Preparation and Applications of Immobilized Microorganisms - A Survey of Recent Reports

8/11/2019 Preparation and Applications of Immobilized Microorganisms - A Survey of Recent Reports

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i s qu i t e sho r t and sca l e -up i s pos s ib l e . Howeve r, m ic rob ia l enzymes , l i ke t hose f romo t h e r s o u r c es , w h e n i s o l a te d f r o m t h e i r n a t u r a l e n v i r o n m e n t a re g e n e r a l ly u n s t a b l ea n d a r e e a si ly d e n a t u r e d u n d e r o p e r a t i n g c o n d i t i o n s . M o r e o v e r, th e i r u s e is r es t ri c te di n t h e m a i n t o t h e c a t a l y s is o f s i n g le r e a c t i o n s i n w h i c h c o f a c t o r r e g e n e r a t i o n is n o tr e q u i r e d . T h e s e p r o b l e m s m a y b e o b v i a t e d , o r a t l e as t m i n i m i z e d , b y d i r ec timmobi l i za t i on o f mic rob ia l ce l l s con ta in ing the des i r ed ac t iv i t y o r ac t i v i t i e s [2 ] .O p e r a t i n g c o s t s ar e lo w e r e d s i g n i fi c a n t ly b e c a u s e e n z y m e e x t r a c t i o n a n d p u r i f i c a t i o nis n o l o n g e r n e c e s s a r y a n d b e c a u s e h i g h e r y i el d s o f a c t i v i t y a r e o b t a i n e d o nimm obi l i z ing ce l l s r a th e r t h an f r ee enz ym es [2]. Imm ob i l i zed v i ab l e ce ll sy s t ems a l soh a v e a d v a n t a g e s o v e r t r a d i t i o n a l b a t c h f e r m e n t a t i o n s . T h e s e i n c lu d e h i g h e r p r o d u c ty i el d s, t h e a b i li ty t o o p e r a t e c o n t i n u o u s l y a n d a t h i g h d i l u t i o n r a te s w i t h o u t c u l t u r ew a s h o u t , t h e c a p a c i t y t o r e c h a rg e t h e s y s t e m b y i n d u c i n g g r o w t h o f r e st in g c e lls , a n dacce l e r a t ed r eac t ion r a t e s because o f i nc reased ce l l dens i ty [2 ] .

T h e f i r s t s u cc e s s fu l i n d u s t r ia l a p p l i c a t i o n o f i m m o b i l i z e d m i c r o b i a l ce llp r e p a r a t i o n s , i.e ., t h e c o n t i n u o u s p r o d u c t i o n o f l: a s p a r t i c a c id , w a s c a r r i e d o u t b yC h i b a t a a n d c o l le a g u e s in 1 97 3 [ 1 ]. I n t h e i n t e r v e n i n g y e ar s th e n u m b e r o f r e p o r t sa n d p a t e n ts o n t h e u se s o f im m o b i l iz e d m i c r o o r g a n i s m s h a s m u s h r o o m e d d e s p it e t h ef a c t t h a t r e s t r i c t e d p e r m e a b i l i t y o f c e l l w a l l s t o s u b s t r a t e a n d p r o d u c t a n d t h eo c c u r r e n c e o f u n w a n t e d s i d e - r e a c t i o n s c o n t i n u e t o p o s e p r o b l e m s . I n o u r s u r v e y o ft h e l i t e r a t u r e f r o m J a n u a r y 1 9 8 3 t o O c t o b e r 1 9 8 7 w e f o u n d t h a t m o r e t h a n 6 0 0p a p e r s o n th e p r e p a r a t i o n a n d a p p l i c a t i o n s e x c l ud i n g a n a l y t i c a l u s es ) o fi m m o b i l i z e d m i c r o o rg a n i s m s h a v e b e e n p u b l i s h e d . A n a l y t i c a l u s e s o f s u c hprepa ra t ions a r e dea l t w i th i n de t a i l e l s ewhere i n t h i s vo lume [3 ] .

Reviews

T h e i m p r e s si v e n u m b e r o f r ev ie w a r ti c le s o n t h e p r e p a r a t i o n a n d a p p l i c a t i o n s o fi m m o b i l i z e d m i c r o o rg a n i s m s t h a t h a v e b e e n p u b l i s h e d s i n c e 1 9 83 o b v i o u s l y re f le c t sthe co ns ide rab l e r e sea rch e f fo r t and gene ra l i n t e r e s t i n t h i s top i c . Such r ev iewsi n c l u d e t h o s e o f a g e n e r a l n a t u r e [2 , 4 - 3 9] a n d i t is p e r h a p s f i tt in g t h a t a m o n g t h e s eis a h i s to r i c a l tr e a t m e n t o f t h e s u b j e c t b y C h i b a t a a n d c o l le a g u e s [ 11 ]. T h e m o s tr ecen t gene ra l r ev iew, t ha t by Ak in [39 ], cove r s such top i c s a s adva n tages an dd i s a d v a n t a g e s o f im m o b i l i z e d ce ll s, s u p p o r t s a n d i m m o b i l i z a t i o n p r o c e d u r e s u s ed ,

a n d a p p l i c a t i o n s a n d p e r f o r m a n c e c h a r a c t e ri s ti c s o f i m m o b i l i z e d c el l b i o c a t a ly s t s .O t h e r r e vie w s c o n c e n t r a t e o n s u p p o r t s o r p r o c e d u r e s f o r i m m o b i l iz a t i o n [ 4 0 - 4 6 ], o nv a r io u s a s p e ct s o f p ro c e s s e n g i n e e ri n g [ 4 7 - 5 4 ] , o n t h e d e s ig n a n d p e r f o r m a n c e o fr e a c to r s [ 5 5 - 5 7 ] a n d o n t h e p r o b l e m s o f e n s u ri n g a n a d e q u a t e s u p p l y o f o x y g e n t othe immobi l i zed ce l l s du r ing ope ra t ion e t c . [ 58 ] .

mmobi l i z a t i on and immob i l i z ed p repa ra t i ons

I m m o b i l i z e d c e ll b i o c a t a ly s t s m a y b e d e f i n e d a s c e ll s t h a t a r e p h y s i c a ll y r e s tr i c te dt o a d e f i n e d s p a c e w i t h r e t e n t i o n o f s o m e o r a ll o f t h e i r c a t a ly t i c a c t iv i t y f o r r e p e a te d

a n d c o n t i n u o u s u s e s ee , e .g ., R e f. 3 9) . O n e m a y a d d t h a t i m m o b i l i z a t i o n is a l sod e s i g n ed t o m a k e t h e c el l c a ta ly s ts m o r e a m e n a b l e t o h a n d l i n g a n d c o n t r o l. A m o n gt h e p r o c e d u r e s u s e d t o i m m o b i l i z e m i c r o o rg a n i s m s a r e a g g r e g a t i o n o r f l o c c u l a t i o n ,


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a d h e s i o n o r a d s o r p t i o n , c o v a l e n t l i n k a g e o f c e ll t o c e ll o r c el l t o s u p p o r t , e n t r a p m e n to r e n c a p s u l a t i o n i n g e l s o r o t h e r p o l y m e r s , a n d p h y s i c a l r e t e n t i o n w i t h i nm e m b r a n o u s s t r u c t u r e s o r h o l l o w f i b r e s . E x a m p l e s o f e a c h o f t h e s e p r o c e d u r e s f o ri m m o b i l i z a t i o n a n d d e s c r i p t i o n s o f n e w s u p p o r t s h a v e b e e n t h e s p e c i f i c s u b j e c tm a t t e r o f a la rge n u m b e r o f p a p e r s [ 5 9 - 110 ] . A s s t a t e d a bo v e , t h e o b j e c t o fi m m o b i l i z a t i o n i s t o r e s t r i c t c e l l m i g r a t i o n w i t h m i n i m a l i n h i b i t i o n o f c a t a l y t i ccapac i ty. However, i t mus t be sa id tha t i n many sys t ems the cond i t ions used ini m m o b i l i z a t i o n a n d t h e r e s u l t a n t h i g h l y l o c al i ze d c o n c e n t r a t i o n o f c e ll s ha v e b e e ns e e n t o a f f e c t g r o w t h a n d m o r p h o l o g y [111 -117 ]. T h e e f f e c t s o f i m m o b i l i z a t i o n o ngenera l m e tabo l i sm o r spec i f i c enzym e ac tiv i ti e s [51 , 118-132] have a l so r eceived thea t t e n t i o n t h e s e a s p e c t s d e se rv e . A n y i m m o b i l i z e d m i c r o b i a l p r e p a r a t i o n d e s t i n e d f o rs e r io u s a p p l i c a t i o n m u s t , a m o n g o t h e r q u a l i ti e s , b e s ta b le i n o p e r a t i o n . I n t h i s c o n te x tsevera l pape r s [133-139] spec i f i ca l ly devo ted to s t ab il it y, s t ab i l i za t ion and ac t iva t iona re o f in t e re s t, a s a re those [140-146] de ta i l i ng p rocedures fo r examin a t ion o f imm obi l -i zed p repa ra t ions .

