Purification of Fermentation Products - American Chemical Society

The loading capacity of cycloheximide recovery has been shown to increase by 30% and the purity of the antibiotic eluted by organic solvents is above ...
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Design of a New Affinity Adsorbent for Biochemical Product Recovery HENRY Y. WANG and KEVIN SOBNOSKY Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109

Conventional biochemical product recovery processes generally contain a solid removal step in which c e l l debris and other solids are being removed from the product containing aqueous broth before further purification. Substantial amount of product loss occurs at this step due to binding and washing. A new affinity adsorbent has been developed by immobilizing small adsorbents in hydrogel beads. The internal mass transfer problems that exist in most commercial adsorbents can be reduced by using smaller size adsorbents. The adsorbent containing hydrogel beads can be quite large (1-3 mm) and can be easily recovered from the fermentation broth. Selective adsorption can also be achieved by changing the composition of the hydrogel and the types of the adsorbents. This concept has been shown to work well for several antibiotics and vitamins. The loading capacity of cycloheximide recovery has been shown to increase by 30% and the purity of the antibiotic eluted by organic solvents is above 90% which is significantly higher than the conventional extraction methods.

The process of recovering and purifying fermentation products in the biochemical industries is generally d i f f i c u l t and costly. The product can exist intracellularly or extracellularly and it may be sensitive to temperature change» extremes of pH, certain chemicals and enzyme activities of the microorganisms. Frequently, the energy and labor costs spent on recovery and purification of the fermentation product far exceed the cost of fermentation. This is especially true for intracellular recombinant protein products. The final concentration of the fermentation product is usually low (less than 0.2 wt%). Therefore, new processing techniques must be developed to improve the existing biochemical product recovery procedures. The conventional biochemical product recovery processes can be divided into four sections: 1. Removal of insolubles 2. Primary isolation of product 3. Purification 0097-6156/85/0271-0123$06.00/0 © 1985 American Chemical Society In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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4. F i n a l product i s o l a t i o n (1,2) . A t l e a s t one o r more u n i t o p e r a t i o n s a r e needed t o a c c o m p l i s h each o f these s t e p s . Increase i n the number o f r e c o v e r y s t e p s w i l l d e f i n i t e l y reduce the o v e r a l l e x t r a c t i o n y i e l d even though t h e p r o d u c t p u r i t y may be i n c r e a s e d ( F i g u r e 1 ) . C e r t a i n amount o f t r a d e o f f w i l l be needed i n a c h i e v i n g b o t h the p r o d u c t y i e l d and the p r o d u c t p u r i t y o f a s p e c i f i c product r e c o v e r y scheme. I n g e n e r a l , t h e d r a s t i c drop o f e x t r a c t i o n y i e l d o c c u r r e d d u r i n g the f i r s t two steps," namely s o l i d s removal and p r i m a r y i s o l a t i o n o f product ( F i g u r e 1 ) . Whole beer p r o c e s s i n g u s i n g e i t h e r s o l v e n t e x t r a c t i o n o r r e s i n a d s o r p t i o n have been r e p o r t e d t o i n c r e a s e e x t r a c t i o n y i e l d ( 3 , 4 ) . T h i s i n c r e a s e i n r e c o v e r y i s because p r o d u c t l o s s e s due t o b