Lect6(Inoculum Preparation and Development).pdf

8/14/2019 Lect6(Inoculum Preparation and Development).pdf

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InoculumPreparation and

Development


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Strain of microorganism - most important part of a

fermentation process. (pure and produce thedesired product at optimal level).

Two most important types of microorganism -bacteria and fungi. More plant and animal cells arealso being grown in bioreactors for production of

highly specialized product.


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General course of fermentation in

the production of primary andsecondary metabolites


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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


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Culture used to inoculate a fermentationsatisfies the following criteria:


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1. must be in a healthy, active state thus minimizing thelength of the lag phase in the subsequent fermentation.

2. must be available in sufficiently large volumes toprovide 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.


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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 - seedmedium may be different composition from theproduction medium.


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However, the lag time in fermentation is minimized bygrowing the culture in the 'final-type' medium.

Inoculum development medium should be sufficientlysimilar 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.


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Inoculum built up in a number of stages (two or three

stages in shake flasks and one to three stages infermenters, depending on the size of the vessel) toproduce sufficient biomass to inoculate to theproduction stage fermenter.


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Throughout this procedure there is a risk ofcontamination and strain degeneration and

necessitating stringent quality-control procedures.

Compromise may be reached regarding the size of theinoculum to be used and the risk of contamination

and strain degeneration.


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Culture purity checks are carried out at each stage todetect contamination as early as possible.

The procedure for the development of inoculum forbacterial 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 wassubdivided, stored in a frozen state and used as inoculafor several months.


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A single colony, derived from a sub-master culture, wasinoculated into liquid medium and grown to maximum logphase.

This culture was then transferred into nineteen time (19) itsvolume of medium and incubated again to the maximum log

phase, at which point it was dispensed in 20-cm3 volumes, plugfrozen and stored at below -20.

At least 3% of the samples were tested for purity andproductivity in subsequent fermentation and, provided these weresuitable, the remaining samples could be used as initial inocula forsub- sequent fermentations.


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One of the thawed samples was used as a 5%

inoculum for a seed culture, which in turn, wasused as a 5% inoculum for the next stage in the

programme.


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This procedure ensured that a proven inoculum wasused for the penultimate stage in inoculum development.

Yeasts, bacteria, fungi and Streptomyceteshavedifferent requirements for inoculum development.

Table 1 inoculum development. (MSword)


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brewing of beer and the production of biomass

(bakers yeast - longest established)


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Brewing

It is common practice in British brewing industry to usethe 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 ofthe strain, the most common degenerations being a

change in the degree of flocculence and attenuatingabilities of the yeast.


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In breweries employing top fermentations these

dangers are minimized by collecting yeast to be usedfor future pitching from middle skimming.


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As head of yeast develops, surface layer (the mostflocculent and highly contaminated yeasts) isremoved and discarded and underlying cells (middle

skimming) are harvested and used for subsequent

pitching.


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Therefore, middle skimming will contain cells which havedesired flocculence and which have been protected fromcontamination by surface layer of the yeast head.


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Pure inocula and devised a yeastpropagation scheme utilizing a 10% inoculum

volume at each stage in the programme andemploying conditions similar to those usedduring brewing has been used.


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However, modern propagation schemes useinoculum volumes of l% or even lower andmay use conditions different from those usedduring brewing.

Therefore, continuous aeration may be usedduring the propagation stage, which seems to

have little effect on the beer produced in thesubsequent fermentation.


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Bakers yeast


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Development of inoculum for productionof bakers yeast and quoted a process

involving five stages, the first two beingaseptic while the remaining stages werecarried out in open vessels.

The first two stages were carried out inclosed vessels without aeration or nutrientfeeds.


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Development of inocula for bacterialFermentation


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

Long lag phase: time wasted and medium isconsumed in maintaining a viable culture prior togrowth.


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Length of lag phase is affected by size of theinoculum and its physiological condition.

