Btec22

Batch Culture
• A type of culture operation in a closed culture
system which contains an initial limited amount
of nutrient. As the inoculated culture or
inoculum cells grow, it will undergo several
phases in the growth curve.
• The bioreactor is first charged with medium,
inoculated with cells, and the cells achieve the
required density or optimum product
concentrations.
• The bioreactor contents are discharged, and
the bioreactor is prepared for a fresh charge of
medium.
The Stages of Batch Fermentation

• Shake Flask
• Seed Fermenter
• Production Fermenter
• All nutrients are provided at the beginning of the
cultivation and products are removed at the end.

Nutrients, cells, reagents-----------------→PRODUCT

Advantages
• Short duration

• Less chance of contamination as nutrients are

added at the beginning

• Separation of batch material for traceability

• Easier to manage
Disadvantages

• Product is mixed in with nutrients , reagents , cell

debris, and toxins.

• Shorter productive time.

• Productivity is low.
 Microbial Growth Phases Associated
with Batch Fermentation

• Lag Phase

• Exponential Phase

• Stationary Phase

• Death Phase

https://www.cs.montana.edu/webworks/projects/stevesbook/contents/chapters/chapter002/section002/black/page001.html
Operation is thus characterized by
three periods of time
• Filling period,

• Cell growth and cell production period

• Final emptying period

Phases of Batch Culture Process:

1. Lag Phase
• It is the first major point of microbial
growth in batch culture process.
• After the inoculation of microorganisms
into the fresh medium, it will undergo a
period that may be suspected that there
is no growth taking place.
• Still, there is only a minimal increase in
cell density.
 1. Lag Phase

• It is a period of adapting or
adjusting to the cells’ new
environment.
• In some fermentation, it is
not taking place.
• On a commercial scale, the lag phase duration
should be shortened as much as possible, and
it can be resolved by using a suitable
inoculum.
• dX/dt and dS/dt are essentially zero.
Yield Coefficient and Specific Growth
Rate
• The lag phase is an adaptation period, where
the bacteria are adjusting to their new
conditions.

• The length of the lag phase can vary

considerably, based on how different the
conditions are from the conditions that the
bacteria came from, as well as the condition of
the bacterial cells themselves.
• Actively growing cells transferred from one
type of media into the same type of media,
with the same environmental conditions, will
have the shortest lag period.

• Damaged cells will have a long lag period,

since they must repair themselves before they
can engage in reproduction.
• This process could include the repair of
macromolecular damage that accumulated during
stationary phase and the synthesis of cellular
components necessary for growth.
2. Log or Exponential Phase

• Growth rate of the cells gradually increases,

• Cells grow at a constant, maximum rate.
• Nutrients are in excess, and the organism is
growing at its maximum specific growth rate,
µmax, for the optimal conditions.
2. Log or Exponential Phase

• The exponential growth of a single-celled

organism is replicated through binary fission.
• The growth of microorganisms will continue
indefinitely in the culture.
• However, when the growth of microorganism
and the excretion of the microbial product
depends on the consumption of nutrients
dramatically affects the organism‘s growth.
• Hence, after a particular time, the growth rate
of the culture decreases until growth stops.
• The termination of growth is due to the
exhaustion of some essential nutrients in the
medium, toxin accumulation (toxin limitation),
or the combination of the two.
2. Log or Exponential Phase
• Cells have adjusted to their new environment
• The cells are dividing at a constant rate resulting in
an exponential increase in the number of cells
present. This is known as the specific growth rate
and is represented mathematically by first order
kinetics as the following:
𝑑𝑥
=(μ-kd)X
𝑑𝑡
where X is the cell concentration,
μ is the cell growth rate, and
kd is the cell death rate.
• The term μ– kd can be referred to as μnet. The
cell death rate is sometimes neglected if it is
considerably smaller than the cell growth rate.
• There are other models used to determine cell
growth rate that depend upon inhibition
• Substrate Inhibition
• Product Inhibition
• Toxic Compounds Inhibition
• The type of inhibition causes mathematical
changes in the previously presented Monod
equation for batch kinetics
Substrate Inhibition
• In batch fermentation, this can occur during the
initial growth phases while substrate
concentrations are high
• If this is a major problem, continuous or fed-batch
fermentation methods should be considered
Product Inhibition
• In batch fermentation, this can occur after
induction of the recombinant gene
3. Stationary Phase
• It is the third major phase microbial growth in a
batch fermentation process.
• The growth rate has declined zero but may still
metabolically active and produce secondary
metabolites.
• It occurs when the number of cells dividing and
dying is in an equilibrium state.
• It can result in depletion of one or more
essential growth nutrients, accumulation of
toxin by product.
• Occurs when the number of cells dividing
and dying is in equilibrium and can be the
result of the following:
• Depletion of one or more essential growth
nutrients
• Accumulation of toxic growth associated by-
products
• Stress associated with the induction of a
recombinant gene
• Primary metabolite, or growth associated,
production stops
• Secondary metabolite, or non-growth
associated, production may continue
 4. Death Phase
• It is the last major phase of microbial growth in
a batch fermentation process, which is also
known as the decline phase.
• The rate of cells dying is greater than the rate
of cells dividing.
• The decrease in growth rate and the cessation
of growth, due to the depletion of the substrate,
may be described by the relationship between µ
and the residual growth-limiting substrate.
4. Death Phase

