127
Example 6.2
A chemostat study was performed with yeast. The medium flow rate was varied and the steadystate concentration of cells and glucose in the fermenter were measured and recorded. The inlet concentration of glucose was set at 100 g/ I: The volume of the fermenter contents was 500 mz'. The inlet stream was sterile. Flow rate F, ml/hr 31 50 71 91 200 Cell Concentration Cx, g/l 5.97 5.94 5.88 5.76 Substrate Concentration
c., g/.e
0.5
1.0
2.0
4.0
o
100
a. Find the rate equation for cell growth. b. What should be the range of the flow rate to prevent washout of the cells?
Solution:
a. Let's assume that the growth rate can be expressed by Monod kinetics. If this assumption is reasonable, the plot of 1/ /L versus l/es will result in a straight line according to Eq. (6.35). The dilution rate for the chemostat is
D=F
The plot of 1/ D versus l/Cs straight line with intercept
IS
V shown in .Figure 6.9 which shows a
_1_
/Lmax
=
3.8
and slope Ks
/Lmax
= 5.2
Therefore, /Lmax cell growth is
0.26 hr ",
r
and Ks

=
1.37 g/l.
The rate equation of
x  1.37 + Cs b. To prevent washout of the cells, the cell concentration should be maintained so that it will be greater than zero. Therefore, from Eq. (6.33)
0.26 Cs Cx
ex
= Yxis (Csi

Tm/Lmax
Ks _
1) > 0
142
,,
~r.
r:
~
,
j
t
air
t
t,,t
,
t
~r.
t
t
~
t
t
y
(a)
(b)
(c)
(d)
Figure 6.20 Loop fermenters: loop, and (d) jet loop.
(a) airlift, (b) leI pressure cycle; (c) stirred.
can maintain a high airflow rate per unit area at the lower section of the fermenter where the cell concentration is high. Several sieve plates can be installed in the column [Figure 6.19( c)] for the effective gasliquid contact and the breakup of the coalesced bubbles. The cylindrical column can be divided into multiple stages which are equipped with stirrers [Figure 6.19(d)]. This configuration will be analogous to the stirredtank fermenter connected in series as explained in aearlier section. To enhance the mixing without internal moving parts, the fermentation broth can be pumped out and recirculated by using an external liquid pump [Figure 6.19(e) and (f)].
6.8.2
Loop Fermenter
A loop fermenter is a tank or column fermenter with a liquid circulation loop, which can be a central draft tube or external loop. Depending on ho\v the liquid circulation is induced, it can be classified into three different types: airlift, stirred loop, and jet loop (Figure 6.20). The liquid circulation of the airlift fermenter is induced by sparged air which creates a density difference between the bubblerich part of the liquid in the riser and the denser bubbledepleted part of the liquid in the downcomer
which wa.
.20(c) and (d).143 as shown in Figure 6. developed for s The leI pressure
the aerobic fermentation requiring heat removal such as the singlecell protein production from methanol. and that the high circulation of the fermentation liquor provides good mixing (Technical Brochure... The air serves two purposes: It provides the oxygen needed for the growth of the microorganisms and the rising air creates natural circulation of the liquid in the fermenter through the loop.20( a). The liquid circulation and mixing can be enhanced by installing a propeller or by circulating liquid externally using a pump as shown in Figure 6. A heat exchanger to cool the liquid medium is installed in the loop. England) is an airlift ferrnenter with an outer loop. adding the propeller or pump diminishes the real advantages of an airlift fermenter for being simple and energy efficient. It was claimed that the fermenter gives a high rate of oxygen absorption per unit of volume. Medium and air are introd uced into the upper and lower parts of the loop as shown in Figure 6. that it uses a high proportion of oxygen in the air passed through the Iermenter. reI Ltd. However.20(b).).
cycle ferrnenter (Imperial Chemical Industries Ltd .
144
Nomenclature B C
flow rate of bleeding stream.)
r. mass per unit volume of culture. mass per unit volume of abiotic phase. extracellular property.
Is
C
kg/rn!
l
m
n
r
T. kg/m3 s rate of jth component formed from ith reaction per unit volume of a system. . 3 kg/m constants cell number density. number of cells/rrr' dilution rate. s specific volume of a system containing only biotic phase. m3 mass of biotic phase. S1 flow rate. I. defined as B I F average cell division rate. kg/m3 s time. intracellular property. m3 system coefficient for the Monod kinetics. kg/m3 residence 'time. kg/m3 intracellular concentration. kg/m3 s rate of jth component formed from ith reaction per unit volume of a system. kg/m3 average mass of cell in a system. kg flow rate of filtrate stream.