i n d i n g w i t h s o l i d s and t h e i r subsequent removal can be e l i m i n a t e d . I n some c a s e s , whole beer p r o c e s s i n g may e l i m i n a t e the need f o r f i l t e r a i d i n the i n i t i a l step thus r e d u c i n g c o s t o r e l i m i n a t e t h e time consuming f i l t r a t i o n step a l t o g e t h e r . Whole b r o t h p r o c e s s i n g i m p l i e s p r o c e s s i n g t h e f e r m e n t a t i o n b r o t h w i t h o u t removal of t h e i n s o l u b l e f r a c t i o n , t h i s p r o c e d u r e t h e r e f o r e e l i m i n a t e s the i n i t i a l s t e p s such as s o l i d removal. Even though t h e major advantage c l a i m e d , so f a r , has been t o e l i m i n a t e the s o l i d removal s t e p , i t i s q u i t e m i s l e a d i n g because the s o l i d s s t i l l have t o be c o n c e n t r a t e d a f t e r w a r d s f o r waste d i s p o s a l . I t i s g e n e r a l l y agreed among t h e p r a c t i t i o n e r s o f whole b e e r p r o c e s s i n g t h a t p r o d u c t removal i n the p r e s e n c e o f c e l l s i s more d i f f i c u l t t o a t t a i n , and r e q u i r e s t h a t a l l p h y s i c o - c h e m i c a l parameters o f t h e o p e r a t i o n such as f e r m e n t a t i o n medium c o m p o s i t i o n t o be s t a n d a r d i z e d . The purpose o f t h i s paper i s t o d e s c r i b e a new approach t o a c h i e v e whole b r o t h p r o c e s s i n g u s i n g i m m o b i l i z e d s o l i d phase a d s o r b e n t s . A d s o r p t i o n a l l o w s t h e s e l e c t i v e c o l l e c t i o n and c o n c e n t r a t i o n onto s o l i d s u r f a c e s o f s p e c i f i c d i s s o l v e d m o l e c u l e s from t h e b r o t h . A d s o r p t i o n can be n o n - s p e c i f i c , f o r those mechanisms based on p o l a r , van der Waals and i o n i c i n t e r a c t i o n s , o r h i g h l y s e l e c t i v e f o r a f f i n i t y b i n d i n g based on b i o c h e m i c a l means ( 1 , 8 ) . Commercially a v a i l a b l e adsorbants a r e g e n e r a l l y g r a n u l a r porous p a r t i c l e s t o p r o v i d e e x t e n s i v e s u r f a c e a r e a , w i t h v o i d volumes approaching 30-50% o f t h e whole p a r t i c l e . Pore diameters a r e u s u a l l y l e s s than 0.01 mm. Many a d s o r b e n t s , such as a c t i v a t e d carbon and ion-exchange r e s i n s , can e f f i c i e n t l y s e p a r a t e a n t i b i o t i c s and o t h e r s m a l l b i o l o g i c a l l y a c t i v e molecules from the f e r m e n t a t i o n b r o t h . U n f o r t u n a t e l y , these adsorbents a l s o i n t e r a c t w i t h the m i c r o b i a l c e l l s and some o f t h e d i s s o l v e d n u t r i e n t s . Thus, t h e use o f ion-exchange r e s i n s and a c t i v a t e d carbon t o remove f e r m e n t a t i o n p r o d u c t s i s f r e q u e n t l y a s s o c i a t e d w i t h problems o f simultaneous removal o f n u t r i e n t s and s i d e p r o d u c t s . S u b s t a n t i a l volume r e d u c t i o n o c c u r s b u t o n l y l i m i t e d p u r i f i c a t i o n can be a c h i e v e d . Commercial adsorbents and ion-exchange r e s i n s a r e a v a i l a b l e i n v a r i o u s m a t r i c e s and s i z e s . L a r g e r p a r t i c l e s a r e p r e f e r r e d f o r easy s e p a r a t i o n from the b r o t h b u t they can be i n t e r n a l mass transfer limited. We have been i n t e r e s t e d i n i m m o b i l i z i n g d i f f e r e n t adsorbents such as a c t i v a t e d carbon powder and ion-exchange r e s i n s i n h y d r o g e l s such as c a l c i u m a l g i n a t e o r p o t a s s i u m carrageenan. The

