THE DEVELOPMENT OF

INOCULA FOR INDUSTRIAL

FERMENTATIONS
Principles of Fermentation Technology
Microbiology Study Program
School of Life Sciences and Technology - ITB

Dr. Dea Indriani Astuti

Criteria for Inocula

It must be in a healthy, It must be available in

active state thus sufficiently large volumes It must be in a suitable
minimizing the length of to provide an inoculum morphological form
the lag phase in the of optimum size
subsequent fermentation

It must be free of It must retain its product-

contamination forming capabilities
Inoculum Development

The process adopted to produce an inoculum

meeting all inocula criteria is called inoculum
development
A critical factor in
obtaining a suitable The quantity of
inoculum is the choice of Major differences in inoculum should be
the culture medium  pH, osmotic pressure large enough ( normally
inoculum development and anion composition 3 to 10%) to minimize
medium should be may result in very the length of the lag
sufficiently similar to the sudden changes in phase and to generate
production medium to uptake rates which, in the
minimize any period of turn, may affect maximum biomass in
adaptation, thus reducing viability the production
the lag phase and the fermenter
fermentation time
Criteria for the transfer of inoculum

The physiological condition of the inoculum when it is

transferred to the next culture stage can have a major
effect on the performance of the fermentation.
The size or the amount of inoculum The optimum time of transfer
• The most widely used criterion for the • Standardization of cultural condition and
transfer of vegetative inocula is biomass monitoring the state of an inoculum culture
and such parameters as packed cell so that it is transferred at the optimum time,
volume, dry weight, wet weight, turbidity, i.e. in the correct physiological state
respiration, residual nutrient concentration • In recent years, probes for on-line
and morphological form assessment of biomass and real-time
expert computer system have been used
to predict the time of inoculum transfer for
industrial scale fermentations
The effect of inoculum age on growth and productivity
(a) The carbon cioxide production rate (CPR) profile of the inoculum culture showing the
points at which inocula were removed
(b) The effect of inoculum age on the CPR of the production fermentation
(c) The effect of inoculum age on productivity in the production fermentation
The development of inocula for yeast processes

The larger industsrial fementation utilizing yeast are the brewing

of beer and the production of biomass
Brewing Industry
• Common practice: back slopping
• The brewing terms: “crop” , referring to the
harvested yeast from the previous
fermentation, and “pitch”, meaning to
inoculate
• Advantages of back slopping: reduced cost
• Disadvantages: introduction of
contaminants and the degeneration of the
strain
• Rarely used for more than five to ten
consecutive fermentation because of strain
degeneration and contamination 
periodical production of a pure inoculum
Bakers’ yeast
• The development of an inoculum through a
large number of aerobic stages
• Although the production stages of the
process may not be operated under strictly
aseptic condition, a pure culture is used for
the initial inoculum
• The process involved batch and fed-batch
fermentation
Brewing yeast
Traditional “open vessels “
fermenters
 During the fermentation the yeast cells
flocculate and float to the surface  the
surface layer (the most flocculent and
highly contaminated yeast) is removed
and the underlying cell (‘middle
skimming’) are used for subsequent
pitching
 The pitching yeast is treated to reduce
the level of contaminating bacteria and
remove protein and dead yeast cell by
reducing the pH of the slurry to 2.5-3,
washing with water, washing with
ammonium persulphate and treatment
with antibiotics such as polymyxin,
penicillin and neomycin
Cylindro-conical fermenters
 Yeast flocculates and collects in the cone
at the bottom of the fermenter (subject to
the stresses of nutrient starvation, high
ethanol conc., low water activity, high
CO2 conc., and high pressure)  viability
and physiological state of the yeast not
be ideal for an inoculum
 Key physiological features of yeast
inoculum is the level of sterol which is
required for membrane synthesis. They
are only produced in the presence of O2
 aerating the wort before inoculation
 Pitching yeast are vigorously aerated
prior to inoculation
The development of inocula for bacterial processes
The main objective of inoculum
development is to produce an active
inoculum which will give as short a lag
phase as possible in subsequent culture
• A long lag phase is disadvantageous
in that not only time wasted but also
medium is consumed in maintaining a
viable culture prior to growth
• Lag phase could be almost
completely eliminated by using
inoculum medium of the sama
composition as used in the production
fermenter
The length of the lag phase is affected
by the size of the inoculum and its
physiological condition
• Inoculum size normally 3-10% of the
culture volume
• Bacterial inocula should be
transferred in the log phase of
growth when the cells are still
metabolically active
• The age of inoculum particularly
important in the growth of
sporulating bacteria  inoculum
containing a high percentage of
spores will result in a long lag phase
The development of inocula for bacterial processes

The inoculum development program for a vitamin B12 pilot scale

fermentation using Pseudomonas denitrificans
The development of inocula for fungal processes

Types of fungal inoculum:

Inoculum development for spore Inoculum development for vegetative
forming fungi fungi
• Majority of industrial important fungi • Some fungi will not produce asexual
and streptomycetes are capable of spores  an inoculum of vegetative
asexual reproduction  It is common to mycelium musst be used, eg. Gibberella
use a spore suspension as seed during an fujikuroi
inoculum development program • Problem: difficulty of obtaining a
• Advantage: a spore inoculum contains uniform, standard inoculum  the
far more propagules than a vegetative procedure may be improved by
culture fragmenting the mycelium in an
homogenizer, prior to use as inoculum
Basic techniques to produce spores

Sporulation on solidified Sporulation on solid Sporulation in

media media submerged culture
• Most fungi will • Many filamentous • Many fungi will
sporulate on suitable organisms will sporulate in submeged
agar media but a sporulate profusely on culture provided a
large surface area the surface of cereal suitable medium is
must be employed to grains (barley, ground employed
produce sufficient maize, rice, etc.) from • This techniques is more
spores. which the spores may convenient because it
• Methods for improving be harvested is easier to operate
an agar surface area: • The sporulation is aseptically and it may
• “roll-bottle” affected by the be applied on a large
techniques in amount of water scale
cylindrical bottles added to cereal • Most actinomycetes do
• Using “roux bottle” before sterilization not sporulate in
and the relative submerged culture
humidity of the
atmosphere
The use of the spore inoculum

The inoculum development program for the production of cxlavulanic acid

from Streptomyces clavuligerus
The use of the spore inoculum

The inoculum development programe for the production of

sagamicin by Micromonospora sagamiensis
The effect of the inoculum on the morphology of
filamentous organisms in submerged culture

The filamentous fungi type of growth when

grown in submerged culture:
Pellet form type Filamentous form
• Consisting of compact discrete • Hyphae form a homogenous
masses of hyphae suspension dispersed through the
• Far less viscous, but also less medium
homogenous broth • Extremely viscous broth which may
• Mycelium at the centre of the pellet be very difficult to aerate
may be starved of nutrients and adequately
oxygen due to diffusion limitations
The effect of the inoculum on the morphology of
filamenous organisms in submerged culture

Morphological form of • Penicillin production by P.chrysogenum → filamentous

growth
the organism • Citric acid production by A.niger → pelleted growth
influences the
• Lovastation production by A. terreus → pelleted
productivitiy of the growth
culture:

Morphology may be
influenced by both the • High spore inoculum → dispersed form of growth
concentration of • Low spore inoculum → pelleted formation
spores in a spore • Rich, complex media → dispersed growth
inoculum and the
• Chemically defined media → pelleted growth
inoculum development
medium