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Cell Kinetics and Fermenter Design

Ref.: Chap 6, Biochemical Engineering, James M. Lee


Prepared by:
Engr. Ray Limuel C. Laureano

Introduction
Understanding the growth kinetics of microbial,
animal, or plant cells is important for the
design and operation of fermentation systems
employing them. Cell kinetics deals with te
rate of cell growth and how it is affected by
various chemical and physical conditions.

Introduction
Cell kinetics is the result of numerous
complicated networks of biochemical and
chemical reactions and transport phenomena,
which involves multiple phases and multicomponent systems. During the course of
growth, the heterogeneous mixture of young
and old cells is continuously changing and
adapting itself in the media environment
which is also continuously changing in physical
and chemical conditions. As a result, accurate

Introduction
mathematical modeling of growth kinetics is
impossible to achieve. Even with such a
realistic model, this approach is usually
useless because the model may contain many
parameters which are impossible to
determine.

Introduction
Assumptions are made to be able to arrive at
simple models which are useful for fermenter
design and performance predictions. The
simplest model is the unstructured, distributed
model which is based on the following
assumptions:
1. Cells can be represented by a single
component, such as cell mass, cell number,

Introduction
or concentration of protein, DNA, or RNA. This is
true for balanced growth, since douling of cell
mass for balanced growth is accompanied by
doubling of all other measurable properties of
the cell population.
2. The population of cellular mass is distributed
uniformly throughout the culture. The cell
suspension can be regarded as homogeneous
solution. The heterogeneous nature of cells can

Introduction
be ignored. The cell concentration can be
expressed as dry weight per unit volume.
The medium is formulated so that only one
component may be limiting the reaction rate.
All other components are present at
sufficiently high concentrations, so that minor
changes do not significantly affect the reaction
rate.

Introduction
Fermenters are also controlled so that
environmental parameters such as pH,
temperature, and dissolved oxygen
concentration are maintained at a constant
level.

Various Models for Cell Kinetics


Cell Components
Population

Unstructured
Distributed Cells are represented
by a single component,
which is uniformly
distributed throughout
the culture.
Segregated Cells are represented
by a single component,
but they form a
heterogeneous
mixture.

Structured
Multiple cell components,
uniformly distributed
throughout the culture
interact with each others.
Cells are composed of
multiple components and
form a heterogeneous
mixture.

Definitions
CX
= cell concentration, dry cell weight per unit volume
CN
= cell number density, number of cells per unit volume

= cell density, wet cell weight per unit volume of cell mass
dCX/dt = change of dry cell concentration with time
rX
= growth rate of cells on a dry weight basis
dCN/dt = change of cell number density with time
rN
= growth rate of cells on a number basis

= division rate of cells on a number basis


= dlog2CN/dt

Definitions
dCX/dt and rX:
dCX/dt change of cell concentration in fermenter, which may include
the effects of the input and the output flow rates, cell recycling, and
other operating conditions of a fermenters
rX actual growth rate of the cells
*rX = dCX/dt in BATCH operation
dCN/dt and dCX and dt:
Growth rate based on the number of cells and that based on cell
weight are not necessarily the same because the average size of
the cells may vary considerably from one phase to another. When
the mass of an individual cell increases without division, the
growth rate based on cell weight increases, while that based on
the number of cells stays the same.

Definitions
During exponential growth period, which is the phase
that we are most interested in from an engineers point
of view, the growth rate based on the cell number and
that based on cell weight can be assumed to be
proportional to each other.
dCX/dt, dCN/dt, dlog2CN/dt ()
Sometimes, the growth rate can be confused with the division
rate, which is defined as the ratio of cell division per unit
time.
N
C N C N0 2

Definitions
Average division rate:

t
1
log2 C N log2 C N0
t

Division rate at time t:

d log2 C N

dt

Definitions
The growth rate defined as the change in cell number with
time is the slope of the CN vs t curve, while the division rate is
the slope of the log2CN vs t curve. As explained later, the
division rate is constant during the exponential growth
period, while the growth rate is not.

