Introduction to Fermentation

Genetically modified Escherichia coli have been chosen as the host

organism for each of the co-proteins to be produced. Each strain of
E. coli will contain a different gene that is responsible for producing
the desired co-protein. The modified E. coli cells will be separately
grown through the process of batch fermentation. This tutorial will
introduce you to the following areas regarding batch fermentation:
• Microbial Growth Phases Associated with Batch
Fermentation
• Lag Phase
• Exponential Phase
• Stationary Phase
• Death Phase
• The Stages of Batch Fermentation
• Shake Flask
• Seed Fermentor
• Production Fermentor
 Microbial Growth in Batch Fermentation

As the cells in a batch

fermentation grow, they
follow a growth curve
similar to the one shown
here. The growth curve
contains four distinct
regions known as
phases. They are as
follows:
1) Lag Phase
2) Exponential Phase
3) Stationary Phase
4) Death Phase

Growth curve is from Shuler p. 161.

 Microbial Growth in Batch Fermentation

Lag Phase

• The first major phase of microbial growth in a batch fermentation

process
• A period of adaptation of the cells to their new environment
• Minimal increase in cell density
• May be absent in some fermentations

Shuler p. 161-162.
 Microbial Growth in Batch Fermentation

Exponential Phase

• The second major phase of microbial growth in a batch fermentation

process
• Also known as the logarithmic growth 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.
Shuler p. 162-163.
 Microbial Growth in Batch Fermentation

Exponential Phase (continued)

• Cell growth rate is often substrate

limited, as depicted in the figure to
the right.
• The growth curve is well
represented by Monod batch
kinetics, which is mathematically
depicted on the following slide.
Shuler, p. 163.
 Microbial Growth in Batch Fermentation

Exponential Phase (continued)

• Monod batch kinetics is represented mathematically in the

following equation:
µ = µ maxS
Ks + S
where µ is the specific growth rate, µ max is the maximum specific
growth rate, S is the growth limiting substrate concentration, and
KS is the saturation constant which is equal to the substrate
concentration that produces a specific growth rate equal to half
the maximum specific growth rate. All specific growth rates
account for the term µ – kd and should be considered to be µnet
values.

Shuler p. 176.
 Microbial Growth in Batch Fermentation

Exponential Phase (continued)

• 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

Shuler, p. 178-180.
 Microbial Growth in Batch Fermentation

Exponential Phase (continued)

• 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

Shuler, p. 178-180.
 Microbial Growth in Batch Fermentation

Stationary Phase

• The third major phase of microbial growth in a batch fermentation

process
• 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

Shuler p. 163-164.
 Microbial Growth in Batch Fermentation

Death Phase

• The fourth major phase of microbial growth in a batch

fermentation process
• Also known as the decline phase
• The rate of cells dying is greater than the rate of cells dividing
• Similar to Exponential phase, it is represented mathematically by
first order kinetics as the following:
dX = -kdX
dt

Shuler p. 164-165.
 Microbial Growth in Batch Fermentation

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

Shuler, p. 161.
 Microbial Growth in Batch Fermentation

Measuring the optical density with a

spectrophotometer is a quick and
easy way to 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
the optical density is that both viable Spectrophotometer pictured above is
and non-viable cells absorb this a copyright of Perkin Elmer

wavelength. As a result, the values

taken are not representative of only
viable cells.
 Batch Fermentation

Now that you understand how microbial cells grow in a batch

process, it is time to see how a general biotechnology fermentation
process works. An example, of a fermentation process is
represented in the block flow diagram shown below. The different
blocks depicted are described in detail in the following slides.

Inoculum
Shake Flask
Vial

1st Seed 2nd Seed Production

Media Prep Purification
Fermentor Fermentor Fermentor
 Batch Fermentation

First, a frozen vial containing a few

milliliters of one recombinant E. coli
strain is taken out of a freezer and
thawed. This vial is sometimes referred
to as an inoculum vial and its’ contents
is known as an inoculum.

