Continuous Bioreactors

Chemostat with Recycle

 Batch Reactors
Cell Growth
dX S
rX X m X
dt KS S

Substrate Utilization
dS X m S X
rS
dt YX / S K S S YX / S

Product (cometabolic contaminants use negative sign)

1 dP
qp YP / X g g
X dt
1 dP m S
qp
X dt KS S
 Continuous culture: the
chemostat
1. Concentration of a limiting nutrient
2. Dilution rate

Results from a batch culture

Monod Kinetics applies!!! F D is dilution rate

D F is flow rate
V V is volume
Substrate depletion kinetics !!
 Continuous Reactors

Chemostat - CSTR - continuous stirred tank reactor for the cultivation of cells.
mixing supplied by impellers and rising gas bubbles
assume complete mixing - composition of any phases do not vary with position
liquid effluent has the same composition as the reactor contents
 Mass Balance on Chemostat
Acc = in - out + gen - cons
dci
VR Fcif Fci VR rfi
dt
VR - reactor volume
F - volumetric flow rate of feed and effluent streams (they are
equal)
ci - concentration of component i in the reactor
cif - concentration of component i in the influent or feed stream
 If we have a steady state reactor - no changes in composition with time
then and

dci
0
dt
Define as the dilution rate -
F
rfi of the mean cif or) residence time
(ci holding
reciprocal
V
detention timeR
F 1
For cell mass, if we assume a sterile feed: D
ci = X and Xf = 0 and VR
rx = X then X = DX

D= at SS
 Chemostat with Monod Kinetics
mS KS
D S
KS S mm 1
The above equations only holds if mmax >1
If mmax < 1 or D>
washout of the cells occurs
Cells leave the reactor faster than they are dividing.
mS f
Dmax Near washout the reactor is very sensitive to
KS S f variations in D
Small change in D large shifts in X and/or S
If max = 0.5 hr-1 then D< 0.4 hr-1
 Cell Growth in Ideal Chemostat
Determination of Monod Parameters
In Chemostat: g=D, varying D obtains D~S m S
g D
KS S
1 K 1 1
S ( Lineweaver - Burk)
D m S m

4 0.3
DX
3.5
0.25
3
0.2 Washed out: If D

DX (g/L-hr)
2.5
S, X (g/L)

m = 0.2 2
X S
0.15
is set at a value
hr-1 1.5
0.1
greater than m
1
0.05
(D > m),
0.5
0 0
the culture
0 0.05 0.1 0.15 0.2 0.25 cannot
D (1/hr)
reproduce
quickly
Chemostat technique: reliable, constant environment, operation enough
may be difficult.
to maintain
Intracellular Product Formation
-Chemostat
dci
VR Fcif Fci VR rfi
dt
Steady State and ci = P

D Pf P YP / X X 0

If Pf = 0
YP / X X
P
D
 Substrate Balance on Chemostat -
Intracellular Product
dci
VR Fcif Fci VR rfi
dt
If ci = S
1 dS
FS 0 FS VR X VR
YX / S dt

At Steady State
X
D S 0 S X YX / S S 0 S
YX / S
With Monod
Ks D
X YX / S S 0
m D
 Chemostat with Extracellular
Product
Cell Mass Balance
dX
VR FX 0 FX VR X
dt
D
Substrate Balance
1 1 dS
FS 0 FS VR X VR q P X VR
YX / S YX / P dt

Solve Substrate at SS for X

 Class Exercise

E. coli is cultivated in continuous culture under

aerobic conditions with glucose limitation. When the
system is operated at D= 0.2 hr-1, determine the
effluent glucose and biomass concentrations assuming
Monod kinetics (S0 = 5 g/l, m= 0.25 hr-1 , KS = 100
mg/L, Y x/s = 0.4 g/g)
 Chemostat with Recycle
FR, XR

F, X0 F, X2
V, X1

F+FR, X1

Fnutrientflowrate
Vreactorvolume
X1xconcentrationinreactor
X2Xconcentrationineffluent
XRXconcentrationinrecycle
FRrecycleflowrate
 Chemostat with Recycle Cell
mass equation
Acc=inout+gen
dX 1
FX0+FRXR(F+FR)X1+VX1=V
dt

