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V, S, P
) ) dM = M i M o + RG RC dt
Mass Balance for batch enzyme catalytic reaction
max S d (VS ) = V dt Km + S
Usually, V is constant, so we have:
S dS = max dt Km + S
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Integrating:
dt =
Km + S dS max S
Km S0 S0 S f tb = ln + max S f max
Enzymes are subject to deativation. Thus, the concentration of active enzyme in the reactor, and therefore the value of max, may change during reaction. When deactivation is significant, variation of max with time can be expressed by:
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max = max 0 e
Therefore,
kdt
max 0 e k d t S dS = dt Km + S
Separating variables gives:
kd t
dt =
Km + S dS max 0 S
d ( xV ) = xV k d xV dt
For V constant,
dx = ( k d ) x dt
x = x0 e (max kd ) t
or
xf 1 tb = ln x0 max k d
If the rate of cell death is negligible, we have:
x = x0 e
maxt
and
tb = 1 max ln xf x0
q dS = ( max + P + m ) x dt Y X / S YP / S
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q dS = ( max + P + m ) x0 e max dt Y X / S YP / S
and
tb =
1 max
ln[1 + (
1 YX / S
S0 S f ] qP m ) x0 + + maxYP / S max
tb =
1 max
S0 S f ln[1 + ] 1 m + ( ) x0 Y X / S max
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tb =
1 max
YX / S ln[1 + ( S 0 S f )] x0
d ( PV ) = P xV dt
If cell death is negligible and V constant,
dP = P x0 emaxt dt
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tb =
1 max
max ln[1 + ( Pf P0 )] P x0
Cell concentration
xf
x0 tp t1 tb t hv tp t1 tb t hv
Figure 10.2 The entire time period needed for batch cell culture
t dn = t p + t1 + t hv
tT = tb + tdn
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Fed-batch culture is used extensively in production of bakers yeast to overcome catabolic repression and control oxygen demand; it is also used routinely for penicillin production. Space must be allowed in fed-batch bioreactors for addition of fresh medium; in some cases a portion of the broth is removed before injection of additional material. The flow rate and timing of the feed are often determined by monitoring parameters such as dissolved-oxygen level or exhaust gas composition. As enzyme reactions are rarely carried out as fed-batch operations, we will consider fed-batch bioreactors for cell cultures or fermentations only.
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Feed stream F xi Si Pi
V, x, S, P
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dV =F dt
A mass balance for cells:
d ( xV ) = Fxi + xV k d xV dt
Expand the differential equation gives:
dV dx x +V = Fxi + ( k d ) xV dt dt
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Therefore,
xF + V
dx = Fx i + ( k d ) xV dt
Dividing by V gives:
dx F F = xi + ( k d ) x dt V V
and
dx = Dxi + ( k d D ) x dt
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Usually, the bioreactor is operated first in batch until a little high cell density is achieved and the substrate virtually exhausted, then, fedbatch operation is started with medium flow rate F. As a result, cell concentration x is maintained relatively high and approximately constant so that dx/dt 0 and D, therefore, Monod expression for cell growth can be written as:
D=
or
max S KS + S
DK S S= max D
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If no product is produced, or the product formation is directly coupled with energy metabolism, we have:
x dS = D( Si S ) dt YX / S
At high cell density, virtually all substrate entering the vessel is consumed immediately; therefore, S << Si and dS/dt 0. Applying these relationships with D, we obtain:
x Y X / S Si
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For product synthesis directly coupled with energy metabolism, at the same time assuming the feed does not contain product:
P YP / S Si
Even though cell concentration remains virtually unchanged with time dx/dt 0, because the broth volume increases with time during fed-batch culture, the total biomass within the bioreactor also increases. Consider the rate of increase of total biomass in the bioreactor dX/dt:
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dV dx dX d ( xV ) = =x +V = Y X / S Si F dt dt dt dt
X = X 0 + (Y X / S Si F )tbf
This indicates that, if YX/S, Si and F constant, the total biomass in fed-batch culture increases as a linear function of time. Under conditions of high biomass density and almost depletion of substrate, a quasi-steady-state condition previals in fed-batch bioreactors in which dx/dt 0, dS/dt 0 and dP/dt 0, and x, S and P are almost constant, but , V, D and X are changing with time.