I. m3 yield constant bleeding ratio. S1 specific growth rate. 81 or kg/m3 s density.)
v
~
y
{3
V
"8
J. s
Is
Is
SUBSCRIPT b
'l
m n
X
p
p
batch fermenter input stream mixed fermenter cell in number basis cell in dry weight basis product plugflow fermenter
. s doubling time. kg number of cells rate of growth per unit volume.. mass per unit volume of bioticphase. m3 concentration.L
P
r
extracellular concentration. m3/kg working volume of fermenter.
0. Find the rate equation for cell growth. The flow rate and inlet substrate concentration were varied and the steadystate concentration of glucose in the fermenter was measured
.51
Steadystate Cell Cone.25
a.9 21.44 4.70 8.1 Derive the relationshi p giving the change with respect to time of the cell concentration in a batch fermenter. (1968) reported the results of a chemostat study on the growth of a specific strain of baker's yeast as shown in the following table. The inlet stream of the chemos tat did not contain any cells or products.3 Andrews (1968) proposed the following model for the growth of microorganisms utilizing inhibitory substrates.7 10. g/ l
7.138 0.
21.100 0.40 2.160 0.97 4.57 8.33 1.226
Steadystate Ethanol Conc. Cx.
J1. 6.8
Steadystate Glucose Cone.186 0. Cr.054 0.
Dilution Rate D. sl f. Eq. g/ f.00 1. Find the rate equation for product (ethanol) formation. hr1
0.145 S
substrate
Problems
6 .. b. gil
2.2 Aiba et al. c.= J1.242
Inlet Glucose Cone.2 20.24). The inlet stream was sterile.max
1 + Ks
VS
+ Cs Ki
Assume that a chemostat study was performed with a microorganism. 6. The volume of the fermenter content was 1e.20 2.
c.079 0.5 10.084 0. (6.198 0.
6 3.24 hr ". Determine the kinetic parameters croorgamsm.
dt
Y'x/s
dCp 1 dCx rp= = dt Y'x/P dt
where Ks
100 gil.46 gig.
dt
dCx
=
fLmax
(Cp)n(C 1sc.
= 0.20 flh?
c. Cpo'
=
= 0.16. Andrews concluded in his paper that the primary result of substrate inhibition in a continuous culture may be process instability.
6.
Y'x/s
= 0.25 0.f Illr 0.1 1.
CXo
=
Y'x/P 0. Explain what might happen if you suddenly increase the substrate concentration from 30 to 60 st £ and why.80 0. If the cell yield. Cp.5 0.146
and recorded Flow Rate F. and n = 2.70 0.20 0. Show the effect of the initial versus t curve.7 1.35 0.
1(50
b. what is the steadystate centration when the flow rate is 0.1 gl £. 'lX/5. and product fermentation process are given as follows:
in
rx
.4 Rate equations
the ethanol
for the cells (yeast).50 0. is 0. substrate concentration on the Cx
. ) CX
rs= 
= 
sc.6
gl t. fLmax = 0. substrate (glucose).06.3
10
30 22 15 and K/) of this micell con
a.
dt
Pm
K s + Cs
1
c. and Cs as a function of time when Gso = 100 gl £.
1.70 as follows (the data are arbitrary): Inlet Glucose Concentration Steadystate Glucose Concentration
Cs" gil
30 30 30 30 30 30 60 60 60 (Pmax.60 0. b.
CSo gil
0. Calculate the change of Cx.50 0.
CPm
=
a.
If you increase the flow rate from 7 to 10 e/hr for these two fermenters connected in series) what will happen and why? Make a recommendation to avoid the problem if there is any. The inlet stream is sterile.
The cell yield Yx/s
is found
a.8 f/hr) what will be the steadystate cell concentration of the outlet stream? The substrate concentration of the inlet stream is 13 g/ e. a. Show the effect of the maximum versus t curve (Gso = 100 g/f!).5 Derive Eqs.6.45. (6.8 Herbert this model and Monod model by
et al. The feed stream is sterile.
6. The initial substrate concentration is 10 g/ f . medium can be expressed by growth rate
({Lmax) on the Cx
6.umaxGsCx
Ks
+ Cs
.365 hr "! and Ks = 6.42). If you connect one more 10e CSTF the cell and substrate concentrations
to the first one) what will be in the second fermenter?
d.eCs/Ks)Cx
where {Lmax = 0. What will be the cell and substrate stream? c. 6. (1956) reported that the growth kinetics of Aerobacter cloacae in a chemically defined medium (glycerol as a limiting substrate) could be expressed by Monod kinetics as follows:
rx . Explain the difference between using p. You are going to cultivate E.935CsCx
0.umax(1 . and the cell yield constant (Yx/s) is 0. 6. versus Cs graph.147
c.39) Monod (6. Wh at will be the doubling the CSTF? time and the division rate of the cells in concentrations of the outlet
b.71
+ Cs
[g/f!hr]
where Cs is the concentration of a limiting substrate) glucose.41) and (6.dt
_ dGx _ .6 The growth
rate of E.8 g/ to be 0. b. If you cultivate this microorganism in a 10 f CSTR with the flow rate of 2.
e.7 Suppose
that the growth the following equation:
rate of a microorganism
can be expressed
as
"x = . coli in synthetic kinetics as Tx
=
0. coli in a steadystate CSTF (working volume: 10 f) with a flow rate of 7 f!/hr.
