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or-Am ylase prod uct ion in aq ueo us two-p h a s e s y s t e m s w i th B a c i l lu s s u b t i l i sE l is A n d e r s s o n , A n n - C h r i s t i n J o h a n s s o n a n d B / i r b e l H a h n - H ~ i g e r d a lApplied Microbiology, Chemical Center, Lund University, PO Box 124, S-211 O0 Lund, Swed en

( R e c e i v e d 1 0 D e c e m b e r 1 9 8 4 )The production of a-amylase (1,4-a-D-glucan glucanohydrolase, EC 3.2.1.1) by Baci l lus subt i l i s hasbeen studied in repeated batch fermentations in aqueous two-phase systems. In a phase systemcomposed of PEG 600, 8% (w/w), PEG 3350, 5% (w/w)/Dextran T 500, 2% (w/w), 82% of theenz yme partitioned to the top phase. The enzym e concentration in the top phase reached 0. 85 -1.35 U m1-1 during the fermenta tions compared with 0.58 U m1-1 in the reference fermenta tion.In the phase system compo sed o f PEG 3350, 9% (w/wJ/Dextran T 500, 2% (w/w), 73% of theenzyme partitioned to the top phase. However, the enzyme concentration in this phase systemreached only 0.35 U m1-1 in the top phase. The bacterial cells were microscopically observed topartition totally to the bo ttom phase in the aqueous two-phase system used. The results are discussedin relation to recirculation of cells by immobilizing to a solid matrix. Extraction o f the produc t tothe top phase and the effect of the phase polymers, especially PEG, on the production are alsodiscussed.Keywords: Fermen tation; c~-amylase;Bacillus subtilis; aqueous two-phase system

I n t r o d u c t i o nCe ll r e c i rc u l a t i o n a n d c o n t i n u o u s o p e r a t i o n ma y r e s u l t i ni mp r o v e d p r o d u c t i v i t i e s i n b i o t e c h n i c a l p r o c e s s e s i n v o l v i n gc e l ls . Th i s c a n b e a c h i e v e d b y , f o r e x a m p l e , i mm o b i l i z i n gc e l ls t o s o l i d s u p p o r t s 1 o r b y u s i n g m e m b r a n e t e c h n i q u e s . 2W i t h t h e i n c r e a s i n g k n o w l e d g e a b o u t i m m o b i l i z a t i o nt e c h n i q u e s, t h e p r o d u c t i o n o f e n z y m e s a n d o t h e r e x tr a -c e l l u l a r p r o d u c t s p r o d u c e d b y Bacillus s p e c i e s w i t h t h e s et e c h n i q u e s h a v e been reported: a - a my l a s e ( 1 A- a - D- g t u c a ng l u c a n o h y d r o l a s e , E C 3 . 2 . 1 . 1 ) a - a b a c i t r a c in s a n d p r o -i n su l in . 6 H o w e v e r , i m m o b i l i z a t i o n t o s o li d s u p p o r t s i m p o s e sr e s t r i c t i o n s i n t e r ms o f s t e r i c h i n d r a n c e s a n d d i f f u s i o nl i mi t a t i o n s , w h i c h l i mi t s i t s a p p l i c a b i l i t y t o l o w mo l e c u l a rwe i g h t s u b s t r a t e s .

A q u e o u s t w o - p h a s e s y s t e m s o f f e r a n o t h e r p o s s i b i l i t yt o r e c i r c u l a t e l i v i n g c e l l s . Th e c e l l s a r e p a r t i t i o n e d t o t h eb o t t o m p h a s e .7 Th e c e l l - f r e e t o p p h a s e c a n t h e n b e u s e dt o a c c u m u l a t e t h e d e s i re d p r o d u c t .A q u e o u s t w o - p h a s e s y s t e m s a r e f o r m e d w h e n w a t e rs o l u t io n s o f t w o p o l y m e r s o r o n e p o l y m e r a n d o n e s a lta r e mi x e d . Th e y h a v e s u c c e s s f u l l y b e e n u s e d f o r t h e l a r g e -s c a le i s o l a t i o n a n d p u r i f i c a t i o n o f i n t r a c e l l u l a r e n z y me s . 8-11Th e y h a v e a l s o b e e n a p p l i e d t o b i o l o g i c a l a n a l y s is 12 an dc o f a c t o r r e g e n e r a t i o n . 1 3 -1 4 E n z y m a t i c b i o c o n v e r s i o n s i na q u e o u s t w o - p h a s e s y s t e m s h a ve b e e n d e m o n s t r a t e d f o r15t h e d e a c y l a t i o n o f b e n z y l p e n i c i l l i n a n d t h e s a c c h a r if i c a ti o n16o f c e l lu l o s e . Se v e r a l mi c r o b i a l s y s t e m s h a v e a l s o b e e ni n v e s t i g a t e d : t o x i n p r o d u c t i o n w i t h Clostridium tetani, 17c o n v e r s i o n o f c e l l u l o se 18 a n d s t a r c h 7 t o e t h a n o l , p r o d u c t i o no f a c e t o n e a n d b u t a n o l u s i n g Clostridium acetobutylicum 19a n d c e l l u l a s e p r o d u c t i o n w i t h Trichoderma reesei. 2