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contamination also hinders CSTRs application in animal and plant cell cultures and other fermentations, such as penicillin, in which much lower growth rate of cells and complete nutrients increase the opportunity for undesirable microorganisms to grow quickly within the system. Different steady-state operating strategies are available for a CSTR. In a chemostat, the liquid volume within the bioreactor is maintained constant by setting the inlet and outlet flow rates equal; the dilution rate is therefore constant and steady state is achieved in the chemostat by adjusting itself to the feed rate. In a turbidostat, the liquid volume is kept constant by setting the outlet flow rate equal to the inlet flow rate; however, the inlet flow rate is adjusted to keep the biomass concentration constant. Thus, in a turbidostat the dilution rate adjusts to its steady-state value corresponding to the set biomass concentration.
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F x S P
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Characteristic operating parameters for CSTRs are the dilution rate D and the average residence time . The following relationship exists:
= 1 V = D F
For a given throughput, the bioreactor size V and associated capital and operating costs are minimized when is made as small as possible. Continuous bioreactor theory allows us to determine relationships between or D and steady-state substrate, product and cell concentrations.
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FS i FS
max S V =0 Km + S
D( Si S ) =
max S Km + S
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If kinetic parameters including max and Km and substrate concentration contained within the feed can be used directly to calculate the dilution rate required to achieve a particular level of substrate conversion, then, the steady-state product concentration and productivity can be evaluated from stoichiometry. For immobilized enzyme system, we have:
D( Si S ) = T max S Km + S
where T is the total effectiveness factor, S is the bulk substrate concentration, and max and Km are intrinsic kinetic parameters.
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x=
D( Si S ) D +m YX / S
x = Y X / S ( Si S )
Therefore,
x = Y X / S ( Si
DK S ) max D
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The dilution rate for the highest biomass productivity is given by:
Dopt = max (1
KS ) K S + Si
Now we consider the CSTR in which immobilized cells are used as illustrated below.
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F xs + xsV + TximV = 0
Rearrange:
or
Dx s = ( x s + T xim )
T xim D = (1 + ) xs
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x s T x im V V =0 F (Si S ) YX /S YX /S
and
D( Si S ) =
also
YX / S
( x s + T xim )
D ( Si S )Y X / S max S = K S + S ( Si S )Y X / S + T xim
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100
80 Substrate conversion, %
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xim = 0
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Dcri
0 0 0.1 0.2 0.3 0.4 0.5 Dilution rate, D h1 Figure 10.7 Steady-state substrate conversion as a function of dilution rate with and without immobilized cells (calculated with max = 0.1 h1, KS = 103 g l1, YX/S = 0.5 and Si = 8 103 g l1)
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We can find for any xim > 0, dilution rate D at steady state is greater than . Accordingly, dilution rate is no longer limited by the maximum specific growth rate max, as discussed before, that means immobilized cell bioreactor can be operated at D considerably greater than Dcrit for free cells without washout happening. We also can find that, at a given dilution rate, presence of immobilized cells improves substrate conversion and reduces the amount of substrate lost in the product stream. However, bioreaction rates with immobilized cells can be significantly affected by the mass transfer in and around the particles.
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Product stream F S f
L z F S
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max S FS Z FS z + z Az = 0 Km + S
Rearrange:
F ( S z + z S z ) A z
and
max S = Km + S
max S = Km + S
u( S z + z S z ) z
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u( lim
That is:
S z + z S z z
z 0
)=
max S Km + S
max S dS u = dz Km + S
Integrating with boundary condition S = Si at z = 0 gives:
Km Si Si S f ln + ) L = u( max S f max
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For
V V L = = A = F F u A
we have:
Km Si Si S f = ln + max S f max
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Only a few enzyme reactions, for example, liquification of starch slurry using -amylase before being saccharified to fermented sugar, are applying this operation mode. If enzyme is immobilized, mass-transfer effects should be considered, therefore:
S dS = T max dz Km + S
Generally, PFR operation is not suitable for cell culture unless the biomass is recycled or there is a continuous inoculation operation. However, for sterilization of medium, PFR is widely used and will be discussed later. If cells are immobilized and packed within the column, PFR modified properly can be applied.
