FERMENTATION TECHNOLOGY

ASSIGNMENT

Name : L.B. Sabarish

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Criteria for Inocula

 It must be in a healthy, active state thus minimizing the length of the lag phase in the subsequent
fermentation
 It must be available in sufficiently large volumes to provide an inoculum of optimum size
 It must be free of contamination
 It must be in a suitable morphological form
 It must retain its productforming 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 inoculum is the choice of the culture medium 
inoculum development medium should be sufficiently similar to the production medium to minimize any
period of adaptation, thus reducing the lag phase and the fermentation time.

The quantity of Major differences in inoculum should be pH, osmotic pressure large enough ( normally
and anion composition 3 to 10%) to minimize may result in very the length of the lag sudden changes in
phase and to generate uptake rates which, in the maximum biomass in turn, may affect the production
viability fermenter

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 transfer of vegetative inocula is biomass and such parameters
as packed cell volume, dry weight, wet weight, turbidity, respiration, residual nutrient concentration and
morphological form

• Standardization of cultural condition and monitoring the state of an inoculum culture so that it is
transferred at the optimum time, i.e. in the correct physiological state • In recent years, probes for on-line
assessment of biomass and real-time expert computer system have been used to predict the time of
inoculum transfer for industrial scale fermentations.

 The carbon cioxide production rate (CPR) profile of the inoculum culture showing the points at
which inocula were removed The effect of inoculum age on the CPR of the production
fermentation The effect of inoculum age on productivity in the production fermentation

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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

Bakers’ yeast

• Common practice: back slopping

• The development of an inoculum through a large number of aerobic stages

• The brewing terms: “crop” , referring to the harvested yeast from the previous

• Although the production stages of the fermentation, and “pitch”, meaning to process may not be
operated under strictly inoculate aseptic condition, a pure culture is used for the initial inoculum.

Advantages of back slopping:

• Reduced cost • The process involved batch and fed-batch

Disadvantages:

• Introduction of fermentation 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

• 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 

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 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.

 The length of the lag phase is affected by the size of the inoculum and its physiological condition.

• 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.

• 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 forming fungi

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Inoculum development for vegetative 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 media

 Sporulation on solid media

 Sporulation in submerged culture

• Most fungi will sporulate on suitable agar media but a large surface area must be employed to produce
sufficient spores. • Methods for improving an agar surface area: • “roll-bottle” techniques in cylindrical
bottles • Using “roux bottle”

• Many filamentous organisms will sporulate profusely on the surface of cereal grains (barley, ground
maize, rice, etc.) from which the spores may be harvested • The sporulation is affected by the amount of
water added to cereal before sterilization and the relative humidity of the atmosphere

• Many fungi will sporulate in submeged culture provided a suitable medium is employed • This
techniques is more convenient because it is easier to operate aseptically and it may be applied on a large
scale • Most actinomycetes do not sporulate in submerged culture

The use of the spore inoculum

The inoculum development program for the production of cxlavulanic acid from Streptomyces
clavuligerus.

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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 medium • Far less viscous, but also less homogenous broth • Extremely viscous broth which
may be very difficult to aerate • Mycelium at the centre of the pellet adequately may be starved of
nutrients and oxygen due to diffusion limitations

The effect of the inoculum on the morphology of filamenous organisms in submerged culture
Morphological form of the organism influences the productivitiy of the culture:

o Morphology may be influenced by both the concentration of spores in a spore inoculum and
the inoculum development medium

• Penicillin production by P.chrysogenum → filamentous growth • Citric acid production by A.niger →

••••

High spore inoculum → dispersed form of growth Low spore inoculum → pelleted formation Rich,
complex media → dispersed growth Chemically defined media → pelleted growth.