Growth Cycle for Batch Cultivation


If you inoculate unicelullar microorganisms into a
fresh sterilized medium and measure the cell
number density with respect to time and plot it,
you may find that there are six phases of growth
and death.
1. Lag phase: A period of time when the change of
cell number is zero.
2. Accelerated growth phase: The cell number
starts to increase and the division rate increases
to reach a maximum.

Growth Cycle for Batch Cultivation


3. Exponential growth phase: The cell number
increases exponentially as the cells start to divide.
The growth rate is increasing during this phase,
but the division rate which is proportional to
dlnCN0/dt , is constant at its maximum value.
4. Decelerated growth phase: After the growth rate
reaches a maximum, it is followed by the
deceleration of both growth rate and division
rate.

Growth Cycle for Batch Cultivation


5. Stationary phase: The cell population will
reach a maximum value and will not increase
any further.
6. Death phase: After nutrients available for the
cells are depleted, cells will start to die and
the number of viable cells will decrease.

Growth Cycle for Batch Cultivation

Lag Phase
The lag phase (or initial stationary, or latent) is an
initial period of cultivation during which the
change of cell number is zero is negligible or zero.
Even though the cell number does not increase,
the cells may grow in size during this period.
Length of this period is affected by:
(a) Type and age of MOs
(b) Size of inoculum
(c) Culture conditions

Lag Phase
If MOs are inoculated from a medium with a
low nutrient concentration to a medium with
a high concentration longer lag phase period
because the cells must produce the enzymes
necessary for the metabolization of the
available nutrients
If the MOs are moved from high to low
nutrient concentration lag phase period is
short

Lag Phase
Size of inoculum if a small amount of cells are
inoculated into a large volume, longer lag phase
period
*For a large-scale operation of the cell culture, it is
our objective to make this lag phase as short as
possible.
At the end of the lag phase, when growth begins,
the division rate increases gradually and reaches
a maximum value in the exponential growth
phase. This is called the accelerated growth
phase.

Exponential Growth Phase


In unicellular organisms, the progressive
doubling of cell number results a continually
increasing rate of growth in the population. A
bacterial culture undergoing balanced growth
mimics a first-order autocatalytic chemical
reaction (Carberry, 1976; Levenspiel, 1972).
Therefore, the rate of the cell population
increase at any particular time is proportional
to the number density (CN) of bacteria present
at that time:

Exponential Growth Phase


dCN
rN
C N
dt

Where:
= specific growth rate (h1)
1 dCN d ln C N

C N dt
dt

Exponential Growth Phase


d ln C N
d log2 C N

ln 2
dt
dt

ln 2

If is constant with time during the exponential


growth period, then
CN

CN 0

dCN
dt
CN

t0

upon integration:
CN CN 0 exp t t0

Exponential Growth Phase


Doubling time, td, is given by
td

ln 2

Classification of microorganisms in
terms of growth-rate on temperature
Group
Thermophiles
Mesophiles
Psychrophiles
Obligate
Facultative

Temperature, C
Minimum
Optimum
Maximum

R. Y. Stanier, M. Doudoroff, and E. A. Adelberg, The Microbial World,


3d ed., p. 316, Prentice-Hall, Inc. Englewood Cliffs, N.J., 1970.

Factors Affecting the Specific Growth


Rate
Substrate Concentration: Monod equation
empirical expression based on the form of
equation normally associated with enzyme
kinetics or gas adsorption:

maxCS
K S CS

KS is equal to the concentration of nutrient


when the specific growth rate is half of its
maximum value (max).

Factors Affecting the Specific Growth


Rate
Several other models have been proposed to
improve the Monod equation:

maxCS

K I 1 CS K I 2CS 2

max 1 e CS / K S
max

1 K S CS

max

n C S

Factors Affecting the Specific Growth


Rate
If several substances can limit the growth of a
microorganism, the following model is
employed:
C1
C2
max
...
K1 C1 K 2 C2

A term ke is used when the limiting nutrient is


the energy source for the culture

maxCS
K S CS

ke

Factors Affecting the Specific Growth


Rate
Product Concentration: As cells grow they
produce metabolic by-products which can
accumulate in the medium. The growth of
microorganisms is usually inhibited by the
products, whose effect can be added to the
Monod equation as follows:
CS
max
K S CS

K P

K P C P

CS
max
K S CS

CP
1

C Pm

Factors Affecting the Specific Growth


Rate
Which CPm is the maximum product
concentration above which cells cannot grow
due to product inhibition
Other conditions: The specific growth rate of
MOs is also affected by medium pH,
temperature, and oxygen supply. The
optimum pH and temperature differ from one
MO to another.