T h e m o s t c o m m o n l y u s e d m e t h o d o f c e ll i m m o b i l iz a t i o n h a s b e en e n t r a p m e n t o re c a p s u l a t i o n i n g e l s a n d p o l y m e r s , b o t h n a t u r a l a n d s y n t h e t i c . H y d r o c o l l o i d g e l s ,e s p e c ia l ly al g i n at e s , c o n t i n u e t o b e t h e f a v o u r i t e s u p p o r t a l t h o u g h w i t h a r a n g e o fva r i a t ions f ro m the o r ig ina l p rocedu re [99 , 137 , 147] . A lg in ic ac id is a po lym er o fD -m an nu ron ic a nd L-guluron ic ac ids jo ine d by ~-1 ,4 an d o~-1 ,4 g lycos id ic l inkages .So lu t ions o f sod ium a lg ina te a re v i scous . By con t ra s t , ca l c ium a lg ina te s a re ge ls , t hec a l c i u m i o n s a c t i n g a s b r i d g e s b e t w e e n p o l y m e r c h a i n s a t g u l u r o n i c a c i d r e s id u e s. A ss t a t ed a b o v e , a lg i n a te s a r e a m o n g t h e m o r e f a v o u r e d s u p p o r t s b e c a u s e t h e y a r e

n o n t o x i c a n d b e c a u s e t h e e a s e w i t h w h i c h g e l f o r m a t i o n t a k e s p l a c e a l l o w s m i l dc o n d i t i o n s t o p r ev a il d u r i n g i m m o b i l i z a t i o n . I n e s se n c e i m m o b i l i z a t i o n is e f f ec t e d b ys u s p e n d i n g c el ls i n a s o l u t i o n o f s o d i u m a l g i n a te w h i c h i s t h e n d r o p p e d , e x t r u d e dor sp rayed in to a so lu t ion con ta in ing ca lc ium [46] . The ge l beads tha t fo rm a reu s u a l l y a l lo w e d t o h a r d e n f o r a p e r i o d i n w h i c h c a l c i u m c r o s s - l in k i n g is m a x i m i z e d .Whi le i t i s poss ib l e to use a r ange o f o the r d iva len t i ons fo r ge l fo rma t ion , ca l c iumis gene ra l ly cons ide red to be the mos t appropr i a t e . Th e en t r ap ped ce ll s have ane l e m e n t o f f r e e d o m w i t h i n t h e g e l s t r u c t u r e a n d c a n m u l t i p l y. H o w e v e r, g r o w t h a to r n e a r t h e s u r f a c e m a y r e s u l t i n l o s s o f c e l l s , w i t h c o n s e q u e n t d e t e r i o r a t i o n o fc a t a l y t i c a b i l i t y a n d c o n t a m i n a t i o n o f t h e s u b s t r a t e / p r o d u c t s t r e a m i n u s e .

D e n a t u r a t i o n o f t h e b e a d s t r u c t u r e c a n o c c u r a s a r es u lt o f c o m p e t i t i o n w i t h o t h e rions fo r b ind ing s i te s and i f ca l c ium i s r emo ved by p rec ip i t a t ion w i th phosph a te . I tis t y p i c a l i n t h e p r a c t i c a l u s a g e o f a l g i n a t e - e n t r a p p e d m i c r o o rg a n i s m s t o m i x c e ll sr a n g in g i n c o n c e n t r a t i o n f r o m 1 0 - 3 0 g / 10 0 m l ) w i t h a n e q u a l v o lu m e o f s o d i u m

aigin ate 4o70, w /v) so tha t a f ina l bead co nc en t ra t io n of 27o w /v) is achieved [46 ,1 4 8 - 1 5 3 ] . I n l a b o r a t o r y - s c a l e t e c h n o l o g y b e a d s m a y c o n v e n i e n tl y b e f o r m e d b yd r o p w i s e a d d i t i o n o f t h e c e l l / s o d i u m a l g i n a t e m i x t u r e f r o m a s y r i n g e i n t o t h ec a l c i u m c h l o r i d e s o l u t io n . T h i s c a n o f c o u r s e b e m e c h a n i s e d a n d b e a d s iz e m a y b ea l t e red by jud ic ious cho ice o f the bo re s i ze o f the sy r inge need le. Bead shape m ay a l sobe a l t e red to su i t t he in t end ed ap p l i ca t io n [99 ]. M oreover, i t has r ecen t ly been

r e p o r t e d t h a t t h e u s e o f r e s o n a n c e t e c h n i q u e s t o b r e a k u p t h e j e t o fb i o c a t a l y s t / a l g i n a t e m i x t u r e i n t o d r o p l e t s o f u n i f o r m s iz e a s t h e y ar e i n t r o d u c e d i n t ot h e d i v a l e n t c a t i o n s o l u t i o n i n c re a se s b y t w o o r d e r s o f m a g n i t u d e t h e s c a le o f


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i m m o b i l i z a t i o n o v e r t h a t w h i c h c a n b e a c h i ev e d b y c o n v e n t i o n a l n o z z l e in j e c t i o n[153I.

A p a r t f r o m a l g i n at e s, t h e m o s t c o m m o n l y f a v o u r e d h y d r o g e l f o r c e llim m ob i l i za t i o n i s ca r r a gee nan . I ts s t ruc tu re i s s imi l a r to t ha t o f agar. I t i sg a l a c t o p y r a n o s y l - b a s e d w i t h a s i g n i f i c a n t p e r c e n t a g e o f s u b s t i t u t e d r e s i d u e s . I t i sc o m p r i s e d o f a n u m b e r o f m a j o r f r a c ti o n s o f w h i c h t h e K , t h e m o s t f r e q u e n t l y u s ed ,a n d t f r a c t i o n s f o r m t h e r m o r e v e r s i b l e g e ls . G e l s t ru c t u r e s a r e a c h ie v e d b y f o r m a t i o no f in t e r c h a i n h el ic e s. O n w a r m i n g t o 5 0 C , 5 ( w / v ) K - c a r ra g e e n a n e x is ts a s as o l u t i o n i n w h i c h t h e p o l y m e r c h a i n s a r e p r e s e n t a s r a n d o m c o i l s . To m a x i m i z e g e lp e r f o r m a n c e , t h e c a r r a g e e n a n c a n b e w a s h e d w i th 3 ( w / v ) s u c ro s e t o r e m o v ec a l c i u m i o n s . T h e c a r r a g e e n a n s o l u t i o n i s t h e n m i x e d w i t h a n e q u a l o r l e s s t h a ne q u a l v o l u m e o f c e ll s a n d t h e m i x t u r e i s d r o p p e d i n t o a c o l d s o l u t i o n o f e i th e r K C Io r C a C1 2. B e a d s f o r m e d o n c o o l i n g u s in g C a C I 2 a r e t h e n h a r d e n e d o r c u r e d u s i n gK C I . U l ti m a t e l y, p o t a s s i u m i s u s e d a s t h e s t a b il iz i n g c a t i o n . T h e t e m p e r a t u r e o f t h ec e l l / c a r r a g e e n a n s o l u t i o n u s e d i s c ri ti c a l in t h a t i t d e t e r m i n e s t h e e f f i c i e n c y o f b e a df o r m a t i o n a n d m a y h a v e a n a d v e r s e a f f e c t o n c e ll v i ab i li ty. N e v e r t h el e s s, t h es t r u c t u r a l i n t e g r i t y o f a g e l s o f o r m e d i s n o t a s s e n s i t i v e a s t h o s e f o r m e d f r o ma l g in a t e s t o th e p r e s e n c e o f a n i o n s a n d c a t i o n s i n th e m a c r o e n v i r o n m e n t [9 8 ].M od i f i ed p roce dures , e . g ., t he i nc lus ion o f s i l ic a , have been inv es t iga t ed [51 , 149 ].F o r m a t i o n o f b e a d s b y su s p e n s i o n o f t h e m o n o m e r i n h y d r o p h o b i c p h a s e s is a ls os h o w i n g p r o m i s e [ 43 , 1 0 4, 1 5 71 . P r e p o l y m e r i z e d s y n t h e t i c m a t e r i a l s h a v e a l s o b e e nused a s en t r app ing o r encapsu la t i ng med ia [72 , 100 , 154 ] a s have a r ange o fm isce l lane ou s na tu ra l ge ls inc lu din g aga r [361 , ch i tos an [67 , 133 , 155], co l la ge n [39 ,77 , 156] , egg w hi te [101] , ge la t in [391 , locu s t be an gu m [158], var iou s m ixed ge lsys t ems [39, 102 , 1581 and ma te r i a l s t ha t can be cop o ly m er i z ed by r ad i a t i on [59 , 61,69 , 71, 1031. O th er m at r ice s a re inc lu de d in the re fere nce li s t [59 , 110] . Ce l i te has bee ni n c l u d e d i n s o m e e n t r a p p e d b i o c a t a l y s t s y s t e m s t o i m p r o v e o n g e l p e r m e a b i l i t y a n drobus tness [391 .

