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**- To keep the cell concentration higher than the normal
**

steady-state level in a chemostat. - To increase the cell and growth-associated product yield. - For low-product-value processes: e.g. waste treatment. fuel ethanol ,

X1, S

v

**Chemostat with Cell Recycle
**

Cell mass balance (qp=0, kd ≈0, X0=0, Monod equation is applied):

where µ=µnet=µg-kd

A chemostat can be operated at dilution rates higher than the specific growth rate when cell recycle is used.

1

Chemostat with Cell Recycle

When kd=0

**Chemostat with Cell Recycle
**

Mass balance on growth-limiting substrate (qp=0, kd ≈0, X0=0, Monod equation is applied):

FS 0 FS V g X 1 1 YX / S

M

(1 ) FS V

dS dt

At steady state, dS/dt 0, D M X1 Y X / S (S0 S )

g

S

Since g [1 (1 C )]D, X1 Y X / S ( S0 S )

M

K s D(1 C ) m D(1 C )

1 C

, X1

YX / S [1 (1 C )]

M

[S0

K s D(1 C ) ] m D(1 C )

2

Chemostat with Cell Recycle

No recycle

µm=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 α=0.5

**Chemostat with Cell Recycle
**

Cell mass balance around the cell separator.

X1, S

v

(1 ) FX 1 FX 2 FCX 1 X 2

3

**Example-Chemostat with Cell Recycle
**

Organisms are cultured in a chemostat with cell recycle. The system is operated under glucose limitation.

**F 100 ml/h, V 1000ml, S 0 10 g glucose/L
**

M YX/S 0.5gdw cells/g substrate; μ m 0.2h 1 ,

Ks 1g/L, C 1.5, α 0.7, X 0 0, K d 0

Determine specific growth rate μnet, S in the reactor effluent, cell concentration in the recycle stream (CX1) and in the concentrator effluent (X2)

**Multi-stage Chemostat System
**

In some fermentations, the growth and productformation steps need to be separated.

e.g. secondary metabolite, culture of genetically engineered cells.

P1≈0 Growth stage

P2 Product formation stage

At steady state, Vn, Xn,Sn,,Pn in the reactor of each stage don’t change with time.

4

Genetically Engineered Cells (cont.)

**Features of Genetically Engineered Cells:
**

• have inserted recombinant DNA (plasmids) which allow for the production of a desired protein product. • GE cells grow more slowly than original nonmodified strain (due to the extra metabolic burden of producing product). • Genetic Instability causes the GE culture to (slowly) lose ability to produce product. The nonplasmid carrying cells or the cells with mutation in the plasmid (revertants) grow faster.

5

Genetically Engineered Cells (cont.):

In the first stage, only cell growth occurs and no inducer is added for product formation. The GE cells grow at the maximum rate and are not outcompeted in the first chemostat by revertant cells. When cell concentrations are high, an inducer is added in the latter (or last) chemostat to produce product at a very high rate.

**Multi-stage Chemostat System
**

X0=0, Vi, i=1,2….n constant. Stage 1: cell growth condition, Kd=0, qp=0 dX Cell mass: FX 0 FX 1 1 X 1V1 V1 1 dt At steady state X0 1 D1 (1 ) X1 Limiting substrate: At steady state

FS 0 FS1 V1

1 X 1

Y XM/ S

V1

dS1 dt

S1 S 0

1 X 1

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.

6

**Multi-stage Chemostat System
**

Stage 2 – product formation conditions, Kd=0, F’=0 dX FX 1 FX 2 2 X 2V2 V2 ‘ 2 Cell mass: dt X1 At steady state 2 D2 (1 ) where D 2 F X2 V2 F

P1≈0

P2

V2 are constant.

**Multi-stage Chemostat System
**

Stage 2 – product formation conditions, Kd=0, F’=0 Limiting substrate: At steady state

FS1 FS 2 V2

2 X 2

Y

M X /S

V2

qp X 2 YP / S

V2

dS 2 dt

S 2 S1

2 X 2

D2YXM/ S

qp X 2 D2YP / S

Product: At steady state

FP FP2 V2 q p X 2 V2 1

dP2 dt

P2 P 1

qp X 2 D2

7

**Multi-stage Chemostat System
**

Stage n – product formation conditions, Kd=0, F’=0

Similarly, equations could be obtained for nth stage. X n Dn (1 n 1 ) Xn

S n S n 1

n X n

DY

M n X /S

qp X n DnYP / S

Pn Pn 1

qp X n Dn

If μn (e.g. Monod model) and qp are known functions, Xn , Pn, and Sn at nth stage could be determined by the above equations.

Fed-Batch

Nutrients are continuously or semi-continuously fed, while effluent is removed discontinuously.

- overcome substrate inhibition or catabolite repression by intermittent feeding of substrate by maintaining low substrate concentration. for production of secondary metabolites e.g. antibiotics, lactic acid, E. Coli making proteins from recombinant DNA technology.

8

Fed-Batch

Analysis of fed-batch with substrate continuously fed and no output: t=0, V0=0, F is constant.

dV F V Vo Ft dt At quasi steady state, S added=S consumed, X, S concentrations are constant.

Volume:

**Cell mass balance:
**

since

FX O V net X

dV d ( XV ) dX V X , then dt dt dt

d ( XV ) dt

FX O V net X V

V net X X

1 dV F dV net D dt V dt V F F net

V

dX dV X dt dt

V0 Ft

Do 1 D0 t

Ks D

net D

m S

Ks S

if K d 0

Then S

m D

(Monod growth model applied)

Fed-Batch

M

where S≈0

assuming X≈Xm

M

where Xt=Xt0 at t=0

9

Fed-Batch

(g) qp (i.e. g product/g cells-min) at Pt=Pt0, t=0

Fed-Batch

10

**Example: Fed- Batch
**

In a fed-batch culture operating with intermittent addition of glucose, the value of V is given at time t=2hr, when the system is at quasi-steady state.

F dV 200 ml/h, V 1000ml, S0 100 g glucose/L dt M YX/S 0.5gdw cells/g glucose; μ m 0.3h 1 ,

Ks 0.1g/L, X t 0 30 g cells

Determine V0 (initial volume of the culture). At t=2 hr, find S, X and Xt at quasi-steady state if qp=0.2 g product/g cells-h, P0=0.

11

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