In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

8. WANG AND SOBNOSKY

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purpose i s t o develop an a f f i n i t y bead designed t o i n c r e a s e b o t h mass t r a n s f e r and s e l e c t i v e p u r i f i c a t i o n f o r whole b e e r p r o c e s s i n g . We a r e a l s o i n t e r e s t e d i n u s i n g these a f f i n i t y beads f o r i n s i t u p r o d u c t removal.

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Design o f t h e A f f i n i t y Bead One o f t h e o b j e c t i v e s o f a b i o c h e m i c a l s e p a r a t i o n system d e s i g n i s to minimize t h e number o f s t e p s ( F i g u r e 1 ) . One way o f a c c o m p l i s h i n g t h i s i s t o p e r f o r m t h e s e p a r a t i o n and c o n c e n t r a t i o n i n s i t u t h a t i s d i r e c t l y w i t h t h e whole f e r m e n t a t i o n b r o t h u s i n g s o l i d s phase a d s o r b e n t s . T h i s type o f s e p a r a t i o n r e q u i r e s t h e d e s i g n o f an a f f i n i t y bead t h a t p r o v i d e s f o r s e l e c t i v e product removal from t h e f e r m e n t a t i o n b r o t h . V a r i o u s types o f s o l i d adsorbents have been used t o c o n c e n t r a t e d i f f e r e n t b i o c h e m i c a l p r o d u c t s from f e r m e n t a t i o n b r o t h s . The s i z e o f t h e s o l i d adsorbent p a r t i c l e i s important because l a r g e m a c r o s c o p i c beads can e a s i l y be s e p a r a t e d and r e c o v e r e d from f e r m e n t a t i o n b r o t h s . However, l a r g e porous beads e x h i b i t i n t e r n a l d i f f u s i o n a l r e s i s t a n c e and depending on p r o c e s s i n g t i m e , a l l t h e b i n d i n g s i t e s o f t h e adsorbent may n o t be u t i l i z e d , r e s u l t i n g i n a lower a d s o r p t i o n c a p a c i t y . A l s o , f o r some adsorbents c e l l d e b r i s and p r o t e i n a c e o u s m a t e r i a l s may tend to adhere t o t h e s u r f a c e o f t h e s o l i d adsorbent and would contaminate t h e product i n t h e subsequent e l u t i o n p r o c e s s (7_). We have been i n v e s t i g a t i n g the p o s s i b l i t y o f i m m o b i l i z i n g s o l i d adsorbents such as a c t i v a t e d carbon, n o n - i o n i c p o l y e r m i c r e s i n s , monoclonal a n t i b o d i e s i n s m a l l h y d r o g e l beads (7), u s i n g b o t h K-carrageenan and c a l c i u m a l g i n a t e t o p r o v i d e t h e h y d r o g e l m a t r i x . Adsorbent c o n c e n t r a t i o n s i n these h y d r o g e l beads ( F i g u r e 2) can be as h i g h as 30-35 wt %. H i g h e r c o n c e n t r a t i o n s than 30 wt % o f t h e adsorbents weakening t h e bead s t r u c t u r e , a l l o w i n g f r a g m e n t a t i o n under shear. These composite i m m o b i l i z e d adsorbents ( F i g u r e 2) p r o v i d e a d d i t i o n a l s e l e c t i v i t y f o r p r o d u c t a b s o r p t i o n from t h e f e r m e n t a t i o n b r o t h . V e r y l a r g e macromolecules w i l l be e x c l u d e d from t h e h y d r o g e l and those t h a t do p e n e t r a t e w i l l d i f f u s e through the g e l a t d i f f e r e n t r a t e s depending on t h e i r s i z e . I f t h e b i n d i n g s i t e s a r e s e l e c t i v e i n n a t u r e , o n l y t h e d e s i r e d p r o d u c t w i l l be adsorbed. The g e l i s r e v e r s i b l e ( w i t h t h e p r e s e n c e o r absence o f exogenous c a t i o n s ) and t h e adsorbents w i t h t h e d e s i r e d p r o d u c t can be r e c o v e r e d from t h e g e l m a t r i c e s through washing and d i s s o l v i n g the g e l . Thus, b o t h c o n c e n t r a t i o n (volume r e d u c t i o n ) and p u r i f i c a t i o n can be a c h i e v e d . The l a r g e h y d r o g e l beads can a l s o e a s i l y be r e c o v e r e d from t h e f e r e m e n t a t i o n b r o t h w i t h o u t d i s r u p t i n g the m i c r o b i a l c u l t u r e . Adsorption o f Cvcloheximide

t o Immobilized

Beads

Recovery o f c y c l o h e x i m i d e , a g l u t a r i m i d e , a n t i f u n g a l a n t i b i o t i c (M. W. 281) produced by Streptomvces g r i s e u s has been used as a model system. The a n t i b i o t i c f e r m e n t a t i o n was c a r r i e d out a c c o r d i n g t o Kominek ( 5 ) . The whole f e r m e n t a t i o n b r o t h was a d j u s t e d t o pH 6.0 and s t o r e d i n 4°C c o l d room. The a n t i b i o t i c

In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Downloaded by UNIV LAVAL on October 19, 2015 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch008

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F i g u r e 1. C o r r e l a t i o n of e x t r a c t i o n y i e l d v e r s u s product p u r i t y f o r a b i o c h e m i c a l product r e c o v e r y scheme. Key: A, a f t e r ferment a t i o n ; B, a f t e r p r o t e i n e x t r a c t i o n ; C, a f t e r c o n c e n t r a t i o n ; D, column 1; E, column 2; F, column 3; G, column 4.

