Chemostats

!" !" !" !"

(constant chemical environment)

continuous feed of fresh nutrient homogeneous mixture in reactor at steady-state constant volume ~ 7 reactor volumes of feed until steady-state is reached

d [ X ] d [S ] d [ P] -0 dt dt dt

F S0 X0=0 P0=0

S, P, X Air

! !
VRX

F S X P VR

Inlet - Outlet + Generation - Degradation = Accumulation FX0 FX + VRkDX = VR

dX dt

( l %( g % ( l %( g % & #& # & #& # ' h $' l $ ' h $' l $

1 %( g % )l *( & #& # ' h $' l $

1 %( g % )l *( & #& # ' h $' l $

)l *( &

g % # 'l !h$

I. divide through by VR II. let D = F/VR (recall that VR/F is the residence time)

dX - DX 0 , ). + k D + D *X dt
at SS = 0 For >> kD X0 = 0

=D

Cells are removed at a rate equal to the average growth rate of the cells.

 is limited by at least one substrate

.-D-

. max S
KS , S

if "F" is set too high and D > max cells wash-out A chemostat can be used to measure KS The material balance on limiting substrate:

FS 0 + FS + V R . X
at SS

dS 1 1 V q X V + R P R dt Y XM Y PM S S

4 . qP 1 D )S 0 + S * - X 2 M , / Y Y P / 2 S 0 3 XS
At steady-state where = D, and you can measure qP in the case of product formation, then with several values of D and S you can find
You can also find kD set D = - kD

Y XM and YP S . S

The Chemostat as a Tool Chemostats are used to determine physiological parameters of cells, in particular !" YATP

!"

Y XM S

!" kD
Difficulties with chemostats include !" reversion of recombinant cultures !" wall growth

Calculation of

Y XM and ms S

From the substrate balance, we can see that

( KS D % # X - Y XM & S + 0 S& .m + D # $ '
M here Y X S is taken as a constant (i.e. no endogenous metabolism or maintenance)

(Endogenous metabolism = reactions in the cell which consume "cell substance.") The value of Y X S is independent of .
M

For steady-state without product formation:

D )S 0 + S * -

X. Y XM
S

and = D + kD

D)S 0 + S * 1 + M )D , k D * - 0 X YX
S

The "apparent yield", or the measured yield is the substrate consumed per cells produced or

)S 0 + S *
X

- Y XApp
S

then
( & 1 D& App & YX ' S 1 Y
App X S

% kD # D + + -0 M # Y XM # YX S S $ 1 Y
M X S

mS -0 D

maintenance is
mS kD Y XM

1 Y
App X S

X S0 + S
intercept =

slope = mS = (gS/gcells/hr)

1 Y XM
S

Y XM

g cells/g substrate

1/D then kD = mS* Y XM or slope/intercept

Production of a product DP = qPX recall that qP = From our substrate balance

1 dP - YP . S X dt

D )S 0 + S * -

1 Y
M X S

)D , k D * X ,

1 qP X YP
S

Solving for X

% ( # & # & D # & M X - Y X )S 0 + S *& M # S Y &D,k , XS q # D P # & YP # & S $ '

Productivity of a Chemostat Productivity of biomass: DX Productivity of a product: DP To determine the maximum productivity based on the dilution rate, find:

d )DP * =0 dD

Chemostats with Recycle Cell recycle enables higher productivity of the biomass. This is used for both continuous and batch reactors. Here we outline how to perform the mass balances with recycle. F Fresh S0 Medium X0=0 P0=0 "F (1+")F, S, X1, P

S, P, X Air

! !
CX1

Cell Separator

F, X2, P, S

VR

The material balance around the bioreactor on biomass

FX 0 , 5FCX 1 + (1 , 5 ) FX 1 , V. X 1 - V

dX 1 dt

" is the recycle ratio that can be user determined. C = the ratio of concentrations of cells in the recycle stream to the reactor effluent. The total flowrate in the recycle loop, which begins after the cell separator is "F. The fresh feed entering the bioreactor has the same flow rate as the flow exiting the cell separator. So the feed into the system is the same as the flow out of the system. Therefore no fluid volume accumulates in the system. The fluid flow from the reactor to the cell separator must then total the sum out of the system plus the amount recycled, or: Feed from reactor = F + "F = (1 + ")F We can solve for the growth rate from the above material balance = (1 + " - "C)D = [1 + "(1 - C)]D From this equation, we can see also that since C > 1, and "(1 - C)<0, then < D. A chemostat with cell recycle MAY have a higher dilution rate than the specific growth rate.

The biomass balance around the cell concentrator (1 + ")X1 = "CX1 + X2 And we can solve for X2: X2 = (1 + ")X1 - "CX1 = X1(1 + " - "C) Why does C ! (X1/X2)?? The material balance on growth-limiting substrate (fed in the liquid)

FS 0 , 5FS + V
At steady state

.X 1
YX
S

+ (1 , 5 ) FS - V

dS dt

X1 -

Y X )S 0 + S *
S

and therefore,

X1 -

Y X )S 0 + S *

)1 , 5 + 5C *

notice that the cell concentration with and without recycle in 1/(1 + " - "C)

the chemostat differ by the factor,

Is (1 + " - "C) greater or less than zero? Is the cell concentration with cell recycle increased or decreased? The substrate in the effluent can be estimated from
SK S D)1 , 5 + 5C * . m + D)1 , 5 + 5C *