T h e p r e s e n t i n v e s t i g a t i o n c o n c e r n s t h e p r o d u c t i o n o fa m a c r o m o l e c u l a r p r o d u c t , t h e e n z y m e a -a m y l a s e , w i t hBacillus subtilis.

M a t e r i a l s a n d m e t h o d sMicroorganisms, media and culture condit ions

Bacillus subtilis w a s k i n d l y d o n a t e d b y D r M a t t h ia sU h l 6 n, R o y a l I n s t i tu t e o f T e c h n o l o g y , S t o c k h o l m , S w e d e n .T h e s t r a i n i s a m u t a n t o b t a i n e d f r o m B. subtilis, Ma r b u r g ,A T C C 6 0 5 1 .T h e o r g a n is m w a s g ro w n o n a m e d i u m c o n t a in i n g p e rl i t re : 1 0 g D- g l u c o s e, 5 g p e p t o n e , 2 g y e a s t e x t r a c t ( b o t hDi f c o , D e t r o i t , M i c h ig a n , USA ) , 1 .5 g Na C1 , 0 . 5 g KHz PO4 ,0 . 5 g Mg SO4 " 7 H 2 0 a n d 0 .1 g Ca C1 2 . To ma i n t a i n t h es t r a i n i t wa s g r o wn f o r 2 4 h a t 3 7 C i n a r e c i p r o c a l s h a k e r( 1 7 5 s t r o k e s mi n - 1 ) i n a 1 l i tr e b a f f l e d E r l e n m e y e r f l a s k .T h e m e d i u m v o l u m e w a s 1 0 0 m l a n d t h e p H w a s a d j u st e dt o 7 . 0 b e f o r e s t e r i l i z a ti o n . S t e r i l e g l y c e r o l wa s t h e n a d d e dt o c o n s t i t u t e 1 5 % o f t h e v o l u m e . Th i s s o l u t i o n wa s f r o z e na n d s to r e d a t - 7 0 C .

I n t h e a - a m y l a s e p r o d u c t i o n me d i u m t h e D- g l uc o se i nt h e m e d i u m a b o v e w a s e x c h a n g e d f o r s o l u bl e s t a r ch ,5 0 g 1 -1 . Fo r t h e c u l t i v a t i o n s i n p h a s e s y s t e ms t h e n u t r i e n t swe r e c a l c u l a t e d a s g p e r k g p h a s e s y s t e m. Th i s g i v e s an e g l ig i b l e d e v i a t io n f r o m t h e c a l c u l a t i o n o n a v o l u me b a s i s .P r o d u c t i o n m e d i u m ( 1 0 0 m l ) w a s in o c u l a t e d w i t h t h es t o c k c u l t u r e ( 5 ml ) a n d c u l t i v a t e d a s d e s c r i b e d a b o v e f o r1 6 h . Th i s s o l u t i o n wa s u s e d a s a se e d c u l t u r e f o r t h ef e r m e n t e r c u l t i v a t i o n s . T h e f e r m e n t e r ( m o d e l F P L - 3 ,C h e m o f e r m A B , H / i g e rs t en , S w e d e n ) h a d a w o r k i n g v o l u m e

0141- -0229 /85 /070333- -06 $03 .00 1 9 85 Bu t te rw o r th & C o . ( Pub li sh e rs ) L td En z y m e a n d M i c r o b . T e c h n o l . , 1 9 8 5 , v o l . 7 , J u l y 3 3 3

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Paperso f 2 . 5 l i t re i n wh i c h 2 . 5 k g p h a s e s y s t e m wa s u s e d . Th es t i rr i n g wa s s e t a t 3 0 0 r e v mi n - ~ a n d t h e a e r a t i o n 1 v v m i na l l c u l t i v a t i o n s , p H wa s ma i n t a i n e d a t 7 . 0 d u r i n g t h ec u l t i v a t i o n s w i t h N a OH a n d H CI , b o t h 3 M. Th e s t r e n g t ho f a l k a l i a n d a c i d wa s c h o s e n t o e l i mi n a t e d i l u t i o n o f t h ep h a s e s y s t e m , wh i c h mi g h t c h a n g e i t s v o l u m e r a t i o . A 3 0 %s i l i c o n e a n t i f o a mi n g a g e n t ( BDH Ch e mi c a l s L t d , Po o l e ,E n g l a n d ) w a s u s ed d r o p w i s e w h e n n e e d e d t o b r e a k t h e f o a m .A q u e o u s t w o -p h a s e s y s t e m

