MODES OF FERMENTATION
There are three main modes of fermentation technique: batch, fed-batch and
continuous.
BATCH CULTURE:
Batch fermentation is the simplest mode of operation, and is often used in the
laboratory to obtain substantial quantities of cells or product for further analysis. A
batch fermentation is a closed system, where all of the nutrients required for the
organism’s growth and product formation are contained within the vessel at the
start of the fermentation process. The vessel can take the form of a shake fl ask,
single use disposable system, or, for tighter control of parameters such as oxygen
transfer, pH, agitation, etc., a bioreactor can be used. After medium sterilisation, the
organism is inoculated into the vessel and allowed to grow. The fermentation is
terminated when one or more of the following has been reached: (i) microbial
growth has stopped due to the depletion of the nutrients or the build of toxic
compounds; (ii) after a fi xed predetermined period of time; (iii) the concentration of
desired product has been achieved.
Diagram of a simple batch fermentation process
The Batch Culture Growth Curve
When cells are grown in a batch culture, they will typically proceed through a
number of distinct phases. The lag phase, which may or may not be present, is
described as ‘little or no growth at the beginning of the fermentation due to the
physiochemical equilibrium between the microorganism and the environment
following inoculation’. The lag phase can be time consuming and costly and so it is
highly desirable to minimise this phase. Nutrient depletion and the formation of
inhibitors (typically excreted products such as ethanol, lactic acid, acetic acid,
methanol, and aromatic compounds) have the effect of decelerating cell growth, and
the cells then enter the stationary phase where the rate of cell growth equals that of
cell death. Later as the nutrients deplete cells enter the death phase where the rate of
cll death overcomes the cell growth
Examples of Batch Fermentation: Submerged batch cultivation can be used for the
production of alcoholic beverages (beer, wine, and distilled spirits such as whisky,
brandy, rum, and others), organic acids used in the food industry either as acidifiers
or as preservatives (citric, acetic (vinegar), and lactic acids), and amino acids used as
flavor enhancers (e.g., monosodium glutamate) or sweeteners (e.g., aspartate).
Advantages of Batch Culture
1. Simplicity of use. A batch culture can be easily readied, and, depending on
the microorganism used, can be fi nished in less than 24 hours.
2. Operability and reliability: less likely to have instrument failure on short
batch runs;
3. Production of secondary metabolites that are not growth-related (i.e.,
produced when the organism enters stationary phase);
4. Fewer possibilities of contamination: all of the materials required for the
bioprocess are present in the vessel and sterilised before the run starts.
5. It is easy to assign a unique batch number to each run, generating high
confidence in the history of each batch of product. This is critically important
in a highly regulated environment
6. The batch culture growth curve gives a good indication of when to stop the
fermentation. Growth-associated products (primary metabolites) are
produced during the exponential phase with their formation decreasing when
growth ceases. Secondary metabolites are produced as the cells enter
stationary phase
Disadvantages of Batch Culture
1. Culture ageing, and more importantly differentiation, can be a specific
problem, especially so with growth-related products
2. Build up of toxic metabolites can restrict cell growth and product formation;
3. Initial substrate concentrations may have to be limited due to problems with
inhibition and repression effects, therefore affecting the amount of product
that can be obtained from such simple systems;
4. Batch-to-batch variability;
5. The use of batch cultures in industrial systems can lead to an increased non-
productive period due to down time required for cleaning, resterilisation,
filling and cooling of equipment;
6. If using the organism from one bioprocess to seed another culture,
degeneration or differentiation may occur, which could affect the bioprocess
and product formation;
7. Cellular autolysis may occur during the decline and stationary phase,
affecting the amount of product, its composition and potentially adding to
downstream processing challenges due to release of autolytic breakdown
products, activation of proteases;
8. From a physiological viewpoint the use of batch cultures actually contributes
greatly to the complexity of the experiments since the cell population is
heterogeneous and constantly changing. This makes the use of such systems
for clearly identifying cause and effect relationships in cell physiology rather
unattractive
FED BATCH CULTURE
Fed-batch culture represents a semi-open system in which one or more nutrients are
aseptically and gradually added to the bioreactor in steps while the product is
retained inside that is, the volume of the culture broth in the bioreactor increases
within this time.