Stationary Phase and Death Phase


The growth of microbial populations is normally limited
either by the exhaustion of available nutrients or by
the accumulation of toxic products of metabolism. As a
consequence, the rate of growth declines and growth
eventually stops. At this point a culture is said to be in
the stationary phase.
The stationary phase is usually followed by a death phase
in which the organism in the population die. Death
occurs either because of the depletion of the cellular
reserves of energy, or the accumulation of toxic
products. Like growth, death is an exponential
function. In some cases, the organisms not only die but
also disintegrate, a process called lysis.

Batch or Plug-Flow Fermenter


An ideal stirred fermenter is assumed to be well
mixed so that the contents are uniform in
compositions at all times. Another ideal
fermenter is the plug-flow fermenter, the
analysis of which is analogous to the ideal
batch fermenter.

Batch or Plug-Flow Fermenter


In a tubular-flow fermenter, nutrients and
microorganisms enter one end of a cylindrical
tube and the cells grow while they pass
through. Since the long tube and lack of
stirring device prevents complete mixing of
the fluid, the properties of the flowing stream
will vary in both longitudinal and radial
direction. However, the variation in the radial
direction is small compared to that in the

Batch or Plug-Flow Fermenter


longitudinal. The ideal tubular-flow fermenter
without radial variations is called a plug-flow
fermenter (PFF). In reality, the PFF fermenter
is hard to be found. However, the packed-bed
fermenter and multi-staged fermenter can be
approximated as PFF.

Batch or Plug-Flow Fermenter


Even though the steady-state PFF is operated in
a continuous mode, the cell concentration of
an ideal batch fermenter after time t will be
the same as that of a steady-state PFF at the
longitudinal location where the residence
time is equal to t.

Batch or Plug-Flow Fermenter

Batch or Plug-Flow Fermenter


If liquid medium is inoculated with a seed
culture, the cell will start to grow
exponentially after the lag phase.
dCX
rX C X
dt

Integrating the above eqn gives


CX

CX 0

dCX

rX

CX

CX 0

dCX
dt t t0
C X

t0

Batch or Plug-Flow Fermenter


According to the prior eqn, the batch growth
time t t0 is the area under the 1/rX versus CX
curve between CX0 and CX as shown below.

Batch or Plug-Flow Fermenter


At this time just note that the curve is U shaped,
which is characteristic of autocatalytic reactions:

SX X X
The rate for an autocatalytic reaction is slow at the
start because the concentration of X which acts
as a biological catalyst is low. It increases as cells
multiply and reaches a maximum rate. As the
substrate is depleted and the toxic products
accumulate, the rate decreases to a low value.

Batch or Plug-Flow Fermenter


If Monod kinetics adequately represents the
growth rate during the exponential period:
CX

CX 0

K S CS dCX
maxCS C X

dt
t0

The growth yield (YX/S) is defined as


YX / S

CX CX 0
C X

CS CS 0 CS

Batch or Plug-Flow Fermenter


Cell concentration change with respect to time:

CX
K S YX / S
K S YX / S
C
t t0 max
1 ln

ln S 0
CS
C X 0 CS 0YX / S
C X 0 C X 0 CS 0YX / S

MM eqn VS Monod eqn


rmaxCS
dCP

dt
K M CS

dCP maxCS C X

dt
K S CS

Batch or Plug-Flow Fermenter


Monod kinetic parameters, max and KS, cannot
be estimated with a series of batch runs as
easily as the MM parameters for an enzyme
reaction. In the case of cell cultivation, the
initial rate of reaction in a batch run is always
zero due to the presence of a lag phase,
during which Monod kinetics does not apply.
It should be noted that even though the
Monod equation has the same form as the
MM equation, the rate eqn is different.

