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CHAPTER 6: CELL KINETICS AND FERMENTER DESIGN

6.10. You are going to cultivate yeast, Saccharomyces Cerevisiae, by using a 10m3 fermenter
your company owns. You want to find out the amount of ethanol the fermenter can produce.
Therefore, a chemostat study was carried out and the Monod kinetic parameters. For the
microorganism grown in the glucose medium at 30oC, pH= 4.8, were found to be Ks= 0.025 g/L
and umax= 0.25h-1. The ethanol yield (Yp/s) is 0.44 (g/g) and cell yield (Y X/S) is 0.019 (g/g). The
inlet substrate concentration is 50 g/l.
a. What flowrate will give the maximum total ethanol production in the continuous fermenter and
what is the maximum ethanol production rate?
b. If you want to convert 95% of the incoming substrate, what must be the ethanol production
rate for the continuous fermenter?
c. If you have two 5m3 fermenters instead of one 10m3 fermenter, what is your recommendation
for the use of these fermenters to convert 95% of the incoming substrate? Would you recommend
connecting two fermenters in series to improve the productivity? Why or why not?
Given: V=10m3; Ks= 0.025 g/L; umax= 0.25h-1; Yp/s= 0.44; YX/S= 0.019 g/g; Cso= 50 g/l
Required:
(a) F
(b) F @ 95% Cso conversion
Solution:

(a) =

Ks+Cso
=
Ks

0.025 g 50 g
+
L
l
=44.73
0.025 g / L

Cso
Cs= CS opt = +1 = 50gL-1/ (44.73 +1) = 1.0934g/L
D= umax.CS/ (Ks+Cs)= 0.25h-1(1.0934g/L)/ (0.025 g/L+1.0934g/L)= 0.2444/h
F= DV= 0.2444/h (10m3)= 2.444 m3/h
(b) For 95% substrate conversion Cs= 0.05Cso = 4.75g/l.
0.25
g
4.75
h
l
D= umax.CS/ (Ks+Cs) =
= 0.0249 /h
g
g
0.025 + 4.75
l
l

( )( )
( )( )

F= DV= (0.0249/h) (10m3) = 0.2490 m3/h

(c) Cxo= YX/S(Cso- CS)= 0.019 (50- 4.75)g/L=


g/L

44.73
Cx1= YX/SCso( +1 = (0.019)(50)( 4.573 )=
0.9292g/L
Cso
Cs1= +1 =

0.8598

50 g/l
(44.73+1) = 1.0934g/L

D1= umax.CS/ (Ks+Cs) = 0.25h-1(1.0934g/L)/ (0.025 g/L+1.0934g/L)= 0.2444/h


F1= D1V1= 0.2444 h-1( 5m3)=1.2220 m3/h

Cx2= YX/SCs1( +1 = (0.019)( 1.0934g/L)=0.0208g/L


Cs2=

Cs 1
+1 =

1.0934
45.73 = 0.0239 g/L

0.02080.9292
YX/S= 0.02391.0934

0.8494

Therefore; a two- 5 cubic meters of fermenters in series will increase the Growth
yield for 95% Ethanol conversion

CHAPTER 6: CELL KINETICS AND FERMENTER DESIGN ; ADDITIONAL PROBLEM


During the growth of E. coli in a batch reactor, the pattern can be modelled using the
Monod expression of the form;
=

max
K S+ S

Where:
= specific growth rate
max =maximum specific growth rate
Ks = Monod constant
S = substrate concentration
For the above process, show how the time required to reach the maximum population of
cells can be estimated.
If the concentration of cells at the start of the exponential growth is 0.08g/L and the
essential substrate concentration is 36g/L. Find the time where the maximum population
of cells is reached given that max = 0:55h1, Ks = 1:2g/L and k = 1:4gsubstrate/(gcellsh)

