Mode of Fermentation

1. Batch Fermentation

Batch fermentation refers to a partially closed system in which most of the materials required are loaded onto the
fermentor, decontaminated before the process starts and then, removed at the end. The microorganisms will grow in a
culture medium where the necessary nutrients are provided in an available form and all other environmental factors are
also suitable. The only material added and removed during the course of batch fermentation is the gas exchange and pH
control solution. In this mode of operation, composition of culture medium, biomass concentration and the metabolic
product concentration gradually change as a result of metabolism of cells.

Advantages

 Easy to set up.

 Environmental conditions are relatively easy to control.
 The types of vessels used can be used for different processes at different time.
 If the culture becomes contaminated, it is only one batch that is lost.
 No blockage can occur.
 Initial capital cost is lower.
Disadvantages

 Less effective for the production of biomass and primary metabolites.

 Greater stress on instruments and probes by increased frequency of sterilization.
 Batch to batch variability.

2. Continuous Fermentation

Continuous culture is a technique involving feeding the microorganisms used for the fermentation with fresh nutrients and
at the same time, removing spent medium plus cells from the system. In this type of fermentation, the exponential growth
phase of microbes may be prolonged by the addition of fresh medium to vessel. The vessel should be designed in such a
way that the added volume displays an equal volume of culture in the vessel. If medium is fed continuously to such vessel
at a suitable rate, a steady state is achieved eventually. Steady state is formation of new biomass in vessel is equivalent to
the loss of cell from vessel. Under these conditions, the rate at which new cells are produced in the culture vessel is
exactly balanced by the rate at which cells are being lost through the overflow from the culture vessel. If the working
volume of fermentor is V m3 and the rate of flow in and out is F m3h-1, then
𝐹
D (dilution rate) = 𝑉

In such a system, the rate of change in biomass concentration in the culture is given by the increase in biomass due to
growth minus the loss of biomass carried out in the outflow. The increase in biomass per unit volume due to growth is
given by µX and decrease due to loss in outflow is DX.
𝑑𝑋
Rate of increase in biomass concentration 𝑑𝑡
= µX - DX

𝑑𝑋
A steady state develops in continuous system, in which biomass concentration remain constant ( 𝑑𝑡 = 0) (as biomass
concentration is constant, rate of increase 0); the amount of biomass lost from fermentor by dilution (diluted out in out-
flowing medium) is replaced exactly by growth, so that µX – DX= 0

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 ⸫ µ = D.

In steady state, growth rate is equal to D. in this way, growth rate can be controlled. The faster the flow rate, the faster the
growth rate.

Advantages

 The non-productive periods are avoided.

 Works all the time.
 Good utilization of the reactor.
 Productivity is high.
 These processes are more easily automated. This helps eliminate human error and thus ensures greater uniformity
in the quality of the products.
 Time and labor savings.

Disadvantages

 Long culture period.

 Complexity of operation.
 Risk of contamination.
 Slow growing contaminants might not have developed to the point where they can be noticed in the 4, 5, or 10
days of a batch fermentation; they can pose a serious threat to production in continuous culture which goes on for
up to 3, 6 or 9 months. If the contaminant is fast growing then the danger while serious in a batch fermentation is
infinitely more so in a continuous fermentation.
 Another problem was that mutants better adapted to the environment of the continuous fermentor are easily
selected.

Chemostat

This system depends on the fact that the concentration of an essential nutrient (substrate) within the culture vessel will
control the growth rate of the cells. The concentration of substrate within the vessel is in turn controlled by the dilution
rate (the rate at which fresh medium is added to the culture (flow rate) divided by the volume of culture vessel).
Therefore, by adjusting the dilution rate the growth rate can be controlled.

If the dilution rate is very low, the cells reach a high density because they are leaving the vessel at a very slow rate;
moreover, they have time to use the substrate almost completely. Therefore, the substrate concentration is maintained at
low level within the vessel. This low substrate concentration permits the cells to grow at only slow rate.

If the dilution rate is high, the cell density is low because the cells are leaving the vessel a high rate. They have little time
to utilize the substrate that is entering the vessel and therefore the substrate concentration is maintained at a high level
within the vessel. This high concentration allows the cells to grow at a high rate.

If the dilution rate is increased to the point where it exceeds the maximum growth rate of cells, then ‘washout’ occurs,
that is the cells cannot grow as fast as the rate at which the culture is being diluted by fresh medium and they are soon
eliminated from the culture vessel.

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Turbidostat

In turbidostat, a photoelectric device continuously monitors the cell density within the culture vessel and controls the
dilution rate to maintain the cell density at a constant value. If the density becomes too high, the dilution rate is increased;
if the density becomes too low, the dilution rate is decreased.