Ideal Continuous Stirred-tank


Fermenter
Chemostat
Cellular growth is usually limited by one
essential nutrient, and other nutrients are in
excess. For a chemostat at steady state, the
nutrient, product, and cell concentrations are
constant. For this reason, the name chemostat
refers to constant chemical environment (for
cell culture).

Ideal Continuous Stirred-tank


Fermenter

Ideal Continuous Stirred-tank


Fermenter
A turbidostat is a CSTF in which the cell
concentration in the culture vessel is
maintained constant by monitoring the optical
density of the culture and controlling the feed
flow rate.

Ideal Continuous Stirred-tank


Fermenter

Ideal Continuous Stirred-tank


Fermenter
Microbial population can be maintained in a state
of exponential growth over a long period of time
by using a system of continuous culture.
Continuous culture can be operated as chemostat
or turbidostat.
Chemostat flow rate is set at a particular value
and the rate of growth of the culture adjusts to
this flow rate
Turbidostat turbidity is set at a constant level by
adjusting the flow rate

Ideal Continuous Stirred-tank


Fermenter

Ideal Continuous Stirred-tank


Fermenter
Material Balance
Input Ouput + Generation = Accumulation
dCX
FC Xi FC X Vr X V
dt

For steady-state condition CSTF: dCX/dt = 0


V C X C Xi
m
F
rX

Ideal Continuous Stirred-tank


Fermenter
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
cells in a CSTF are growing exponentially (rX =
CX)
1

1
m
D

Ideal Continuous Stirred-tank


Fermenter
The specific growth rate of a microorganism can be
controlled by changing the medium flow rate and
CS can be calculated with a known residence time
and the Monod kinetic parameters as:
CS

KS

m max 1

Note: eqn above is only applicable when mmax > 1.


If mmax < 1, the growth rate of the cells is less
than the rate of cells leaving the outlet stream.
Consequently, all of the cells in the fermenter will
be washed out.

Ideal Continuous Stirred-tank


Fermenter
If the growth yield is constant, then
C X YX / S CSi CS
Correlation of CX:
For CP:

KS

C X YX / S CSi
m max 1

C P C Pi

KS

YP / S CSi
m max 1

Evaluation of Monod Kinetic


Parameters
If a certain microorganism follows Monod
kinetics, the plot of 1/ versus 1/CS yields the
values of max and KS by reading the intercept
and the slope of the straight line. However,
1/ approaches infinity as the substrate
concentration decreases. This gives undue
weight to measurements made at low
substrate concentrations and insufficient
weight to measurements at high substrate
concentrations.

Evaluation of Monod Kinetic


Parameters
Linear Equations in the determination of Monod
kinetic parameters:
1

KS

CS

KS

max max CS

max

max

max K S

CS

CS

Example 6.2
A chemostat study was performed with yeast.
The medium flowrate was varied and the
steady-state concentration of cells and glucose
in the fermenter were measured and
recorded. The inlet concentration of glucose
was set at 100 g/L. The volume of the
fermenter contents was 500 mL. The inlet
stream was sterile.

Example 6.2
Flow rate
F, mL/h

Cell Concentration
CX, g/L

Substrate Concentration

31
50
71
91
200

5.97
5.94
5.88
5.76
0

0.5
1.0
2.0
4.0
100

CS, g/L

a. Find the rate equation for cell growth.


b. What should be the range of the flow rate to
prevent washout of the cells?

Example 6.2
Given:
F
CSi = 100 g/L

V = 500 mL
*inlet stream is
sterile
CS
CX

Example 6.2
Given:
Data:
Flow rate
F, mL/h
31
50
71
91
200

Cell Concentration Substrate Concentration


CS, g/L
CX, g/L
5.97
0.5
5.94
1.0
5.88
2.0
5.76
4.0
0
100

Example 6.2
Required:
a. rate eqn for cell growth
b. flow rate to prevent washout of cells

Example 6.2
Solution:
a. Making use of the linear form of Monod eqn to
solve for the kinetic parameters max and KS:
1

Note:

max
1
V
m

KS

1
max CS

*Exclude point when F = 200 mL/h because we


need to consider only the exponential growth
phase.