The yield was found to be 0. a.23 gj t instead of 1.23 x 102 gj I!. For the following three questions. volume) which can handleIflf [jhr in the feed stream. what is the best fermenter system (fermenter type. the concentration of glycerol in the feed stream and that of glycerol in the outlet stream should be .1maxCsCX Ks + Cs where J. where F dCx dt
=
total rate of cell produc
.53 g dry weight of organismjg glycerol used.23 X 102 gj f. You are a biochemical engineer who has been assigned the task of designing the most effective continuous fermentation system to grow the microorganism (Aerobacter cloacae) with glycerol as its limiting substrate. 3 gj t and 0. You were quite sure of this because the substrate concentration in the outlet stream has to be so low. Who is right? Prove whether you are right or wrong by drawing the Ijrx versus Cs curve for this microorganism. Since you have learned that the 1j r x versus C 5 curve for Monod kinetics has a U shape. respectively. a. you have recommended that the most effective system would be the combination of a continuous stirredtank fermenter (CSTF) and a plugflow fermenter (PFF).1 gj E.) b.. your boss is insisting that the use of second PFF in addition to the first CSTF will not improve the productivity very much. think about how you can nicely correct you boss's wrong idea.1max = 0.5 hr=' and Ks = 2 gj t The organism is being cultivated in a steadystate 100 £/hr. Recommend the best fermenter system (fermenter type and volume) which can handle 100 fjhr of feed stream. How is this case different from the case of part (a) and why? 6. If you are wrong. CSi = 50 g/ £. If Ks = 1.5. (If you are right.85hr1 and ]{s = 1. What size vessel will give the maximum tion? CSTF. However. Does it have a U shape? Discuss why you are right or wrong. it will teach you that you have to be careful nut to make a quick conclusion without adequate aualysis. The best fermenter system is defined as that which can produce the maximum amount of cells per unit time and volume.9 Suppose you have an organism that obeys the Monod equation: f.148 where f. Draw the block diagram of the fermenter system with the concentrations of the substrate and the cells in the inlet and outlet "streams of each fermenter. c. and Yx/s = 0.1max = 0.
Therefore. what must the ethanol production rate be for the continuous fermenter? c. were found to be: Ks = 0. If the exiting flow from the first fermenter in part (a) is fed to a second fermenter whose size is the same as the first. You 'want to find out the amount of ethanol the fermenter can produce. a. What are the substrate fermen ter in part (a)? and cell concentrations of the optimum
c. Saccharomyces cerevisiae. If you want to convert 95 percent of the incoming substrate.10 You are going to cultivate yeast. If you have two 5 m+ferrnenters instead of one 10 m3fermenter.12 Consider an organism with the following data for a If chemostat. pH 4. what should be the size of the second fermenter to reduce the substrate concentration to 1g/.e.11 You are a biochemical engineer in a pharmaceutical
company.149 b. a chemostat study was carried out and the Monod kinetic parameters for the microorganism grown in the glucose medium at 30°C. Currently. what kind of fermenter is your company using for penicillin production? Why? Your boss asked you to study the possibility of using an airlift fermenter as a replacement since it has many advantages. If the exiting flow from the fermenter in part (a) is fed to a second fermenter (CSTF). What is your recommendation?
6. what will be the cell and substrate concentrations leaving the second fermenter? 6.? d. by using a 3fermenter 10 m your company already owns.8.25h1.025glf and /Lmax = 0. what is your recommendation for the use of these fermenters to convert 95 percent of the incoming substrate? Would you recommend connecting two fermenters in series to improve the productivity? Why or why not?
6.44 (gig) and cell yield (YXjs) is 0. What flow rate will give the maximum total ethanol production in the continuous fermenter and what is the maximum ethanol production rate? b. Your company is a major producer of penicillin.019 (gig). The ethanol yield (Ypjs) is 0. using
. The inlet substrate concentration is 50 g] f.
