated into 120.000-dm3 production fermenter

6 hours Grown to the greatest production of cells

Table.The inoculum development programme for the clostridial acetone-butanol fermentations Stage Cultural conditions Medium 1. Reconstitution of the spore stock Potato glucose broth culture-24 hour incubation 2. Stage 1 culture inoculated into 600 4% sugar (as invert molasses) cm3 of medium. Incubated for 20-24 hours 5% (NH4)2SO4 6% calcium carbonate 0.2% phosphorus pentoxide (as superphosphate) 3. 90cm3 of stage 2 culture inoculated As for stage 2 into 3000cm3 medium in a 4000-cm3 Erlenmeyer flask

Stage

Cultural conditions

4

Culture inoculated into 25,000-dm3 fermenter

5

culture inoculated into 300,000to 2,500,000-dm3 fermenters at a 0.5 to 3% inoculum

Medium As for stage 2 but with 6% sugar As for stage 4 but with ammonia feed

Development of inoculum for fungal Fermentation

Majority of industrially important fungi are capable for asexual sporulation, so it is common practice to use a spore suspension as seed during an inoculum development programme. Three basic techniques to produce a high concentration or spores:

1. Sporulation on solidified media Most fungi will sporulate on suitable agar media but a large surface area must be employed to produce sufficient spores.

A 'roll-bottle' technique for production of spores of Penicillium chrysogmum: 300 cm3 quantities or medium containing 3% agar were sterilized in 1-dm3 cylindrical bottles, which were then cooled to 45°and rotated on a roller mill so that agar set as a cylindrical shell inside the bottle.

The bottles were inoculated with a spore suspension from a sub-master slope and incubated at 24°for 6 to 7 days.

Involved some sacrifice in ease of visual examination but it provided a large surface area for cultivation of spores in a vessel of a convenient size for handling in laboratory.

In some fermentations, large-scale inoculum must consist of spores. To obtain a spore crop, the preserved culture is cultivated on a solid substrate in 2-10 liter glass vessels under conditions of constant temperature and sterile aeration for 8-24 days.

Substrate for production of large amounts of spores is a granular material such as bran, peat, rice, or barley. In order to ensure continued aeration, the substrate must be shaken daily, which makes maintenance of aseptic conditions difficult.

Roller bottle for large-scale spore collection

2. Sporulation on solid media

Many fungi will sporulate profusely on the surface of cereal grains from which the spores may be harvested. Substrates such as barley, hard wheat bran and ground maize are suitable for sporulation of a wide range of fungi.

Sporulation of a given fungus is particularly affected by amount of water added to the cereal before sterilization and relative humidity of the atmosphere, which should be as high as possible, during sporulation.

System for sporulation of Aspergillus ochraceus in which a 2.8 dm3 Fernbach flask containing 200 grams of 'pot' barley or 100 grams of moistened wheat bran produced 5 X 1011 conidia after six days at 28° and 98% relative humidity has been described.

This was 5 times the number obtainable from a Roux bottle batched with Sabouraud agar and 50 times the number obtainable from such a vessel batched with Difco Nutrient Agar, incubated for same time period. Mass production of spores of several Aspergillus and Penicillium species could also obtained on whole loaves of white bread.

3. Sporulation in submerged culture

Many fungi will sporulate in submerged culture provided a suitable medium is employed. Example of use of this technique for production of inoculum for an industrial fermentation is by the griscofulvin process. The conditions for submerged sporulation of the griseofulvin-producing fungus, Penicillium patulum, and the medium utilized is given in Table.

These authors found that for prolific sporulation, nitrogen level had to be limited to between 0.05 and 0.1% w/v and that good aeration had to be maintained.

Also, an interaction was demonstrated between nitrogen level and aeration. Lower the degree of aeration, the lower the concentration of nitrogen needed to induce sporulation. Submerged sporulation was induced by inoculating 600 cm3 of the above medium, in a 2-dm3 shake flask, with spores from a well sporulated Czapek-Dox agar culture and incubating at 25°for 7 days.

The resulting suspension of spores was then used as a 10% inoculum for a vegetative seed stage in a stirred fermenter, the seed culture subsequently providing a 10% inoculum for the production fermentation.

More convenient technique compare to solid and solidified media; easier to operate aseptically and it may be applied on large scale.

Table. Medium for the submerged sporulation of P. patulum Whey powder, to give

Lactose Nitrogen

3.5% 0.05%

KH2PO4

0.4%

KCL

0.05

Corn-steep liquor solids to give approx. 0.04%N

0.38%

Table The development of inoculum of Penicillium chrysogenum for the production of penicillin. 1st stage

Master culture – mould spores in sterile sand

2nd stage

Working slope culture – spore culture on agar medium in test tube

3rd stage

“Roll bottle” culture – spore culture on agar medium in 1 L bottles.