Soln:
Substrate consumption during the growth can be described by;
dS
=kX
dt

and the cell growth is given by;


dX
=X
dt
where
X=

1 dX
dt

Substitute X into

dS
dt and integrating gives;
dS
1 dX
=k (
)
dt
dt

dS=

But
=

k
dX

max
K S+ S

hence;

dS=

k
dX
max
K S+ S

rearranging gives;
0

XS

S
k
K + S dS= dX
S
max X
S
0

Integrating both sides leads to;


X S=X 0+

max
KS
(S0 + K S ln
)
k
K S+ S0

The average can is determined by dividing the specic growth rate with the total
substrate in the reactor, which gives;
0

dS
avg=

S0

dS
S0

avg=

K S+S dS
S0

dS
S0

avg=

max [ S 0+ Ksln

KS
]
K S +S0

S0

Therefore, the new cell growth expression should be in the form of;
dX
= avg X
dt
and integrating,
XS

1
1
dt= X dX
avg X
0
0

gives;
t=

X
1
ln S
avg X 0

substituting avg previously lead to;


X
max [ 0+

max
KS
X
(S 0 + K S ln
) ]ln S
k
K S + S0
X0
S
t= 0

Consider the amount of cell produced at the given substrate concentration:


Using;
KS
S 0 + K S ln
KS+ S0
)
max
X S =X 0 +

k
1.2
36+1.2
36+1.2 ln
0.55
X S=0.08+

1.4

XS=12.604
and using the expression for avg;
0.55[36+1.2 ln
avg=

1.2
]
36+ 1.2

36
avg=0.487

thus, the time taken for the cell population to reach a maximum value;
X
1
t=
ln S
avg X 0
t=

1
12.604
ln
0.487
0.08
t = 10.34 hrs.

CHAPTER 6: CELL KINETICS AND FERMENTER DESIGN ; ADDITIONAL PROBLEM


Biochemical Engineering: A Textbook for Engineers, Chemists and Biologists
Shigeo Katoh and Fumitake Yoshida, 2009
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?

Given: E. coli
td=0.5 h
Cn=10 Cno=0.1

Reqd: a)
b) t

Soln:
a)
ln 2
td=
ln 2
= 0.5
=1.3868 h-1
b)
Cn
ln (
)
Cno =(t-to)

t=

ln (

Cn
)
Cno

10
)
.1
t=
1.3863
t= 3.3219 h
ln (

CHAPTER 6: CELL KINETICS AND FERMENTER DESIGN ; ADDITIONAL PROBLEM


Biochemical Engineering: A Textbook for Engineers, Chemists and Biologists
Shigeo Katoh and Fumitake Yoshida, 2009
E. coli grows from 0.10 kg dry cell/m3 to 0.50 kg dry cell/m3 in 1 h.

1. Assuming the exponential growth during this period, evaluate the specific growth rate.
2. Evaluate the doubling time during the exponential growth phase.
3. 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.
Given: E. coli
Cn= 0.50 kg dry cell/m3 Cno=0.10 kg dry cell/m3
t=1 h
Reqd: a)
b) td
c) t (from 0.1 to 1)
Soln:
a)
ln

Cn
Cno
t
.5
)
.1
1

ln (
=

=1.6094/h
b)
td=

ln 2

td= ln 2 / 1.6094
td= 0.4307 h = 25.8412 min
c)
ln

Cn
Cno

1
.1
=
1.6094
ln

t =
CHAPTER
6: CELL KINETICS AND FERMENTER DESIGN ; ADDITIONAL PROBLEM
Bioprocess
1.4307 h Engineering Principles by Pauline M. Duran, pp. 364- 367

Steady state Concentration in a Chemostat


The Zymomonas mobilis cells are used for chemostat in a 60 m 3 fermenter. The feed contains
12g/l glucose; Ks= 0.2 g/L; Yxs=0.06 g/g; Ypx=7.7 g/g, umax=0.3/h; ms=2.2g/g.h;
Yps=Ypx.Yxs=0.46g/g.
a. What flowrate is required for a steady-state substrate concentration of 1.5 g/L?
b. At the flowrate of (a), what is the cell density?
c. At the flowrate of (a), what concentration of ethanol is produced?
Given: Ks= 0.2 g/L;
Yps=Ypx.Yxs=0.46g/g.

