Foundations of Biochemical Engineering - American Chemical Society

17 Immobilized Cells Catalyst Preparation and Reaction Performance J. KLEIN and K.-D. VORLOP

Downloaded by CORNELL UNIV on October 5, 2016 | http://pubs.acs.org Publication Date: January 18, 1983 | doi: 10.1021/bk-1983-0207.ch017

Technical University of Braunschweig, Institute of Chemical Technology, Federal Republic of Germany Immobilized cells have proven to be effective cata lysts i n the enzymatic conversion of organic com pounds. Such catalysts are typically prepared by entrapment of cells i n polymeric carriers, and the methods of ionotropic gelation and polycondensation of epoxids w i l l be described. Depending on enzymatic activity and particle size the transformation may proceed i n the reaction or diffusion controlled re gime. Quantitative estimation of the effectiveness factor-Thiele modulus relation w i l l be presented for different reaction types. This includes the experi mental determination of the catalytically active c e l l concentration and the effective d i f f u s i v i t y i n the porous polymeric carrier. Transport limitation can also be a controlling factor i n the experimental determination of the operational s t a b i l i t y of such biocatalysts.

A large number of products i n the pharmaceutical and food industry i s obtained from fermentation processes. Examples are amino acids, stereoregular organic acids, antibiotics, ethanol, etc. In a classical fermentation process the product formation is s t r i c t l y coupled to c e l l growth resulting i n a possibly unfav orable byproduction of biomass. Furthermore these processes are typically performed as batch operations. As has been shown already on an industrial scale, fermenta tion can be substituted by heterogeneous catalysts with resting microbial cells immobilized i n polymeric carriers. Repeated use of the once formed biomass, continuous process operation, and elim ination of costly separation steps of product solution from bio mass are obvious advantages of this new technology. Some p r i n c i pal aspects of a) immobilization methodology, b) catalyst effect iveness, and c) operational s t a b i l i t y shall be outlined i n this contribution. 0097-6156/83/0207Ό377$06.00/0 © 1983 American Chemical Society

Blanch et al.; Foundations of Biochemical Engineering ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