A m o r e p r a g m a t i c a p p r o a c h t o i m m o b i l i z a ti o n o f b i o ca t a ly s t s is t o e n c o u r a g e t h eg r o w t h o f c e ll s i n v o id s i n p o r o u s m a t e r ia l s . T h e s u c c es s o f s u c h s y s te m s d e r i v es f r o mt h e n a t u r a l t e n d e n c y o f m i c r o b i a l c el ls t o a d h e r e t o s u r fa c e s ( a d s o r p t i o n ) o r t o o n ea n o t h e r ( f l o c c u l a t i o n ) . I n a t y p i c a l e x a m p l e y e a s t c e l l s h a v e b e e n i m m o b i l i z e d i n1 c m 3 r e t ic u l a t e d p o l y u r e t h a n e c u b e s b y g r o w i n g t h e c e ll s i n c o n t a c t w i t h t h es u p p o r t f o r 2 0 h [ 15 91 . T h e c u b e s c a n t h e n b e p a c k e d i n c o l u m n s o r u s e d i n t o w e rf e r m e n t e r s y st e m s . S u c h c a t a l y s ts d o n o t s u f f e r t h e d i s a d v a n t a g e o f a l g i n a t e b e a d si n b e i n g s e n si ti v e t o t h e p r e s e n c e o f a n i o n s a n d c a t i o n s t h a t m i g h t d i s r u p t t h e i rs t r u c t u r a l i n t e g r i t y n o r a r e t h e c e l l s s u b j e c t e d t o p o s s i b l e h e a t s h o c k d e n a t u r a t i o na s c o u l d b e t h e c a s e i n th e p r e p a r a t i o n o f c a r r a g e e n a n s y s te m s . T h e y d o n o t , h o w e v er,a f f o r d t h e s a m e d e g r e e o f c o n t r o l o v e r c o n t a m i n a t i o n o f p r o d u c t s tr e am s . Va r io u sc l o th s , f o a m s , s p o n g e s a n d m e t a ll ic m e s h e s h a v e b e e n u s e d a s i m m o b i l i z a t i o ns u p p o r t s . S i n c e t h e s e a r e u s u a l l y b o t h r o b u s t a n d i n e x p e n si v e, s c a le - u p o f s u c hsys t ems i s re l a t ive ly ea sy [115 , 124 , 160 -1631 . M oreove r, t he p roces se s o fi m m o b i l i z a t i o n a r e g e n e r a ll y q u i te s i m p le . A s a n e x a m p l e w e m a y c i te t h ei m m o b i l i z a t i o n o f r w i n i a o n D E A E - c e l l u l o s e w h i c h i s a c h i e v e d m e r e l y b y m i x i n gce l l s w i th a s lu r ry o f t he r e s in (2 g ce l l s /10 ml r e s in ) a t pH 7 [152] . Th i s p rocedureis a n a l o g o u s t o i m m o b i l i z a t i o n o f p r o te i n s b y i o n i c b in d i n g . I n g e n e ra l , im m o b i l i z e d


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cell systems depend not only on physical entrapment factors resulting from colonygrowth but also on a variety of adhesion forces. The latter may be enhanced inporous or non-porous support matrices by any one of a variety of possiblemodifications to the cells or to the supports [42 44 75 78 105 106 164-166]. Onesuch modification involves the adsorption of aluminium ions onto the surface of

accharomyces cerevisiae [75 167 168]. The a luminium ions neutralize repulsivecharges and so facilitate adhesion of the cells to glass plates.

Aggregation and flocculation a form of cell immobilization is frequently used toexploit the catalytic abilities of certain microorganisms in established towerfermenters. Various examples of systems operated in this mode have been reported[169-171]. Flocculation may be stimulated or enhanced by the use ofpolyelectrolytes various gas treatment techniques or by covalent bonding [172 173].One example is the cross-linking of permeabilized yeast cells using albumin and

glutaraldehyde [109]. A disadvantage of this system and those like it is that thebifunctional reagent used in cross-linking by reacting with essential cellularcomponents may effect significant loss o f catalytic activity.

Various membrane systems not only provide a means of immobilizing cells theyalso provide the reactor vessel. Such systems may however experience problems asa result of excessive cell growth and biomass build-up [174]. A typical example ofthe use to which such systems may be put is the immobilizat ion of yeast cells withinultrafiltration hollow fibres [175]. The fibres used had a nominal molecular weightcut-off value of 10000 and to prevent the build-up of biomass a nitrogen-deficientmedium was used.

e a c t o r s

In theory immobilized cell catalysts may be used in stirred-tank reactors. Inpractice however this mode of operation has not been favoured. Membrane systemsincluding ultrafiltra tion cells and hollow fibre reactors have enjoyed considerable useas have those dictated by specific requirements e.g. in biosensors [107 174 176177]. However a survey of the literature shows that two major systems plug-flowand air-lift bioreactors continue to predominate [36 39]. A problem associated withbioreactors destined for use with living cells is that of gas production. In the packed-

bed type gas generation leads to the development of considerable pressures on thereactor walls. In the case of air-lift types the accumulation of gases within beadsnecessitates the incorporat ion of limiting mesh systems that prevent the loss of beadsfrom the top of the reactor. In attempts to overcome these problems a bioreactorsystem consisting of cascades of fluidized-beds with side loops for aeration andstripping of carbon dioxide has been devised [51]. An alternative approach involvesthe in troduction of a basket containing immobilized cells into an appropriate vesselso as to create a bioreactor. Such a system has been investigated for the continuousacidif ication of milk and for the production of ethanol [150 178]. Further variationshave included the use of rotating biological surface reactors [179-181] and the use

of film fermenters. In one such fermenter cotton cloth provides the support [172].Immobilization of microorganisms and incorporation of the preparation into a

reactor creates a complex dynamic situation in which the potential for cell growth


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and the diffusion of gases, substrates and products must be considered. Reactordesign and the problems associated with mass transfer of substrates and productshave been the subject of a number of papers and review articles [48, 51, 55, 108, 110,114, 118, 133, 143, 163, 182-206]. Other papers have dealt with procedures forensuring an adequate rate of oxygenation during operation [207-210].

pplications

Current applications of immobilized microorganisms on an industrial-scale are,with few exceptions, restricted to the production of antibiotics, amino acids andorganic acids. Nevertheless, research in many different countries shows that thepotential of these preparations for other applications is enormous. Thus, apart fromthe above, they may find use in the production of ethanol and other fuels, in theexploitation of biomass, in organic syntheses, in wastewater treatment and in thefood, dairy and drinks industries. Current and potential applications have been wellreviewed [132, 154, 211-221]. In the text to follow, we have tried to sort into relatedgroups the papers dealing with specific uses o f immobilized microorganisms. Howev-er, strict categor ization is not possible. For example, immobilization of an organismwith a view to producing cellulase could be included under protein synthesis orbiomass exploitation. Similarly, biomethanation of organic nutrients in aqueouseffluents could be listed under wastewater treatment or fuel production. Despite thiswe trus t that the reader will readily be able to locate the references of interest.