The addition of the feed can be over a short or long period, starting immediately
after inoculation or at a predetermined point during the run. The feeding strategy
can be continuous over a long period of time or incremental, with the addition of
fixed volumes at given time points. The following feeding regimes are followed
Fed-batch is advantageously used in processes
(a) where substrate inhibition or catabolic repression is expected; this problem can be
overcome by using a “safe” concentration of the substrate in batch mode followed by
feeding the remaining substrate within fed-batch operation,
(b) where a Crabtree effect (repression of yeast respiratory enzymes by high
concentrations of glucose) is expected (de Deken, 1966); by gradual feeding of the
substrate, the production of ethanol by yeasts can be eliminated under aerobic
conditions,
(c) where a high cell density is required; a high and constant specific growth rate can
be maintained by exponential feeding of the substrate,
(d) where a high production rate should be achieved; cell metabolism can be
regulated by precise sequential feeding of nutrients,
(e) where a high viscosity of culture broth is expected (e.g., production of dextran or
xanthan); a gradual dilution of the medium can overcome the problems of mixing
and oxygen transfer.
The typical food fed-batch fermentations are large-scale production of baker’s
yeast, pure ethanol, which is further utilized for alcoholic beverages produced by
mixing ingredients such as liquors or cordials, and submerged acidification for
vinegar production. Yeast production is the only technology in which the respiratory
metabolism of S. cerevisiae, leading to high biomass yield, is stressed. Nevertheless,
due to the Crabtree effect, that is, the formation of ethanol under aerobic conditions
in the presence of excess substrate, the only alternative for producing baker’s yeast is
fed-batch cultivation.
Types of Fed Batch Fermentation:
Fixed Fed-Batch Fermentation: Within fixed volume fed-batch fermentation, the
limiting substrate is fed without diluting the culture. The volume within the
bioreactor is maintained at around the same level by feeding either a super-
concentrated medium containing the limiting substrate or the addition of a gas such
as oxygen. Advantage can also be taken of the loss of volume via evaporation, since
the volume can be kept constant by addition of an equal volume of liquid feed. A
draw and re-fi ll strategy can also be adopted where a predetermined volume of the
fermenter contents are drawn off at a selected time, and replaced with an equal
volume of fresh medium. In this way, the reactor volume remains constant and the
fresh nutrients have the effect of decreasing the concentrations of inhibitory species
present, allowing extended processing.
Variable fed-batch fermentation: is one where the addition of the feed alters the
working volume in the bioreactor. The feed can be the same medium as that used in
the vessel at the beginning of the process, or it can be a concentrated solution of the
limiting substrate. This allows the organism to continue growing at its maximum
specific growth rate resulting in a higher concentration of fi nal biomass.
Advantages of Fed-batch Control
1. Controlling the concentration of the limiting substrate prevents the repressive
effects of high substrate concentration and avoids catabolite repression.
2. By careful feeding strategy the organism’s growth rate and subsequent
oxygen demand can be controlled.
3. High cell density (up to ten times greater) can be achieved by use of fed-batch
over batch culture. Batch culture limits the final cell growth due to no extra
carbon being added during the run. Using fed-batch with a careful feeding
strategy, E. coli and Pichia can achieve very high cell densities of over 100
gL−1.
4. Increased production of non-growth-related secondary metabolites. Many
secondary metabolites are produced from intermediates and end products of
primary metabolism. Others are formed after introduction of key precursors
after the growth phase, as is seen in penicillin production with phenylacetic
acid or phenoxyacetic acid, precursors of penicillins G and V respectively,
being added prior to the stationary phase to allow the formation of penicillin.