BIOCHEMICAL

378

ENGINEERING


Polymer Entrapment V a r i o u s methods h a v e b e e n p r o p o s e d f o r w h o l e c e l l i m m o b i l i z a t i o n i n c l u d i n g a d s o r p t i o n and c o v a l e n t a t t a c h m e n t t o a p r e f o r m e d c a r r i e r , c r o s s l i n k i n g , f l o c c u l a t i o n , m i c r o e n c a p s u l a t i o n , and e n t r a p m e n t . P h y s i c a l e n t r a p m e n t i n a p o r o u s m a t r i x i s by f a r t h e most f l e x i b l e and most commonly u s e d t e c h n i q u e . Considering the f a c t t h a t t h e p o l y m e r n e t w o r k has t o be f o r m e d i n t h e p r e s e n c e o f t h e f i n a l l y entrapped b i o l o g i c a l m a t e r i a l , the performance c r i t e r i a of c h e m i c a l and p h y s i c a l n a t u r e a r e as f o l l o w s : (1) The n e t w o r k f o r m a t i o n has t o p r o c e e d u n d e r m i l d c o n d i t i o n s (pH and t e m p e r a t u r e ) i n an aqueous e n v i r o n m e n t ; (2) t h e n e t w o r k has t o be c h e m i c a l l y s t a b l e u n d e r v a r i o u s r e a c t i o n c o n d i t i o n s (pH, b u f f e r s o l u t i o n , i o n i c and n o n i o n i c s u b strates, etc.); (3) t h e s i z e and t h e p o r o s i t y o f t h e p o l y m e r i c c a r r i e r ( p r e f e r a b l y as b e a d s ) has t o be c o n t r o l l e d ; (4) t h e p o s s i b i l i t y f o r a l a r g e v a r i a t i o n o f b i o m a s s c o n t e n t i n t h e c a t a l y s t s h o u l d be g i v e n ; (5) t h e c a t a l y s t b e a d s s h o u l d be m e c h a n i c a l l y s t a b l e t o be used i n v a r i o u s r e a c t o r c o n f i g u r a t i o n s (packed bed, f l u i d i z e d bed, s t i r r e d tank). A p p r o p r i a t e p o l y m e r i c c a r r i e r s c a n be o b t a i n e d f r o m p o l y m e r i c , o l i g o m e r i c , and monomeric p r e c u r s o r s . Due t o unwanted c h e m i c a l i n t e r a c t i o n of such chemicals w i t h the c e l l m a t e r i a l l a r g e r s i z e of t h e s e p r e c u r s o r s i s f a v o r a b l e . The i o n o t r o p i c g e l a t i o n , s t a r t i n g f r o m p o l y e l e c t r o l y t e s and t h e p o l y c o n d e n s a t i o n , s t a r t i n g f r o m o l i g o m e r i c epoxy r e s i n s , a r e t y p i c a l p r o b l e m s o l u t i o n s . I o n o t r o p i c G e l a t i o n of P o l y e l e c t r o l y t e s T h i s method o f n e t w o r k f o r m a t i o n i s d e f i n e d as a c r o s s l i n k i n g r e a c t i o n of p o l y e l e c t r o l y t e s w i t h lower molecular weight m u l t i v a lent counterions. C o n s i d e r i n g t h e p o l y m e r i c component, a n i o n i c ( e . g . , a l g i n a t e , CMC (I) o r c a t i o n i c ( c h i t o s a n (2)) substances can be u s e d . T h i s v a r i e t y o f p o l y m e r s and t h e a p p r o p r i a t e c o u n t e r i o n s a r e s u m m a r i z e d i n F i g u r e 1. The c h o i c e o f t h e p o l y m e r i s d e t e r m i n e d by t h e pH r e g i o n o f t h e r e s p e c t i v e b i o c a t a l y t i c r e a c t i o n , s i n c e a l l i o n o t r o p i c g e l s a r e r e v e r s i b l e s t r u c t u r e s w h i c h c a n be r e d i s s o l v e d by i n c r e a s e ( a l g i n a t e ) o r d e c r e a s e ( c h i t o s a n ) o f pH b e yond c e r t a i n l i m i t s . A second important c r i t e r i o n i s the i o n i c c o m p o s i t i o n o f t h e r e a c t i o n medium and t h e p o s s i b i l i t y o f i n s o l u b l e byproduct o r complex f o r m a t i o n w i t h the network forming i o n s . I n a t y p i c a l a l g i n a t e entrapment process the c e l l s are sus pended i n a 3% s o d i u m a l g i n a t e s o l u t i o n and t h i s v i s c o u s s u s p e n s i o n i s p r e c i p i t a t e d d r o p w i s e i n a 1% C a C l 2 s o l u t i o n . A f t e r 30 m i n u t e s s t a b l e C a - a l g i n a t e g e l s a r e f o r m e d where t h e c e l l s a r e i m m o b i l i z e d i n a macroporous s t r u c t u r e . F o l l o w i n g to t h i s p r e c i p i t a t i o n p r o c e s s a p a r t i a l d r y i n g s t e p c a n be a p p l i e d w h i c h r e s u l t s i n a homogeneous s h r i n k i n g o f t h e p a r t i c l e s , t h u s i n c r e a s i n g c o n -



KLEIN

A N D VORLOP

Immobilized

POLYELECTROLYTES

Cells

379

as Catalysts

MULTIVALENT COUNTERIONS

POLYANIONS ALGINATE '""cOO^

Χ

CARBOXYMETHYLCELLULOSE

φ

CARBOXY-GUARGUM

Ca . Fe . Zn 2 +

Al \ 3

2 +

Fe

2 +

...

3 +

COPOLY-STYRENEMALEIC ACID POLYCATIONS CHITOSAN

Fe(CN)

6

4

\

Fe(C^

3

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POLY-PHOSPHATE /

^ΝΗ3

Φ

Χ

θ

POLY-ALDEHYDO-CARBONIC ACID P0LY-1-HYDR0XY-1SULF0NATE-PR0PEN-2 ALGINATE

Figure 1. Summary of polymer-counterion systems to be used in ionotropic gelation for whole cell entrapment. Reprinted, with permission, from Ref. 13. Copy right 1982, Plenum Publishing Corp.


