Chapter 9: Operating Bioreactorsdrshonna/cm4710f03/lectures/chapter9.pdf · Chapter 9: Operating Bioreactors David Shonnard Department of Chemical Engineering Michigan Technological

1

1Michigan Technological UniversityDavid R. Shonnard

Chapter 9: Operating Bioreactors

David ShonnardDepartment of Chemical Engineering

Michigan Technological University


Presentation Outline:

� Choosing Cultivation Methods

� Modifying Batch and Continuous Reactors

� Immobilized Cell Systems

2


The Choice of Bioreactor Affects Many Aspects of Bioprocessing.

1. Product concentration and purity2. Degree of substrate conversion3. Yields of cells and products4. Capitol cost in a process (>50% total capital expenses)

Further Considerations in Choosing a Bioreactor.1. Biocatalyst. (immobilized or suspended)2. Separations and purification processes

Choosing the Cultivation Method


Batch or Continuous Culture?

These choices represent extremes in bioreactor choices

Productivity →for cell mass or growth-associated products

Batch Culture: assume kd = 0 and qp = 0

��

UE� �UDWH�RI�FHOO�PDVV�SURGXFWLRQ�LQ��EDWFK�F\FOH

UE� �

;P� ;

WF

� �<

;�6

0 �6R

WF

WF� �EDWFK�F\FOH�WLPH� �

�

µPD[

lnX m

X o

� � �WO

Exponential growth time

Lag timeHarvest &Preparation

3


Batch or Continuous Culture? (cont.)

Continuous Culture: assume kd = 0 and qp = 0

��

UF� �UDWH�RI�FHOO�PDVV�SURGXFWLRQ�LQ�FRQWLQXRXV�FXOWXUH

UF� �'RSW;RSW

VHW�G';

G'� ��⇒ ��'RSW� �µPD[��

.6

.6� 6R

)

;RSW� �<;�6

0 ��6R �.6�'RSW

µPD[ � 'RSW

)� �<; �6

0 ��6R � �.6 � � �.6�6R �.6� ��

'RSW;RSW� �<; �60 �µPD[��

.6

.6� 6R

)��6R � �.6 � � �.6�6R � .6 ��

�� ≈ �<;�6

0 �µPD[�6R��ZKHQ�.6 � �� 6R



Comparing Rates in Batch and Continuous Culture

��

UF

UE

� �<

;�6

0 �µPD[�6

R

<; �6

0 �6R� �

�

µPD[

OQ;

P

;R

� WO

� �OQ;

P

;R

� WOµmax

$�FRPPHUFLDO�IHUPHQWDWLRQ�ZLWK�

;P

;R

� �� WO� ��KU� �DQG�µ

PD[ ��KU ��

UF

UE

� �� ⇒ �� Continuous culture method is ~ 10 times more productive for primary products(biomass & growth associated products

4



Why is it that most commercial bioprocess are Batch??

1. Secondary Product Productivity → is > in batch culture(SPs require very low concentrations of S, S << Sopt)

2. Genetic Instability → makes continuous culture less productive(revertants are formed and can out-compete highly selected and

and productive strains in continuous culture.)

3. Operability and Reliability (sterility and equipment reliability > for batch culture)

4. Market Economics (Batch is flexible → can product many products per year)



Most Bioprocesses are Based on Batch Culture(In terms of number, mostly for secondary, high value products)

High Volume Bioprocesses are Based on Continuous Culture(mostly for large volume, lower value, growth associated products --ethanol production, waste treatment, single-cell protein production)

5


Modified Bioreactors: Chemostat with Recycle

To keep the cell concentration higher than the normal steady-state level, cells in the effluent can be recycled back to the reactor.

Advantages of Cell Recycle

1. Increase productivity for biomass production

2. Increase stability by dampening perturbations of input stream

properties


Chemostat with Recycle: Schematic DiagramFigure 9.1

• Centrifuge• Microfilter• Settling Tank

Mass balance envelope

α = recycle ratio

C = cell concentration ratio

X1 = cell concentration in reactor effluent

X2 = cell concentration in effluent from separator

Recycle Stream“Bioprocess Engineering: Basic Concepts”Shuler and Kargi, Prentice Hall, 2002

6


Chemostat with Recycle: Biomass Balance

��

);R� � �α)&;

�� α �);

�� 9

5µ;

�� 9

5

G;�

GW

DW�VWHDG\ � VWDWH��G;

�

GW ��DQG�VWHULOH�IHHG��;

R ��

α)&;�� α �);

�� 9

5µ;

� �

DQG�VROYLQJ�IRU�µµ� �>� �α �� &�@'6LQFH�& ! ��DQG�α �� &� � ��WKHQ�µ� � �'

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


Chemostat with Recycle: Biomass Balance

��

µ� �>� �α �� &�@'

0RQRG�(TXDWLRQ��µ� � µmax�6

.6

+ 66XEVWLWXWH�0RQRG�(TQ��LQWR�DERYH��VROYH�IRU�6

6� �.