Classification of continuous microbial cultivation

a) Single state continuous fermentations

There are fermentations in which the entire operation is carried out in one vessel, the nutrient being added simultaneously
with broth outflow. This system is suited for growth related fermentation such as yeast, alcohol or organic acid
production.

b) Multiple-stage continuous fermentation

This consists of a battery of fermentation tanks. The medium is led to the first and the outflow into the second, third or
fourth as the case may be. This is most frequently used for the fermentation involving metabolites. The first tank may be
used for the growth phase and subsequent tanks for production, depending on the various requirements identified for
maximal productivity.

c) Recycled single or multiple stage continuous fermentation

The outflowing broth may be freed of the organisms by centrifugation and the supernatant returned to the system. This
system is particularly useful where the substance is difficult to degrade or not easily miscible with water such as
hydrocarbon. Recycling can be applied in single stage fermentor. In multiple stage fermentors, recycling may involve all
or some of the fermentation vessels in the series depending on the need.

d) Semi-continuous fermentation

In semi-continuous fermentation, simultaneous nutrient addition and outflow withdrawal are carried out intermittently,
rather than continuously. There are 2 types of semi-continuous fermentation.

In cyclic continuous, a single vessel is usually employed, although a series of vessels may be used. For products which
are growth linked, such as ethanol or biomass, most of the product will be formed towards the end of the growth phase,
when most growth occurs. If at the end of this phase, a portion of the culture medium is removed from the fermentor and
replaced by fresh medium, then growth of the organisms will continue. This process can be repeated at appropriate
intervals and is known as semi-continuous fermentations. By using this system, down time and lag phase are avoided and
high biomass concentration can be maintained.

In cell reuse, cells are centrifuged from the fermentation broth and used to re-inoculate fresh medium. It is continuous
only in the sense that cells are reused; in essence it is batch fermentation.

3. Fed-batch fermentation

Fed-batch cultivation is a modification of batch cultivation in which the nutrient is added intermittently to a batch culture.
Nutrients can be added at the same rate as they are used up, so excess of nutrient (and any inhibition resulting from this)
can be avoided. An example of this is in the production of alkaline protease by species of Bacillus; batch feeding of
nitrogen sources (ammonia, ammonium ions or amino acids) keeps these substrates at low concentrations and thus avoids
the repression that these nitrogen sources exert on protease synthesis. Some diffusion capsules are also used which are
cylindrical capsules to one end of which a semi-permeable membrane is fixed. The nutrient diffuses slowly out through
the membrane to the medium.
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Fed-batch fermentations can be the best opinion for some systems in which the nutrients or any other substrate are only
sparingly soluble or are too toxic to add the whole requirement for a batch process at the start.

Finally, in fermentations such as mycelia culture the increase of viscosity with time can be compensated by the addition of
relatively small quantity of water during the fermentation time, although the efficacy of this protocol is controversial
among researchers.

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Effect of substrate concentration on growth rate:

During the growth of microorganisms in batch culture, no nutrients are added except acid and alkali for maintaining pH
and air for the growth of aerobic microbes. Normally, the carbon substrate present in the medium serves as the limiting
substrate for growth. The relationship between the specific growth rate (µ) and the concentration of growth limiting
substrate can be described by Monod equation, where
µmS
µ = 𝐾𝑠+𝑆 ……………(1)

µ= specific growth rate (day-1)

µm= maximum specific growth rate (day-1)

S= growth rate limiting substrate concentration (mg/L)

1
Ks = 2 (saturation coefficient), substrate concentration at one half of maximum growth rate/ Monod constant.

It corresponds to the substrate concentration at which µ is one half of its maximum.

If Ks = S, equation (1) becomes

µmS µmS µm
µ= 𝑆+𝑆
== 2𝑆
= µ) = 2
…………. (2)

The Monod equation provides a continuous transition between the 1st and 0 order kinetics based on growth limiting
substrate concentration.
µmS
If Ks ˃˃˃S, µ= 𝑆+𝑆
= KˊS

The model would be reduced to a 1st order expression.

µmS
If Ks ˂˂˂S, µ= = µm
𝑆

The model would become 0 order expression.

1
From equation (2), it can be inferred that Ks = S, when µ is the value of µm
2

It is a measure of affinity of the organism for the substrate. The maximum value of µ can be realized only when the
substrate concentration is very very large. Therefore, when substrate concentration is in excess, the growth of microbes
takes place at a rate equal to its µm (equation 4).

When the substrate concentration is growth limiting, it will not support the growth of organisms at its µ m value. Whether
the deceleration phase (the stage of retarding the growth to reach to stationary phase) would be long or short, depends on
Ks. if the value of Ks is low (high affinity for S) then the growth rate will not be affected until the substrate concentration
has reduced significantly and therefore the deceleration phase of such culture would be short. On the other hand, if the
value of Ks is high, then the growth rate will be deleteriously affected at a relatively higher substrate concentration and
accordingly the deceleration phase for such a culture would be relatively long.