HARAMAY UNIVERSITY

HARAMAYA INSTITUTE OF TECHNOLOGY

Introduction to Biochemical
Engineering

By: Workisa .B
 Chapter 5

CELL KINETICS AND FERMENTER DESIGN

 Introduction
 Understanding of the cell kinetics of microbial, animal, or
plant cells is important=> design

 Cell kinetics deals with the rate of cell growth and how it is
affected by various chemical and physical conditions.

 Unlike enzyme kinetics , cell kinetics is the result of

numerous complicated networks of:

Biochemical

Chemical reactions

Transport phenomena
 Accurate mathematical modelling of growth kinetics is impossible
(due to heterogeneous mixture of old cell with young cells..) to achieve.

 Therefore, we must make assumptions to be able to arrive at simple

models which are useful for fermenter design and performance predictions.

 Unstructured, distributed is the simplest model based on the following

assumptions:

1. Cells can be represented by a single component, such as cell mass ,

cell number, or the concentration of protien, DNA, or RNA.

2. The population of cellular mass is distributed uniformly

throughout the culture. The heterogeneous nature of the cells can
be ignored.
 Besides the assumptions for the cells, the medium is formulated so
that only one component may be the limiting the reaction rate.

 Minor changes do not significantly affect the reaction rate.

 Fermenters are also controlled so that the environmental parameters

such as PH, temperature, and dissolved oxygen concentration are
maintained at constant level.
Terminologies in Microbial growth
The definitions of terminologies

The growth rate can be defined in several different ways:

• e.

 dCx/dt is the change of the cell concentration in a fermenter , which

may include the effect of

Input and output flow rates

Cell recycling

And other operating conditions of the fermenter

o rx=dCx/dt  only for batch operations

 The growth rate based on the number of cells and that based on
cell weight are not necessarily the same b/c the average size of the
cells may vary considerably from one phase to another.
• At exponential phase the growth rate

 based on the cell number and based on cell weight are

proportional.

The growth rate can be confused with the division rate, which is
defined as the rate of cell division per unit time.

• if all of the cells in a vessel at time t=0(CN=CN0) have divided once

after a certain period of time ,

CN=CNo*2N

And the average division rate is

 The division rate at time t is

 Growth rate  the slope of the CN vs t curve

 Division rate  the slope of

 The division rate is constant during the exponential growth period,

while the growth rate is not.
 Growth cycle for Batch cultivation
 If you inoculate unicellular microorganisms into a fresh sterilized medium

 The plot of cell number density with respect to time has six phases

1. Lag phase – no cell number change

2. Accelerated growth phase– CN starts to increase and d increases to reach

maximum.

3. Exponential growth phase- CN increases exponentially and d is

constant at its max. value.

4. Decelerated growth phase- deceleration of both growth rate and the

division rate.

5. Stationary phase- CN reach maximum

6. Death phase- the cell starts to die

1. Lag phase
• The change of cell number is negligible or zero

• The cells may grow in size.

• It is intialy stationry or latent.

The length of lag phase depend on

The type and age of the microorganisms

The size of inoculum

The culture conditions

The lag phase usually occurs

• When the cells must adjust to the new medium

If MO are inoculated from a medium with low concentration

to a medium with high concentration length the lag period
is usually long

If the MO are moved from high to low nutrient

concentration no lag phase

If a small amount of cells are inoculated into a large volume

a long lag phase
For large scale (large fermenter) production to minimize the
length of lag phase

 we need to have a series of progressively larger

seed tanks to minimize the effect of lag phase

At the end of the lag phase, when the growth begins, the
division rates increases gradually and reaches a maximum
value I the exponential growth period
 2. Exponential growth phase
 The progressive doubling of cell no. results in  increase the rate of
growth

 If a bacterial culture undergoing Balanced growth(1st order)

 The rate of cell population increase at any particular time

where m is the specific growth rate(hr-1)

 The growth rate is d/t from the specific growth rate

 The specific growth rate

• If m is constant with time during the exponential growth
period

• CN0 is the cell number concentration at t0 (at the beginning of

exponential growth)
• The doubling time (td), by setting CN=CNo and to=0

• The td is inversely proportional to the specific growth rate and

is reciprocal of the division rate
Factors affecting the specific growth rate
a. Substrate concentration the effect of substrate concn. On
m is the Monod equation, which is an empirical expression

where Ks is the system coeff

• m= 1/2mmax  the value of Ks=Cs

• The Monod equation is an over simplifications of the

complicated mechanisms of cell growth

• An increase of Cs , m reaches mmax and ,further increase doesn’t

affect the specific growth rate
However, it has been observed that the specific growth rate
decreases as the Cs is increased beyond a certain level.

Several other model is proposed to improve the Monod

equation.