27 1. your boss would like you to design a continuous reactor system with an inlet flow and substrate concentration of 250 f/hr and 25 sl E.025.0 14. Report reactor volumes and effluent cell. The inlet substrate concentration is 100 g/ f and the flow rate is 20 f/hr. Assume no cells or product in the system inlet. respectively.150 an inlet substrate concentration
Cs.0 5..13 A strain of yeast is being cultivated in a 30. [Contributed by Brian S.56 3.32 12. whereas the substrate concentrations of the two streams are the same. and cell concentration CXi is 0.37 1.0 gil.1 18. j. if inlet substrate concentration Cs.8
Concentration Substrate Cell Cx (g/ f) Cs (g/ f) 10.7
Product
Cp (gl e)
1. of 30 g/ E:
Flowrate F (me/hr) 27. and product concentrations for the proposed ferrnenterj s).LmtJ1: = 0.. perfectly mixed vessel at steady state with no cell death.04 1. which will produce an overall yield of product of 2100 kg/yr.8 16. and YXjS = 0.7 15.0 g/ E. In a continuous..3 h ". Would you recommend a single fermenter of two fermenters in series? Design a system which will meet production constraints while minimizing total fermenter volume.f CSTF with a cell recycling system (cell settler) as shown in the following figure. Using the preceding organism.05 g/I. Calculate the steadystate substrate and cell concentrations in the fermenter. given an operating time of 300 day /yr at 24 hr / day. The cell settler was designed so that the cell concentration of its outlet stream is 30 percent of that of its inlet stream.70 2. what dilution rate D will give the maximum total rate of cell production? What are the outlet cell and substrate concentrations at this dilution rate? b.
I
. Hooker) TriState University. now equals 25.5 24.] 6.2 22.44
a. substrate. The feed stream is sterile. The growth rate of the cells can be represented by the Monad kinetics with the parameters: Ks = 0.
Determine the recycle ratio needed to minimize reactor size for fractional conversion of XSf = 0.995. 6. and the fraction of Gmass with time during a batch cultivation of a microorganism which has the following parameters
. The optimal recycle rate corresponds to the minimumsized reactor needed to attain a desired level of conversion.XSf)
R(l .XSf)
R[l
+ R(l
R+l
. b. The recycle rate (R) is defined as
R = volume of fluid returned
to entrance volume leaving the system
a. and inhibitor. show the change of the concentrations [in g dry weight/f) of Gmass. (1967). Show that an optimal recycle rate must satisfy
In 1 + R(1 . It has been determined that the fermenter efficiency can be improved' by recycling a portion of the product stream so that it returns to the entrance for an additional pass through the fermenter.XSf)]
where XSj is the fraction of a limiting substrate S that is converted to cell mass.15' By using the structured model proposed by Ramkrishna et al.14
A plugflow fermenter is to be used to cultivate microbial cells. Hmass.151
F = 20 e fhr Cs = 100gj£
I
Cx Cs
6.
(1967).0 X 105 g/ £ C'X GO = 1.6 g/ £
Compare your' simulation result with Figure 14 of the paper by Ramkrishna et al. = 0. = 3.1 g/ f J{ = 150 f/ ghr J{' = 70 f/ ghr «.L
=
a~
aT
=2
0.0267
a~l = 0
g/ £
as
=8
g/ £
C'so=10g/£ C'To = 0 g/ e C'XHO = 8.5 hr1 f. What are the differences? Explain.0 x 105 I{~ = 0.2 g/ f
=
=0 a~ = 0. the shape of the curves are quite different from those in the paper.0 x 10.5 hr1
f. If you showed the change of the cell concentrations in g dry weight/liter.5 X 105
J{~
«. Do the parameter values predict realistic growth curves? What new features can this model predict which the Monod model cannot?
.L' = 2.152 and initial conditions: 0.2 x 104 aT1 = 0.
covalently attached to the l' carbon atom of the sugar by an Nglycosylic bond as shown in Figure 7. nucleotides.
3
a polymer. A nitrogenous base of either purine or pyrimidine derivation. nucleotide. especially one composed of more than 100 repeated
. 3 ..Chapter 7 Genetic Engineering
The central tool for the new biotechnology is the recombinant DNA technique.? The monomeric unit. has the following three components (Figure 7. By inserting foreign genetic information into fastgrowing microorganisms. This technology is also known as genetic engineering because it involves the manipulation of genetic materials.
The name genetic engineering should not mislead the readers that it is a field of engineering.
1 2
Read Chapter 1 for a general introduction
to the new biotechnology.? In this chapter.1
DNA and RNA
Deoxyribonucleic acid (DNA) is the most important molecule in living cells and contains all of the information that specifies the cell. A cyclic fivecarbon (pentose) sugar: deoxyribose for DNA. it is a field of biological science.1): 1. A phosphate attached to the 5' carbon of the sugar by phosphoester linkage. b. The purines: adenine (A) and guanine (G). and ribose for RNA. we can produce foreign gene prod ucts (proteins) with higher rates and yields that have not been possible with any other cellular systems. 2.! It allows direct manipulation of genetic material of individual cells. thymine (T) for DNA only. basic principles involved in recombinant DNA technology and problems involved in cultivating the genetically engineered cells are briefly described. DNA and ribonucleic acid (RNA) are macromolecules that are linear polymers built up from simple subunits. a. The pyrimidines: cytosine (C). and uracil (U) for RNA only.1. Macromolecules: monomers.