Yxs=0.06

g/g;

Ypx=7.7

g/g,

umax=0.3/h;

ms=2.2g/g.h;

Cs= 1.5 g/L, V= 60m3


Required:
a. F
b. Cx
c. Cp
Solution:
(a) D= umax.CS/ (Ks+Cs)= (0.3h-1)(1.5g/L)(0.2g/L + 1.5 g/L)= 0.26 h-1
F= DV= 0.26/h (60m3) = 15.6 m3h-1.
(b) When synthesis of the product is coupled with energy metabolism as for ethanol, Cx is
evaluated; therefore;

Cx =

D ( CsoCS )
D
=
+ms
Yxs

( 0.26h ) ( 121.5) g/l


0.26
h
2.2
+
0.06 h

= 0.42 g/L

(c) Assuming ethanol is not present in the feed, Cpo=0. Steady-state product concentration is
3.4
g
0.42
qpx
h
L
Cp= Cpo+ D = 0 +
= 5.5 g/L
0.26
(
)
h

( )(

CHAPTER 6: CELL KINETICS AND FERMENTER DESIGN ; ADDITIONAL PROBLEM


Bioprocess Engineering Principles by Pauline M. Duran, pp. 364- 367
Substrate conversion and Biomass Productivity in a Chemostat
A 5 m3 fermenter is operated continuously with feed substrate concentration of 20 kg/m 3. The
microorganism cultivated in the reactor has the following characteristics: umax= 0.45/h; Ks= 0.8
kg/m3; Yxs= 0.55 kg/kg.
(a) What feed flow rate is required to achieve 90% substrate conversion?
(b) How does the biomass productivity at 90% substrate conversion compare with the
maximum possible?
Given: umax= 0.45/h; Ks= 0.8 kg/m3; Yxs= 0.55 kg/kg
Required: (a) F; (b) productivity comparison
Solution:
(d) For 90% substrate conversion Cs= 0.1 Cso = 2kg/m3.
0.45
kg
2
h
m3
D= umax.CS/ (Ks+Cs) =
= 0.32 /h
kg
kg
0.8
+ 2
m3
m3

( )( )
( )( )

F= DV= (0.32/h) (5m3) = 1.6 m3/h


(e) Assuming maintenance requirements and product formation are negligible:

Qx= D (Cso-

KsD

-1
3
umaxD Yxs = 0.32h (20kg.m -

3.17 kg.m-3.h-1
Maximum biomass @ Dopt
Dopt= umax (1-

KS
KS+Cso

)= umax(1- -1)

( 0.8m3kg )( 0.32h )
(

0.45 0.32

)
h
h

)(0.55kg/kg)=

Dopt= 0.45h-1(1-

0.8 kg
m3
0.8 kg
kg
+20
m3
m3

) = 0.36h-1

Maximum biomass productivity is determined with D= Dopt


Qxmax= D (Cso-

KsD

umaxD Yxs

Dopt= 0.36/h (20kg.m-3-

(0.8 m3kg )( 0.36h )


0.45 0.32
(

)
h
h

) (0.55 kg/kg) = 3.33 kg.m3.h-1

Therefore, biomass productivity at 90% substrate conversion is

3.17
3.33 x100 = 95%

of the theoretical maximum.