A m i n o a c i d sThe production of amino acids, is, as stated earlier, one of the most successfulapplications of immobilized microbial cells [26]. Procedures for the formation ofamino acids generally from ketoacids [222] and for the specific production o f alanine[223-229], arginine [230], aspartate [231-244], glutamate [245], N-acetylmethionine [246], phenylalanine [247-250], tryptophan [251] and tyrosine[252-256] have also been reported. Chibata and co-workers at the Tanabe SeiyakuCo. in Osaka have developed a process whereby asparate and alanine can beproduced from ammonium fumarate by sequential exploitation of the aspartaseactivity of immobilized E. co l i and the aspartate decarboxylase activity of

immobilized P d a c u n h a e [223-225]. The 1984 production rates of alanine andaspartate were 10 tonne s/month and 100 tonnes /month , respectively [223]. Theoperational stability of the cellular aspartase T,/2 4 months), by contrast with theinstability of the free enzyme, and the use of mutants of E. col i lacking fumaraseactivity thereby eliminating the conversion of fumarate to malate) contributedsignificantly to these impressive production figures [235]. The interfering fumaraseE. colt) and alanine racemase P.. d a c u n h a e ) activities could also be selectively

eliminated by appropriate treatment of the cells prior to immobilization [218]. TheWu-Sxi Solvent Factory in China and Kyowa Hakko Kogyo Co., Ltd. in Japan alsoproduce aspartate using a similar procedure [150]. Evans and colleagues [250] have

recently reported the isolation of a strain of C o r y n e b a c t e r i u m e q u ithat producesmore than 33 g/l o f L-phenylalanine from c~-acetamidocinnamic acid ACA) in molaryields greater than 997o using immobilized cells. The inducible cellular activities


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ACA translocase

Acetamidocinnamate (exogenous) ~ Acetamidocinnamate (endogenous)

ACA acylase II

Phenylpyruvate ~ ~

NH4+ I ~ - ' NADH

~ Phenylalanine dehydrogenase

~ x,...__~ NAD +

L-Phenylalanine

a-Aminocinnamate

exploited in this process include a permease, an acylase and a dehydrogenase.An oxidizable substrate such as lactate, pyruvate or formate is included to ensure a

supply of NADH. A mutant, OARI-16, produces phenylalanine 2.5-times fasterthan does the parent strain [250]. Moreover, it is insensitive to endproduct inhibitionand accumulates more than 30 g/1 of the amino acid. The authors discuss the

commercial feasibility of this system for the continuous production of L-phenylalanine under mild conditions. Note than L-phenylalanine is importan t notonly as an essential amino acid in human nutrition but like asparate is also a rawmaterial for synthesis of the synthetic sweetener, aspartame. Thus, demand for theamino acid is rising. One may also note that the feedstock containing a-acetamidocinnamate, lactate and NH4OH is cheap and that reducing equivalentsare recycled by the sequential operation of lactate and phenylalaninedehydrogenases. Nishida and co-workers [257] have also developed a procedure forthe production of L-phenylalanine from acetamidocinnamic acid usingcoimmobilized cells of Corynebac te r iumsp. and Para cocc us denitrificans This two-

step reaction (outlined below) is catalysed by the acetamidocinnamateamidohydrolase activity of Corynebac te r iumsp. and the amino-transferase activityof Paracoccus deni t r i ficans[257].

Amidohydrolase Aminotransferase

Acetamidocinnamate Phenylpyruvate f ~ L-Phenylalanine

+ L-amino acid

Ant ib io t icsThe use of cell-free immobilized enzymes has had considerable impact on theindustrial production of antibiotics especially in those reactions involving only a


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single step (see, e.g., Ref. 258). However, because of the diff iculties encounteredwith complex tota l enzymic synthesis using such preparations [258] it was perhapsinevitable that the possible utility of immobilized whole cells in these processeswould receive attention. Indeed, several companies including Bayer and Beecham,since 1969, and Taiyan Pharmaceutical and Shanghai Pharmaceutical, since 1978, havehad pilot plant if not industrial-scale facilities for 6-aminopenicillanic acidproduction [218]. The potential of immobilized microorganisms in antibioticproduction has been reviewed by several investigators [258-262]. Specific reportshave also demonstrated that the total synthesis or modification of candidicin [263],cephalosporins [264- 266], chlorotetracycline [267- 269], cyclosporin [270],nikkomycin [271, 272] , oxytetracycline [273], patulin [274-276], penicillin[277-293], rifamycin [294-297], thienamycin [298-301] and tylosin [2721 byimmobilized whole cells is possible.

B i o m a s s c o n v e r s i o nBiomass may be defined as everything excluding fossil fuels that has been derived

as a result of photosynthesis. Whatever the definition used, biomass, whetherprimary (trees account for 90%) or secondary (viz. wastes or residues of forest,agricultural, domestic or industrial origin), is potentially a vast and renewablereservoir of fuel and chemical feedstocks. Realization of this potent ial requires tha tthe major components o f biomass, the polysaccharides cellulose and hemicellulose,be degraded to their monomeric forms and sequentially or simultaneously fermentedto desired end products such as ethanol. The major bottleneck in achieving these goalsis the fact that cellulose, an insoluble crystalline material, is not very amenable tohydrolysis. Thus, large amounts of cellulase are required for its conversion. To thisend various groups of investigators are actively searching for improved cellulaseproducing organisms or are attempting by mutation or recombinant DNAtechniques to improve on the cellulase-producing abilities of known organisms or toconfer on non-cellulolytic species the ability to utilise this substrate. In the contextof the present survey we note that procedures for immobilization of Tr i c h o d e r n areesei one of the most powerful cellulase-producing fungi, have been investigated[302, 303] as has the ability of this and other organisms to produce cellulase whenimmobilized [304-306]. Other reports deal with the ability of immobilizedcellulolytic organisms to saccharify cellulosic substrates [306-310] including sugarcane bagasse [306], sawdust [309] and cereal cha ff [310]. The hydrolysis ofcellobiose, a B-1,4-1inked dimer of glucose and the major endproduct of cellulaseaction [311-313] and inulin, a polymer of fructose found, for example, in high yieldsin Jerusalem artichoke [314, 315] by immobilized microorganisms exhibiting /3-glucosidase and inulinase, respectively, has also been investigated. Anaerobicdigestion of 'wastes' such as pig slurry by immobilized consortia capable ofproducing methane [316-318] may also be cited as examples o f the use ofimmobilized microorganisms in exploitation of the potential of biomass.

E t h a n o l a n d o t h e r s o lv e n t s o r f u e l sEven a cursory perusal o f the pertinent literature would show that the production

of ethanol is the most actively researched area of application of immobilized


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microorganisms. Advances in this area were reviewed in 1984 by Margaritis andMerchant [319] and more recently by Godia and colleagues [320]. Many papers andpatents relating to ethanol production deal with the organisms used, methods ofimmobilization, as well as with process and reactor considerations [155, 164, 165,169, 170-173, 175, 181, 321-396]. As fur ther examples of the potential of biomassas a source of fuels one notes that the possibility of using immobilizedmicroorganisms to produce ethanol from hydrolysates of wood [397], bagasse [398]and other cellulosic raw materials [399] has been demonstra ted as has that fromcellobiose [400-402], sucrose [403-405], apple juice [406], beet juice [407], canejuice [147], molasses [408], sweet sorghum juice [409], banana fruit pulp sugar [410],fructose [411], Jerusalem artichoke [412-415], starches [148, 159, 416-418], sugars,such as xylose or xylulose, derived directly or indirectly from hemicellulose [191,402,419-422] and from whey permeate or lactose [423-434].

The production of other solvents such as acetone and or butanol [151,435-444],2,3-butanediol [445-448] and glycerol [449-452] and o f gaseous fuels such asmethane [453-461] and hydrogen [193, 462-470] has also been examined. Twopapers deal with immobilized microorganisms as fuel cells [471, 472]. We may alsoinclude in this section the report on the continuous transforma tion of benzaldehydeto benzyl alcohol [177].