7.
>
Therefore. so a smaller amount of medium passes (through.128
20~~~~~
15
1 D
10
).
Solving the preceding equation for V F
Tm
yields
Tm = .2.Lmax
F<
V?Sif.5
1. as shown in Figure 6. On the other hand) at point B the cell concentration of the outlet stream is high.Lmax
). but the residence time is long.0 1 Cs
1.128 f/hr
6.3
Productivity of CSTF
Normally. + CSt
= 0. Tm curve.5
2. The productivity at point A is equal to that at point B. the cell concentration of the outlet stream is low but the residence time is short.
Ks +
Cs
!
CSif. the productivity of the fermenter is expressed as the amount of a product produced per unit time and volume.26) 1.Lmax
o
0.5(100)(0. more medium can pass through. the productivity of cell mass is equal to CX/Tm. Point A is an unstable region because it is very close to the washout point D. which is equal to the slope of the straight line OAB of the Cx vs.10.0
Figure 6. If the inlet stream is sterile (CXi = 0).9 The plot of 1/ D versus s l Cs for Example
6.Lmax
ic. and because a small fluctuation in the residence time can bring about a large change in
. At point A.37 + 100
= 0. therefore.5.
and Cx = (Cx)opt.· 129
lOl
I
i
Cs
J
2
i sf
rX.. as shown in Figure 6.935. the value of the maximum productivity is equal to the slope of line OC..71 g/I!. Since the residence time is equal to the area of the rectangle of width Cx and height l/Tx' on the l/Tx versus Cx curve. CSi = 109/I!. The cell productivity for a steadystate CSTF with sterile feed is
=TX=
Cx Tm
j. The curve is drawn by using the Monod model with f.6. it is the minimum when the l/Tx is the minimum.38)
. the productivity increases. Productivity is equal to the slope of the straight line OAB. The operating condition for the maximum productivity of the CSTF can be estimated graphically by using l/Tx versus Cx curve.11. The slope of the line will have its maximum value when it is tangent to the Cx curve. As the slope of the line increases.. The maximum productivity will be attained when t. The maximum productivity can be attained when the residence time is the minimum.p'
gil
V.10.
the cell concentration. = (Tm)opt.. as shown in Figure 6. It would be interesting to derive the equations for the cell concentration and residence time at this maximum cell productivity. Therefore...max
ex ::rn
=
rx
ex.
hr
6
Figure 6.LmaxCsCX
Ks
+ Cs
(6. Yx/s = 0.'max = 0. J(s = 0.
ex
o
D
~
Trn
2
sopt
Tm. and the length of AB decreases.10 The change of the concentrations of cells and substrate as a function of the residence time.
4
Comparison of Batch and CSTF
As discussed earlier. +
(6.
rGxo
T
dCx
x
(6. Cs. we obtain the optimum cell concentration for the maximum productivity as
CX.=. Ks = 0.6.935.opt
= Yx/sCs
j
a
+1
a
(6.11 A graphical illustration of the CSTF with maximum productivity.5.38) for Cs yields the optimum resiTm. cX. the residence time required for a batch or steadystate PFF to reach a certain level of cell concentration is Tb.opt
6
Cx
Figure 6. After substituting Cs = CSj .Lmax
6.130
4
3
1
1
o
2
4
CX.71gil.opt
=
J. (6.43)
. The solid line represents the Monod model with jJmax = 0. (6.42)
Substituting dence time:
Eq.39)
where
Q'
= VI Ks Ks CSi .
CS.
The productivity is maximum when dTx/ dCx = O. and setting the resultant equation to zero.to
+ l.41)
1)
(6.41) into Eq. differentiating with respect to Cx.opt
=
a +'1
c:
(Q'
(6. = 10 g] t.40)
Since Cs
= CSi

Cx /Yx/s. = O. Yx)» = 0.Cx/Yx/s into the preceding equation.
