Chapter 8

Microbial Growth Kinetics

in Fed-Batch Culture
SITI NOR SYAIRAH BINTI ANIS
BTC 661
 Definition of
Fed-Batch Culture
and Cell Growth in
Fed Batch Culture
Definition of Fed-Batch Culture
• Fed-batch fermentation is a semi-batch operation.
• An enhancement of the closed batch process.
• In the fed-batch process one or more substrates, nutrients, and/or
inducers are introduced into the bioreactor.
• The fermentation starts as a batch fermentation but at a determined point,
one or more components is fed to the operation either continuously or
intermittently.
• Sources of carbon, nitrogen, phosphates, nutrients, precursors, or
inducers are fed either into the culture by manipulating the feed rates
during the run.
• So the volume of the culture usually increases during the process, unless
concentrated substrate is used to keep the volume relatively constant.
• The aim of it is for the concentrations of limiting substances to remain
constant (steady-state) or for them to follow a predetermined
profile.
• This leads to a maximized yield of the product.
• The products are harvested only at the end of the process.
• A dynamic operation. By manipulating the feed rates, the
concentrations of limiting nutrients in the culture can be manipulated
either to remain at a constant level or to follow a predetermined
optimal profile until the culture volume reaches the maximum, and
then a batch mode is used to provide a final touch.
• In so doing, the concentration of the desired product or the yield of
product at the end of the run is maximized.
• Then cells containing the product are harvested.
Question in Fed-Batch Culture?
• The most important questions to ask in fed-batch culture operations
are:
What compounds(s) should be fed?
How they should be added?

• The answers:
Depend on the characteristics of the organisms used.
Feb-Batch Fermenter
Batch vs Fed-Batch Culture
A fed-batch is useful in achieving high concentration of products as a result
of high concentration of cells for a relative large span of time.
Two cases can be considered:

1. The production of a growth associated product

• These are those products formed simultaneously with microbial growth.

• Fed batch increase the production of biomass
• The specific rate of product formation is proportional to the specific rate of
growth.
• In this case, it is desirable to extend the growth phase as much as possible,
minimizing the changes in the fermenter as far as specific growth rate,
production of the product of interest and avoiding the production of by-
products.
• Example: Production of constitutive enzyme.
2. The production of a non-growth associated product.

• Products are formed during the stationary phase when the net growth rate
is zero.
• The specific rate of product formation is constant.
• The fed-batch would be having two phases: a growth phase in which the
cells are grown to the required concentration and then a production phase
in which carbon source and other requirements for production are fed to
the fermenter.
• This case is also of particular interest for recombinant inducible systems:
the cells are grown to high concentrations and then induced to express the
recombinant product.
• Example: Secondary metabolites like antibiotics (penicillin).
Fed-batch cultures can be run in different ways:

Different feeding regimes in fed-batch processes, (a) Variable feeding regime; (b) continuous feeding
regime; (c) intermittent feeding regime; (d) incremental feeding regime
Advantages and disadvantages of various
feeding strategies
A simple fed-batch fermentation set-up.
Feeding begins at a predetermined point during the process, often at the
end of the exponential growth phase.
The feed consists of a super-concentrated version of the process medium,
allowing control of the growth rate and carbon flux through the system.
 Physiology and
Kinetics of
Fed-Batch Culture
Kinetics of Fed-Batch Culture
1. The exponential cell growth stage is of interest.
2. The fed-batch reactor is perfectly mixed.
3. Heat effects are small so that isothermal reactor operation can be
assumed.
4. The liquid density is constant.
5. The broth in the bioreactor consists of liquid plus solid material, the mass
of cells. This heterogeneous mixture can be approximated as a
homogenous liquid.
6. The rate of cell growth is given by the Monod equation in:
7. The rate of product formation per unit volume rp can be expressed
as:

where the product yield coefficient YP/X is defined as:

8. The feed stream is sterile and thus contains no cells.

• Normally, the feed concentration is very high (i.e., the feed is a very
concentrated solution) and the feed flow is low, giving a low dilution
rate.
• For the fed-batch reactor, the dilution rate is given by:

• To keep D constant, there needs to be an exponentially increasing

feed flow to the bioreactor, which is normally practically impossible as
it may lead to oxygen limitations.
• The feed flow is therefore adjusted or increased until limitations in
the oxygen supply set in, at which point the feed flow is kept
constant.
• This will give a decreasing specific growth rate.
• However, because the biomass concentration usually increases, the
volumetric uptake rate of substrates (including oxygen) may be
kept approximately constant.
• From the above it is quite clear that there may be many different
feeding strategies in a fed-batch process, and optimization of the
operation is a complex problem that is difficult to solve empirically.
• Even when a very good process model is available, calculation of the
optimal feeding strategy is a complex optimization problem.
• In an empirical search for the optimal feeding policy, the two most
obvious criteria are:
1. Keep the concentration of the limiting substrate constant.
2. Keep the volumetric growth rate of the biomass (or uptake of a
given substrate) constant.
Limitation of Fed-Batch
Culture and Application
of Fed-Batch Culture.
Advantages and Applications
1. Suitable for primary metabolites products.
2. Substrate is metabolized rapidly.
3. Overcome problems found in continuous culture: contamination,
mutation and plasmid instability.
4. Allow to feed small quantities of substrate instead of having it all
inside from the beginning to ensure the microorganisms grow better.
• Large amount of substrates can be toxic to the growth of the
microorganisms or biomass required for harvesting later
• Example:
- Baker’s yeast which is collected as a biomass at the end of the process.
- Yeast require glucose substrate to grow and multiply, but if there is too
much glucose, ethanol can be produced as a byproduct which is harmful to
the growth of yeast.
- It is important to maximize biomass production first and reduce byproduct
production, even if the byproduct is desirable for us to harvest later.
- Hence, fed batch allows the glucose to be given in small amounts to
promote yeast growth but minimize ethanol production which is harmful to
the yeast.
5. Allows the viscosity and oxygen level in the system to be controlled
for ideal growth
• Some substrates cause the solution in the fermenter to have higher
viscosity, which reduces the amount of oxygen that can be absorbed
and dissolved inside.
• If the substrate concentration is too high, the oxygen levels inside the
tank get low and the microorganisms will die faster than grow which
is undesirable.
6. Allows the production of by products efficiently.
• For the harvesting of byproducts (not biomass), adding too much substrate
will cause the cells to use the nutrients to multiply and grow instead of
creating the desired byproduct for harvestation.
• Example 1:
- In the production of ethanol, if too much glucose is given to the chamber,
the cells will experience an exponential phase of growth and will not seek
alternative pathways like anaerobic respiration in the stationary phase to
produce glucose since there is no stress introduced for the system for them
to undertake different metabolic pathways.
• Example 2:
-This scenario is similar for penicillium bacteria, where the cell culture has to
receive stress with low glucose content to produce penicillin as a secondary
metabolite.
Disadvantages of Fed-Batch Culture
1. Additional instruments for feedback control may be costly (compare with
batch culture).
2. Its fermentation time is high as compare to the batch culture
fermentation.
3. We have to take the samples at regular intervals to check the
concentration of the substrates.
4. In systems without feedback control, where the feed is added on a pre-
determined fixed schedule, it is difficult to deal with any deviations in the
organisms growth pattern (i.e. time courses may not always follow
expected profiles).
5. Requires a substantial amount of operator skill.
 Examples of Fed Batch Fermentation
1. The first well-known fed-batch fermentation application was used to
grow yeast cells by incrementally adding sugar during fermentation.
• This kept the glucose concentration low to suppress alcohol formation and
maximize the yield of yeast cells.
• High glucose concentration would accelerate the cell growth, which in turn
would cause anaerobic conditions that favoured ethanol formation and
lowered the yield of yeast cells.
• Additional sugar was added at a rate that was always less than the rate at
which the yeast cells could use it.
• Intermittent or incremental feeding of nutrients to an initially dilute
medium was introduced thereafter in large-scale yeast production to
improve the yeast yields while obviating the production of ethanol.
• However, there is some speculation that a small amount of ethanol may be
necessary to ensure the quality of the baker’s yeasts.
2. Penicillin production. This process is compartmentalized into two
separate phases:
• Phase 1 is the rapid growth phase, where the culture for penicillin grows at
the maximum specific growth rate.
- In this phase, excess glucose in the culture results in the
accumulation of acid. The demand of oxygen is also greater than the
fermenter's capacity for aeration. Inadequate glucose will lead to the organic
nitrogen in the culture to be used as a carbon source, which results in a high
pH which causes inadequate biomass production.

• Phase 2 is the slow growth phase, where the penicillin is produced.

- This is the starvation phase where penicillin is able to propagate by
killing off competition for the scarce resources as the energy (pyruvates)
available get scarce. The feed rates used should be optimized such that the
growth rate and oxygen consumption of the system is at the level where a
highest rate of penicillin synthesis can be achieved and with adequate
oxygen available in the medium for growth.
Thank You …