In Purification of Fermentation Products; LeRoith, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Downloaded by UNIV LAVAL on October 19, 2015 | http://pubs.acs.org Publication Date: January 8, 1985 | doi: 10.1021/bk-1985-0271.ch008

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has been shown t o be s t a b l e f o r a t l e a s t two weeks under such condition. XAD-4, a porous n o n i o n i c p o l y m e r i c r e s i n o f p o l y s t y r e n e i n n a t u r e (Rohm and Haas, PA.) has been shown a b l e t o c o n c e n t r a t e c y c l o h e x i m i d e from t h e f e r m e n t a t i o n b r o t h (6,7) . XAD-4 r e s i n s can be i m m o b i l i z e d i n t o K-carrageenan o r c a l c i u m a l g i n a t e by adding the r e s i n s (up t o 30 wt%) i n d i s s o l v e d h y d r o g e l s o l u t i o n and pump the s l u r r y through s m a l l t u b i n g ) d r o p l e t s form b y d r i p p i n g i n t o a r e s p e c t i v e g e l l i n g s o l u t i o n . The r e s i n s a r e entrapped i n t h e hardened h y d r o g e l beads. The s i z e o f t h e h y d r o g e l beads can be c o n t r o l l e d by the s i z e o f the t u b i n g . Smaller r e s i n p a r t i c l e s can be o b t a i n e d by p u l v e r i z i n g t h e r e s i n p a r t i c l e s b y m e c h a n i c a l means. The a d s o r p t i o n k i n e t i c s o f c y c l o h e x i m i d e (CH) from e i t h e r aqueous s o l u t i o n o r whole f e r m e n t a t i o n b r o t h were c a r r i e d out u s i n g both p l a i n XAD-4 r e s i n s and i m m o b i l i z e d XAD-4 r e s i n s . By d e t e r m i n i n g the b u l k c o n c e n t r a t i o n o f CH (Ρ), the batch a d s o r p t i o n r a t e o f CH u s i n g XAD-4 r e s i n can be e s t i m a t e d . The a d s o r p t i o n r a t e can be modeled as f o l l o w s : (1) Q i s t h e l o a d i n g c a p a c i t y o f CH by t h e p o l y m e r i c r e s i n and m i s the amount o f the p o l y m e r i c r e s i n added. Κ i s t h e s p e c i f i c a d s o r p t i o n r a t e c o n s t a n t o f CH and P* i s t i e s u r f a c e c y c l o h e x i m i d e c o n c e n t r a t i o n w h i c h i s i n e q u i l i b r i u m t o Q: P* = f(Q)

(2)

A F r e u n d l i c h type a d s o r p t i o n i s o t h e r m can be used as long as Ρ be kept below 500 mg/1. As shown i n F i g u r e 3, t h e a d s o r p t i o n isotherms o f XAD-4 r e s i n i n both aqueous CH s o l u t i o n and f e r m e n t a t i o n have been determined. I n the fermentation b r o t h , the maximum a d s o r p t i o n c a p a c i t y was reduced due t o a d d i t i o n a l i m p u r i t i e s b e i n g adsorbed onto the r e s i n s . C l o s e t o 3 0 % r e d u c t i o n was observed. When i m m o b i l i z e d XAD-4 r e s i n s i n K-carrageenan a r e u s e d , s i m i l a r a d s o r p t i o n p a t t e r n s a r e observed. The maximum l o a d i n g c a p a c i t y o f 110 mg CH/gm r e s i n can be a c h i e v e d even i n t h e f e r m e n t a t i o n b r o t h i n d i c a t i n g e x c l u s i o n o f v a r i o u s i m p u r i t i e s from the XAD-4 r e s i n s which a r e i m m o b i l i z e d i n s i d e the h y d r o g e l . By i m m o b i l i z i n g t h e s o l i d phase adsorbents such as XAD-4 r e s i n i n t h e h y d r o g e l , we a c t u a l l y add an a d d i t i o n a l d i f f u s i o n r e s i s t a n c e t o t h e a d s o r p t i o n . The p r o d u c t m o l e c u l e s need t o d i f f u s e through t h e h y d r o g e l b e s i d e d i f f u s i n g through t h e stagnant s u r f a c e l a y e r and p e n e t r a t i n g through t h e r e s i n p a r t i c l e t o t h e r e s i n s u r f a c e . As shown i n F i g u r e 4, t h e s p e c i f i c a d s o r p t i o n r a t e c o n s t a n t s o f t h e f r e e XAD-4 r e s i n i s much h i g h e r than t h e i m m o b i l i z e d XAD-4 r e s i n . T h i s i s p r i m a r i l y due t o t h e a d d i t i o n a l d i f f u s i o n a l r e s i s t a n c e through t h e h y d r o g e l . I n b o t h c a s e s , t h e s p e c i f i c r a t e c o n s t a n t s decrease as t h e r e s i n s become s a t u r a t e d w i t h t h e p r o d u c t , c y c l o h e x i m i d e . The s p e c i f i c a d s o r p t i o n r a t e o f the i m m o b i l i z e d XAD-4 r e s i n s can be i n c r e a s e d s u b s t a n t i a l l y when p u l v e r i z e d XAD-4 r e s i n s (dp