5.28) which is equal to the area of the rectangle of width ex and height 11/Tx. The area under the l/rx versus curve between CXo and Cx is equal to Tb . such as the final conversion. if the final cell concentration is less than CX. If the final cell concentration to be reached is in the stationary phase.6
Multiple
Fermenters
Connected in Series
A question arises frequently whether it may be more efficient to use multiple fermenters connected in series instead of one large fermenter.12 Graphical illustration of the residence time required (shaded area) for the (a) CSTF and (b) Batch fermenter. because it requires the smallest residence time. Since the l/rx versus curve is U shaped. (6.12b is smaller than that for the CSTF. On the other hand.
ex
ex. because two CSTFs connected in series require more residence time than one
ex
. we can make the following conclusions for single fermenter: 1..131
1
rx
Cx
Cx (b)
(a)
Figure 6. as shown in Figure 6. In the l/Tx versus ex curve.12a.
where to is the time required to reach an exponential growth phase. as shown in Figure 6. Choosing the optimum fermenter system for maximum productivity depends on the shape of the l/Tx versus curve and the process requirement. 2. one fermenter is better than two fermenters connected in series. the residence time for the CSTF is expressed as Eq.
ex
6.opt. The most productive fermenter system is a CSTF operated at the cell concentration at which value of l/rx is minimum.to. the batch fermenter 'is a better choice than the CSTF because the residence time required for the batch as shown in Figure 6.
6. the concentrations of the substrate. However.
6.13 Graphical illustration of the total residence time required (shaded area) when two fermenters are connected in series: (a) CSTF and PFF.
CSTF does. = 0).13a).
Figure 6.opt followed by another CSTF connected in series is also better than one CSTF (Figure 6.132
1
rx
Cx (a)
Cx
(b)
Figure 6.
. A CSTF operated at CX.13b). 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. as shown in (Figure 6. If the input stream is sterile (Cx. connected in series.1
CSTF and PFF in Series
Figure 6.14 Schematic diagram of the two fermenters. and (b) two CSTFs. The result of the material balance for the first fermenter is the same as the single CSTF which we have already developed. CSTF and PFF. CSTF followed by PFF.14 shows the schematic diagram of the two fermenters connected in series.s« followed by a PFF.
47) will result. the cultivation of microorganisms in the PFF is limited to several experimental cases. as follows: CSI = 
Ks TmlJ.CS2
(6.1

(6. such as the tubular loop batch fermenter (Russell et al.133 cell and product can be calculated from Eqs. Another approach is to integrate Eq. and (6. (6. as explained already.. the residence time can be estimated by
7p2
=
lcx
r
CX2
dCx =
TX
1
lcx
r
CX2
1
(Ks + Cs)dC.48)
1n CX2 CXI
+
KsYx/s 1n . The residence time of the second fermenter can then be calculated using Eq. Furthermore.34).opt.opt followed by a PFF.
(6. (6.48) have to be solved simultaneously to estimate the cell and substrate concentrations. Eqs.I CS CXl + CSI Yx/s CS2
If the final cell concentration (Cx2) is known.LmaxCsCX
(6.31).42)
CXl = Y:y/s (CSi
T
Ks mlJ. 1974) and 'scraped tubular fermenter (MooYoung et al.6. 1979).46)
Since growth yield can be expressed as
YXIS
=
CX2 . the growth kinetics in a PFF can be significantly different from that in a CSTF.47) and (6.48).45 )
For the second PFF.47)
Integration of Eq. However.44)
(6.47).46) after the substitution
Ip2J. (6.
6. the best combination of two fermenters for a minimum total residence time is a CSTF operated at eX. If the residence time of the second fermenter is known. (6. since a CSTF operated at CX. (6..Lmax .CX1 CSI .46) numerically until the given Ip2 value is reached.opt followed another CSTF connected in series
. the final substrate concentration (CS2) can be calculated from Eq. (6.x J.Lmax
_( 
KsYx/s CXl + CSI Yxts
+
1)
of Eq.2
Multiple CSTFs in Series
If the final cell concentration is larger than CX.33). (6.Lmax 
1)
(6. Another more practical approach is to use multiple CSTFs in series. (6.
Cx . For the nth steadystate CSTF.
is also better than one CSTF. (6.53)
which is the slope of the line connecting (CxpO) and (CXl'lx)' Therefore. (6.49).50)
CXn . 1967).
.LmaxCSnCXn x.49 ) where f. we can calculate
either dilution rate with the known cell concentration.15 shows the schematic diagram of the multiple CSTFs connected in series. Similarly. D. the dilution rate of the first reactor when the inlet stream is sterile is . or vice versa.CX1
TX2
I (6. or vice versa.CXn_1 YXIS = CSn_1 .16. Ks + CSn Growth yield can be expressed' as
T 
(6.114
F CS.49). (6. The estimation of the cell or substrate concentration with the known dilution rate can be done easily by using graphical technique as shown in Figure 6. From Eq.52)
which can be represented by the slope of the straight line connecting the origin and (CXlJTxJ in Figure 6.51) simultaneously.CSn
(6. you can estimate the cell concentration of each fermenter.
Figure 6. = F =
Vi
TXl
CX1
(6. for the second fermenter
D2
=
V2
F
=
CX2 .51)
By solving Eqs. by knowing the dilution rate of each fermenter. the material balance for the microorganisms can be written as (6.15
Schematic diagram of the multiple CSTFs connected in series. Figure 6.50). and (6.16 (Luedeking.