Bacterial inocula should be transferred in logarithmic

phase of growth, when cells are still metabolicallyactive.


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5% inoculum of a logarithmically growing culture ofa thermophilic Bacillus has been used for productionof proteases.

Two-stage inoculum-development programme hasalso been used in production of proteases byBacillus subtilis:


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Inoculum for a seed fermenter was grown for 1 to 2days on a solid or liquid medium and thentransferred to a seed vessel where the organism wasallowed to grow for a further ten generations before

transfer to production stage.

The inoculum development programme for the production of


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The inoculum development programme for the production ofbacitracin by Bacillus subtilis

Stage Cultural conditions Incubation time

1. 4-dm3shake flask inoculated 18 to 24 hourswith a stock culture

2. Stage 1 culture inoculated into 6 hours

750-dm3 fermenter3. 750-dm3 culture inoculated into Grown to the

point of 6000-dm3 fermenter greatest productionof cells

4. 6000-dm3 culture inoculated into

120.000-dm3 production fermenter


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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

cm3 of medium. Incubated for molasses)

20-24 hours 5% (NH4)2SO46% 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


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Stage Cultural conditions Medium

4 Culture inoculated into 25,000-dm3 As for stage 2 but

fermenter with 6% sugar

5 culture inoculated into 300,000- As for stage 4 but

to 2,500,000-dm3 fermenters at a 0.5 with ammonia feed

to 3% inoculum


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Development of inoculum forfungal Fermentation


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Majority of industrially important fungi are capable forasexual sporulation, so it is common practice to use aspore suspension as seed during an inoculumdevelopment programme.

Three basic techniques to produce a high concentrationor spores:


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1. Sporulation on solidified media

Most fungi will sporulate on suitable agar media but

a large surface area must be employed to producesufficient spores.


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A 'roll-bottle' technique for production ofspores of Penicillium chrysogmum: 300 cm3

quantities or medium containing 3% agar weresterilized in 1-dm3 cylindrical bottles, which

were then cooled to 45and rotated on a rollermill so that agar set as a cylindrical shellinside the bottle.


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The bottles were inoculated with a spore

suspension from a sub-master slope andincubated at 24for 6 to 7 days.


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Involved some sacrifice in ease of visualexamination but it provided a large surface area

for cultivation of spores in a vessel of a convenientsize for handling in laboratory.


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In some fermentations, large-scale inoculum mustconsist of spores.

To obtain a spore crop, the preserved culture is

cultivated on a solid substrate in 2-10 liter glass vesselsunder conditions of constant temperature and sterileaeration for 8-24 days.


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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, thesubstrate must be shaken daily, which makesmaintenance of aseptic conditions difficult.


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Roller bottlefor large-scalespore collection


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2. Sporulation on solid media


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Sporulation of a given fungus is particularly affectedby amount of water added to the cereal before

sterilization and relative humidity of theatmosphere, which should be as high as possible,during sporulation.


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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 28and 98%relative humidity has been described.


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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 NutrientAgar, incubated for same time period.

Mass production of spores of severalAspergillus and

Penicillium species could also obtained on whole loaves of

white bread.


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3. Sporulation in submerged culture


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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.


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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.


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Also, an interaction was demonstrated between nitrogen

level and aeration. Lower the degree of aeration, the lowerthe 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 25for 7 days.


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More convenient technique compare to solid

and solidified media; easier to operateaseptically and it may be applied on largescale.


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Table. Medium for the submerged sporulation ofP.

patulum

Whey powder, to give LactoseNitrogen

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 ofPenicillium chrysogenum for

the prod ction of penicillin


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1st stage Master culture mould spores in sterile sand

2nd stage Working slope culture spore culture on agar medium intest tube

3rd stage Roll bottle culture spore culture on agar medium in 1L bottles.