6'�� α�� &��

µmax � �'��α�� &��

7


FSo + αFS - (1 + α )FS - VR

µX 1

Y X / SM = V R

dSdt

at steady - state (dSdt

= 0)

FSo + αFS - (1 + α )FS - VR

µX 1

Y X / SM = 0

and solving for X 1

X 1 = Dµ

Y X /SM (So - S); But

Dµ

= 1

[1 + α (1 - C)]

X 1 = Y X /S

M (So - S)[1 + α (1 - C)]

= Y X /S

M

[1 + α(1 - C)]So -

K SD(1 + α (1 - C))µmax - D(1 + α(1 - C))

Chemostat with Recycle: Substrate Balance


Chemostat with Recycle: ComparisonFigure 9.2

α=0.5, C=2.0, µmax=1.0 hr-1, KS=0.01 g/L, YMX/S=0.5

X1 = cell concentration in reactor effluent with no recycle

X1(recycle) = cell concentration in effluent with recycle

=DX1

=DX1

“Bioprocess Engineering: Basic Concepts”Shuler and Kargi, Prentice Hall, 2002X1(recycle)

8


Multiple Chemostat Systems

Applicable to fermentations in which growth and product formation need to be separated into stages: .

Growth stage Product formation stage

P1 P2

“Bioprocess Engineering: Basic Concepts”Shuler and Kargi, Prentice Hall, 2002


Multiple Chemostat Systems (cont.)

1. Genetically Engineered Cells:

Recombinant

DNA

Translate to protein product


9



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 non-modified strain (due to the extra metabolic burden of producing product).

→ Genetic Instability causes the GE culture to (slowly) lose ability to produce product. The non-plasmid carrying cells or the cells with mutation in the plasmid (revertants) grow faster.



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 out-competed 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.

10



2-Stage Chemostat System Analysis

Stage 1 - cell growth conditions, kd=0, qp=0, steady-state

��

µ1� �µPD[�6

�

.6� � �6

�

� �'��IURP�ELRPDVV�EDODQFH

UHDUUDQJLQJ��6��

.6�'

�

µPD[� � �'

�

��ZKHUH�'��

)

9�

;�� <

;�6

0 ��6R� 6

��IURP�VXEVWUDWH�EDODQFH




Stage 2 - product formation conditions, kd=0, F ❘ =0, steady-state

��

);�� );

�� 9

�µ2;�

� �9�

G;�

GW� ��ELRPDVV�EDODQFH

µ2� �µPD[�6

�

.6� � �6

�

� �'��

;�

;�

��ZKHUH�'��

)

9�

)6�� )6

�� 9

�

µ2;�

<;�6

0� � �9

�

T3;

�

<3�6

� �9�

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)3�� )3

�� 9

�T3;

�� 9

�

G3�

GW� ��SURGXFW�EDODQFH

11




Stage 2 - product formation conditions, kd=0, F ❘ =0, steady-state

��

µ2� �µPD[�6

�

.6� � �6

�

� �'��

;�

;�

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6�� 6

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'��<

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3�;

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�� 9

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XVH�;��LQ�SURGXFW�EDODQFH�WR�VROYH�IRU�3

�


Fed-Batch Operation

Useful in Antibiotic Fermentation

→ reactor is fed continuously (or intermittently)

reactor is emptied periodically

→ purpose is to maintain low substrate concentration, S

→ useful in overcoming substrate inhibition or catabolic repression, so that product formation increases.

12


Fed-Batch Operation (cont.)

Before t = 0, almost all of the substrate,

So, in the initial volume, Vo, is converted

to biomass, Xm, with little product form-

ation (X=Xm§YX/SSo) and P§ 0.