4th stage

Plant inoculum culture – spores or mycelium grown in first stage of plant fermentation units

5th stage

Penicillin production culture – mycelium grown in the 2nd stage of plant fermentation units.

Use of Spore as An Inoculum

When considering production of gluconic acid by Aspergillus niger, the merits of inoculating the final fermentation directly with a spore suspension as compared with germinating the spores in a seed tank to give a vegetative inoculum. Direct spore inoculation would avoid cost of installation and operation of the seed tanks.

However, use of germinated spores reduced fermentation time of final stage, thus allowing a greater number of fermentations to be carried out per year.

Labour costs for production of vegetative inoculum could be almost as high as for final fermentation although some of these costs may be recovered. In that gluconic acid produced in the penultimate stage would be recoverable from the final fermentation broth and would contribute to the buffering capacity throughout the fermentation.

Choice of inoculum for production stage depends on length of the cycle of the fermentation process, plant size and capacity and availability and cost of labour.

Inoculum development for vegetative fungi Some fungi will not produce asexual spores and therefore an inoculum of vegetative mycelium must be used.

Gibberella fujikuroi is a fungus used for commercial production of gibberellin. An inoculum development programme for gibberellin fermentation is as follows:

Cultures were grown on long slants (25 X 150 mm test tubes) of potato dextrose agar for I week at 24°. Growth from three slants was scraped off and transferred to a 9dm3 carboy containing 4 dm3 of a liquid medium composed of 2% glucose, 0.3% MgSO4.7H20, 0.3% NH4CI and 0.3% KH2PO4. The medium was aerated for 75 hours at 28°before transfer to a 100-dm3 seed fermenter containing the same medium.

Major problem in using vegetative mycelium as initial seed is difficulty obtaining a uniform, standard inoculum. The procedure may be improved by fragmenting the mycelium in an homogenizer, such as a Waring blender, prior to use as inoculum. This method provides a large number of mycelial particles and therefore a large number of growing points.

Effect of inoculum on morphology of fungi in submerged culture

When filamentous fungi are grown in submerged culture, the type of growth varies from 'pellet' form, (compact discrete masses of hyphae) to filamentous form (hyphae) in which form a homogeneous suspension dispersed through the medium. Morphology of fungus in submerged culture is critical in many industrial fermentations. Two factors determining fungal form: medium composition and concentration of spores in a spore inoculum.

High spore inoculum will tend to produce a disperse form of growth whilst a low one will favour pellet formation. Effect of concentration of a spore inoculum on the morphology of Penicillium chrysogenum is given in Table

Citric acid, penicillin, submerged mushroom culture and fungal protein processes are affected by morphology of the producing fungus and this is summarized in Table Therefore, in commercial production of these products, it is critical to grow the fungus in the desired morphological form which necessitates the use of an inoculum which will achieve this end.

Table The effect of spore concentration and medium on the morphology of P. chrysogenum in liquid culture Medium

Spore concentration in the medium

Morphology

Corn-step dextrin

More than 10 x 10dm-3 Less than 10 x 10dm-3

Filamentous Pellets

Czapek-dox

More than 3.0 x 10dm-3 Less than 3.0 x 10dm-3

Filamentous Pellets

Glucose-lactoseammonium lactate

More than 2.0 x 105dm-3 Less than 2.0 x 105dm-3

Filamentous Pellets

Table. The effect of fungal morphology on the performance of some industrial fermentations Fermentation

Organism

Optimum morphological form

Penicillin

P.chrysogenum

Filamentous

Citric acid

A.niger

Pellets

Submerged mushroom culture

Agaricus campestris

Pellets

Fungal protein

No species quoted

Filamentous

ASEPTIC INOCULATION OF FERMENTERS

Inoculation of plant scale fermenters may involve transfer of culture from a laboratory fermenter, or spore suspension vessel, to a plant fermenter, or the transfer from one plant fermenter to another. To prevent contamination during the transfer process, it is essential that both vessels be maintained under a positive pressure and the inoculation port be equipped with a steam supply.

Example of the apparatus is shown in Fig. For inoculation, the total culture (spores plus culture medium) is suspended with the aid of a surface-active agent (e.g., Tween 80) and transferred into the fermenter.

Fig. Inoculation of inoculum into the 2 L fermenter using inoculum flask and Herbert connector.

INOCULUM CELL COUNTS

In order to obtain an accurate data collection, initial number of cells or spores should be obtained. This can be done using total cell count using a device called a haemocytometer. A haemocytometer provide estimates of number of cells or spores in a suspension.

•For optimal yields, not only number of cells and spores have an influence but also nutrient medium used for the inoculum, temperature of growth, and inoculum age. •Induction or repression phenomena in the culture used for inoculum may also affect the rate of production.

Fig. Grid of a Haemocytometer

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