Example 6.2
Solution:
Upon linear regression:
max = 0.2514 h1
KS = 1.5256 g/L
Therefore:
maxCS
rX C X

K S CS

CX

0.2514CS
g
rX
CX

1.5256 CS
L h

Example 6.2
Solution:
b. To prevent washout of cells, the cell
concentration should be maintained so that it
will be greater than zero.

KS
0
C X YX / S CSi
m max 1

Solving for m gives:


m

V K S CSi

F CSi max

Example 6.2
Solving for F
VC Si max
F
K S C Si

0.5L 100 g/L 0.2514h1


F
1.5256g/L 100 g/L

F 0.1238 L/h

Example 11-1/p. 561] Bioprocess Engineering: Kinetics,


Biosystems, Sustainability, and Reactor Design by Shijie Liu

A strain of mold was grown in a batch culture on


glucose and the following data were obtained as
shown in the table. Do the following:
(a) Calculate the maximum net specific growth rate.
(b) Calculate the apparent growth yield.
(c) What maximum cell concentration could one
expect if 150 g of glucose were used with the
same size inoculum?
(d) How many generations of cells are there in the
culture for part (c)?

Example 11-1/p. 561] Bioprocess Engineering: Kinetics,


Biosystems, Sustainability, and Reactor Design by Shijie Liu

Example 11-1/p. 561] Bioprocess Engineering: Kinetics,


Biosystems, Sustainability, and Reactor Design by Shijie Liu
(a) To obtain the maximum net specific growth rate, we
calculate the rates using the finite difference scheme as
illustrated in the following table:

Example 11-1/p. 561] Bioprocess Engineering: Kinetics,


Biosystems, Sustainability, and Reactor Design by Shijie Liu

From the table, we find that the maximum net


specific growth rate, max 0.0752/h

CX
20.9 1.0
(b)YX/S

0.4004
CS 50.0 0.3
(c) CX,max CX0 YX/SCS0 1.0 0.4004150 61.0604 g cells/L
lnCX CX0 ln61.0 1.0

5.93 6
(d) n
ln2
ln2

Problem 11.1/p. 601] Bioprocess Engineering: Kinetics,


Biosystems, Sustainability, and Reactor Design by Shijie Liu

A simple, batch fermentation of an aerobic


bacterium growing on methanol gave the results
shown on the table. Calculate:
(a) Maximum growth rate, max
(b) Specific growth rate of the cells, YX/S
(c) Mass doubling time, td
(d) Saturation constant, KS
(e) Specific growth rate, net at t = 10 h

Problem 11.1/p. 601] Bioprocess Engineering: Kinetics,


Biosystems, Sustainability, and Reactor Design by Shijie Liu

Problem 11.2/p. 601] Bioprocess Engineering: Kinetics,


Biosystems, Sustainability, and Reactor Design by Shijie Liu
The data in Table P11.2 were obtained for Pyrodictium occultum
at 98C. Run 1 was carried out in the absence of yeast extract
and run 2 with yeast extract. Both runs initially contained Na 2S.
The vol% of the growth product H2S collected above the broth
was reported as a function of time as shown in Table P11.2.
(a) What is the lag time with and without the yeast extract?
(b) What is the difference in the maximum specific growth rates,
max, of the bacteria with and without the yeast extract?
(c) How long is the stationary phase?
(d) During which phase does the majority production of H2S
occur?

Problem 11.2/p. 601] Bioprocess Engineering: Kinetics,


Biosystems, Sustainability, and Reactor Design by Shijie Liu

Problem 11.4/p. 604] Bioprocess Engineering: Kinetics,


Biosystems, Sustainability, and Reactor Design by Shijie Liu

The data in Table P11.4 were obtained for the


effect of temperature on the fermentative
production of lactic acid by a strain of
Lactobacillus delbruekii. From these data,
calculate the value of the activation energy for
this process.