BIOCHEMICAL


380

ENGINEERING

s i d e r a b l y t h e m e c h a n i c a l s t a b i l i t y as w e l l a s t h e p a c k i n g d e n s i t y of the entrapped c e l l s i t s e l f . A l l these f a c t o r s are very advanta geous f o r t h e b i o c a t a l y t i c a p p l i c a t i o n . The f l e x i b i l i t y o f t h e a l g i n a t e - m e t h o d c a n be d e m o n s t r a t e d a c c o r d i n g t o t h e f o l l o w i n g p a r a m e t e r b o u n d a r y v a l u e s : p o l y m e r c o n c e n t r a t i o n f r o m 0.5 t o 8%, CaC^ c o n c e n t r a t i o n f r o m 0.05 t o 2%, c e l l c o n c e n t r a t i o n (on wet w e i g h t b a s i s ) f r o m 0.1 t o 100%, b e a d d i a m e t e r s f r o m 0.1 t o 5 mm, and p r e p a r a t i o n t e m p e r a t u r e s f r o m 0 t o 80° C. Cells of different s t r u c t u r e ; e.g., a e r o b i c (1) o r a n a e r o b i c m i c r o b e s ( 3 ) , p l a n t c e l l s ( 4 ) , mammalian c e l l s ( 5 ) , c a n be e n t r a p p e d and t h u s s t a b i l i z e d without c o n s i d e r a b l e t o x i c i t y problems. A p r o b l e m o f p r a c t i c a l i m p o r t a n c e i s t h e s c a l e up o f t h e i m m o b i l i z a t i o n p r o c e s s f r o m amounts o f s e v e r a l grams t o s e v e r a l hundred l i t e r s . W h i l e s m a l l amounts c a n e a s i l y be p r e p a r e d u s i n g one c a p i l l a r y o r i f i c e , a b u n d l e o f s u c h c a p i l l a r y i n a s i e v e p l a t e t y p e c o n s t r u c t i o n w i l l g i v e l a r g e r amounts o f i d e n t i c a l p a r t i c l e s , i f the c a p i l l a r y c h a r a c t e r i s t i c s a r e not changed. These d e v i c e s a r e shown i n F i g u r e 2. Polycondensation

o f Epoxy

Resins

I n t h i s c a s e c o v a l e n t n e t w o r k s o f h i g h m e c h a n i c a l and c h e m i c a l s t a b i l i t y a r e o b t a i n e d as a r e s u l t o f c r o s s l i n k i n g r e a c t i o n of e p o x i d e s w i t h m u l t i f u n c t i o n a l a m i n e s ( 6 ) . The m a i n p r o b l e m s o f t h i s t e c h n i q u e a r e t h e t o x i c i t y o f t h e amino-component and t h e u s u a l l y l o w p o r o s i t y o f t h e p o l y m e r i c n e t w o r k . The t o x i c i t y , mea s u r e d by t h e v i a b i l i t y o f i m m o b i l i z e d y e a s t c e l l s , c o u l d be m i n i m i z e d a) by p r o p e r s e l e c t i o n o f e p o x y and amino components and b) by i n t r o d u c t i o n o f a p r e g e l l i n g t i m e i n t h e o r d e r o f 15 m i n u t e s b e f o r e m i x i n g t h e c e l l s w i t h t h e c o n d e n s a t i n g o l i g o m e r s ( 7 ) . The p o r o s i t y o f t h e m a t r i x i s i n t r o d u c e d by t h e i m m o b i l i z e d c e l l s i t s e l f and by an i n t e r m e d i a t e p r e p a r a t i o n o f an i n t e r p e n e t r a t i n g n e t w o r k w i t h an i o n o t r o p i c g e l . The i o n o t r o p i c g e l a t i o n i s a l s o u s e d t o c o n t r o l t h e p a r t i c l e s h a p e and s i z e . A c o m p l e t e scheme o f s u c h a n i m m o b i l i z a t i o n p r o c e s s i s shown i n F i g u r e 3. A g a i n q u i t e h i g h c o n c e n t r a t i o n s (up t o 70% on wet w e i g h t b a s i s ) o f c e l l s c a n be f i n a l l y i n c o r p o r a t e d i n s u c h a p o l y m e r i c s t r u c t u r e . The v i a b i l i t y o f t h e y e a s t c e l l s , and t h u s t h e r e d u c e d t o x i c i t y o f t h e e n t r a p m e n t m e t h o d , c a n be d e m o n s t r a t e d by c e l l g r o w t h i n the m a t r i x , which gives r i s e to a corresponding a c t i v i t y i n crease f o r ethanol p r o d u c t i o n from glucose ( 7 ) . This behavior i s shown i n F i g u r e 4. The f a c t o r o f a c t i v i t y i n c r e a s e compared t o t h e i n i t i a l v a l u e i n c r e a s e s w i t h d e c r e a s i n g i n i t i a l l o a d i n g ; however, i t i s o b v i o u s t h a t an u p p e r l i m i t o f a c t i v i t y w i l l f i n a l l y be reached. The r e a s o n f o r t h i s phenomenon, as w e l l as f o r t h e a c t i v i t y d e c r e a s e w i t h i n c r e a s i n g i n c u b a t i o n t i m e , w i l l become obvious from the d i s c u s s i o n s o f the f o l l o w i n g chapter.


