Batch,
Fed Batch and Continuous Fermentation
Fermentation may be carried out as batch, continuous or fed-batch processes. Batch growth involves a closed system where all nutrients are present at the start of the fermentation in a fixed volume. There may be further additions that limit to acids or bases for pH control, or gases for aeration, etc.
In fed-batch systems fresh medium or
medium components are fed continuously, intermittently or are added as a single
supplement. Here the volume of the
system increases with time.
Continuous fermentations are open
systems where fresh medium is continuously fed into the fermentation vessel and
spent medium and cells are removed at the same rate and thus the volume remains
constant.
Batch fermentation
This is a closed culture system which contains an
initial, limited amount of nutrients. The microorganisms from the inoculum will
pass through a number of phases, lag phase, log or exponential phase,
stationary phase and death or decline phase.
Lag phase is immediately after inoculation when no
apparent growth takes place and this is considered as a time of adaptation. The
length of the lag phase should be reduced as much possible in a commercial
process to be economical. Following lag
phase, the logarithmic or log phase ensues, this is the period during which the
growth rate of cells gradually increases, the cells grow at a Constant,
maximum, rate possible. The exponential
phase may be described as
dx/dt = µx
Where x is the concentration of microbial biomass, t
is time in hours and µ is the specific growth rate.
The production of primary metabolites occurs during
the log phase and this phase is known as the trophophase. Examples of primary metabolites are amino
acids, nucleotides, vitamins, citric acid, acetic acid, ethanol, etc.
The growth of the organism results in the consumption
of nutrients and the excretion of microbial products. Thus, after a certain
time the growth rate of the culture decreases until growth ceases. This may be
due to the depletion of essential nutrients in the medium (substrate
limitation) or accumulation of toxic products by organism (toxin limitation) or
both these factors.
The nature of the limitation of growth can be studied by growing the organism in the presence of different concentrations of substrates and plotting the biomass concentration at stationary phase against the initial substrate concentration.
It
can be seen that with initial increase in substrate concentrations a
proportional increase in the biomass produced occurs. The situation may be explained as
x = Y(S-SR)
Where x is the concentration of biomass produced,
Y is the yield factor (g biomass produced/ substrate
consumed),
S is the initial substrate concentration, and
SR is the residual substrate concentration.
The yield factor is a measure of the efficiency of
substrate conversion into biomass.
After stationary phase, death phase occurs. The
decrease in growth rate and the cessation of growth due to the depletion of
substrate, may be explained using Monod equation,
µ = µmax s/(Ks+s)
Where s is the residual substrate concentration, µ is
the specific growth rate, µmax is the maximum specific growth rate
and Ks is substrate utilization constant, which is equal to substrate
concentration when µ is half µmax and this is a measure of the
affinity of the organism for the substrate.
Secondary metabolites are produced
during the stationary phase and this phase is also known as
idiophase. Secondary metabolites are
organic compounds that are not directly involved in the normal growth, development
or reproduction of an organism. Microbial secondary metabolites include
antibiotics, pigments, toxins, etc.
A primary metabolite is considered as growth-linked
and secondary metabolites are non-growth-linked products.
Application of batch fermentation
Batch fermentation may be used to produce biomass,
primary metabolites and secondary metabolites. For biomass production, cultural
conditions supporting the fastest growth rate and maximum population would be
used. For primary metabolite production
conditions to extend the exponential phase and for secondary metabolite
production, conditions giving a short exponential phase and an extended
production phase will be used.
A batch fermentation possess disadvantages
since several distinct practical stages are associated with the operation of a
batch fermentation as follows,
- Charging
or filling the fermenter with fresh medium;
- Sterilization
of the fermenter and medium;
- Inoculation
of the fermenter;
- Production
of microbial products;
- Harvesting
of biomass and spent fermentation broth; and
- Cleaning
of the vessel.
This has major economic implications since
for a considerable period of time, the fermenter vessel is not producing
microbial products, but is being cleaned, filled, sterilized, etc. The non-productive
period is referred to as the down-time of the fermenter. The down time is very high for a batch fermentation.
