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 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
r fi  ( ci  c if )
V
– reciprocal of the mean holding or residence time
– 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  m m 1
• The above equations only holds if mmax >1
• If mmax < 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
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

•  F - nutrient flow rate

• V - reactor volume
• X1 - x concentration in reactor
• X2 - X concentration in effluent
• XR - X concentration in recycle
• FR - recycle flow rate
 Chemostat with Recycle Cell
mass equation
Acc = in - out + gen
dX 1
F X0 + FR XR - (F+ FR) X1 + VX1 = 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
recycle ratio • FRXR term
C = XR /X1 concentration FR = F
factor
XR = CX1
FRXR = CFX1

dX 1
F X0 + FR XR - (F+ FR) X1 + VX1 = V
dt
dX 1
F X0 + CFX1- (1 + )F X1 + VX1 = V
dt
 Recycle cont
• Assume
– steady state dX
= 01 Chemostat can be
dt operated at higher
– sterile feed X0 = 0 dilution rates than the
Then specific growth rate
(C - 1 -)F + V = 0 when cell recycle is
If D = F/V for recycle used.
   = D(1+ (1 -C))
 
if C > 1 (concentration of cells) then (1 - C) < 0
then  < D
 Substrate balance - Recycle
X 1 dS
FS0  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 ) 
 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?
 Chemostat in Series

F, S0 F’, S’0
V1, X1,
F, S1, X1
S1

V2, X2,
S2

F2, S2, X2
 Chemostat in Series
(no additional feed)
• First stage (assuming Monod)

K S 1 D1
S1 
 m1  D1
X 1  YX / S  S 0  S1 
• Second Stage
dX 2
FX 1  FX 2  V2  2 X 2  V2
dt
 Chemostat in Series cont.
• At Steady State
 X1 
 2  D2 1  
 X2 
X1
 1,  2  D2
X2

• Substrate Balance
1 dS
FS1  FS 2  V2  2 X 2  V2
YX / S dt
 Chemostat in series
• At Steady State
2 X 2
S 2  S1 
D2 YX / S

• D2 = F/V2 and could have Monod growth for 2

 
• Solve S and   equations simultaneously for X2 
and S2  once the value of 2 is known
 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
• At steady state the two equations can be
solved simultaneously for S2 and V2
• Major advantage is to separate production
from growth
 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 qP = 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

Differentiation the above equation using chain rule, and substitute

for dV/dt
dci F (t )
 rfi  [cif  ci ]
dt V
 Fed-batch cont.
• Cell balance – sterile feed
rfi  X
KS D
dX S
 (   D) X m  D
dt
• This can be a steady state reactor if substrate is
consumed as fast as it enters (quasi-steady-state).
Then dX/dt = 0 and  = D, like in a chemostat.
Recall, D = F / V
 Fed batch cont
• Substrate balance – no outflow (Fcout = 0), sterile feed
• St = SV and Xt = XV (mass of substrate or cells in reactor at a
given time)
• S0 = substrate in feed stream
substrate substrate
in consumed

dS t
X t
no substrate out
Substrate  FS 0  (Flow out = 0)
balance dt YX / S
t
Cell dX
balance  X t

dt
 Fed batch cont.
• Quasi steady state for St – change in substrate
is very small in reactor and is consumed as
rapidly as fed
X t
FS 0 
YX / S
t
dX
 X  FYX / S S 0
t
Integrating from t=0 to t
dt
X  X 0  FYX / S S 0t
t t
 Fed batch cont.
• Quasi steady state for St – change in substrate
is very small in reactor and is consumed as
rapidly as fed (then, dSt/dt = 0)
X t
FS0 
YX / S
YX / S FS0
X 
t


dX t
 X t  FYX / SS0 Integrating from t=0 to t
dt
X  X 0  FYX / SS0 t
t t
Note, "t" was missing.
 Fed batch cont.
X  X  FYX / SS0 t
t t
0

• What this means

– the total amount of cells in the reactor increases
with time
– dilution rate and  decrease with time in fed
batch culture
– Since  = D, the growth rate is controlled by
the dilution rate.
 Product profiles in fed batch
• Product profiles can be obtained by using the definitions
of Yp/s or qp.
• When Yp/s is constant (at quasi-steady-state with S <<S0):
P ≈ Yp/s S0, and
FP ≈ FYp/s S0,
• When qp is constant,
t
dP
 q pX t
dt

• where Pt is the total amount of product in culture

• Substituting
X  X  FYX / SS0 t
t t
0
• yields t
dP
 q p X m  V0  Ft 
dt
 Ft 
P  P  q p X m  V0   t
t t
0
 2
• in terms of product concentration
V0  V0 Dt 
P  P0  q pX m   t
V V 2 
 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 P0 = 0.1 g/L, determine the
product concentration at t = 10 h