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2 Time (h)
Figure 10.9 Variation of temperature with time for batch sterilization of medium
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For operation of batch sterilization systems, we must be able to estimate the holding time required to achieve the desired level of cell destruction. As well as destroying contaminant organisms, heat sterilization also destroys nutrients in the medium. To minimize this loss, holding times at high sterilization temperature should be kept as short as possible. Cell death occurs at all times during batch sterilization, including the heating-up and cooling-down periods. The holding time thd can be minimized by taking into account cell destruction during these periods.
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In a batch vessel where cell death is the only process affecting the number of viable cells, we have:
dN = k d N dt
N1 ln = k d t hd N2
or
N1 N2 kd
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ln thd =
where
kd =
therefore
E d Ae RT
d dN = Ae RT N dt
for heating
d t1 N0 ln = Ae RT dt 0 N1 E
T = Ts (1 +
T0 Ts e Ts
UAt M mC p
)
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M s ht M m C p T0 ) T = T0 (1 + Ms t 1+ Mm
electrical heating
T = T0 (1 +
for cooling
Qt ) M m C p T0
d tf N2 ln = Ae RT dt t2 Nf
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where
UA M wC pwt M wC pw [( )(1e )] T0 Tci M mC p T = Tci 1 + e T ci
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Heating Temperature
Holding
Fermentation temperature
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Normally, cell death below 100 C is minimal; however, when heating and cooling are relatively slow, temperatures remain close to the maximum for considerable periods of time, cell numbers can be reduced significantly outside the holding period. Usually, holding time is of the order of minutes whereas heating and cooling of large medium volumes take hours. The design procedures outlined in this section apply to batch sterilization of medium when the temperature is uniform throughout the vessel. However, if the medium contains contaminant particles in the form of flocs or pellets, temperature gradient within the
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particles may develop and the temperature at the center of the particles will lower than that in the bulk medium. As a result, cell death inside the particles is not as effective as in the bulk medium. Longer holding time is required to treat solid-phase substrates and media containing particles. When batch sterilization is scaled up to larger volumes, much longer times are needed to achieve the same sterilization results as that of small-scale tanks, the destroy of nutrients is exacerbated extremely. Therefore, continuous sterilization process is developed for large-scale use.
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(a)
Fermenter
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(b)
Condensed water
Steam
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Continuous sterilization, particularly a high-temperature, shortexposure-time process, can significantly reduce damage to medium nutrients while achieving high levels of cell destruction. Other advantages include improved steam economy and more liable scale-up. The amount of steam needed for continuous sterilization is only 20 ~ 25% that used in batch processes; the time required is also obviously reduced because heating and cooling are virtually instantaneous.
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Steam injection
Heat exchange
Heat exchange
Time
Time
Figure 10.13 Variation of temperature with time in the continuous sterilisers of Figure 11.12
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An
sterilisers is the characteristics of medium flow in the system. Ideally, all medium entering the system at a particular instant should spend the same time in the steriliser and exit the system at the same time also, that is plug-flow. Otherwise, if mixing occurs within the holding pipe, a risk of contamination will be transferred to the outlet of the sterilized medium from the inlet of raw material. Deviation from plug-flow behavior is characterized by the degree of axial dispersion along the pipe which will be further discussed later.
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Summary
After studying this chapter, you should: understand the concept of well-mixed flow and plug-flow modes, two extremely flowing conditions; be able to predict batch bioreaction time required to achieve designed substrate conversion for enzyme reaction and cell culture; be able to predict the performance of fed-batch bioreactors operated at quasi-steady-state conditions; for CSTR, know how to control its operation in order to avoid washout of cells and to obtain the optimum cell productivity; and finally be able to compare the performance of batch, CSTR and PFR and select proper operation method for a designed bioprocess.
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