Immobilized Microbial Cells in Complex Biocatalysis - ACS Publications

1 Immobilized Microbial Cells in Complex Biocatalysis WOLF R. VIETH and K. VENKATSUBRAMANIAN

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Downloaded by 79.110.19.233 on October 5, 2016 | http://pubs.acs.org Publication Date: August 16, 1979 | doi: 10.1021/bk-1979-0106.ch001

Department of Chemical and Biochemical Engineering, Rutgers—The State University, New Brunswick, NJ 08903

Continuous heterogeneous catalysis by fixed microbial cells represents a new approach to established fermentation processes. Immobilization of isolated (and purified) enzymes and microbial cells mediating simple, monoenzyme reactions has already been reduced to industrial practice. However, the development of immobilized cell systems to carry out complex fermentation processes--characterized by multiple reactions and complete reaction pathways involving coenzymes--is still in its infancy. Drawing upon our rather concerted effort in this area over the past several years, we are appraising the prospects and problems of such a technological advancement in this brief communication. The Approach In earlier papers from this laboratory, we have proposed the terms "Controlled Catalytic Biomass" and "Structured Bed Fermentation" to describe immobilized cell systems effecting complex biocatalysis (1,2). The meaning of these terms is obvious when one considers the biocatalyst in relation to its microstructure, predesigned catalytic reactor design, and controlled catalytic activity vis-a-vis cellular reproduction. Some of the potential advantages of such a catalytic system are summarized in Table I. Examining the character of microbial cells in classical fermentation, i t is clear that they possess the desired catalytic machinery in a highly structured form. The controlled conditions of fermentation permit retention of this meticulous structural 1

Also with: H.J. Heinz Company, World Headquarters, P.O. Box 57, Pittsburgh, Pennsylvania 15230.

Presented at the Symposium on "Immobilized Cells and Organelles," ACS National Meeting, Miami Beach, September, 1978.

0-8412-0508-6/79/47-106-001$05.00/0 © 1979 American Chemical Society Venkatsubramanian; Immobilized Microbial Cells ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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IMMOBILIZED MICROBIAL CELLS


TABLE 1 POTENTIAL ADVANTAGES OF IMMOBILIZED WHOLE CELL SYSTEMS OVER CONTROLLED FERMENTATIONS

1.

Placement o f Fermentation on Heterogeneous Basis

C a t a l y s i s Design

2.

Higher Product Y i e l d s

3.

A b i l i t y t o Conduct Continuous Operations As Opposed t o T r a d i t i o n a l Batch Fermentation

4.

Operation a t High D i l u t i o n Rates Without Washout

5.

A b i l i t y to Recharge System by Inducing Growth and Reproduct i o n o f Resting C e l l s

6.

Decrease o r E l i m i n a t i o n o f Lag and Growth Phases f o r Product Accumulation A s s o c i a t e d With the Non-Growth Phase of the Fermentation

7.

P o s s i b i l i t y o f A c c e l e r a t e d Reaction Rates Due t o Increased C e l l Density

Venkatsubramanian; Immobilized Microbial Cells ACS Symposium Series; American Chemical Society: Washington, DC, 1979.