At t=0, feed is started at a low flow rate

such that substrate is utilized as fast as

It enters the reactor. Therefore, S remains

very low in the reactor and X continues to

maintain at §YX/SSo over time. The volume

increases with time in the reactor and

Product formation continues.

t

increasing

t=0

tw cycle time




Behavior of X, S, P, V, and µ over time


13



Analysis of Fed-Batch Operation

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� �)�� ⇒ ��9� �9R� � �)W

%LRPDVV��);R� � �9µ;� �

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GW� �9

G;

GW� ;

G9

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9

G9

GW� �

)

9� �'

��µ� �)9� �

)9R� )W

� �'

R

�� 'RW

0 0



Analysis of Fed-Batch Operation (cont.)

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G;GW

� ��RU�G;

W

9

GW� �

9G;

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GW

9��

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W

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9

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;�66R)

LQWHJUDWLQJ��;W� �;

WR� � �<

;�66R)W�� 9

R� �)W�;

P

14



Analysis of Fed-Batch Operation (cont.)

��

3URGXFW�)RUPDWLRQ� �WRWDO�SURGXFW� �3W� �39

)RU�PDQ\�VHFRQGDU\�SURGXFWV� �WKH�VSHFLILF�UDWH�RI

SURGXFW�IRUPDWLRQ�LV�D�FRQVWDQW� �T3�

G3W

GW� �T

3�;

W�� T

3��9

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P

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WR� � �T

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��W�

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9� � �T

3;

P�9R

9�'W

��W

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�9R� )W�

� � �T3;

P�

9R

�9R� )W�

�)W

��9R� )W�

�W


Immobilized Cell Systems; 9.4

Restriction of cell mobility within a confined space

Potential Advantages:

1. Provides high cell concentrations per unit of reactor volume.

2. Eliminates the need for costly cell recovery and recycle.

3. May allow very high volumetric productivities.

4. May provide higher product yields, genetic stability, and shear damage

protection.

5. May provide favorable microenvironments such as cell-cell contact,

nutrient-product gradients, and pH gradients resulting in higher yields.

15


Immobilized Cell Systems; 9.4

Potential Disadvantages/Problems:

1. If cells are growing (as opposed to being in stationary phase) and/or

evolve gas (CO2), physical disruption of immobilization matrix could

result.

2. Products must be excreted from the cell to be recovered easily.

3. Mass transfer limitations may occur as in immobilized enzyme

systems.


Methods of Immobilization

Active Immobilization:

1. Entrapment in a Porous Matrix:

cellsInert/solid core

porous polymer matrix

Polymers:agar, alginateκ-carrageenanpolyacrylamidegelatin, collagen

Polymeric Beads:

16


Methods of Immobilization (cont.)

liquid core with cells

hollow spherical particle

semipermeable membraneMembrane:nylon, collodion,polystyrene,polylysine-alginate hydrogelCellulose acetate-ethyl acetate

Encapsulation:

“less severe mass transfer limitations”



nutrients

liquid in shell side

semi-permeable membrane

Hollow Fiber Membrane Reactor:

products

shell

nutrients products

tube

cells

17



2. Cell Binding to Inert Supports:

microporesdP>4 dC• cells in micropores

porous glass, porous silica, aluminaceramics, gelatin, activated carbonWood chips, poly propylene ion-exchange resins(DEAE-Sephadex, CMC-), Sepharose

Micro-porous Supports:

“mass transfer limitations occur”



Binding Forces:

ion exchangesupport

Electrostatic Attraction --

-- -- --

+

+++

Hydrogen BondingsupportHO

C-O-

||O

cell

18



Binding Forces:

Covalent Bonding: (review enzyme covalent bonding)

Support materials: CMC-carbodiimidesupport functional groups-OH, -NH2, -COOH

Binding to proteins on cell surface



Overview of Active Cell Immobilization Methods:

Adsorption AdsorptionSupport Capacity Strength

Porous silica low weak

Wood chips high weak

Ion-exchange resins high moderate

CMC high high

19



Passive Immobilization:

liquid phasesupport

Biofilm(biopolymer + polysaccharides)

• wastewater treatment• mold fermentations• fouling of processing equipment


Analysis of Biofilm Mass TransferFigures 9.1, 9.12

O2 diffusion in biofilms Substrate/product diffusion in biofilms


20


Analysis of Biofilm Mass Transfer (cont.)