• Similar to exponential phase, it is

represented mathematically by first
order kinetics as the following:
𝑑𝑋
=-kdX
𝑑𝑡
Determination of Microbial Growth
Curve
• There are a two main methods primarily used to
establish a growth curve. Both of which are
represented on the previously shown growth curve.
• Viable Cell Count
• Initially lower curve representing the number of cells that are
actually viable
• Determined by plating a sample from the culture
• Optical Density
• Initially higher curve representing the number of cells that are both
viable and non-viable
• Determined by taking an optical measurement using a
spectrophotometer
Optical Density

• Measuring the optical density with a

spectrophotometer is a quick and easy way to
develop a growth curve.
• One takes a sample of the fermentation broth
and measures the absorbance at a particular
wavelength in the spectrophotometer.
• For E. coli cells in a typically LB medium, the
wavelength used in 600 nm.
• The measured value can be compared to
previous measurements made in conjunction
with cell plating or cell counting. The negative
side of using and non-viable cells absorb this
wavelength.
• As a result, the values taken are not
representative of only viable cells.
Fed-Batch Culture
Characteristics of Fed-Batch Culture

• It is similar to the batch fermentation process

• However, there is an extension of the batch

culture by feeding periodically with medium
with no removal from the vessel.

• A volume of medium is inoculated with the

organism and allowed to grow in a period of
time.
• Subsequently, a fed is initiated into the
fermenter when a ‘quasi-steady state is
attained in which the growth limiting substrate
has exhausted.

• The supply of growth-limiting substrate or

medium will sustain the microorganisms’
growth in fed-batch reactor.
• In terms of medium, the initial concentration is
relatively low.

• Then the reactor will then continually be fed

with medium concentrated with carbon and
nitrogen in increments.
 Pump 1
Separator
Pump 2

(Biomass X)

Feedstock
vessel
(sterile)

Collection vessel
Comparison of Batch vs. Fed-batch
Process
Advantages of Fed-batch culture
than batch culture
• The maintenance of the fed-batch culture is in the
low substrate level.

• The most problem of fermentation is when

substrates are fed at a high concentration,
repression occurs due to high concentration of
the substrate.

• The productive phase of the fermentation will be

extended.
• In the cycle fed-batch system, a periodic
shift of the growth rate enhances
secondary metabolite production as
many secondary metabolites are not
growth-associated.

• Kojic acid production is an example of a

secondary metabolite produced by
Aspergillus oryzae.
Advantages of Fed-batch culture
than batch culture
• The product formation in this system depends
on the specific growth rate of
microorganisms.

• Nutrients are fed exponentially to obtain

optimum desired products by monitoring the
specific growth rate of the microorganism.

• Example: antibiotic production, which is mostly

secondary metabolites.
• There is a drastic reduction of oxygen transfer
and heat because of high viscosity culture
broth.

• Using fed-batch culture, the fed of nutrients is

controlled pulse wise followed by the output of
a certain volume from the fermenter.
• This technique reduces the high viscosity
effect of the culture.

• Biopolymer production usually encounters this

problem and fed-batch culture is suited in the
fermentation.
 Advantages of Fed-batch culture
than Batch Culture
• In extracellular enzyme production, it is
controlled by catabolite repression.

• Enzyme synthesis is inhibited by the presence of

readily utilized carbon source or inhibitory
carbon source.

• Using fed-batch culture technique, the inhibitory

carbon source is fed gradually to overcome the
inhibitory effect for the production of
extracellular enzymes.
Disadvantages

• Provides another point of ingress for contamination

• May produce high cell density numbers and product

yields which are difficult to deal within downstream,
creating bottlenecks in the whole process.
Classification of Fed-Batch Culture

• Fix volume Fed-Batch Culture

• Variable volume fed-batch Culture

Fix volume fed-batch culture

• In this type of fed-batch, the limiting substrate is

fed without diluting the culture.

• The culture volume can also be kept constant by

feeding the growth limiting substrate in undiluted
form, such as a very concentrated liquid.

• Additionally, the feed is provided at constant rate.

• The production of mass of biomass per mass of
substrate is constant during the fermentation time.
Variable volume fed-batch culture

• Volume changes with fermentation time due to the

substrate feed.

• The substrate is provided in a manner that maximizes

the specific growth rate.

• The substrate is added exponentially for biomass

production to maintain a constant growth rate in a
culture growing exponentially.
• Meanwhile, this culture considers the
limitation of one substrate concentration.

• The conditions of variable fed-batch culture

are;
• (a) the input of substrate is equal to the
consumption of cell, and
• (b) the input of substrate depends on the specific
growth rate and total cell in the culture.
References:

• https://aasnig.com/fermentor-bioreactor-design-and-
its-functions/
• https://projects.ncsu.edu/project/actionagenda/copr
otein/media/Fermentation.pdf
 Thank you and God bless!
If any of you lacks wisdom, you should ask God, who gives
generously to all without finding fault, and it will be given to
you.
James 1:5