They are:
• If several substances can limit the growth of a microorganism, the following
model ca be employed

• If the limiting nutrient is the energy source for the culture, a certain amount
of substrate can be used for purposes other than growth. Some model
include a term, Ke (maintenance of cells)
b. Product concentration

• The effect of product inhibition on the growth of MO is added to

Monod equations as follows:

or

Cpm is the max. Cp above which cells cannot grow due to product
inhibition.
c. Other conditions
• The specific growth rate of microorganisms is affected by

 Medium PH

 Temperature

 Oxygen supply

The optimum PH and temperature differ from one

organism to another
 3. Stationary and death phase
• the growth of microbial population is normally limited
 by exhaustion of available nutrients or
 by accumulation of toxic products of metabolism
• cells in the stationary phase have a chemical composition different from that of
cells in the exponential phase.
• death occurs b/c of
-the depletion of the cellular reserves of energy
- the accumulation of toxic products
• like growth , death is an exponential function
• in some case, the organisms not only die but also disintegrate, a process called
lysis.
 Stirred-tank Fermenter
The bioreactor is frequently called a fermenter , if the transformation is
takes place by a living cells or in vivo cellular components (enzymes).

-fermenter employing living cells

-enzyme reactor employing enzyme

 In laboratories cells are cultivated in Erlenmeyer flasks on shaker.

 The gentle shaking is very effective

-to suspend the cells

-to enhance the oxygenation through the liquid

surface and

-to aid the mass transfer of nutrients

without damaging the cells

 for large-scale production , the stirred tank fermenter is the
most widely used design in industrial fermentation.

 the STF can be used for high viscosity media

 widely used in pharmaceuticals industries

 STF usually built with stainless steel and operated in mild

operating conditions, and the life expectancy of the fermenter
is also long
• the disadvantages of STF

the agitator consumes a large amount of energy

 can damage a shear sensitive cells

 As the blade width to diameter ratio increases the
velocity profile becomes a more blunt, less parabolic
shape, which yields a lower amount of shear due to a more
gradually velocity gradient

 By increasing the width to diameter ratio STF is

employed in cultivating animal and plant cells
 Large Fermenters
 made of stainless steel

 h/d ratio of vessel-2:1 or 3:1

 agitated with 2 or 3 turbine impellers

 the agitator shaft enter from the top or bottom of the vessel

 impeller diam./tank diamet. is 0.3 to 0.4

 in the case of 2 impeller the distance b/n two impellers is 1.5

impeller diameters

 in the case of 3 the distance is equal to one impeller

diameter
 4 equally spaced baffles are placed/installed to prevent the
vortex

 the width of the baffle is usually 1/10 of tank diameter

 for aerobic fermentation, a single orifice is needed in b/n the

bottom impeller and the bottom vessel to aerate the fermenter

 PH controller

 the heating and the cooling system  to control the

temperature
 Batch or Plug-Flow Fermenter
 Ideal stirred fermenter is assumed to be well mixed

 In tubular flow fermenter, nutrients and MOs enter one end of a

cylindrical tube and the cells grow while they pass through

 The fluid properties variation in radial direction is small as

compared to the variation in the longitudinal direction

 The ideal tubular flow fermenter without radial variations is

called a plug flow fermenter (PFF).

 The packed bed fermenter and multistage fermenter can be

approximated as PFF.

 The batch fermenter is the same as a steady state PFF

 the change of cell concentration in batch fermenter is equal
to the growth rate of cells in it

 To derive the performance equation of a batch

fermentation,
 Ideal continuous stirred tank fermenter
 The growth chamber is connected to a reservoir of sterile
medium

 Once growth has been initiated, fresh medium is continuously

supplied from the reservoir
 Continuous culture systems can be operated as chemostat or
as turbidostat.

 In chemostat the flowrate is set at a particular value and the

rate of growth of culture adjusts to this flowrate.

done by setting the pump at a constant flowrate

 In turbidostat the turbidity is set at constant level by

adjusting the flowrate

requires an optical sensing device and a controller

 Therefore, chemostat is easier to operate than turbidostat

 The material balance for MO in CSTF:
FCxi – FCx + Vrx=VdCx/dt

where rx=the rate of cell growth in the fermenter

dCx/dt=the change of cell concentration in

the fermenter with time.

• For steady state operation of a CSTF,

dCx/dt=0

The above equation shows that :

the residence time=the area of the rectangle of the width

Cx-Cxi, and height 1/rx on the 1/rx versus Cx curve.
• The shorter the residence time in reaching a certain cell
concentration, the more effective the fermenter.

• If the input stream is sterile (Cxi=0), and the cell in the

CSTF are growing exponentially(rx=mCx), then the above
equation simplified to
Evaluation of monod kinetic parameters
• For steady state CSTF

the specific growth rate is equal to the dilution rate

It is helpful in studying the effects of various components of the medium on

the specific growth rate.

by measuring the steady state substrate concentration at various flow

rates, various kinetic models can be tested and the value of the kinetic
parameters can be estimated.

• By rearranging the monod equation

where, m=D for chemostat

 Undue weight to measurementsat low Cs

 Insufficient weight measurementsat high Cs

 For better estimation, we can rearrange as follows

 However, the limitation of this approach to determine the

kinetic parameters is in the difficulty of running a CSTF.