Differential Substrate Balance:

∆x

∆y∆z

Material volume in biofilm, ∆V = ∆x ∆y ∆z

Rate of diffusion in through the area ∆x ∆z

��'H

G6

G\ �\

�∆[�∆]yy+∆y

Rate of diffusion out through the area ∆x ∆z

��'H

G6

G\ �\ �∆\

�∆[�∆]

Rate of substrate consumption in the volume ∆V = ∆x ∆y ∆z(due to cell growth, orproduct formation)

��

�

<; � 6

0�

µPD[

�6

.6�6

�;��∆[�∆\�∆]

��

�

<3 �6

�qS�6

.6 � 6�;��∆[�∆\�∆]



Differential Substrate Balance at Steady-State:

��

�'HG6G\ �\

�∆[�∆]� � � �'HG6G\ �\�∆\

�∆[�∆]

� � �

�<;� 6

0 �µPD[ �6.6 �6

�;��∆[�∆\�∆]� ��

'LYLGH�WKURXJK�E\�∆[�∆\�∆]�DQG�VZLWFK�RUGHU�RI�ILUVW��WHUPV

�

'HG6G\ �\ �∆\

� 'HG6G\ �\

∆\� �

�<; �6

0 �µPD[ �6.6 �6

�;� ��

Rate of diffusion in through the area ∆x ∆z

Rate of diffusion out through the area ∆x ∆z

Rate of substrate consumption in the volume ∆V = ∆x ∆y ∆z

- - = 0

21



Differential Substrate Balance at Steady-State:

��

'H

G�6G\ �

� ��

<; � 6

0�

µPD[�6

.6�6

�;��HTQ��

%RXQGDU\�&RQGLWLRQV

6� �6RL��DW�\� ��DW�WKH�ELRILOP� � �OLTXLG�LQWHUIDFH�

G6G\

� �� DW�\� �/��DW�WKH�ELRILOP� � �VXSSRUW�LQWHUIDFH�



Dimensionless Substrate Balance at Steady-State:

��

G�6�G\��

� �φ��6�� β�6�

��HTQ��

ZKHUH��6� 6

6R

,��\� \

/� ��β

6R

.6

� ��

DQG�φ� �/µ

PD[;

<; � 6

0 'H.

6

��7KLHOH�0RGXOXV�


6�� DW�\�� DW�WKH�ELRILOP� � �OLTXLG�LQWHUIDFH�

G6�G\�

� �� DW�\�� DW�WKH�ELRILOP� � �VXSSRUW�LQWHUIDFH�

A numerical solution is required

Analyticalsolution is possible for0 order and 1st order kinetics

22



Zero Order Substrate Consumption Kinetics:

��

G�6�G\��

� �φ��6�� β�6�

�� IRU�β !!�� DQG�φ ��

G�6�G\��

� �φ��β��]HUR � RUGHU�VXEVWUDWH�FRQVXPSWLRQ�NLQHWLFV

��

GG\�

G6�G\�

� �φ��β�

�� ⇒ �� GG6�G\�

∫ � �

φ��β�G\�∫

��

G6�

G\��

φ��

β�\�� &

�




��

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� ��

� �φ��

β�� &

�� ⇒ ��&

��

φ��

β�

��

G6�

G\��

φ��

β�\��

φ��

β� ��LQWHJUDWH�DJDLQ�� G6�∫ � �

φ��β�\��

φ��β�

∫ G\�

��6� �

φ��

2β�\��

φ��

β�\� � �&

�

23




��

%RXQGDU\�FRQGLWLRQ��DW�\�� 6��

� �φ��

β��

φ��

β�� &

�� ⇒ ��&

��

��

6� �φ��2β�

\�� φ��β�

\� � ��RU��6� �φ��β�

\��2 �

� � �\

� � ��

IRU�φ��

β��


Biofilm Effectiveness

The effectiveness factor is calculated by dividing the rate of substrate diffusion into the biofilm by the maximum substrate consumption rate.

Solve for the Effectiveness Factor, η

��16$6� � � $6'H

G6

G\ �\ �

� �η� µPD[ �6R�;

<; � 60 ��.6 �6R)

($6/�

Rate of substrate diffusion into biofilm through an area AS at the surface at y = 0

Volumetric rate of substrate consumption within the biofilm in a volume (ASL)

24


Effectiveness Factor

Biofilm is most effective for β >>1

η increases as φdecreases for any value of β



Spherical Particle of Immobilized CellsFigure 9.14

VP is particle volumeAP is particle area


25


Analysis of Mass Transfer in Spherical Particle

Dimensionless Substrate Balance at Steady-State:

��

G�6�GU��

� � ��U�G6�GU��

φ��6�� β�6�

��HTQ��

ZKHUH��6� 66R

,��U� U5� ��β

6R

.6

� ��

DQG�φ� �5µ

PD[;

<; � 6

0 'H.