CHAPTER 6: CELL KINETICS AND FERMENTER DESIGN ; ADDITIONAL PROBLEM
Bioprocess Engineering Principles by Pauline M. Duran, p. 375
Plug-Flow Reactor for immobilized enzymes:
Immobilised lactase is used to hydrolyze lactose in dairy waste to glucose and galactose. Enzyme
is immobilized in resin particles and packed into a 0.5 m 3 column. The total effectiveness factor
for the system is close to unity; Km for the immobilized enzyme is 1.32 kg/m 3; umax is 45 kg.m3 -1

h . The lactose concentration in the feed stream is 9.5 kg.m -3; a substance conversion 98% is

required. The column is operated with plug Flow for a total of 310 days a year.
a. At what flowrate should the reactor be operated?
b. How many tonnes of gluctose are produced a year?
Given: Cs= 0.02Cso= 0.19 kg.m-3; Cso= 9.5 kg.m-3; umax= 45 kg.m-3h-1; Km= 1.32 kg.m-3;
VPFR=0.5 m3
Required: a. F, b. mass glucose (Tons)
Solution:
(a) For 98% substrate onversion:
Km
Cso CsoCs
= umax ln Cs + umaz = 0.32 h

F=

V
3
= 1.56 m /h

(b) The rate of lactose conversion is equal to the difference between inlet and outlet mass
flow rates of lactose= F(Cso-Cs)= 1.56 m3(0.5-0.19) kg/m3= 14.5 kg/h
kg 24 h 310 d 1 kmol
kmol
Lactose converted= 14.5 h . 1d . 1 yr . 342 kg =315 yr
The enzyme reaction is:
Lactose+ H20 --) glucose + galactose
Therefore, from reaction stoichiometry, 315 kmol glucose are produced a year. The
molecular weight of glucose is 180; thus,
Mass glucose= 315

kmol 180 kg
1T
Tonnes
.
.
=56.7
yr 1 kmol 1000 kg
yr

CHAPTER 6: CELL KINETICS AND FERMENTER DESIGN ; ADDITIONAL PROBLEM


Chemical Engineering by J. M. Coulson and J. F. Richardson
A continuous fermenter is operated at a series of dilution rates though at constant, sterile, feed
concentration, pH, aeration rate and temperature. The following data were obtained when the
limiting substrate concentration was 1200 mg/l and the working volume of the fermenter was 9.8
l. Estimate the kinetic constants Km,m and kd as used in the modified Monod equation:

and also the growth yield coefficient Y.


Feed flowrate Exit substrate concentration Dry weight cell density
(l/h)

(mg/l)

(mg/l)

0.79

36.9

487

1.03

49.1

490

1.31

64.4

489

1.78

93.4

482

2.39

138.8

466

2.68

164.2

465

Solution
The accumulation = input output + rate of formation,
which for the biomass gives:
V(dX/dt) = FX0 FX + V(mSX)/(Ks + S) kdXV

(equation 5.126)

and for the substrate:


V(dS/dt) = FS0 FS VmSX/Y(Ks + S)

(equation 5.127)

At steady state, dS/dt = 0.


Taking the dilution rate, D = F/V , then the balance for the substrate becomes:
S0 S {mSX/[DY(Ks + S)]} = 0
or:

X/D(S0 S) = (KsY/m)/S + Y/m

(i)

Similarly, for the biomass:


X0 X + {mSX/[D(Ks + S)]} kdX/D = 0
Since the feed is sterile, X0 = 0 and:
DX = [mSX/(Ks + S)] kdX.
From the material balance for substrate:
(mSX)/(Ks + S) = DY(S0 S)
and substitution gives:
DX = DY(S0 S) kdX
or:

(S0 S)/X = kd/DY 1/Y

(ii)

From equation (i), it is seen that a plot of X/D(S0 S) and 1/S will produce a straight line of
slope KsY/m and intercept Y/m.