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AND VENKATSUBRAMANIAN

Complex Biocatalysis

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i n t e g r i t y but ,the r e s u l t i n g c e l l u l a r suspensions are u s u a l l y at low c o n c e n t r a t i o n . Considering f r e e enzymes d e r i v e d from these c e l l s , i t i s p o s s i b l e t o concentrate them by e x t r a c t i o n processes, but l a c k i n g the a n c i l l a r y s t r u c t u r e which s t a b i l i z e s them i n the c e l l , they are r e l a t i v e l y unstable. Some s t r u c t u r a l r e c o n s t i t u t i o n i s p o s s i b l e by immobilization, l e a d i n g to higher concentrat i o n and b e t t e r s t a b i l i t y but one i s then r e s t r a i n e d t o c o n s i d e r a t i o n of s i n g l e step or two-step r e a c t i o n s . With immobilized c e l l s , one has the concentrated form, there i s s t r u c t u r a l p r e s e r v a t i o n and s t a b i l i t y together with the p o s s i b i l i t y of improved r e a c t o r design, based upon the c h a r a c t e r i s t i c s of the c a r r i e r . Thus, immobilized c e l l systems c o n s t i t u t e an important o p t i o n w i t h i n the framework of biochemical technologies (Table 2). The o v e r a l l r a t i o n a l e f o r whole c e l l immobilization i s o u t l i n e d i n Table 3. In a l l our work, we have used r e c o n s t i t u t e d bovine hide c o l l a g e n as the c a r r i e r matrix of c h o i c e . The b i o m a t e r i a l , c o l l a g e n , o f f e r s a number of unique advantages as a support f o r m i c r o b i a l c e l l immobilization. Other p u b l i c a t i o n s from our l a b o r a t o r y describe these advantages as w e l l as the procedures to prepare f i x e d c e l l s i n d e t a i l (3, 4). We have attached many d i f f e r e n t microorganisms i n t h i s manner; some of the complex r e a c t i o n s mediated by such f i x e d c e l l p r e p a r a t i o n s are shown i n Table 4. Process V a r i a b l e s Several important c o n s i d e r a t i o n s i n the p r e p a r a t i o n and use of collagen-bound c e l l systems are adumbrated here with c i t r i c a c i d production by immobilized A s p e r g i l l u s n i g e r as an example. The c o l l a g e n membrane must be c r o s s l i n k e d t o make i t s t r u c t u r a l l y strong enough to withstand the shear f o r c e s i n r e a c t o r o p e r a t i o n . I t was found that post-tanning the c o l l a g e n - c e l l membrane by exposing i t t o a 5% glutaraldehyde s o l u t i o n f o r one minute r e s u l t e d i n an optimal r e t e n t i o n of c a t a l y t i c a c t i v i t y which was a l i n e a r f u n c t i o n o f the c e l l l o a d i n g . We can l o a d the s t r u c t u r e up t o 70% c e l l s (by dry weight) and the amount of expressed a c t i v i t y i n batch assay i n c r e a s e s p r o p o r t i o n a t e l y . However, the mechanical strength drops o f f too d r a s t i c a l l y , and a good compromise i s 50% c e l l s on a dry weight b a s i s . In the course of these s t u d i e s , we came t o r e a l i z e t h a t the dehydration of c e l l s i s d e l e t e r i o u s ; even under r e f r i g e r a t e d c o n d i t i o n s c e l l a c t i v i t y could reduce s i g n i f i c a n t l y . T h i s has l e d us to new d i s p e r s i o n techniques and/or d r y i n g or s o l i d i f i c a t i o n techniques to preserve these f r a g i l e s t r u c t u r e s which can so e a s i l y denature (6). Maximal c a t a l y t i c a c t i v i t y of the c e l l s i s r e t a i n e d upon immobilization when the c e l l s are i n the proper p h y s i o l o g i c a l s t a t e . T h i s corresponds to an optimal i n d u c t i o n o f enzyme a c t i v i t i e s p a r t i c i p a t i n g i n the d e s i r e d r e a c t i o n sequence;


Venkatsubramanian; Immobilized Microbial Cells ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 2

BIOCONVERSION NETWORK

TABLE


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VIETH AND VENKATSUBRAMANIAN

Complex

BiOCdtalysis


TABLE 3

RATIONALE FOR WHOLE CELL IMMOBILIZATION

1.

Obviates Enzyme E x t r a c t i o n / P u r i f i c a t i o n

2.

G e n e r a l l y Higher O p e r a t i o n a l S t a b i l i t y

3.

Lower E f f e c t i v e Enzyme Cost

4.

High Y i e l d o f Enzyme A c t i v i t y on Immobilization

5.

C o f a c t o r Regeneration

6.

Retention of S t r u c t u r a l and Conformational

7.

Greater P o t e n t i a l f o r Multi-Step Processes

8.

Greater Resistance t o E n v i r o n t a l P e r t u r b a t i o n s

Integrity



Klebsciella pneumoniae

Mammalian erythrocyte

11.

Anacystis nidulans Anacystis nidulans Streptomyces griseus Pseudomonas aeruginosa

Serratia marcescens Acetobacter sp. Corynebacterium lilium Aspergillus niger Chloroplast

10,

9.