6



6�� DW�U�� DW�WKH�SDUWLFOH� � �OLTXLG�LQWHUIDFH�

G6�GU�� DW�U�� DW�WKH�SDUWLFOH�FHQWHU�


Particle Effectiveness

If all of the particle cells “see” substrate at a concentration So or high enough to grow maximally, then the particle is said to have an effectiveness of 1.

Rate of S consumption by a single particle

��16$3� � � $3'H

G6

GU �U 5� �η� µPD[ �6R�;

<; �60 ��.6 � 6R)

93

Rate of substrate diffusion into particle through an area AP at the surface at r = R

Volumetric rate of substrate consumption within the particle in a volume (VP)

26


Particle Effectiveness (cont.)

Equation 9.58 can be solved analytically for limiting cases:

Case 1, for So<<KS (very dilute substrate)

��

η� ��φ�

�WDQK��φ

− ��φ

φ� �93

$3

µmax�;<

; �6

0 'H.

6





Case 2, for So>>KS (very concentrated substrate)

��

'H

G�6

GU��

�

U

G6

GU

� �

µPD[6

<; � 6

0 �.6

+ 6);� �

µPD[;

<; � 6

0


6� �6R��DW�U� �5��DW�WKH�SDUWLFOH� � �OLTXLG�LQWHUIDFH�

G6GU� ��DW�U� ��DW�WKH�SDUWLFOH�FHQWHU�

27




Case 2, for So>>KS

Use a variable transformation, S’=S/r

��

�

U

G �6

GU��

µPD[;

<; �6

0 'H

6ROXWLRQ�IRU�6�LV�

6� �6R� � �

µPD[;

��<; � 6

0 'H

(5� � U �)

��

$W�D�FULWLFDO�UDGLXV��UFU�� 6� ��

�� 6R� � �

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��<; �6

0 'H

(5� � UFU

�)

UFU

5

2

� �� 6�'

H�6

R�<

; � 6

0

µPD[�;�5�




Case 2, for So>>KS

��

η� �

µPD[;

<; � 6

0�

43

π��5� � UFU

� )

µPD[;

<; �6

0�4

3π�5�

� �� UFU

5

3

RU

η�� 6 �'

H�6

R�<

; � 6

0

µPD[�;�5�

3

2

28


Bioreactors Using Immobilized CellsFigure 9.15

The single particle analysis for η can be used in the analysis of bioreactors having immobilized cells:

Consider a plug flow reactor filled with immobilized cell particles



Bioreactors Using Immobilized Cells (cont.)

A differential balance on a thin slice of particles within the reactor:

Soi

So

0

z

H

F, So

F, So-dSo

z

z+dz

differential volume element

��)6R ]� )6

R ] �G]� �1

6�D�$�G]�

Rate of substrate flow into element

Rate of masstransfer intoparticles within element

Rate of substrate flow out of element

=-

29



Using the definition of η:

��

16$

3� ��η� µ

PD[�6

R�;

<; �6

0 ��.6�6

R)

93

�)G6

R

G]� �η� µ

PD[�6

R�;

<; � 6

0 ��.6�6

R)

9

3

$3

�D�$

ZKHUH�D� �VXUIDFH�DUHD�RI�SDUWLFOH�SHU�XQLW�YROXPH�RI�EHG��FP� � FP��EHG�

��$� �FURVV � VHFWLRQDO�DUHD�RI�WKH�EHG��FP� �



At z = 0, So = Soi: integrating assuming η is constant

��

.6�OQ

6RL

6R

� � ��6RL � 6R�� η�

µPD[�9

3�;�D�$

<; �6

0 �)�$3

+

IRU�ORZ�VXEVWUDWH�FRQFHQWUDWLRQ��6RL��.

6�

OQ6R

6RL

�� η� µ

PD[�9

3�;�D�$

<; �6

0 �)�$3�.

6

+

QRWH�[�� [93

$3

D��DYHUDJH�FHOO�PDVV�FRQF��LQ�WKH�EHG�

Documents

Chapter 9: Operating Bioreactorsdrshonna/cm4710f03/lectures/chapter9.pdf · Chapter 9: Operating Bioreactors David Shonnard Department of Chemical Engineering Michigan Technological