Food, dairy and drinks industriesThe fermentative properties of microorganisms have long been used in the making

of those staples of life, bread, cheese, beer and wine. It is not surprising therefore

to find that much effort has been expended on examination of the possibility ofimproving on the efficiency of some of these applications by using immobilizedmicrobial cells. For a review of advances in this area up to 1983 the reader is referredto the paper by Weetall and Zelko [473]. Examples of specific applications ofimmobilized cells include the reduction of the limonin and nomilin contents of citrusjuices so as to prevent the development of bitterness [474, 475]. However, the greatestapplication of these preparations is in the production of high fructose syrups.Approximately 6 million tons of high fructose syrup, representing 457o of the totalindustrial sugar market, were produced in the U.S. in 1982 [218]. While it must besaid that the bulk of this was manufactu red in processes using immobilized cell-free

glucose isomerase the use of immobilized whole cell enzyme is increasing. Indeed,several companies including Gist Brocades, N.V. Actinoplanes missouriensis),Reynolds Tobacco Co. and ICI Arthrobacter sp. , Novo Industri A/S Bacilluscoagulans), Agency of Industrial Science and Technology Streptomyces albus),Miles Laboratories Inc. and Car-Mi Streptomyces olivaceus),Denki Kogatsu Kogyo,Nagase Sangyo and Nabisco Brands Inc. Streptomycessp. currently produce highfructose syrups using immobilized preparations o f the named organisms [218]. Theprocess developed by Novo lndus tri A/S in which pelleted cross-linked Bacilluscoagulans is the catalyst was, and perhaps still is, the most widely used commercially[218]. In 1978 plants using this technology had a capacity of 2 million tonnes/year.

The majority of the reports during 1983-1987 on the use of immobilizedmicroorganisms for the production of high fructose syrups are concerned withexploiting the glucose isomerase activity of the immobilized cells [476-482]. Howev-


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er, several papers also deal with the inversion of sucrose by immobilized whole cellinvertase [483-486] or with the hydrolysis of inulin from Jerusalem artichoke[487-490].

Isomaltulose, a natural component of honey, is a sugar of reduced cariogeneityused in foodstuffs and pharmaceuticals. Its production using immobilized cells hasrecently been investigated [152]. Indeed, in 1982 Tate Lyle developed a pilot-scaleprocess for isomaltulose production using immobilized Erwinia rhapontici [218].Sorbose, much of which is used in the synthesis of vitamin C, may be produced bythe oxidation of sorbitol using immobilized preparations of Gluconobactersuboxydans or cetobacter melanogenum [491-493]. Soy sauce, long produced bytraditional fermentation procedures, may also be prepared using immobilized celltechnology [494]. Osaki et al. using column type reactors containing variousimmobilized microorganisms reduced the time taken for soy sauce production from6 months to 2 weeks. However, it must be said that while the resultant sauceresembled the real thing it had a quantitat ively different pattern of organic acids andaromatics than that enjoyed by the purists [494].

In the dairy industry immobilized microbial cells are also finding increasing use.Examples include a glucose oxidase/E, coli coimmobilisate for the preservation ofmilk [495], lactic acid production/prefermentat ion of milk for cheese making [178,496-499], the production o f yoghurt [500], Emmental [501] and Roquefort [502]flavours and whey protein concentrates [503]. Immobilized microorganisms appearto have great promise in brewing, i.e. in speeding up the time taken to make beer orin improving the quality of the finished product [150, 504-514]. Other applicationsinclude mead production [515, 516], wine-making, e.g. the production of sparklingwines [517-519] and malolactic fermentation [520-524].

Organic acidsSeveral organic acids are important in the food, soft drink and pharmaceutical

industries and are essential intermediates in chemical syntheses [218]. Many of thesehave been produced by traditiona l fermentat ion procedures. However, for others theonly economically feasible process is by the use of immobilized microorganisms. Onereport, which again underscores the potentia l of biomass, details a procedure for theproduction o f organic acids by continuous fermentation of lignocellulosic substrates

[525]. Other papers provide details of processes for the production of acetic acid[526-531], chorismic acid [532], citric acid [533-542], gluconic acid [543], itaconicacid [544-547], lactic acid [548-552] and malic acid [553-556]. The latter isproduced on an industrial scale by the enzymic conversion of fumaric acid usingimmobilized cells of Brevibacterium flavum [553]. The current process, patented bythe Tanabe Seiyaku Co. [554], is the result of a number of modifica tions designedto improve on that first developed in the early 1970s. It is 21 times more efficientthan the original process and is capable of producing about 1 tonne of malic acidper day from a 1000 litre column with 7007o of the maximum theoretical yield [553].The historical development of this process has been related by Chibata and

colleagues [553]. The original process developed in 1974 utilised Brevibacteriumammoniagenes with high fumarase activity immobilized in polyacrylamide gel. How-ever, unconverted fumaric and succinic acids accumulated in considerable


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quantities in the reaction mixture. The former could easily be precipitated byacidification of the reaction mixture. However, separation of succinic and malicacids is difficult. Fortunately it was found that pretreatment of the cells with bileacids prior to immobilization significantly reduced the amount of succinic acidproduced. Moreover, such treatment increased the permeability of the cellmembranes to substrate and product so that the yield of malic acid was improvedconsiderably. The half-life of the fumarase activity of the immobilized cell columnunder operating conditions was 53 days. However, the fumarase was partiallydenatured during immobilization of the cells on polyacrylamide. A 5-foldimprovement in the process was subsequently made by changing from B.ammoniagenes to B flavum and by changing the support from polyacrylamide toK-carrageenan. Subsequently, it was found that the addit ion of polyethyleneimine tothe immobilization medium increased the operational stability of the fumaraseactivity in the immobilized preparation. The column could then be operated athigher temperatures, 50 -5 5 C, for long periods with the result that the current proc-ess, in which unconsumed fumarate is recycled, is 21-fold more efficient than theoriginal [553].

Protein productionMost large-scale protein production processes involve either traditional liquid

cultivation or solid-state fermentation [218]. However, a number of investigatorshave examined the possible potential of using immobilized microbial cells for thesepurposes [176, 557-562]. Other reports since 1983 include the production o fcellulase by Trichoderma reesei [302-305] or by Sporotrichum cellulophilum [306],amylases [563 -565], chlorperoxidase [566], lipase [567] and proteases [568, 569]. Animmobilized rDNA E coli system was claimed to produce 407o pure ~3-1actamasefor several months without significant loss of activity [194, 570]. One presumes thatthis means tha t t3-1actamase accounted for 407o of the total protein produced by thesystem during operation.

Steroid transformationTransform ation of steroids has in the past been carried out by conventional fer-

mentation procedures because of the difficulties involved in chemical transformati-

on [218]. The possibility of using immobilized cell-free enzymes is impractical sincethe more important steroid-transforming enzymes, dehydrogenases and hydroxyla-ses are highly labile and difficult to obtain in quantity [571]. Furthermore, manyof the desired transformations involve multi-step reactions in which cofactor rege-neration is essential. For these reasons steroid trans formation using immobilized mi-crobial cells is becoming the state-of-the-art. Indeed, the first such transformationwas carried out in 1970 [571]. In their excellent review on the use of immobilizedcells in steroid transformation published in 1985, Koshcheyenko and Sukhodolskaya[571 ] discussed the more important enzyme and multienzyme reactions involved andgave details of the procedure for immobilization and utilisation of the appropriate

microorganisms. Since 1983 several papers on this subject have been published. Theseinclude descriptions of immobilizat ion procedures, reactors or processes [572-576]and those on applications [577- 599] including ALdehydrogenation [16 7,585 - 590],


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1 o~-hydroxylation [591 -5 93 ], 16e~-hydroxylation [594, 595], 17~-hydroxylation [596,597] and sequential reactions involved in the synthesis of prednisolone f rom cortexo-lone [598].