6. 7. 8.

4. 5.

2. 3·

1.

Microorganism

Nitrogen

Water Nitrate Glucose

Sucrose Water

Ethanol Glucose

Glucose

Substrate

Ammonia

Oxygen Ammonia Candieidin

C i t r i c acid Oxygen

2-Keto g l u c o n i c acid Acetic acid Glutamic a c i d

Product Comments

Model s t u d i e s of in_ v i v o enzyme a c t i o n

M i c r o b i a l f i x a t i o n of atmospheric n i t r o g e n

Primary metabolite Immobilized o r g a n e l l e ; f i r s t step i n b i o p h o t o l y s i s of water Immobilized a l g a l c e l l s B i o l o g i c a l nitrogen f i x a t i o n A n t i b i o t i c synthesis; secondary metabolite Concentration of plutonium from waste waters (bioadsorption)

Multi-enzyme; c o f a c t o r Pathway (primary metabolite)

Multi-enzyme

COLLAGEN-IMMOBILIZED CELL SYSTEMS

TABLE 4



1.

vrjETH AND VENKATSUBRAMANIAN


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i t i s manifested i n peak product s y n t h e s i s r a t e i n the fermentat i o n . For c i t r i c a c i d p r o d u c t i o n with A. n i g e r , i t turns out to be 72 t o 96 hours i n batch fermentations. Of course, i n a t y p i c a l fermentation process one has to repeat t h i s p a t t e r n each time. A b e t t e r a l t e r n a t i v e , i t would seem, would be t o harvest the c e l l s at t h e i r peak a c t i v i t y , followed by t h e i r immobilization so as t o r e t a i n them i n a v i a b l e s t a t e f o r reuse u n t i l t h e i r s t a b i l i t y has decreased t o an uneconomical p o i n t . Once immobilized, the c e l l s must be kept i n a v i a b l e s t a t e i n the membrane without f u r t h e r excessive r e p r o d u c t i o n . T h i s i s necessary t o channel the s u b s t r a t e i n t o the d e s i r e d product r a t h e r than t o a d d i t i o n a l c e l l mass. Besides, i t would minimize c e l l e l u t i o n from the c a r r i e r matrix as w e l l as preserve the mechanical i n t e g r i t y of the c a r r i e r . We have found t h a t one way to accomplish t h i s i s by l i m i t i n g the c o n c e n t r a t i o n of one o f the e s s e n t i a l n u t r i e n t s i n the medium; f o r example, n i t r o g e n c o n c e n t r a t i o n . An i n d i r e c t b e n e f i t o f t h i s approach i s lowering the growth o f contaminating organisms. Ease o f r e a c t o r scale-up i s an important process engineering c o n s i d e r a t i o n ; maximizing the e f f i c i e n c y o f c o n t a c t between the c a t a l y s t and i t s s u b s t r a t e i s an e q u a l l y c r i t i c a l i s s u e . We have determined t h a t where the bound-cell membrane can be r o l l e d i n t o a s p i r a l wound r e a c t o r c o n f i g u r a t i o n (Jo) , i t provides e x c e l l e n t contact e f f i c i e n c y . The c o l l a g e n membrane i s wound together with a polyolefin Vexar spacer m a t e r i a l . The r e s u l t i n g open m u l t i channel system promotes p l u g flow contact with very low pressure drop even when o p e r a t i n g with p a r t i c u l a t e s u b s t r a t e matter which would cause p l u g g i n g problems i n the conventional type o f f i x e d bed o p e r a t i o n . Fermentation s u b s t r a t e s are o f t e n c h a r a c t e r i z e d by p r e c i s e l y t h i s type o f substrates; so t h i s i s a l a r g e p l u s f a c t o r i n f a v o r o f t h i s type o f design. Furthermore, i t i s p o s s i b l e t o d e s i g n - i n high a c t i v i t y p e r u n i t volume, as a r e s u l t of the c o i l i n g o f a l a r g e amount o f membrane i n t o a confined volume. The b a s i s f o r scale-up becomes then simply the membrane surface area. Presented i n F i g s . 1 and 2 are data r e l a t i n g t o e x t e r n a l and i n t e r n a l mass t r a n s f e r f o r the case o f c i t r i c a c i d s y n t h e s i s . The e f f e c t o f l i n e a r v e l o c i t y on the observed r e a c t i o n r a t e (Fig. 1) shows, f o r t h i s case, the presence o f a s i g n i f i c a n t boundary l a y e r r e s i s t a n c e below a flow r a t e o f 235 ml/min. The existence o f n o n - n e g l i g i b l e pore d i f f u s i o n a l r e s i s t a n c e i s ded u c i b l e from F i g . 2, i n which the dependence o f observed r e a c t i o n r a t e on f i l m t h i c k n e s s i s d e p i c t e d . O v e r a l l the immobilized c e l l s e x h i b i t e d about 50% o f the s p e c i f i c a c t i v i t y o f the f r e e c e l l s (in fermentation) toward the p r o d u c t i o n of c i t r i c a c i d . With regard t o other s i g n i f i c a n t f a c t o r s , oxygen t r a n s f e r can be s i n g l e d out as o f paramount importance. To enhance t h i s t r a n s p o r t step, we operated the s p i r a l wound r e a c t o r counterc u r r e n t l y . In other words, a s p e c i a l p r o v i s i o n was incorporated i n t o the r e a c t o r design t o allow flow o f pure oxygen countercurrent