Problem 11.4/p. 604] Bioprocess Engineering: Kinetics,


Biosystems, Sustainability, and Reactor Design by Shijie Liu

Supplementary Problems
1. Escherichia coli grows with a doubling time of 0.5 h in
the exponential growth phase. (a) What is the value
of the specific growth rate? (b) How much time would
be required to grow the cell culture from 0.1 kg dry
cell/m3 to 10 kg dry cell/m3?
2. E. coli grows from 0.10 kg dry cell/m3 to 0.50 kg dry
cell/m3 in 1 h. (a) Assuming the exponential growth
during this period, evaluate the specific growth rate.
(b) Evaluate the doubling time during the exponential
growth phase. (c) How much time would be required
to grow from 0.10 kg dry cell/m3 to 1.0 kg dry
cell/m3? You may assume the exponential growth
during this period.

Productivity of CSTF
Normally, the productivity of the fermenter is
expressed as the amount of product
produced per unit time and volume. If the
inlet stream is sterile (CXi = 0), the
productivity of the cell is equal to CX/m.

Productivity of CSTF
Cell Concentration and Residence Time for
Maximum Productivity
The cell productivity at steady-state CSTF
CX

rX

maxCS

K S CS

CX

The productivity is maximum when

drX
0
dC X

Productivity of CSTF
Optimum cell concentration for maximum
productivity
C X ,opt

YX / S CSi
1

Where:

K S CSi
KS

Productivity of CSTF
Optimum substrate concentration:
CS ,opt

CSi

Optimum residence time:


m,opt

max 1

Comparison of Batch and CSTF


Since 1/rX versus CX curve is U shaped, the following
assumptions are applied:
1. The most productive fermenter system is a CSTF
operated at the cell concentration as which
value 1/rX is minimum because it requires the
smallest residence time.
2. If the final cell concentration to be reached is in
the stationary phase, the batch fermenter is a
better choice than the CSTF because the
residence time required for the batch is smaller
than that for CSTF.

Comparison of Batch and CSTF


The residence time required for a batch or
steady-state PFF to reach a certain level of
cell concentration is
b t0

CX

CX 0

dC X
rX

Multiple Fermenters Connected in


Series
Choosing the optimum fermenter system for
maximum productivity depends on the shape of
the 1/rX versus CX curve and the process
requirement, such as the final conversion.
In the 1/rX versus CX curve, if the final cell
concentration is less than CX,opt, one fermenter
is better than two fermenters connected in
series, because two CSTFs connected in series
require more residence time than one CSTF
does.

Multiple Fermenters Connected in


Series
If the final cell concentration is much larger than
CX,opt, the best combination of two
fermenters for a minimum total residence
time is a CSTF operated at CX,opt followed by a
PFF. A CSTF operated at CX,opt followed by
another CSTF connected in series is also
better than one CSTF.

Multiple Fermenters Connected in


Series

Multiple Fermenters Connected in


Series

CSTF and PFF in Series

CSTF and PFF in Series


Working Equations
For the CSTF
C S1

KS

m max 1

KS

C X 1 YX / S CSi
m1 max 1

C P1 C Pi

KS

YP / S CSi
m1 max 1

CSTF and PFF in Series


Working Equations
For the PFF
P2

CX 2

CX 1

YX / S

dCX

rX

CX 2

CX 1

K S CS dCX
maxCS C X

CX 2 CX1
C S1 C S 2

C
K S YX / S
K S YX / S
C
1 ln X 2
ln S1
C X 1 CS1YX / S
C X 1 C X 1 CS1YX / S CS 2

P 2 max

Multiple CSTFs in Series

Multiple CSTFs in Series


Working Equations
Material balance on the nth steady-state CSTF

F C Xn1C Xn Vn rXn 0
rXn

YX / S

maxCSnC Xn
K S CSn

C Xn C Xn1

CSn1 CSn

Dilution rates:
F rX 1
D1
V1 C X 1

rX 2
F
D2

V2 C X 2

Example 6.3
Suppose you have a microorganism that obeys
Monod equation:
dCX max C SC X

dt
KS CS

where max = 0.7 h1 and KS = 5 g/L. The cell yield


(YX/S) is 0.65. You want to cultivate this
microorganism in either one fermenter or two in
series. The flow rate and the substrate
concentration of the inlet stream should be 500
L/h and 85 g/L, respectively. The substrate
concentration of the outlet stream must be 5 g/L.