FR, XR

F, X0 F, X2
V, X1
F+FR, X1
 Chemostat with Recycle cont.
Define Substitutions
=FR/F F+FR=(1+)F
recycleratio FRXRterm
C=XR/X1concentration FR=F
factor XR=CX1
FRXR=CFX1

dX 1
FX0+FRXR(F+FR)X1+VX1=V
dt
dX 1
FX0+CFX1(1+)FX1+VX1=V
dt
 Recycle cont
Assume
dX 1
steadystate=0 Chemostatcanbe
dt operatedathigher
sterilefeedX0=0 dilutionratesthanthe
Then specificgrowthrate
(C1)F+V=0 whencellrecycleis
IfD=F/Vforrecycle used.
=D(1+(1C))

ifC>1(concentrationofcells)then(1C)<0
then<D
 Substrate balance - Recycle
X 1 dS
FS 0 FS V (1 ) FS V
YX / S dt
At Steady state and substituting for

D YX / S ( S 0 S )
X 1 YX / S ( S 0 S )
(1 C )
 Recycle Substrate cont.
Assuming Monod

K S D(1 C )
S
max D(1 C )
YX / S K S D(1 C )
X1 S0
(1 C ) max D(1 C )
 Chemostat with Cell Recycle

No recycle

m=1.00h-1, S0=2.0g/l, Ks=0.01 g/l, Yx/s=0.5 g/g,

concentration factor C=2.0 and recycle ratio =0.5
 In Class Exercise -
Consider a 1000 L CSTR in which biomass is being
produced with glucose as the substrate. The
microbial system follows a Monod relations with m
= 0.4 hr 1, KS = 1.5 g/L, and yield factor = 0.5 g/g.
If S0 = 10g/L glucose and F = 100 L/h:
What is the specific biomass production rate (g/l-h) at
SS?
If recycle is used with a recycle stream of 10 L/h and a
recycle biomass concentration five times as large as that
in the reactor exit, what would be the new specific
biomass production rate?
 Multi-stage Chemostat System
In some fermentations, the growth and product-
formation steps need to be separated.
e.g. secondary metabolite,
culture of genetically engineered cells.

P2
P10
Growth stage Product formation stage
At steady state, Vn, Xn,Sn,,Pn in the reactor of each stage dont change with time.
 Multi-stage Chemostat System
X0=0, Vi, i=1,2.n constant.
Stage 1: cell growth condition, Kd=0, qp=0
dX 1
Cell mass: FX 0 FX 1 1 X 1V1 V1
dt
At steady state X0
1 D1 (1 )
X1
Limiting substrate: 1 X 1 dS1
FS 0 FS1 V1 M V1
YX / S dt
At steady state
1 X 1
S1 S 0
D1YXM/ S

1 , 2 ... and n are net specific growth rates in Stage 1, 2,..., and n, recspectively.
When Kd 0, they are equal to the respective specific gross growth rates in each stage.
 Multi-stage Chemostat System
Stage 2 product formation conditions, Kd=0, F=0
dX 2
Cell mass: FX 1 FX 2 2 X 2V2 V2
dt
X F
At steady state 2 D2 (1 1 ) where D 2
X2 V2

P2
P10 V2 are constant.
 Multi-stage Chemostat System
Stage 2 product formation conditions, Kd=0, F=0

Limiting substrate: 2 X 2 qp X 2 dS 2
FS1 FS 2 V2 M V V2
YX / S YP / S dt
At steady state
2 X 2 qp X 2
S 2 S1 M

