Inoculum development
The culture used for seeding the fresh medium in the culture vessel is
called inoculum.
The inoculum should satisfy following criteria,
Must be healthy and active, so as to minimize the lag phase in
subsequent fermentation.
Must be available in sufficient quantity to meet the required size
Must be of suitable morphological form.
Must be free of contamination
Must retain its product forming abilities.
The process adapted to produce inoculum which meets above said
criteria is called “inoculum development”.
Medium used for inoculum development: As product formation is not
intended, seed medium could be of different composition from
production medium.
However it should be “sufficiently similar” to final production
medium.
Major differences in pH, osmotic pressure and anion composition
affects the viability.
In general, inoculum development medium is less nutritious compared
to production medium.
The quantity of inoculum: 3-10% v/v
Inoculum development: 2-3 stages in shake flasks and 1-3 stages in fermenter.
Greater the number of stages, greater is the risk of contamination and strain
degeneration.
10 % inoculum must justify in terms of productivity as it requires considerable
financial resources.
Hence, in a continuous fermentation process inoculum size of 10 % is justified
as it utilizes inoculum once in 100-120 days.
Culture for inoculum development:
Master culture is streaked on the solidified medium and colonies of typical
high productive morphology are selected and inoculated on the slopes
called as sub-master cultures.
From sub-master culture, a liquid medium is inoculated and grown to a
late log phase and then it is added to 19 times its volume and again grown
to late log phase. Then it is dispensed to 20 ml volumes and kept frozen
at -20*C.
At least 3% of the samples are tested for purity and productivity and if
found satisfactory, they shall be used as “working cultures”.
Stored cultures are used as 5 % inoculum.
Physiological conditions of the inoculum: Major effect on the performance of
the fermentation. Correct physiological conditions are evaluated by
o Packed cell volume
o Dry weight
o Wet weight
o Turbidity
o Respiration
o Residual nutrient concentration etc.
Bacterial and yeast process: Inoculum development.
o Eg. 1,00,000 L production fermenter. 1] 30 ml in 250 ml flask. 2]
Transferred to a shake flask of 2 L holding 600 ml. 3] 625ml is used
as inoculum for 11.875 L medium in 20 L fermenter. 4] 12.5 L is
used as inoculum for 237.5 L medium in 400 L fermenter. 5] 250 L
is used to inoculate 4,750 L in a 10,000 L fermenter. 6] 5000 L is
the inoculum for the 1,00,000 L medium.
Fungal culture: Spore inoculum.
Fungal inoculum
Some molds: Don’t produce asexual spores – homogenized filaments
Gibberella fujikuroi for Gibberelin production.
Some molds: Asexual spores – spore inoculum is used.
Morphological form – Filamentous & pelleted form
Filamentous : Highly viscous
Pelleted: Less viscous
P. chrysogenum : Filamentous is productive
A. niger (citric acid) and A. terreus (lovastatin) : Pelleted form is
productive
More spores (x. 105 spores per liter) gives filamentous growth and
lesser gives the pelted growth in a chemically defined medium.