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X

50

100

SUBSTRATE

Figure 1.

RATE

200 ( ML / MIN

250

300

)

Dependence of reaction rate on linear velocity

.

I WET

Figure 2.

FLOW

150

5 MEMBRANE

.

a__

10 15 THICKNESS (MILS)

1

20

Effect of membrane thickness on citric acid production rate. (O) Shake flask, (Q) reactor.



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to the flow of s u b s t r a t e ; i n t h i s sense, the o v e r a l l system operated as a combined absorber-reactor. D i s s o l v e d oxygen conc e n t r a t i o n s of 80 to 90% of s a t u r a t i o n value were maintained throughout the course of the r e a c t o r runs. R e f e r r i n g back t o F i g . 2, the i n c r e a s e d s p e c i f i c a c t i v i t y of the c a t a l y s t observed i n the r e a c t o r compared to t h a t i n a shake f l a s k i s a t t r i b u t a b l e , at l e a s t i n p a r t , to improved oxygen t r a n s f e r i n the r e a c t o r . Thus, the simple, e f f e c t i v e , f l e x i b l e use of the membrane form i n t h i s type of reactor.has demonstrated s e v e r a l a d d i t i o n a l p o s i t i v e features. From a p r a c t i c a l standpoint, the two most important chara c t e r i s t i c s of an immobilized c e l l c a t a l y s t are i t s a c t i v i t y and i t s operational s t a b i l i t y . The l a t t e r parameter i s u s u a l l y expressed i n c a t a l y s t h a l f - l i f e . The amount of a c t i v i t y , say i n I n t e r n a t i o n a l U n i t s (I.U.), would be a f u n c t i o n of c e l l - t o c a r r i e r r a t i o . As mentioned e a r l i e r , a 50% l o a d i n g r a t i o has been found to be optimal. A s p e r g i l l u s niger c e l l s attached t o c o l l a g e n e x h i b i t good a c t i v i t y r e t e n t i o n , as shown i n Table S · Please note t h a t r a t e comparisons have been made on the b a s i s of maximal'rate. I f one uses an i n t e g r a t e d average r a t e obtained over the e n t i r e p e r i o d o f the batch fermentation c y c l e , the comparison becomes even more favorable f o r the immobilized c e l l system, s i n c e i t experiences a very small l a g p e r i o d preceding c i t r i c a c i d s y n t h e s i s . In a d d i t i o n t o s p e c i f i c p r o d u c t i v i t y r a t e s , i t i s a l s o necessary to examine the r e l a t i v e concentrations of the product i n both cases, as the t i t e r value i s very c r u c i a l with regard t o product i s o l a t i o n and p u r i f i c a t i o n . Data obtained thus f a r i n d i c a t e t h a t bound c e l l s y i e l d 8 t o 40% of the f i n a l c o n c e n t r a t i o n obtainable i n fermentation. H a l f - l i f e of the c a t a l y s t was 138 hours. Chromatographic a n a l y s i s of r e a c t i o n products of c i t r i c a c i d synthesized by f i x e d c e l l s r e v e a l s the presence of products generated from s i d e r e a c t i o n s . They i n c l u d e i s o c i t r i c a c i d , o x a l i c a c i d and t r a c e q u a n t i t i e s o f g l u c o n i c a c i d . I s o c i t r a t e i s perhaps the major one, amounting to as much as 15 t o 20% of citrate. O x a l i c a c i d formation i n c i t r i c a c i d fermentations i s reported t o be dependent both on pH and on the extent of a e r a t i o n . By proper c o n t r o l of pH and d i s s o l v e d oxygen l e v e l s , i t might be p o s s i b l e t o reduce the formation of oxalate. Conclusion Immobilized c e l l and o r g a n e l l e systems o f f e r a great d e a l of promise i n mediating many r e a c t i o n schemes to produce commercially important products. U n l i k e bound mono-enzyme systems, c a t a l y s i s by f i x e d c e l l s i s q u i t e complex and many b a s i c aspects are yet to be understood. However, t e c h n i c a l f e a s i b i l i t y of r a t h e r elaborate immobilized c e l l processes, as exemplified by c i t r i c a c i d production through