 The batch runs in a stirred fermenter is not difficult to carryout

• For CSTF runs

we need to have nutrient and product reservoirs which are

connected to the fermenter aseptically

we need to control input and output stream

the control of outlet flow rate is difficult due to the foaming

there is high risk for the fermenter to be contaminated

it is difficult to reach a steady sate b/c of cell’s mutation

and adaptation to new environment
Example
Solution
 Productivity of CSTF
 The productivity of fermenter is expressed as the amount of a
product produced per unit time and volume.

 If the inlet stream is sterile (Cxi=0), the productivity of cell mass is

equal to Cx/tm, which is equal to the slope of straight line of the Cx
vs tm curve.
• At point A,
the Cx of the outlet stream is low
the residence time is short
therefore, more medium passes through
Point A is unstable region because it is very close to the washout
point D, and a small fluctuation of residence time will cause a large
change in the cell concentration.
• At point B,
the Cx of the outlet stream is high
the residence time is long
so a smaller amount of medium passes through
 As the slope of the line increases, the productivity
increases
 The slope of line will have its maximum value when it is
tangent to Cx (point C).
 The max. value of productivity is equal to the slope of
line OC.
 The operating condition for the max. productivity of the
CSTF can be estimated graphically by using 1/rx vs Cx
curve.
 Max. productivity can be attainedat min. residence time
 residence time=area of rectangle under Cx and 1/rx on
the curve
The cell productivity for steady-state CSTF with a sterile feed
is

The productivity is max. when drx/dCx=0

• The optimum cell concentration for the maximum productivity as
 Comparison of Batch and CSTF
The residence time required for batch or steady state PFF to reach a certain
level of concentration is

Where, to is the time required to reach an exponential growth phase

Since the 1/rx vs Cx curve is U shaped, we can make the following

conclusions for a single fermenter.

1. the most productive fermenter system is a CSTF operated at the cell

concentration at which the value of 1/rx is minimum b/c it requires the
smallest residence time.

2. if the final Cx to be reached is in the stationary phase, the batch fermenter

is better choice than the CSTF b/c the t required for Batch is smaller than
that for the CSTF.
 Multiple fermenters connected in series

• In the 1/rx vs Cx curve,

if the final Cx < Cx,opt, one fermenter is better than two
fermenters connected in series(2 ferm. Needs more t)

if the Cx>>Cx,opt. , the combinations of two fermenters for a

minimum total t is a CSTF operated at Cx,opt. followed by a PFF

or a combinations of two CSTF operated at Cx,opt,

followed by another CSTF connected in series is also better

than one CSTF.
 CSTF and PFF in series

• For the 1st CSTF , if the input stream is sterile (Cxi=0),

For the 2nd PFF, the residence time can be estimated by

 if Cx2 is known, Cs2 can be calculated from Yx/s

 Multiple CSTFs in series

• For the nth steady state CSTF, the material balance for microorganisms
can be
By solving the above three equation simultaneously, we can calculate
either dilution rate D, with the known Cx, or vice versa.

The estimation of the Cx or Cs with known dilution rate can be done

easily by using graphical technique.

The D1 of the first reactor when the inlet stream is sterile is

• For the 2nd fermenter

• By knowing the D1 and D2 of each fermenter, you can

estimate the Cx of each fermenter, or vice versa.
Example
• dsd
 Cell Recycling
 If the cells are discharged with the outlet stream which

limits the productivity of fermenters

 We can improve the productivity by recycling the cells

from the outlet stream

 PFF with Cell Recycling
A PFF requires the initial presence of microorganisms in the
inlet stream as a batch fermenter requires initial inoculum.

Unlike the CSTF, the PFF doesn’t require the cell separator
in order to recycle.
• The performance equation of the PFF with Monod kinetics can be
written as:

• If the growth yield is constant,

• By substituting for Cs and integrating will result

Where C’x and C’s can be estimated from the cell and
substrate balance at the mixing point of the inlet and the
recycle stream as
 The cell concentrations of the outlet stream can be
estimated from the overall cell balance

 The cell concentrations of the recycle stream can be

estimated from the overall cell balance over the filter as

 Where b is the bleeding rate which is defined as

 CSTF with Recycling
 The cellular productivity in CSTF increases with an increase in the
Dilution rate D, and reaches a maximum value.

 If the increment D is above the maximum point, the productivity

decreased abruptly and the cells will start to be washed out.

 One way to improve the fermenter productivity is to recycle the cell by

separating the cells from the product stream using a cross flow filter unit
 If all cells are recycled to the fermenter, Cx will increase
continuously with time and a steady state will never be
reached.

 If we need to operate CSTF at a steady state mode, we

need to have a bleeding stream

 The material balance for cells in the fermenter with a cell

recycling unit is
• For a steady state CSTF with cell recycling and a sterile feed,

• If the growth rate can be expressed by Monod kinetics,

• As b is reduced from 1(no recycling) to 0.5, the cell
productivity is doubled.
Alternative Fermenters

Reading assignment

1. Column fermenter
2. Loop fermenter