De x t r a n T 5 0 0 ( P h a r ma c i a F i n e Ch e mi c a l s, Up p s a l a ,S w e d e n ) a n d p o l y ( e t h y l e n e g l y c o l ) ( P E G ) o f t w o d i f f e r e n tm o l e c u l a r w e i g h t s w e r e u s e d t o o b t a i n a q u e o u s t w o - p h a s es y st em s . C a r b o w a x P E G 3 3 5 0 (M W 3 0 0 0 - 3 7 0 0 ) w a so b t a i n e d f r o m U n i o n C a r b i d e ( D a n b u r y , C T , U S A ) a n dP E G 6 0 0 ( M W 5 7 0 - 6 3 0 ) w a s pu r c h as e d f r o m M e rc k( H o h e n b r u n n , F R G ) .

T h e p r e p a r a t i o n o f t h e a q u e o u s t w o - p h a s e s y s t e mc o n s is t in g o f P E G 3 3 5 0 / D e x t r a n T 5 0 0 h a s b e e n d e s c r ib e db y A l b e r t s s o n .2 1 Fo r t h e p h a s e s y s t e m PEG 6 0 0 , PEG3 3 5 0 / D e x t r a n T 5 0 0 t h e to p - p h a s e c o m p o s i t i o n w a sd e t e r m i n e d b y m e a s u r in g t h e d e x t r a n c o n t e n t i n a p o l a ri -m e t e r ( P e r k in - E l m e r ) a t 5 8 9 n m . D r y w e i gh t d e t e r m i n a t i o ng a v e t h e t o t a l p o l y m e r c o n t e n t . T h e r e la t i o n b e t w e e n t h ec o n t e n t o f t h e t w o t y p e s o f P E G w a s a s s u m e d t o b e t h es a me i n t h e t o p p h a s e a s i n t h e t o t a l p h a s e s y s t e m.

De x t r a n , wh i c h i s a c a r b o h y d r a t e , wa s s t e r il i z e d s e p a r a t e l yw i t h t h e s t a rc h t o a v o i d f o r m a t i o n o f u n w a n t e d M a i ll ar dp r o d u c t s w i t h t h e n i t r o g e n s o u r c e s . PEG wa s s t e r il i z e dwi t h s a l ts a n d n i t r o g e n s o u r c e s . Th e p h a s e s y s t e m wa smi x e d d i r e c t l y i n t h e f e r me n t e r a f t e r t h e s t e r i l i z a t i o n ,b e c a u s e m i x i n g o f p h a s e c o m p o n e n t s p r i o r t o s t e r i l i z a t i o nr e s u l t s i n v i s c o u s s o l u t i o n s , wh i c h i mp o s e h e a t t r a n s f e rp r o b l e ms a n d , f u r t h e r mo r e , a r e d i f f i c u l t t o h a n d l e .R e p e a t e d b a t c h f e r m e n t a t i o n s u s i n g a q u e o u s t w o - p h a s es y s t e m s w e r e c a r r i e d o u t a s f o l l o w s : w h e n t h e e n z y m ep r o d u c t i o n c e a s e d , t h e a e r a t i o n a n d t h e s t i r r in g we r et u r n e d o f f a n d t h e p h a s e s we r e a l l o we d t o s e p a r a t e f o r3 0 m i n . T h e t o p p h a s e w a s t h e n p u m p e d o u t t h r o u g h at u b e i n t h e t o p o f t h e f e r m e n t e r . F r e s h t o p p h a s e a n d as m a ll v o l u m e o f t o t a l p h a s e s y s t e m ( c a l c u l a te d t o c o m p e n -s a t e f o r s a mp l e s t a k e n o u t d u r i n g t h e c u l t i v a t i o n ) we r ea d d e d a s e p t i c a l l y t o g e t h e r w i t h n u t r i e n t s . F e r m e n t a t i o nwa s s t a r t e d a g a i n b y a e r a t i n g a n d s t i rr i n g t h e f e r m e n t e r .