4th stage Plant inoculum culture spores or mycelium grown infirst stage of plant fermentation units

5th stage Penicillin production culture mycelium grown in the 2nd

stage of plant fermentation units.

the production of penicillin.


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When considering production of gluconic acid by

Aspergillus niger, the merits of inoculating the finalfermentation directly with a spore suspension ascompared with germinating the spores in a seed tankto give a vegetative inoculum.

Direct spore inoculation would avoid cost of

installation and operation of the seed tanks.


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However, use of germinated spores reducedfermentation time of final stage, thus allowing a

greater number of fermentations to be carried outper year.


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Labour costs for production of vegetative inoculumcould be almost as high as for final fermentation

although some of these costs may be recovered.

In that gluconic acid produced in the penultimate stagewould be recoverable from the final fermentation brothand would contribute to the buffering capacitythroughout the fermentation.


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Choice of inoculum for production stage

depends on length of the cycle of thefermentation process, plant size andcapacity and availability and cost of labour.


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Inoculum development for vegetativefungi

Some fungi will not produce asexual sporesand therefore an inoculum of vegetativemycelium must be used.


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Gibberella fujikuroi is a fungus used for commercial

production of gibberellin. An inoculum development

programme for gibberellin fermentation is as follows:


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Cultures were grown on long slants (25 X 150 mm test

tubes) of potato dextrose agar for I week at 24. Growthfrom three slants was scraped off and transferred to a 9-

dm3 carboy containing 4 dm3 of a liquid medium composedof 2% glucose, 0.3% MgSO4.7H20, 0.3% NH4CI and 0.3%

KH2PO4.

The medium was aerated for 75 hours at 28before

transfer to a 100-dm3

seed fermenter containing the samemedium.


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Major problem in using vegetative myceliumas initial seed is difficulty obtaining auniform, standard inoculum.

The procedure may be improved by

fragmenting the mycelium in an homogenizer,such as a Waring blender, prior to use asinoculum. This method provides a large

number of mycelial particles and therefore alarge number of growing points.


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Effect of inoculum on morphology of fungi

in submerged culture


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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 manyindustrial fermentations.

Two factors determining fungal form: medium composition

and concentration of spores in a spore inoculum.


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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


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Citric acid, penicillin, submerged mushroom culture and

fungal protein processes are affected by morphology of theproducing 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


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morphology ofP. chrysogenum in liquid culture

Medium Spore concentration in themedium

Morphology

Corn-step dextrin More than 10 x 10dm-3

Less than 10 x 10dm-3FilamentousPellets

Czapek-dox More than 3.0 x 10dm-3

Less than 3.0 x 10dm-3FilamentousPellets

Glucose-lactose-

ammonium lactate

More than 2.0 x 105dm-3

Less than 2.0 x 10

5

dm

-3

Filamentous

Pellets

Table. The effect of fungal morphology on the


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performance of some industrial fermentationsFermentation Organism Optimum

morphological form

Penicillin P.chrysogenum Filamentous

Citric acid A.niger Pellets

Submerged mushroomculture

Agaricus campestris Pellets

Fungal protein No species quoted Filamentous


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ASEPTIC INOCULATION OF FERMENTERS


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Inoculation of plant scale fermenters may involvetransfer of culture from a laboratory fermenter, orspore 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 apositive pressure and the inoculation port beequipped with a steam supply.


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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.


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


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INOCULUM CELL COUNTS


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In order to obtain an accurate data collection,initial number of cells or spores should be

obtained.

This can be done using total cell countusing a device called a haemocytometer. Ahaemocytometer provide estimates of number

of cells or spores in a suspension.


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For optimal yields, not only number of cells andspores have an influence but also nutrient medium

used for the inoculum, temperature of growth, andinoculum age.

Induction or repression phenomena in the cultureused for inoculum may also affect the rate ofproduction.


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Fig. Grid of aHaemocytometer


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Thank You for Your Attention

Lect6(Inoculum Preparation and Development).pdf

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