Example 6.3
a. If you use one CSTF, what should be the size of
the fermenter? What is the cell concentration of
the outlet stream?
b. If you use two CSTFs in series, what sizes of the
two fermenters will be most productive? What
are the concentration of cells and substrate in
the outlet stream of the first fermenter?
c. What is the best combination of fermenter types
of volumes if you use two fermenters in series?

Example 6.3
Given:
(a) For a single CSTF
F = 500 L/h
CSi = 85 g/L

max = 0.7 h1
KS = 5 g/L
YX/S = 0.65

CS = 5 g/L

Example 6.3
Required
(a) V
(b) CX
Solution
For a single CSTF
F
D
V

Solving for V

FK S C S
F
F
V

max C S
max C S
KS CS

500L/h5 5 g/L
V
0.7h1 5 g/L
V 1 428.5714L 1 429L

Example 6.3
The outlet cell concentration is

CX YX/S CSi CS

CX 0.6585 5 g/L
CX 52 g/L

Example 6.3
Given
(b)

Example 6.3
Required
(a) V1 and V2
(b) CX1 and CS1

Example 6.3
(a) For two CSTFs in series, the first fermenter
must be operated at CX,opt and CS,opt.
K S C Si
5 85

4.2426
KS
5
CX1 CX,opt YX/S CSi
C S1 C S,opt

4.2426
0.6585
44.7113g/L
1
4.2426 1

C Si
85

16.2133g/L
1 4.2426 1

m1 m,opt

4.2426

1.8691h
max 1 0.74.2426 1

Example 6.3
V1 m1F 1.8691h500L/h 934.55L 935L

For the second fermenter:


Input Output + Generation = Accumulation

FCX1 CX2 V2rX 0

Example 6.3
Rearranging the OMB for V2 gives

FC X2 C X1 KS CS2 50052 455 5


V2

0.7552
max CS2CX2
V2 192.3077L 193L

The total volume of the two CSTFs is


V = V1 + V2 = 935 L + 193 L = 1 128 L
The total volume of the two CSTFs in series is 20%
smaller than a single CSTF.

Example 6.3
Solution for Requirement (c)
The best combination is a CSTF operated at the
maximum rate followed by a PFF.
P2

C X2
K S YX/S
C
1 K S YX/S
S1

1 ln

ln

max C X1 C S1YX/S
C X1 C X1 C S1YX/S C S2

P2

52

1 50.65
50.65
16

1 ln
ln 0.32h

0.7 45 160.65 45 45 160.65 5

V2 P2F 0.32500 160L

Example 6.3
V = V1 + V2 = 950 L + 160 L
V = 1 110 L
The total volume employing a CSTF followed by
a PFF is 22% smaller than a single CSTF. The
difference in volume of a CSTF-CSTF series
versus CSTF-PFF series is negligible.

Problem 6.6
The growth rate of E. coli in synthetic medium can
be expressed by Monod kinetics as
rX

0.935CSC X
0.71 C S

[g/L h]

where CS is the concentration of limiting substrate,


glucose. You are going to cultivate E. coli in a
steady-state CSTF (working volume: 10 L) with
flow rate of 7 L/h. The initial substrate
concentration is 10 g/L and the cell yield constant
(YX/S) is 0.60. The feed stream is sterile.

Problem 6.6
a. What will be the doubling time and the division rate
of the cells in the CSTF? =0.7950 h; =1.2595 h
b. What will be the cell and substrate concentrations
of the outlet stream? C =2.1148 g/L; C =4.7311 g/L
c. If you connect one more 10-L CSTF to the first one,
what will be the cell and substrate concentration in
the second fermenter? CS2=
d. If you increase the flow rate
e. from 7 to 10 L/h for these two fermenters
connected in series, what will happen and why?
Make a recommendation to avoid the problem if
1

Problem 6.9
Suppose you have an organism that obeys the Monod
equation:
dCX max C SC X

dt
KS CS

where max = 0.5 h1 and KS = 2 g/L.