D2YX / S D2YP / S
dP2
Product: FP1 FP2 V2 q p X 2 V2
dt
At steady state qp X 2
P2 P1
D2
 Multi-stage Chemostat System
Stage n product formation conditions, Kd=0, F=0
Similarly, equations could be obtained for nth stage.
X n 1
n Dn (1 )
Xn

n X n qp X n
S n S n 1 M

DnYX / S DnYP / S
qp X n
Pn Pn 1
Dn
If n (e.g. Monod model) and qp are known functions, Xn , Pn, and Sn at
nth stage could be determined by the above equations.
 Chemostat in Series
(Additional Feed in Second Stage)
Cell balance around second stage
dX 2
F1 X 1 F ' X '( F ' F1 ) X 2 V2 2 X 2 V2
dt
At Steady State with X = 0
F1 X 1
2 D '2 Growth rate does not
V2 X 2
typically follow Monod
F1 F ' in Second Stage if
D '2
V2 additional feed.
 Chemostat in Series cont.
Substrate Balance if Additional Feed

1 dS
F1S1 F ' S ( F1 F ' ) S 2 V2 2 X 2
'
0 V2
YX / S dt
Atsteadystatethetwoequationscanbe
solvedsimultaneouslyforS2andV2
Majoradvantageistoseparateproduction
fromgrowth
 In Class Example 9.2
In a two stage chemostat system, the volumes of the first and second
reactors are 500 L and 300 L respectively. The first reactor is used
for biomass production and the second is for a secondary metabolite
formation. The feed flow rate to the first reactor is F = 100 L/h, and
the glucose concentration is 5.0 g/L. Use the following constants for
the cells.
m = 0.3 h-1, Ks = 0.1 g/L Y X/S= 0.4 g/g
Determine the cell and glucose concentrations after the first stage.
Assume that growth is negligible in the second stage and the
specific rate of product formation is q P = 0.02 gP/g cell hr, and Y P/S
= 0.6 gP/gS. Determine the product and substrate concentrations in
the effluent of the second reactor.
Fed Batch Reactor
Reactor Design Equation
V dN A
FA0 FA rA dV
dt
No outflow FA = 0
Good Mixing rA dV
term out of the integral
dN A d C A V
FA0 rA V
dt dt
 Fed Batch Continued
Convert the mass (NA) to concentration. Applying
integration by parts yields
dC A dV
FA0 rAV V CA
Since dt dt
dV
FA0
Then dt

dC A
FA0 rAV V C A FA0
Rearranging dt

dC A FA0 C A FA0
rA
dt V V
 Fed Batch Continued
Or dC A FA0
1 C A rA
dt V

Used when there is substrate inhibition and

for bioreactors with cells.
 Fed-batch Reactors

d (Vci )
Vr fi F (t )c fi
dt
dV
F (t )
dt

Differentiationtheaboveequationusingchainrule,andsubstitute
fordV/dt
dci F (t )
rfi [cif ci ]
dt V
 Fed-Batch
Analysis of fed-batch with substrate continuously fed
and no output: t=0, V0=0, F is constant.
Volume: dV
F V Vo Ft
dt
At quasi steady state, S added=S consumed,
X, S, P concentrations are constant.
Cell mass balance: d ( XV )
FX O V net X
dt
d ( XV ) dX dV
since V X , then
dt dt dt
dX dV
FX O V net X V X
dt dt
dV 1 dV F
V net X X net D
dt V dt V
F F Do
net
V V0 Ft 1 D0 t
m S Ks D
net D if K d 0 Then S
Ks S m D
(Monod growth model applied)
 Fed-Batch

M
where S0
assuming XXm
M

where Xt=Xt0 at t=0

Fed-Batch
(g)

qp (i.e. g product/g cells-min)

at Pt=Pt0, t=0
 Class Exercise 9.4
Penicillin is produced in a fed-batch culture with the intermittent
addition of glucose solution to the culture medium. The initial
culture volume at quasi-steady state is V0= 500 L, and the glucose
containing nutrient solution is added with a flow rate of F = 50 L/h.
X0 = 20 g/L, S0 = 300 g/L, m = 0.2 h-1, Ks = 0.5 g/L and Y x/s= 0.3
g/g
Determine culture volume at t = 10 h
Determine concentration of glucose at t = 10 h
Determine the concentration and total amount of cells at t = 10 h
If qp = 0.05 g product.g cells h and P 0 = 0.1 g/L, determine the
product concentration at t = 10 h