KLEIN

Immobilized

A N D VORLOP

Cells as Catalysts

381


PRESSURE

Figure 2.

Scheme for catalyst bead formation by ionotropic gelation, including scale-up device.

10g tpoxy resin • 33g curing agent (30%-soUn H^O) * 7 ml H 0

-±

2

±

precondensotion (15m. n.)

mixing periodical injection

6g bake i*s yeast • 2ml • 20g β % alginate- sol.

, Qoooonnnnnooo drying (24h)

0

crosslink!ng (20min)

ο.ο

9r^-> ol

2wt%CaCl2-solution

EP0XY-BEADS alginate-dissolution (40min) in 0.1« phosphate-buffer (pH> 6.0)

Figure 3. Process scheme for preparation of biocatalysts by cell entrapment in epoxy beads. Reprinted, with permission, from Ref. 14. Copyright 1982, Science and Technology Letters.



382

BIOCHEMICAL

ENGINEERING

Figure 4. Dependence of biocatalytic activities for the batch fermentation of ethanol from glucose with immobilized yeast cells as a function of incubation time (time for cellgrowth in the carrier) for various initial cell loadings in epoxy carriers.


17.

KLEIN A N D VORLOP

Immobilized

Cells


Effectiveness o f Immobilized C e l l

as Catalysts

383

Catalysts

I t i s a w e l l known f a c t i n h e t e r o g e n e o u s c a t a l y s i s , t h a t t h e c a t a l y t i c a c t i v i t y i s generally not d i r e c t l y proportional to the c o n c e n t r a t i o n o f a c t i v e s i t e s b u t depends a l s o o n h y d r o d y n a m i c c o n d i t i o n s i n t h e s u r r o u n d i n g o f t h e p a r t i c l e s , on p a r t i c l e s i z e and m a t r i x p o r o s i t y . I t i s furthermore w e l l understood, that v a r i o u s t r a n s p o r t phenomena h a v e t o be t a k e n i n t o a c c o u n t , m a i n l y d i f f u s i o n a l transport processes which n e c e s s a r i l y a r e preceding to the r e a c t i o n step i t s e l f . A d i m e n s i o n l e s s number, u s u a l l y c a l l e d T h i e l e - m o d u l u s , c a n be used t o q u a n t i t a t i v e l y account f o r t r a n s p o r t - r e a c t i o n c o u p l i n g phenomena. A s s u m i n g t h e v a l i d i t y o f M i c h a e l i s - M e n t e n r a t e e q u a t i o n - which i s j u s t i f i e d f o r simple enzymatic r e a c t i o n s i n whole c e l l s t o o - t h e f o l l o w i n g e x p r e s s i o n f o r t h e T h i e l e modulus has been d e r i v e d ( 8 ) : 1/2

f

where R i s t h e p a r t i c l e r a d i u s , v t h e r a t e o f r e a c t i o n , the M i c h a e l i s c o n s t a n t , S t h e s u b s t r a t e c o n c e n t r a t i o n and D the e f f e c t i v e s u b s t r a t e d i f f u s i v i t y i n the porous c a t a l y s t p a r t i c l e . On t h e o t h e r hand t h e e f f e c t i v e n e s s f a c t o r η i s d e f i n e d a s t h e r a t i o o f t h e e f f e c t i v e r e a c t i o n r a t e ν t o t h e maximum r e a c t i o n rate ν w h i c h w o u l d be o b s e r v e d w i t h o u t t r a n s p o r t l i m i t a t i o n max e