From equation (ii), it is seen that a plot of (S0 S)/X against 1/D will produce a straight line of
slope kd/Y and intercept 1/Y.
The data are calculated as follows:
Feed
Exit
1/S
flowrate
substrate
(l/mg)
(F l/h)
(S mg/l)
0.79
36.9
0.0271
5.19
1.03
49.1
0.0204
4.05
1.31
64.4
0.0155
3.22
1.78
93.4
0.0107
2.40
2.39
138.8
0.0072
1.80
2.68
164.2
0.0061
1.64
The data are then plotted in Figure 5b from which:
KsY/m = 170,

1/D
(h)
12.41
9.51
7.48
5.51
4.10
3.66

Y/m = 0.59, kd/Y = 0.0133 and 1/Y = 2.222.


slope, KsY/m = 170

(h
)
S

0
S
(
D
/

6
5

Y/ m= 0.59

4
3
X

2
1
0

(
X
/)
S

2.4

slope,kd / Y = 0.0133/h
2.3
(

X
2.2

1/ Y = 2.222

0.01 0.02 0.03


1/S (l/mg)

2.388
2.349
2.322
2.296
2.277
2.228

0 2 4 6 8 10 12
1/D (h)

Figure 5b. Graphical work


Thus:
yield coefficient, Y = (1/2.222) = 0.45 endogenous respiration coefficient,

m = (0.45/0.59) = 0.76 h1

and:

Ks = (170 0.76/0.45) = 300 mg/l

CHAPTER 6: CELL KINETICS AND FERMENTER DESIGN ; ADDITIONAL PROBLEM


Chemical Engineering by J. M. Coulson and J. F. Richardson
Two continuous stirred-tank fermenters are arranged in series such that the effluent of one forms
the feed stream of the other. The first fermenter has a working volume of 100 l and the other has
a working volume of 50 l. The volumetric flowrate through the fermenters is 18 h 1 and the
substrate concentration in the fresh feed is 5 g/l. If the microbial growth follows Monod kinetics

with m = 0.25 h1,Ks = 0.12 g/l, and the yield coefficient is 0.42, calculate the substrate and
biomass concentrations in the effluent from the second vessel. What would happen if the flow
were from the 50 l fermenter to the 100 l fermenter?
Solution
A material balance for biomass over the first fermenter, leads to the equation:
D1 = 1
(equation 5.131)
where D1 is the dilution rate in the first vessel and 1 is the specific growth rate for that vessel.
Assuming Monod kinetics to apply:
D1 = mS1/(Ks + S1)

(equation 5.132)

where S1 is the steady-state concentration of substrate in the first vessel, where:


S1 = D1Ks/(m D1)

(equation 5.133)

If D1 = F/V1 = (18/100) = 0.18 h1, then:


S1 = (0.18 0.12)/(0.25 0.18) = 0.309 g/l
Since the feed to this fermenter is sterile, X0 = 0 and from equation 5.134, Volume 3 the steadystate concentration of biomass in the first vessel is given by:
X1 = Y(S0 S1)

(equation 5.134)

= 0.42(5 0.309) = 1.97 g/l

In a similar way, a mass-balance over the second vessel gives:


D2 = 2X2/(X2 X1)

(equation 5.167)

where D2 is the dilution rate in the second vessel, 2 is the specific growth rate in that vessel and
X2 the steady-state concentration of biomass.
The yield coefficient to the second vessel is then:
Y = (X2 X1)/(S1 S2)
where S2 is the steady-state concentration of substrate in that vessel.

Thus:

X2 = X1 + Y(S1 S2)
= 1.97 + 0.42(0.309 S2)
= 2.1 + 0.42S2

Substituting this equation for X2 into equation 5.167, together with values for D2,2 and X1 leads
to a quadratic equation in S2:
0.
from which:

S2 = 0.0113 and:
g/l

X2 = 2.1 + (0.42 0.0113) = 2.1 g/l

When the tanks are reversed, that is with fresh feed entering the 50 litre vessel, then the dilution
rate for this vessel will be as before, 0.36 h1, although the critical dilution rate will now be:
Dcrit = mS0/(Ks + S0)

(equation 5.148)

= (0.25 5)/(0.12 + 5) = 0.244 h1


This is lower than the dilution rate imposed and washout of the smaller vessel would take place.
The concentrations of substrate and biomass in the final effluent would eventually be those
attained if only the 100 litre vessel existed, that is:
concentration of biomass = 1.97 g/l

concentration of substrate