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 Cultural conditions Incubation time

1. 4-dm3shake flask inoculated 18 to 24 hours

with a stock culture
2. Stage 1 culture inoculated into 6 hours
750-dm3 fermenter
3. 750-dm3 culture inoculated into Grown to the
point of 6000-dm3 fermenter greatest production
of cells
4. 6000-dm3 culture inoculated into
120.000-dm3 production fermenter
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)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 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
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 3.5%
Nitrogen 0.05%

KH2PO4 0.4%

KCL 0.05

Corn-steep liquor solids to give approx. 0.38%

0.04%N
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 9-
dm3 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 Morphology
medium

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

Less than 10 x 10dm-3 Pellets

Czapek-dox More than 3.0 x 10dm-3 Filamentous

Less than 3.0 x 10dm-3 Pellets

Glucose-lactose- More than 2.0 x 105dm-3 Filamentous

ammonium lactate Less than 2.0 x 105dm-3 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 Agaricus campestris Pellets

culture

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
Thank You for Your Attention