Environmental decontaminationEnvironmental decontamination is one area in which immobilized microorganisms

already have application. Indeed, one aspect of this topic, i.e., wastewater treatment,is currently the major large-scale application of surface-bound microbia l cells [218].For example, the Norton Co. in Oak Ridge, TN, have since 1976 operated a large-scale wastewater treatment unit involving microorganisms adsorbed on anthracitein a tapered up-flow anaerobic bioreactor [218]. Advances in the aerobic and anae-robic treatment of wastewaters, effluents and condensates using immobilized wholecells have been reviewed by several investigators [179, 184, 186, 187 ,600 - 609]. Pro-cesses for removal/recovery of heavy metals have been documented [610-617] ashave those for removal of phenols [618- 624], chlorinated phenols, benzoates andhydrocarbons [625 -629]. Procedures for nitrification and denitrif ication [163, 172,630- 633] and for the elimination of cyanide [634], triazine [635], ammonium andphosphate ions [636] and hydrogen sulphide [637] have also been reported. Dehy-droabietic acid, a component of many pulp mill effluents, is toxic to fish. However,it can be converted to harmless products using mycelia of Mortierella isabellina freeor entrapped in alginate [638]. Entrapment stabilized the hydroxylase activity invol-ved in detoxication. A procedure, based on the use of immobilized Pseudomonasindigofera has been developed for the degradation of anabasine, an insecticide con-taminating soils [639]. MacRae [640] has demonstrated the potentia l of using Rho-dosphaeromonas sphaeroides or Alcaligenes eutrophus adsorbed to magnetite for theremoval of the pesticides lindane, 2,4-dichlorophenoxy acetic acid 2,4-D) and2,4,5- trichlorophenoxy acetic acid 2,4,5-T) from contaminated waters.

Nitrogen fixationThe possibility of using alginate beads as inoculant carriers for the slow release

of bacteria that af fect plant growth is under investigation [641]. Immobilized prepa-rations capable o f fixing nitrogen [642] and of photoproduc ing amm onia [643,644]have also been subjects of recent research.

MiscellaneousIncluded in this section are procedures for the production o f acrylamide [645 - 648]

alkaloid synthesis or metabolism [649- 652], conversion of alkenes to epoxides [149,653 - 656], synthesis of 3,4-dihydroxyphenylalanine [DOPA; 657 - 661], synthesis orconvers ion of lipids or lipid-soluble materials [662 - 666], NAD regeneration [ 192,667, 668] and selective reduc tion of oxo acid esters [669, 670]. Other papers providedetails of processes for the production of cyclopropane carboxylic acids [671], hy-drocarbons [672], anthraquinone [673], 2-keto-3-deoxygluconate [674], polysaccha-ride [675], pigment [676], gibberellic acid [677], glutathione [678], water-solublecompounds [679], cytidine diphosphocholine [156] and vitamins [680, 681]. Severalother transformation or conversion reactions catalysed by immobilized cells have alsobeen reported. These include hydroxyla tion of indolyl-3-acetic acid [682], conversi-


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on of chenodeoxycholic to ursodeoxycholic acid [683], conversion of dihydrouracilto uracil [684], stereospecific hydrolysis of d-I-menthyl acetate [685], cathecol syn-thesis from benzene [686], degradation of n-valeric acid [687], depolymeriza tion ofchondroit in C sulphate [688], reduction of nitro derivatives of 1,2-dihydro-3H-1,4-benzodiazepin-2-ones [689], oxidation of secondary alcohols to methyl ketones [690],reduction of terpenic ketones [691] and the preparation of peptides [692].

Prospects for the fu ture

Immobilized cell technology is already established in certain areas such as aminoacid production. The techniques of immobilization and o f reactor design and utilisa-tion have been well documented in many hundreds of review articles and researchpapers. This should encourage those who wish to produce specialized materials for

which no alternative route is available as well as those who see possibilities for im-provement on tradit ional fermentation methods. Most reviewers are agreed that re-s e a r c h i n t h i s a r e a w il l c o n t i n u e a p a c e a n d t h a t r e c o m b i n a n t D N A p r o c e d u r e s c o u p l e d

w i t h t h e i m m o b i l i z a t i o n o f m i c r o b i a l , p l a n t a n d a n i m a l c e l ls w i l l a d d a n e w d i m e n s i -

o n t o a f i e l d t h a t a l r e a d y s h o w s g r e a t p o t e n t i a l .

cknowledgements

Our thanks to Roger Fensom and Carmel O Sullivan for their expert assistance.

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i m m o b i l i z e dGluconobacter oxydans cells. Izv. Aka d. Na uk. SSSR Ser. Biol . 6 , 85 1 - 861.117 Rob inson , P.K. , Gould ing , K.H . , M ak , A. L . and Trevan , M.D . (1986) Fac tors a ffec ting the g rowth

charac te r i s t i c s o f a lg ina te -en t rappedChloreUa. E n z y m e M i c r o b . Te c h n o l . 8 , 7 2 9 - 7 33 .118 M at t i asson , B . , Larsso n , M. and Hah n-H / igerda l , B . (1984) Metabo l ic beh av io ur o f imm obi l i zed

cel ls . Effec t s o f some env i ronm enta l f acto rs . Ann . N.Y. Acad . Sc i. 434 , 47 5- 47 8 .119 Alderc reu tz , P. , Ho is t , O . and Mat t i asson , B . (1985) Ch arac te r iza t ion o fGluconobacter oxydans

immobi l i zed in ca lc ium a lg ina te . Appl . Microb io l . B io technol . 22 , 1 - 7 .120 P ie rce , G .E . , Hayes , T. D . , Bose , A . , Al len , B . R . and Gar re t t , G .E . (1983) Imm obi l i za t ion o fKluy

vera s p e c i e s : e f f e c t o f s u p p o r t m a t e r i a l u p o n f e r m e n t a t i o n . D e v. I n d . M i c r o b i o l . 2 4 , 4 9 3 - 4 9 7 .121 DiLucc io , R .C. and Ki rw an , D. J . (1984) Effec t o f d i s so lved oxygen on n i t rogen f ixa t ion byAzoto

bacter vinelandii. I I . Ion ica l ly -adso rbed cel ls . B io tech nol . B ioeng . 26 , 8 7 - 91 .122 Tak a ta , I . , Tosa , T. and Ch iba ta , I . (1984) S tab i l i ty o f fumarase ac t iv ity o fBrevibacteriumflavum

imm obi l i zed wi th K-car rageenan and Chinese ga l lo tann in . Ap pl . M icrob io l . B io technol . 19 , 85 - 90 .123 A no n (1984) F ixed ce l ls ma ke uraem ia d rug . Bio techn ol . New swatch (McG raw-H i l l ) , 4 (7), 8.124 Ku ma kura , M . and Kae tsu , I . (1984) Increased enzyme ac tiv i ty in imm obi l i zed cel l comp os i tes wi th

a h i g h l y h y d r o p h i l i c p o l y m e r m a t r i x . J . C h e m . Te c h . B i o t e c h . 3 4B , 3 9 - 4 4 .125 W hea t ley, M .A . and Ph i l l ips , C .R . (1984) Enzy mic p roper t i es o f imm obi l i zedAlcaligenesfaecalis

ce l l s wi th ce l l -assoc ia ted /3-g lucos idase ac t iv i ty. B io technol . B ioeng . 26 , 583- 589 .1 26 H a y a s h i , T. , T a j i m a , M . a n d K a w a s h i m a , K . (1 9 8 6) A l c o h o l d e h y d r o g e n a s e ac t iv i ty o f i m m o b i l i -

z e d c e l l s . S h o k u h i n S o g o K e n k y u s h o K e n k y u H o k u k u , 4 9 , 2 4 - 2 8 .


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127 Angelova, B.A., Sukhodl skaya, G. V. and Koshcheenko , K.A. (1986) Comparative study of growthand enzyme activity patterns of free and immobilized Curvularia lunata BKM F 644 mycelium. Izv-Akad. Nauk. SSSR Ser. Biol. 5, 753-761.

128 Monbouq uett e, H.G. and Ollis, D.F. (1986) A structured model for immobilized cells kinetics. Ann .N.Y. Acad. Sci. 469, 230-244.

129 Johan sen, A. an d Flink, J. M. (1986) Influence of alginate properties and gel reinforcement on fer-mentation characteristics of immobilized yeast ceils. Enzyme Microb. Technol. 8, 737-748.

130 Fujimura, T. and Kaetsu, I. (1987) Nature of yeast cells immobil ized by radia tion polymerisati on.Activity dependence on the water cont ent of pol ymer carriers. Biotechnol. Bioeng. 29, 171 - 175.