n = ~ -

(2)

max F o r i m m o b i l i z e d c e l l c a t a l y s t s t h e r e a r e two p o s s i b i l i t i e s t o ob t a i n t h i s f a c t o r . F i r s t l y , t h e p a r t i c l e s o f l a r g e r r a d i u s can be g r i n d e d down t o s u c h a s m a l l s i z e t h a t p o r e d i f f u s i o n becomes neg ligible. I n t h i s case = v ^ + . Due t o t h e s i z e and t h e s i m p l e entrapment o f t h e c a t a l y t i c s p e c i e s l o s s from t h e m a t r i x may b e c o n s i d e r a b l e . Therefore, secondly, the free c e l l a c t i v i t y can be used i n t h e denominator, i f t h e e x a c t c o n c e n t r a t i o n o f c a t a l y t i c a l l y a c t i v e immobilized c e l l s ( X ) known. Since e

Q

i

s

a c t

v.-I.ilfi

(3)

i s t h e s p e c i f i c r e a c t i o n r a t e o f t h e f r e e l y suspended c e l l s , t h e equation ν = ν ' X = v* (4) max act 1

h o l d s , w h i c h f u r t h e r m o r e d e f i n e s v i n E q n . ( 1 ) . B a s e d o n numer i c a l calculations t y p i c a l functions


384

BIOCHEMICAL ENGINEERING


η - f (Φ)

(5)

h a v e b e e n d e v e l o p e d , w h i c h i n t e r r e l a t e t h e two d i m e n s i o n l e s s p a r ameters and w h i c h c a n be checked e x p e r i m e n t a l l y . To e v a l u a t e t h e a p p l i c a b i l i t y o f E q n . ( 5 ) , t h e p a r a m e t e r s i n Eqns. (1-4) have t o be determined i n d e p e n d e n t l y . T h i s has been done f o r t h e c l e a v a g e r e a c t i o n o f P e n i c i l l i n G t o 6APA w i t h immo b i l i z e d E. Qoli c e l l s i m m o b i l i z e d i n e p o x i d e b e a d s ( 9 ) . The r a d i u s R: The r a d i u s o f p a r t i c l e s o b t a i n e d f r o m i o n o t r o p i c g e l a t i o n i s u s u a l l y c o n t r o l l e d w i t h i n v e r y narrow l i m i t s and t h e s i z e c a n e a s i l y b e d e t e r m i n e d by m i c r o s c o p i c m e a s u r e m e n t s . Reaction K i n e t i c s ; The r e a c t i o n r a t e s h a v e b e e n m e a s u r e d a t pH = 7.8 a n d Τ = 37o C, u s i n g a 5% P e n G s u b s t r a t e c o n c e n t r a t i o n . T i t r a t i o n w i t h 0.1 m o l a r NaOH h a s b e e n u s e d t o d e t e r m i n e t h e amount o f p r o d u c t f o r m a t i o n . The K^j v a l u e s o f f r e e a n d i m m o b i l i z e d c e l l s h a v e b e e n o b t a i n e d f r o m t h e L i n e w e a v e r - B u r k p l o t s a s shown f o r some e x a m p l e s i n F i g u r e 5. F o l l o w i n g t o i r r e v e r s i b l e d e a c t i v a t i o n o f enzymes d u r i n g t h e p r o c e s s o f i m m o b i l i z a t i o n , t h e i n e q u a l i t y X £ < ^ m m o b i l . °lds. f o l l o w i n g approach has been developed f o r the determination o f X : i n a c e r t a i n experiment, i n a f i r s t ap proximation X - Ximm.; i . e . , 100% o f a l l i m m o b i l i z e d c e l l s a r e assumed t o b e a c t i v e . I n t h i s case, η 0.23 h a s b e e n d e t e r m i n e d . act ^mm. » become l a r g e r , due t o t h e d e c r e a s e o f t h e a C

n

a c t

a c t

β

I

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Foundations of Biochemical Engineering - American Chemical Society

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