The organism is being cultivated in a steady-state CSTF,
where F = 100 L/h, CSi = 50 g/L, and YX/S = 0.5.
a. What size of vessel will give the maximum total rate
of cell production?
b. What are the substrate and cell concentrations of
the optimum fermenter in part (a)?

Problem 6.9
c. If the existing flow from the first fermenter in part
(a) is fed to a second fermenter (CSTF), what should
be the size of the second fermenter to reduce the
substrate concentration to 1 g/L?
d. If the existing flow from the first fermenter in part
(a) is fed to a second fermenter whose size is the
same as the first, what will be the cell and substrate
concentrations leaving the second fermenter?

CSTF with Cell Recycling


The cellular productivity in a CSTF increases with an
increase in the dilution rate and reaches a
maximum value. If the dilution rate is increased
beyond the maximum point, the productivity will be
decreased abruptly and the cells will start to be
washed out because the rate of cell generation is
less than that of the cell loss from the outlet
stream. Therefore, the productivity of the
fermenter is limited due to the loss of cells with the
outlet stream. One way to improve the reactor
productivity is to recycle the cell by separating the

CSTF with Cell Recycling


cells from the product stream using a cross-flow filter
unit.

CSTF with Cell Recycling


The high cell concentration maintained using cell
recycling will increase the cellular productivity since
the growth rate is proportional to he cell
concentration. However, there must be a limit in
the increase of the cellular productivity with
increased cell concentration because in a high cell
concentration environment, the nutrient-transfer
rate will be decreased due to overcrowding and
aggregation of cells. The maintenance of the
extremely high cell concentration is also not
practical because the filter unit will fail more
frequently at the higher cell concentrations.

CSTF with Cell Recycling


If all cells are recycled back into the fermenter, the cell
concentration will increase continuously with time
and a steady-state will never be reached. Therefore,
to operate a CSTF will recycling in 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
dCX
FC Xi BC X Vr X V
dt

CSTF with Cell Recycling


It should be noted that actual flow rates of the streams
going in and out of the filter unit do not matter as
far as overall material balance is concerned. For a
steady-state CSTF with cell recycling and a sterile
feed,

where , the bleeding ratio, is defined as


B

CSTF with Cell Recycling


Now, D instead of D is equal to the specific growth
rate. When = 1, cells are not recycled, therefore, D
= .
If the growth rate can be expressed by Monod kinetics,
CS is given by
K S
CS
m max

which is valid when mmax > . The cell concentration


in the fermenter can be calculated from the value of
CS as
YX S
CSi CS
CX

Problem 6.13
A strain of yeast is being cultivated in a 30-L CSTF with a
cell recycling system (cell settler) as shown in the
following figure. The cell settler was designed so hat
the cell concentration of its outlet stream is 30
percent of that of its inlet stream, whereas the
substrate concentrations of the two streams are the
same. The growth rate of the cells can be
represented by the Monod kinetics with the
parameters: KS = 0.05 g/L, max = 0.3 h1, and YX/S
= 0.025. Calculate the steady-state substrate and
cell concentrations in the fermenter. The inlet

Problem 6.13
substrate concentration is 100 g/L and the flow rate is
20 L/h. The feed stream is sterile.

For 7/14/2015 (ST_SY14-15)


Solve:
1. Problem 6.3
2. Problem 6.7
3. Problem 6.10
4. Problem 6.12

Supplementary Problems
Problem 12.1/p. 652] Bioprocess Engineering: Kinetics,
Biosystems, Sustainability, and Reactor Design by Shijie Liu
Pseudomonas sp has a mass doubling time of 2.4 h when grown
on acetate. The saturation constant using this substrate is
1.3 g/L (which is unusually high), and cell yield on acetate is
0.46 g cell/g acetate. If the feed stream to a chemostat
contains 38 g/L acetate, determine:
(a) Maximum dilution rate,
(b) Cell concentration when the dilution rate is one-half of the
maximum,
(c) Substrate concentration when the dilution rate is 0.8D max.