The cell yield (Yx/s) is 0. You want to cultivate this microorganism in either one fermenter or two in series. If you use two CSTFs in series. CSi = 10 gil.LmaxCS Ks + Cs
=
0..7(5) 5+5
= 0. CX.3
Suppose you have a microorganism that obeys the Monod equation:
dCx dt
j. Ks = 0.LmaxCsCX Ks + C.
Example 6. 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. If you use one CSTF.
where J.6. what should be the size of the fermenter? What is the cell concentration of the outlet stream? b. respectively. The line represents the Monod model with /lmax= 0.7hr1 and Ks = 5g/e.Lmax = 0. What is the best combination of fermenter types and volumes if you use two fermenters in series?
Solution:
a.16 Graphical solution for a twostage continuous fermentation. YXjS = 0. the dilution rate is equal to specific growth rate: D
=
F V
=
j. The flow rate and the substrate concentration of the inlet stream should be 500f/hr and 85 gil..135
ex
Figure 6.935. For a single steadystate CSTF with a sterile feed.71 gjP. a.65. The substrate concentration of the outlet stream must be 5 gil.35
. = o.
42).sCS
= =
CX.
F
c.opt.
F(C
Xl
.5) = 52g/e
CX.
Cs) = 0.7(5)(52)/(5 + 5) = 192£ The total volume of the two CSTFs is
v = Vi + Vi = 950 + 192 = 1.7(4.35
=
1 429 '
e
of the outlet stream is

Cx = YXIS(CS.opt
i
= )5 ~ 85 = 4. For two CSTFs in series. from Eq.65(85) 4. from Eq.opt = J.opt = a + 1
=
85 4..C ) + ViJ.9(500)
=
4.LmaxCs2CX2
X2
Ks
+ CS
2
By rearranging the preceding equation for Vi.5(85 . (6.6.. (6.
'" =
Cx} CSI
Tml
)Ks .2 1)
= 1.Lmax(a _ 1) = 1.
500(52 .142£
. (6.2 + 1
=
16 gl e
a ='Tin.oyt
b.
9h
r
Vi
= TmlF
= 950 £
=0
For the second fermenter.~·: 1
= 45 gil
Cs. the first fermenter must be operated at and CS.49).2
j
=
Yx/sCs
CSj
a:
1
=
0.2 0. c«
Therefore.136
v=
The cell concentration
F D
=
500 0.39) through Eq.45) 0.
For the second PFF. (6. with Cell Recycling
The cellular productivity in a CSTF increases with an increase in the dilution rate and reaches a maximum value.
6. there must be a limit in the increase of the cellular productivity with increased cell concentration because in a high cell concentration environment. the productivity of the fermenter is limited due to the loss of cells with the outlet stream.32
Therefore. However.65)
= 0.65)
+1
52 In 45
+ 45 + 16(0. The best combination is a CSTF operated at the maximum rate followed by a PFF.65) 0.
V2 = Tp2F
= 0. If the dilution rate is increased beyond the maximum point. c.7
CSTF. If all cells are recycled back into the fermenter. 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 cell loss from the outlet stream. Therefore. 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.137
which is 20 percent smaller than the volume required when we use a single CSTF.32(500) =
= 950
The total volume of the CSTF and PFF is
v
= Vi
+ 112
+ 160
= 1.
. the nutrienttransfer rate will be decreased due to overcrowding and aggregation of cells. The volume of the first fermenter is 950 e as calculated in part
(b).7 45 + 16(0. from Eq.48)
Tp2
= 
1 [(
KsYx/s
CXl
JLmax
+ CSl Yx/s
+
)
1)
12 n CX
CX1
+
KsYx/s
CX1
1 . One way to improve the reactor productivity is to recycle the cell by separating the cells from the product stream using a crossflow filter unit (Figure 6.17). The high cell concentration maintained using cell recycling will increase the cellular productivity since the growth rate is proportional to the cell concentration.65)
160 e
5(0. The additional saving by employing the second PFF instead of a second GSTF is not significant in this case.1] n Cs + CS1 Yxts CS2 16] In 5
=
1 [( 5(0. 110
e
which is 22 percent smaller than the volume required when we use a single CSTF. the cell concentration will increase continuously with time and a steady state will never be reached.
55) where
/3.58) Figure 6. The material balance for I cells in the fermenter with a cell recycling unit is FCx. the bleeding ratio.17
Schematic diagram of CSTF with cell recycling. For a steadystate CSTF with cell recycling and a sterile feed. therefore. (6. to operate a CSTF with recycling in a steadystate mode.138
F Cs.5. Cx . As /3 is reduced from 1 to 0. /3D instead of D is equal to the specific growth rate. (6.
