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Biochem: Cell Kinetics and Fermenter Design

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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.

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

= 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

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.

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.

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.

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.

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.

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:

dCN

rN

C N

dt

Where:

= specific growth rate (h1)

1 dCN d ln C N

C N dt

dt

d ln C N

d log2 C N

ln 2

dt

dt

ln 2

growth period, then

CN

CN 0

dCN

dt

CN

t0

upon integration:

CN CN 0 exp t t0

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

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

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

when the specific growth rate is half of its

maximum value (max).

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

Rate

If several substances can limit the growth of a

microorganism, the following model is

employed:

C1

C2

max

...

K1 C1 K 2 C2

the energy source for the culture

maxCS

K S CS

ke

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

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.

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.

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.

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

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.

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.

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

CX

CX 0

dCX

rX

CX

CX 0

dCX

dt t t0

C X

t0

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.

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.

If Monod kinetics adequately represents the

growth rate during the exponential period:

CX

CX 0

K S CS dCX

maxCS C X

dt

t0

YX / S

CX CX 0

C X

CS CS 0 CS

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

rmaxCS

dCP

dt

K M CS

dCP maxCS C X

dt

K S CS

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.

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).

Fermenter

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.

Fermenter

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

Fermenter

Fermenter

Material Balance

Input Ouput + Generation = Accumulation

dCX

FC Xi FC X Vr X V

dt

V C X C Xi

m

F

rX

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

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

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.

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

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.

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

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

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

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

m

V K S CSi

F CSi max

Example 6.2

Solving for F

VC Si max

F

K S C Si

F

1.5256g/L 100 g/L

F 0.1238 L/h

Biosystems, Sustainability, and Reactor Design by Shijie Liu

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)?

Biosystems, Sustainability, and Reactor Design by Shijie Liu

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:

Biosystems, Sustainability, and Reactor Design by Shijie Liu

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

Biosystems, Sustainability, and Reactor Design by Shijie Liu

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

Biosystems, Sustainability, and Reactor Design by Shijie Liu

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?

Biosystems, Sustainability, and Reactor Design by Shijie Liu

Biosystems, Sustainability, and Reactor Design by Shijie Liu

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.

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

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

m,opt

max 1

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.

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

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.

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.

Series

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

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

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

(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

Input Output + Generation = Accumulation

Example 6.3

Rearranging the OMB for V2 gives

V2

0.7552

max CS2CX2

V2 192.3077L 193L

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

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]

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

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?

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

cells from the product stream using a cross-flow filter

unit.

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.

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

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,

B

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

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.

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.

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