131 Sen, S. and Chakrabarty, S.L. (1987) Amylase from Lactobacillus cellobiosus D-39 isolated fromvegetable wastes: Characteristics of immobilized enzyme and whole cell. Enzyme Microb . Technol.9, 112-116.

132 Fukui, S. and Tanaka, A. (1984) Applicatio n of biocatalysts immobilized by prepolymer methods.Adv. Biochem. Eng./Biotechnol. 29, 1- 30.

133 Ozeredenko, V.G ., Struchaliha, T. I. and Berezin, M.V . (1983) Activation of immobilized cells ofBacillus subtilis 339 by organic solvents. Vestn. Mosk. Univ. ser. 2: Khim. 24, 597- 601.

134 Chee tham, P. S. J. (1984) Stabiliza tion of immobilized enzymes with glycerol. US Patent US 4, 443,538.

135 Schuller, C., van der Meer, A. B., van Lelyveld, P. H. and Jo osten, G. E. H. (1984) Long-term sta-bility of immobilized Pseudomonas oleovorans cells in the product ion of fine chemicals. Prog. Ind.Microbiol. 20, 85- 92.

136 Bajpai, P. and Margaritis, A. (1985) Improvemen t of inulinase stability of calcium alginate-immobilized Kluyveromyces marxianus by treatment with hardening agents. Enzyme Microb. Technol.7, 34- 36.

137 Brouers, M.D. and Hall, D.O. (1985) Drying pretreatme nt enhances the photosyntheti c stabilityof alginate-immobilized Phormidium laminosum Biotechnol. Lett. 7, 567 - 572.

138 Chao, K. C. , Haug en, M. M. and Royer, G. P. (1986) Stabilizat ion of K-carrageenan gel with poly-meric amines: Use of immobilized cells and biocatalysts at elevated temperatures. Biotechnol. Bi-oeng. 28, 1289- 1293.

139 Doran, P .M . and Bailey, J. E. (1986) Effects of hydroxyurea on immobilized and suspended yeastfermentation rates and cell cycle operation. Biotechnol. Bioeng. 28, 1814-1831.

140 Deo, Y.M. , Co sterton, J. W. and Gaucher, G.M. (1983) Examination of immobilized fungal cellsby phase contrast and scanning electron microscopy. Can. J. Microbiol. 29, 1642- 1649.

141 Jone s, A., Razniewska, T., Lesser, B.H. , Siqueira, R., Berk, D., et al. (1984) An assay for themeasurement of the protein content of cells immobilized in carrageenan. Can. J. Microbiol. 30,475 - 481.

142 Kuek, C. and Armitage, T.M. (1984) Scanning electron microscopic examination of calcium algi-nate beads immobilising growing mycelia of Aspergillus phoenicus Enzyme Microb. Technol. 7,129- 133.

143 Brodelius, P. and Vogel, H. J. (1984) Non-invasive 31p NMR studies of the metabol ism of suspen-ded and immobilized plant cells. Ann . N.Y. Ac ad. Sci. 434, 4 96- 500 .

144 Sano, H., Souma, Y. and Toyama, I. (1986) Meth od for estimation of biostability of support mate-rials for immobil ization of anaerobic microbes. Osaka Kogyo Gijutsu Shikensho Kiho, 37, 99 - 103.

145 Paquo t, M. and Lesage, V. (1986) Electrokinetic characterization of immobilized Aspergillus nigerfl-galactosidase. Belg. J. Foo d Chem. Biotechnol. 41, 16 0- 165.

146 Reardon , K. F. , Scheper, T. and Bailey, J.E . (1986) In situ fluorescence monitorin g of immobilizedClostridium acetobutylicum Biotechnol. Lett. 8, 817- 822.

147 Fukushima, S. and Hatakeyama, H. (1983) Novel immobilized bioreactor for rapid continuous ethanolfermentation of cane juice. Ann. N.Y. Acad. Sci. 413, 483-485.

148 McGhee, J. E. , Carr, M. E. and St. Julian, G. (1984) Continu ous bioconversion of starch to etha-nol by calcium alginate-immobilized enzymes and yeasts. Cereal Chem. 61, 4 46- 44 9.

149 Habets-Creutzen, A. Q. H., Brink, L.E. S., van Ginkel, C.G., de Bont, J.A .M . and Tramper, J.(1984) Produc tion of epoxides from gaseous alkenes by resting cell suspensions and immobilizedceils of alkene-utilising bacteria. Appl. Microbiol. Biotechnol. 20, 245-250.

150 Onaka, T. , Nakanishi , K., Inoue, T. and Kubo, S. (1985) Beer brewing with immobilized yeast.Bio/Technol. 3, 467- 470.


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151 Largier, S.T. , L ong, S., Santangelo, J .D. , Jo nes, D .T. an d W oods, D.R . (1985) Imm obil izedClostridium acetobutylicum P262 mu tan t s fo r so lven t p rod uc t ion . A ppl . En vi ron . Microb io l . 50(2) ,477 - 481.

152 Ch eeth am , P. S. , i. , Ga rret t , C. an d Clark, J . (1985) lsom altulose produ ct ion using imm obil ized cells.

B i o t e c h n o l . B i o e n g . 2 7 , 4 7 1 - 4 8 1 .1 53 H u l s t , A . C . , Tr a m p e r, J . , v a n T R i e t, K . a n d We s t e r b e e k , J . M . M . (1 98 5) A n e w t e c h n i q u e f o rthe p roduc t ion o f immobi l i zed b ioca ta lys t in l a rge quan t i t i e s . B io technol . B ioeng . 27 , 870- 876 .

154 Ta nak a , A. , S ono mo to , K. and Fuku i , S . (1984) Var ious app l ica t ions o f l iv ing ce ll s imm obi l i zedb y p re p o l y m e r m e t h o d s . A n n . N . Y. A c a d . S ci. 4 3 4, 4 7 9 - 4 8 2 .

155 Lan te ro , O .J . J r. (1985) Immo bi l i za t io n o f b ioca ta lys t s . Eu rope an Pa ten t 0 133 531.156 Qiu , W. , W ang , W. , Z hang , X. and J i , Y. (1983) S tudy on the b iosyn thes i s o f cy t id ine d iph osp ho-

cho l ine in the co l lagen m embrane- immob i l i zedSaccharomyces cerevisiae cells. Yiyao G ong ye, 4, 1 - 4.157 Ni l s son , K. , B i rnba um , S . , F lygare , S . , L inse , L . , Schroeder, U . , Jepp sson , U. , Larsson , P. -O,

M osb ach , K. an d B rode l ius , P. (1983) Gen era l metho d fo r the im mo bi l i za t ion o f ce ll s wi th p rese r-ved v iab i l i ty. Eur. , i . Appl . Microb . Bio technol . 17 , 319-326 .

158 Link o , P. , Sorvar i , M. and Linko , Y.Y. (1983) E th ano l p rod uc t ion wi th imm obi l i zed cel l r eac to rs .

A n n . N . Y. A c a d . S c i . 4 1 3 , 4 2 4 - 4 3 4 .159 Am in , G. , De M ot , R . , Van Di jck , K . and Verach te r t , H . (1985) Di rec t a lcohol ic fe rm enta t io n o fs ta rchy b iom ass us ing amylo ly t i c yeas t s tra ins in ba tch and imm obi l i zed cel l sys tems . Appl . Micro-b i o l . B i o t e c h n o l . 2 2 , 2 3 7 - 2 4 5 .

160 Ve nka tasu bram ania n , K. (ed . ) . (1979) Imm obi l i zed Microb ia l Ce l l s, AC S Symp. Ser. 106 , Am er i -c a n C h e m i c a l S o c i e t y, Wa s h i n g t o n , D . C .

1 61 A t k i n s o n , B . a n d M a v i t u n a , F. (1 98 3) B i o c h e m i ca l E n g i n e e r i n g a n d B i o t e c h n o l o g y H a n d b o o k , N a -tu re Press , New York .

162 Josh i , S . and Ya mazak i , H . (1985) Use o f bac te ria l fi lms as res iden t inocu la fo r repea ted ba tch fe r-m e n t a t i o n . B i o t e c h n o l . L e t t . 7 , 7 5 3 - 7 5 8 .

163 Du nn , I . J . , Tan aka , H. , U zm an, S . and D enac , M. (1983) Biof ilm f lu id ized bed reac to rs and the i rapp l ica t io n to was tewate r n i t r if i ca t ion . An n . N .Y. Aca d . Sc i. 413 , 16 8- 183.