Fed batch Fermentation
Fed-batch culture is a batch culture which is fed
continuously, or sequentially, with medium, without the removal of culture
fluid. It is established initially in batch mode and is then fed according to either
one of the following strategies:
i) The same medium used to establish the culture is
added - result in an increase in volume.
(ii) A solution of the limiting substrate at the
same concentration as that in the initial medium is added - result in an
increase in volume.
(iii) A concentrated solution of the limiting
substrate is added - result in an increase in volume.
iv) A very concentrated solution of the limiting
substrate is added – does not result in an increase in volume.
Fed-batch systems employing strategies i) and (ii) are
variable volume fed batch system. System employing strategy (iv) is fixed
volume fed batch system. The use of strategy (iii) gives a culture intermediate
between the variable and fixed volume systems.
Continuous
Fermentation
Exponential
growth in batch culture may be prolonged by the addition of fresh medium to the
vessel. Fresh medium is continuously added and an equal
volume of spent fermentation broth and cells are displaced at same rate. Exponential
growth will proceed and steady state will be achieved, that is, formation of
new biomass by the culture is balanced by the loss of cells from the vessel.
The flow of medium into the vessel is related to the
volume of the vessel by the dilution rate, D, defined as
D=F/V
where F is the flow rate and V is the volume of the
vessel.
The net change in cell concentration over a time would
be
dx/dt = growth - output
dx/dt = µx - Dx
Under steady state concentrations, dx/dt will be zero,
then
µx = Dx
µ = D
So under steady state conditions the specific growth rate
is controlled by the dilution rate, up to a maximum value equal to µ max. If the dilution rate is increased above µmax,
complete washout of the cells occurs, as the cells have insufficient time to
divide before being washed out of the reactor via the overflow. The dilution
rate at which this problem of washout is just avoided is termed as the critical
dilution rate (Dcrit).
The controlling effect of the dilution rate on microbial
growth can be explained by the Monod equation,
µ = µmax s/(Ks+s)
At steady state, µ = D, so
D = µmax ŝ / (Ks+ŝ),
where
ŝ is steady state concentration of substrate
ŝ = KsD / (µmax - D)
So
the substrate concentration is influenced by the dilution rate. Thus biomass growth depends upon substrate
concentration and thereby on dilution rate.
Growth of cells is controlled by the
availability of a rate-limiting nutrient. Th system, where the concentration of
the rate-limiting nutrient entering the system is fixed, is termed as a chemostat. In a chemostat the substrate concentration
is held constant. The other type is a turbidostat, where
nutrients in the medium are not limiting. In this case turbidity or absorbance
of the culture is monitored and maintained at a constant value, here the cell
concentration is held constant.
The concentration of cells in the chemostat at steady
state is described by the equation:
ẍ = Y (SR- Sr)
where ẍ is the steady-state cell concentration in
the chemostat, SR is the substrate concentration of inflowing medium, Sr is the steady-state residual
substrate concentration in the reactor and Y is the yield factor.
Therefore, the biomass concentration
under steady state conditions is controlled by the substrate feed concentration
and the operating dilution rate.
Advantages and disadvantages of continuous fermentation system
The down time of fermenter is much
less and is thus more economic. The fermenter can be more easily automated
thus requiring less labor. But the
chances of contamination and loss of productivity of the microorganism (strain
deterioration) is more. The control
operations are more complicated. There will
be problems in the licensing of a
continuous process product since it may not be always possible to trace a
consignment of product to a batch of raw materials. This is due to the fact that in a long
continuous process several different batches of media will be used and thus associating
product with a batch of raw material will be impossible.
These three modes of fermentations are used in various microbial fermentative productions. The choice of the mode of operation, that is batch, fed-batch or continuous fermentation, depends upon the product being produced.
References
Industrial Microbiology: An Introduction, M J. Waites, N L. Morgan, J S. Rockey, G Higton
Principles of fermentation technology, PF Stanbury, A Whittakker, SJ Hall, 1995, Butterworth-Heinemann publications
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