After thawing, the inoculum is

transferred in a sterile manner to a
shake flask containing growth media.
This process is known as inoculation.
For E. coli, the initial pH of the media is
typically around 7 and is controlled by
using a buffering agent in the media. A
picture of a shake flask is depicted to
the right. The volume of media in the Shake flask photo above is a copyright
shake flask is usually on the order of of Kimax Kimble USA
magnitude of hundreds of milliliters.
 Batch Fermentation

After inoculation, the shake flask is

placed in an incubator shaker so the
cells can grow and reproduce. The
shaker is operated at a constant
temperature, which is around 37 °C for
E. coli. The shake flask holders in the
shaker are attached to an orbital plate
that rotates horizontally at a
programmable rate. This shaking
motion has two purposes:
• Keep the cells and the nutrients
in the growth media homogeneous
• Increases the rate of oxygen uptake
by the media for the aerobic E. coli
cells. The cells are grown to a
particular density near the end of
their exponential phase and used to Incubator shaker photo above is a copyright
of New Brunswick Scientific
inoculate a small fermentor known as
a seed fermentor.
 Batch Fermentation

A schematic of a fermentor is shown on the following slide. It is

representative of both a seed and a production fermentor. The E.
coli cells are supplied with filtered oxygen through the sparger
located at the bottom of the fermentor. The agitator is used to keep
the mixture of cells and growth media inside the fermentors
relatively homogeneous. It also increases oxygen mass transfer by
decreasing the size of the oxygen bubbles. The fermentor is
operated at a constant growth temperature to achieve the required
growth rate. Since cells liberate heat during growth, a constant
temperature is maintained using either cooling jackets surrounding
the fermentors, coils inside the fermentor, or a combination of both.
In addition, the cells secrete acids as they metabolize, which
decrease the pH level within the fermentor. As a result, a base is
usually added to the fermentor whenever the pH drops below its
optimum value.
 Batch Fermentation
Aspetic
inoculation pipe

Stirrer shaft seal

Working level

Baffle

Sample point
H

Impellar

Sterile air
line
Drain Point
Air sparger

D
Fermentor schematic is adapted from Stanbury, p. 168.
 Batch Fermentation

Once the cells are transferred to the

seed fermentor, they are grown to a
particular density near the end of
their exponential phase. The
picture presented to the right is of a
2.2 L glass laboratory scale seed
fermentor. The devices associated
with the fermentor and their function
are listed from left to right:
• Peristaltic Pump for pH
control through base addition
• Fermentor for cell growth Fermentor and associated equipment in the photo above is
a copyright of Applikon, Cole Parmer, and Chemcadet.
• Valves for oxygen flow rate
• Electronic devices for pH and
dissolved oxygen measurements
and controls for agitator speed
 Batch Fermentation

This picture shows several different sized laboratory scale fermentors.

Fermentors picture is from Stanbury, p. 173.

 Batch Fermentation

After the cells reach the required optical density in the seed
fermentor, the cells can either be used to inoculate several
increasingly larger seed fermentors until the required volume and
density is reached, or the cells can be transferred directly to the
production fermentor to where they will eventually synthesize the co-
protein. Typically, genetically engineered E. coli cells are grown to a
particular volume and density through a series of increasingly sized
fermentors. This group of seed fermentors is sometimes referred to
as a seed train.
 Batch Fermentation

After the cells reach their required volume

and density, they are transferred to the
production fermentor where they are grown
to a particular density. The density in
which they are grown to depends upon the
desired product being growth or non-
growth associated. For growth associated,
the cells are grown to their mid to late
exponential phase. At this point, a
chemical is added that induces the cells to
begin over-expressing the gene
responsible for the recombinant protein.
The over-expression of the particular gene
and the depletion of nutrients eventually
cause the cells to enter their stationary
growth phase. At this point, the cells are
no longer capable of producing appreciable 14 L Fermentor photo above is a
amounts of the desired protein and the copyright of New Brunswick Scientific
fermentation is ended.
 Fermentation Conclusion

Now that the fermentation process is over, the fermentation broth

containing the cells and the extracellular media is removed from the
production fermentor. This is called harvesting and that completes
the upstream process of fermentation. After the cells are harvested,
the recombinant protein needs to be separated from the cells that
produced them. This is accomplished through the downstream
process of purification.
 Fermentation Conclusion

The following is a list of references that can further explain the topics
discussed in this tutorial:
• Bailey, J. E. and D. F. Ollis, Biochemical Engineering
Fundamentals, 2nd ed., McGraw-Hill Book Co., New York, 1986.
• Brown, T. A., Gene Cloning and DNA analysis, 4th ed., Blackwell
Science Ltd, Oxford, 2001.
• Shuler, M. L. and F. Kargi. Bioprocess Engineering Basic
Concepts, 2nd ed., Prentice Hall, Upper Saddle River, NJ, 2002.
• Stanbury, P. F., A. Whitaker, and S. J. Hall, Principles of
Fermentation Technology, 2nd ed., Butterworth Heinemann,
Oxford, 2000.

This concludes the upstream biotechnology process known as

fermentation and brings us to the end of the fermentation tutuorial.
Please proceed to the Purification Tutorial for information regarding
downstream processing.