5. Reduction of broth viscosity. This is particularly important in filamentous
fungal fermentations, or where the product is highly viscous, such as the
polysaccharide products of Sphingomonas elodea – gellan gum; and
Xanthomonas campestris – xanthan gum. The addition of fresh medium during
the fermentation run, leads to the broth being ‘diluted’, and a brief viscosity
drop, allowing better aeration and agitation within the system.
Disadvantages of Fed-batch Control
1. Detailed knowledge of the organism’s growth and product formation pattern
is required, especially when no feedback loop is used.
2. Defi ciency of reliable online sensors for accurate substrate determination in
near real time.
3. Without feedback control, the feed is predetermined and therefore does not
allow for any fluctuations within the bioprocess. This can potentially lead to
mismatches between feed rates and culture metabolism, in turn leading
substrate levels to become depleted or rise to undesirable levels.
4. The process operator must be fully trained and highly skilled.
CONTINOUS FERMENTATION
Continuous culture represents an open system in which nutrients are
aseptically and continuously added to the bioreactor, and the culture broth
(containing cells and metabolites) is removed at the same time that is, the
volume of the culture broth is constant due to a constant feed-in and feed-out
rate.
There are two types of operations under continous fermentation
The Chemostat
The feed medium contains an excess of all but one of the nutrients required
for growth of the culture. The supply of the nutrient that is not in excess
therefore determines growth rate of the microorganism. Here, a constant
specific growth rate of cells is maintained which is equal to the dilution rate
and is controlled by the availability of the limiting nutrient. Most chemostat
cultures become progressively more unstable as the dilution rate approaches
the critical dilution rate (above which washout occurs). Washout refers to
complete removal of cells. Hence, physiological and other studies involving
this type of continuous culture system should not be operated near this
region.
The Turbidostat
In a turbidostat, the feed medium contains all of the required nutrients in
excess. Growth is therefore not substrate limited as it is in the chemostat, and
the microorganism can grow at its maximum specific growth rate (mmax).
The system can be controlled at a desired cell density by monitoring the
turbidity, and therefore the biomass, continuously. This can be achieved by
measuring optical density using a spectrophotometer. If the monitor detects a
deviation from the cell density set-point value, a signal is relayed to the
controller, and so the rate at which the feed medium is added to the
bioreactor can be adjusted. When the turbidity increases above a set point, the
feed rate is increased in order to dilute the culture and bring the turbidity
back to its set point. If the density of the reactor population falls, the feed rate
is decreased, allowing the population to grow until the turbidity set point is
reached, thus avoiding washout of the organism.
Advantages of Continous Fermentation
1. Productivity and growth rate can be optimised by changing the feed
flow rate during production;
2. A continuous process has to be operated indefinitely; however,
because long periods of operation can result in mechanical failure, the
process must be stopped occasionally to allow for system maintenance;
3. It can take advantage of cell immobilization, which allows the
maintenance of high cell concentrations in the bioreactor at low
substrate concentrations.
4. The effects of environmental or physical factors are more easily
analysed in a continuous system, where any changes in the constant
steady state are observed and can be attributed solely to the change in
those factors.
5. Evolution in these cultures can be readily studied. Basically,
continuous culture of a given strain allows us to ‘direct the evolution’
of the strain.
Disadvantages of Continous Fermentation
1. Increased risk of contamination due to the pumping of the medium
in and out of the bioreactor,
2. The danger of genetic mutations in the production strain in a long-
term operation,
3. Additional investments may be required for technical facilities.
4. Not all products are produced optimally in continuous processes,
e.g. some fermented foods and beverages require cellular products
released from different phases of batch culture growth for full
flavour development
Continous Perfusion Culture:
In these systems, cells are perfused via a membrane with a steady and
continuous flow of fresh medium. The cells are supplied with oxygen and
nutrients, while waste and desired products are continuously removed. The
cells are retained by means of the membrane barrier and are recycled into
the bioreactor. Another option is to retain the cells by binding them to a
substrate (capillary fibers, membranes, micro carriers in fixed bed, and so
on) in the bioreactor.