Therefore. we need to have a bleeding stream.
L Cs
v
''C:>
B Cs Cx
Figure
6.11) into Eq. The cell concentration in the fermenter can be calculated from the value of Cs as (6. If the growth rate can be expressed by Monod kinetics.
.56)
Now. .54)
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. (6. is defined as /3 = B
F
(6. cells are not recycled.18 shows the effect of bleeding ratio on the cell concentration and productivity.17. D = JL. as shown in Figure 6.BCx
+ V JL Cx = V dt
dCx
(6.57)
which is valid when TmJLmax > /3. substitution of Eq. When /3 = 1. the cell concentration and productivity is doubled.55) and rearrangement for Cs yields
Cs =
/3Ks
'TmJLmax 
/3
(6.
such as better aeration. Another way to classify fermenters is based on how the fermenter contents are mixed: by compressed air.0 D = J:_
1m
1.
6. The tank and column fermenters are both constructed as cylindrical vessels. column.5
2.139
0. Fermenters are usually classified based on their vessel type such as tank.
. They can be distinguished based on their heighttodiameter ratio (HI D) as (Schiigerl. or loop fermenters. effective heat removal. These fermenters were designed to improve either the disadvantages of the stirred tank fermenter .4. .0
. immobilization of cells.18 The effect of bleeding ratio on the cellular productivity.5
1. or to meet a specific requirement of a certain fermentation process. by a mechanical internal moving part) or by external liquid pumping. which can be a central draft tube or an external loop. and unusually large designs. 1982):
HI D < 3 HI D > 3
for the tank and for the column fermenter
A loop fermenter is a tank or column fermenter with a liquid circulation loop. Figure 6. the reduction of equipment and operating costs for inexpensive bulk products.8
Alternative
Fermenters
Many alternative fermenters have been proposed and tested.high power consumption and shear damage.3 and the advantages and disadvantages of three basic fermenter types are listed in Table 6. Representative fermenters in each category are listed in Table 6. cell separation or retention.
Excessive foaming. No moving parts 2. Limited to low viscosity system
Airlift
3. No moving parts Bubble Column
3. Simple
4. Low equipment
1. Ability to handle high
viscosi ty media l._r~
Compressed! Azr
i
Primary
Source
of Mixing
I
i
Internal Af ovmg Parts
External
P7. which is usually composed of a long cylindrical vessel with a sparg
.3 Classifications of Ferrnenters
Vessel Type Tank Column Loop
~.
1. Excessive foaming
4.4 and Disadvantages Fermenter
i
multistage propeller loop
sieve tray packedbed
I
I (or cascade)
iI
jet loop
Advantages Type Stirredtank
of Three Basic Disadvantages l.8. High cell concentration
3. Good heat transfer
6. Flexible intensity and adaptable 2. High gas absorption
efficiency
3.140
Table 6. High equipment costs
1. High power consumption 2. Poor mixing
2. stirredtank bubble column tapered airlift pressure column cycle Table 6.1
Column Fermenter
The most simple fermenter is the bubble column fermenter (or tower fermenter). Wide range of mixing
3. Limited to low
costs viscosity system
3.Lmping
. Poor mixing 2. Damage shear sensitive cells
Configurations
Advantages 1. Simple
2.
high airflow rates are required to maintain the cell suspension and mixing... and (f) packedbed column with external pumping. several alternative designs have been proposed. Since it does not have any moving parts. high cell concentrations can be maintained in the lower portion of the column without any separation device.(c)]. . A tapered column fermenter [Figure 6. (e) sievetray column with external pumping..141
j . (c) sievetray bubble column. As bubbles rise in the column) they can coalesce rapidly leading to a decrease in the oxygentransfer rate.. However.19 Column fermenters: (a) bubble column. As the cells settle... Therefore. which leads to the rapid initial fermentation followed by a slower one involving less desirable substrates.
ing device at the bottom [Figure 6. Only the lower part of the column can be maintained with high cell concentrations.. However..19(a) ... column fermenters are inflexible and limited to a relatively narrow range of operating conditions.. The fermenter contents are mixed by the rising bubbles which also provide the oxygen needs of the cells. As the cell concentration increases in a fermenter. (d) multistage stirred column. it is energy efficient with respect to the amount of oxygen transfer per unit energy input. To overcome the weaknesses of the column ferrnenter..... (b) tapered bubble column.19(b)]
.
j
air
(a) (b)
(c)
(d)
(e)
(f)
Figure 6... the increased airflow rate can cause excessive foaming and high retention of air bubbles in the column which decreases the productivity of the fermenter. the bubble column fermenter is usually limited to aerobic fermentations and the rising bubbles may not provide adequate mixing for optimalgrowth.