164 Krug , T.A. a nd Daugu l i s , A . , I . (1983) E th ano l p rod uc t io n us ingZymomonas mobilis i m m o b i l i z e don an ion-exchange res in . B io technol . Le t t . 5 , 159- 164 .1 65 D a u g u l i s , A . , K r u g , T. A . a n d C h o m a , C . E . T. (1 98 5) F i l a m e n t f o r m a t i o n a n d e t h a n o l p r o d u c t i o n

b y Zymomonas mobilis. B i o t e c h n o l . B i o e n g . 2 7 , 6 2 6 - 6 3 1 .166 Reuveny, S . , Mizrah i , A . , Ko t le r, M. and Freem an, A. (1983) A new microcar r ie r fo r cu l tiva t ing

m a m m a l i a n c e l l s . A n n . N . Y. A c a d . S c i . 4 1 3 , 4 1 3 - 4 1 5 .167 Mozes , N . and Rou xhe t , P. G (1984) Deh ydro gena t ion o f cor t i so l byArthrobactersimplex i m m o b i -

l i z e d a s s u p p o r t e d m o n o l a y e r. E n z y m e M i c r o b . Te c h n o l . 6 , 4 9 7 - 5 0 2 .168 Mozes , N . and Rouxh e t , P.G . (1985) Metab o l ic ac t iv i ty o f yeas t imm obi l i zed as supp or ted mo no-

l a y e r. E n z y m e M i c r o b . Te c h n o l . 6 , 4 9 7 - 5 0 2 .1 69 B u l o c k , . I .D . , C o m b e r b a c h , D . M . a n d G h o m m i d h , C . (1 98 4) A st u d y o f c o n t i n u o u s e t h a n o l p r o -

duc t io n us ing a h igh ly f loccu lan t yeas t in the gas li f t tower fe rm ente r. Ch em. Eng . J . 29 , B9 - B24.170 Net to , C . B . , De s t ruhau t , A . and Gom a, G. (1985) E th ano l fe rm enta t io n by f loccu la t ing yeas t . Per-

fo rmance and s tab i l i ty dependence on c r i t i ca l f e rmenta t ion ra te . B io technol . Le t t . 7 , 355- 360 .171 Kur iy ama, H. , Se iko , Y. , Mu r ikam i , T. , Kobayash i , H . and Sono da , Y. (1985) Con t inuo us e thano l

fe rmenta t ion wi th ce l l r ecyc l ing us ing f loccu la t ing yeas t . , i . Fe rment . Technol . 63 , 159- 165 .172 Ciz inska , S . , Voj t i sek , V. , Maixn er, J . , Bar ta , J . and K rum phan z l , V. (1985) Cel l aggrega tes exh i -

b i t ing den i t r ify ing ac t iv i ty. B io technol . Le t t . 7 , 73 7- 74 2 .173 Devere l l , K .F . and Cla rk , T.A . (1985) Indu ced f loccu la t ion o fPachysolen tannophilus us ing the

t o w e r f e r m e n t o r. B i o t e c h n o l . B i o e n g . 2 7 , 1 6 0 8 - 1 6 11 .174 Blanch , H.W . , Vickroy, T.B . and Wi lke , C .R . (1984) Gro wth of p rocaryo t ic ce ll s in ho l low f ib re

r e a c t o r s . A n n . N . Y. A c a d . S c i . 4 3 4 , 3 7 3 - 3 8 1 .175 In loes , D . S . , Michae ls , A .S . , Rober t son , C . R . and M at in , A . (1985) E tha nol p roduc t ion by n i t rogen-

def ic ien t yeas t ce l ls imm obi l i zed in a ho l low-f ib re mem bran e b io reac to r. A ppl . M icrob io l . B io tech-n o l . 2 3 ( 2 ) , 8 5 - 9 1 .

176 ln loes , D .S . , S mi th , W. J . , Tay lor, D .P. , Coh en , S . N . , Michae l s , A . S . and Rob er t son , C .R . (1983)H o l l o w - f i b r e m e m b r a n e b i o r e a c t o r s u s in g i m m o b i l i z e dEscherichia coli fo r p ro te in syn thes i s . B io-t e c h n o l . B i o e n g . 2 5 , 2 6 5 3 - 2 6 8 1 .


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177 Wisniewski, J., Winnicki, T. and Majewska, K. (1983) Continu ous transform ation of benzaldehy-de to benzyl alcohol by Rhodotoru la mucilaginosa immobilized in an ultrafiltration cell. Biotech-nol. Bioeng. 25, 1441- 1452.

178 Kim, K., Naveh, D. and Olson, N. F. (1985) Continuous acidification of milk before ultrafiltration

by an immobilized cell bioreactor. I. Development of the bioreactor. Milchwissenschaft, 40, 605 - 607.179 Kinner, N.E . and Eighmy, T.T. (1985) Biological fixed-f ilm systems. J. Water Pollut. Cont. Fed.57, 526-531.

180 Suga, K. and Boongorsran g, A. (1984) A new model of mass transfer in a rotating disc contactor.Chem. Eng. Sci. 39, 767-773.

181 Del Borghi, M., Con verti, A., Parisi, F. and Ferraiola, G. (1985) Continuo us alcohol fermentati onin an immobilized cell rotating disc reactor. Biotechnol. Bioeng. 27, 761 -7 68 .

182 Radovich, J. M. (1985) Mass transfer effects in fermen tati ons using immobi lized whole cells. Enzy-me Microb. Technol. 7, 2-10.

183 Andrews, G. and Trapasso, R. (1985) The optimal design of fluidized bed bioreactors. J. WaterPollut. Control Fed. 57, 143- 150.

184 Rodrigues, A., Grasmick, A. and Elmaleh, S. (1983) Modeling of biofilm reactors. Biochem. Eng.J. 27, B39-B48.

185 Rittmann , B.E. and Brunner, C .W. (1984) The nonsteady state biofilm process for advanced orga-nics removal. J. Water Pollut. Cont. Fed. 56, 874-880.

186 Benefield , L. and Molz, F. (1985) Mathematica l simula tion of a biofilm process. Biotechnol . Bi-oeng. 27, 921- 931.

187 Tanaka, H. , Mats umura , M. and Veliky, I. A. (1984) Diffusional characteri stics of substrates incalcium alginate gel beads. Biotechnol. Bioeng. 26, 53-58.

188 Klein, J., Stock, J. and Vorlo p, K. D. (1983) Pore size and properties of spherical calcium alginatebiocatalysts. Eur. J. Appl. Microbiol. Biotechnol. 18, 86-91.

189 Furui, M. and Yamashita , K. (1985) Diffusional coefficien ts of solutes in immobil ized cell cata-lysts. J. Ferment. Technol. 63, 167-173.

190 Kabel, J .J ., Robinson, C.W. and Moo-Young, M. (1983) Modelling of xylose fermentation to ethanolby sequential isomerization and fermentation. Biotechnol. Bioeng. Symp. 13, 315-329.

191 Ergan , F., Thoma s, D. and Chang, T. M. S. (1984) Selection and microencapsula tion of an NAD H-oxidising bacterium and its use for NAD regeneration. Appl. Biochem. Biotechnol. 10, 60-72.

192 Day, J.G. and Codd, G.A . (1985) Photosyn thesis and glycolate excretion by immobilized Chlorella emersonii. Biotechnol. Lett. 7(8), 573-576.

193 Georgiou, G., Chalmers, J. J., Shuler, M.L. and Wilson, D. B. (1985) Contin uous immobilized re-combi nant protei n product ion f rom Escherichia coli capable of selective prot ein excretion. A feasi-bility study. Biotechnol. Prog. 1, 75- 79.

194 Black, G. M., Webb, C., Matthews, T. M. and Atki nson , B. (1984) Practical reactor system for yeastcell immobilization using biomass support particles. Biotechnol. Bioeng. 26, 13 1- 141.

195 Chotani , G. K. and Constantin ides , A. (1984) Immobil ized cell cross -flow reactor. Biotechnol. Bi-oeng. 26, 217-220.

196 Anon. (1984) Improvements in bioreactors boost amino acid production. Biotechnol. News, 4(6), 4 - 5.197 Furui, M. and Yamashita, K. (1985) Ho

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