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TABLE 5

CITRIC ACID SYNTHESIS BY IMMOBILIZED CELLS

Sample

Maxiumum s p e c i f i c productivity (g a c i d / g dry c e l l s - h )

R e l a t i v e maximum specific p r o d u c t i v i t y (%)

Fermentation

0.0043

100

Resting C e l l s

0.0045

104

Immobilized C e l l s

0.0021

48.4

Fermentation data obtained from 5-Z s t i r r e d fermentor; others from shake f l a s k s . Sucrose a t 40—£ was used as the s u b s t r a t e i n a l l cases.


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i n t a c t f u n c t i o n o f the TCA c y c l e enzymes, has been demonstrated. I n v e s t i g a t i o n o f the b a s i c problems o f structured-bed fermenta t i o n systems ( c e l l p h y s i o l o g y , c e l l v i a b i l i t y , t r a n s p o r t r e s i s tances, oxygen t r a n s f e r , m i c r o b i a l contamination) i s now being pursued i n our c u r r e n t work. Perhaps the g r e a t e s t p o t e n t i a l f o r immobilized c e l l systems l i e s i n r e p l a c i n g complex fermentations such as secondary m e t a b o l i t e p r o d u c t i o n . Some o f the f u r t h e r developments i n t h i s f i e l d should c l e a r l y be steered i n t h i s direction.


Acknowledgements The authors are g r a t e f u l t o the c o n t r i b u t i o n s o f Mr. Charles B e r t a l a n ; some o f the r e s u l t s reported here have been drawn from h i s t h e s i s . F i n a n c i a l a s s i s t a n c e f o r our work i n t h i s area was p r o v i d e d i n p a r t by a N a t i o n a l Science Foundation Grant (AER 7618816), E t h y l Corporation and H.J. Heinz Company.

Literature

Cited

1.

Venkatasubramanian, Κ., and Vieth, W.R., Progress In Industrial Microbio. (in press).

2.

Vieth, W.R., Annals New York Acad. S c i . (in press)

3.

Vieth, W.R., and Venkatasubramanian, Κ., Methods Enzymol. 34, 243 (1976).

4.

Venkatasubramanian, Κ., Vieth, W.R., and Constantinides, A. i n E.K. Pye and H.H. Weetall (Editors), Enzyme Engineering, Vol. 3, Plenum Press, New York (1978) pp. 29-42.

5.

Vieth, W.R., and Venkatasubramanian, Κ., Enzyme Engineering (Vol. 4), G. Broun and G. Manecke (editors), Plenum Press, New York (in press).

6.

Vieth, W.R., Gilbert, S.G., Wang, S.S., and Venkatasubramanian, Κ., U.S. Patent 3,809,613 (1974).

RECEIVED February 15, 1979.


Immobilized Microbial Cells in Complex Biocatalysis - ACS Publications

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