Professional Documents
Culture Documents
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.
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
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
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.
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.
Where:
= specific growth rate (h1)
1 dCN d ln C N
C N dt
dt
ln 2
dt
dt
ln 2
CN 0
dCN
dt
CN
t0
upon integration:
CN CN 0 exp t t0
ln 2
Classification of microorganisms in
terms of growth-rate on temperature
Group
Thermophiles
Mesophiles
Psychrophiles
Obligate
Facultative
Temperature, C
Minimum
Optimum
Maximum
maxCS
K S CS
maxCS
K I 1 CS K I 2CS 2
max 1 e CS / K S
max
1 K S CS
max
n C S
maxCS
K S CS
ke
K P
K P C P
CS
max
K S CS
CP
1
C Pm
CX 0
dCX
rX
CX
CX 0
dCX
dt t t0
C X
t0
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.
CX 0
K S CS dCX
maxCS C X
dt
t0
CX CX 0
C X
CS CS 0 CS
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
dt
K M CS
dCP maxCS C X
dt
K S CS
1
m
D
KS
m max 1
KS
C X YX / S CSi
m max 1
C P C Pi
KS
YP / S CSi
m max 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
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
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
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
V K S CSi
F CSi max
Example 6.2
Solving for F
VC Si max
F
K S C Si
F 0.1238 L/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
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
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
max 1
CX
CX 0
dC X
rX
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
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
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
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
Example 6.3
Rearranging the OMB for V2 gives
0.7552
max CS2CX2
V2 192.3077L 193L
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
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]
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
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?
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.
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.