Nu t r i e n t s w e r e a d d e d a t t h e c o n c e n t r a t i o n s g iv e n a b o v e .H o w e v e r , w h e r e i n d i c a te d i n t h e t e x t t h e v o l u m e o n w h i c ht h e c a l c u l a t i o n s we r e b a s e d c o u l d b e e i t h e r t h e v o l u m eo f t h e a d d e d f r e sh p h a s e s y s t e m o r th e t o t a l v o l u m e int h e f e r m e n t e r .Sampl ing and ana ly s i s

S a m p l e s t a k e n f r o m t h e f e r m e n t e r w e r e c e n t r i f u g e df o r 1 mi n a t 5 5 0 0 r e v mi n - 1 i n a b e n c h - s c a l e c e n t r i f u g e( W i fu g D o c t o r , S t o c k h o l m , S w e d e n ) t o s e p a r a t e t h e p h a s es ,wh i c h we r e a n a l y s e d s e p a r a t e l y .Ce ll ma s s wa s m e a s u r e d a s a b s o r b a n c e a t 6 2 0 n m i n t h ef e r m e n t a t i o n b r o t h a n d i n t h e b o t t o m p h a s e o f th e p h a s es y s t e m, r e s p e c t i v e l y . V i a b l e c o u n t s we r e a l s o d e t e r mi n e df o r t h e b o t t o m p h a s e . T r y p t o n e G l u c o s e E x t r a c t A g a r( Ox o i d , Ba s i n g s t o k e , En g l a n d ) wa s u s e d . Th e i n c u b a t i o nw a s p e r f o r m e d f o r 2 4 h a t 3 7 C . D r y m a s s d e t e r m i n a t i o n sw e r e n o t p e r f o r m e d , b e c a u se o f i n t e rf e r e n c e f r o m t h ea d d e d p o l y me r s . Th e c e l l ma s s c o n s t i t u t e s o n l y a f e wp e r c e n t o f t h e p o l y m e r m a s s.

a - A m y l a s e w a s a n a l y s e d w i t h P h a d e b a s ( P h a r m a c i aDi a g n o s t ic s , Up p s a l a , Sw e d e n ) .

T h e s t a r c h w a s a n a l y s e d w i t h a m e t h o d i n w h i c h t h es t a r c h wa s f i r s t d e g r a d e d w i t h a my l o g l u c o s i d a s e ( S i g maCh e mi c a l Co . , S t Lo u i s , MO, U SA) f o r 1 5 r a i n a t 5 5 C .Th e D- g l u c o s e f o r me d wa s a n a l y s e d b y t h e me t h o d o fTr i n d e r 2 2 u s i n g 4 - a m i n o a n t i p y r i n e ( S i g ma Ch e mi c a l Co . ,S t Lo u i s , MO, USA) a s a n e l e c t r o n a c c e p t e r . So l u b l es t a r c h wa s u s e d a s a s t a n d a r d .

Th e p a r t i t i o n c o e f f i c i e n t f o r 0 ~ -a my la se i n t h e a q u e o u st wo - p h a s e s y s t e m s wa s d e f i n e d a s K = a c t i v i t y ( t o p p h a s e ) /a c t i v i t y ( b o t t o m p h a s e ) . C o n c e n t r a t i o n w a s u s e d f o rd e t e r m i n a t i o n o f t h e p a r t i t i o n c o e f f i c i e n t f o r s o l u b l es t a r c h .

T h e t o t a l a m o u n t o f a - a m y l a s e i n a n a q u e o u s t w o -p h a s e s y s t e m wa s c a l c u l a t e d f r o m t h e p a r t i t i o n c o e f f i c i e n ta n d t h e v o l u m e r a t i o . T h e t o t a l c o n c e n t r a t i o n o f s - a m y l a s eg i v e n i n t h e f i g u r e s i s t h e r a t i o b e t we e n t h e t o t a l a mo u n ta n d t h e t o t a l v o l u m e .

R e s u l t sPar t i t i on o f B a c i l l u s s u b t i l i s c e ll s in t he aque ou st w o - p h a s e s y s t e m

Bacillus subtilis c e l l s p a r t i t i o n t o t h e b o t t o m p h a s e i na n a q u e o u s t w o - p h a s e s y s t e m o f P E G 3 3 5 0 , 9 % ( w / w ) /D e x t r a n T 5 0 0 , 2 % ( w / w ) (Figure 1). The s a m e p a r t i t io nwa s o b s e r v e d i n t h e p h a s e s y s t e m P EG 6 0 0 , 8 % ( w/ w ) ,P E G 3 3 5 0 , 5 % ( w / w ) / D e x t r a n T 5 0 0 , 2 % ( w / w ) a l s o u s e di n t h is s t u d y . T h e e m u l s i o n o f t h e b o t t o m p h a s e i n th e t o pp h a s e c o a l e s c e s d u r i n g t h e m i c r o s c o p i c a l o b s e r v a t i o n , wh i c hma k e s i t h a r d t o d e t e r m i n e wh e t h e r t h e c e l ls c o l le c t a tt h e i n t e r f a c e o r i n t h e b o t t o m p h a s e . H o w e v e r , Figure 1c l e a r l y sh o ws t h a t t h e c e ll s a re o n t h e b o t t o m - p h a s e si d eo f t h e i n t e r f a c e a n d t h a t t h e t o p p h a s e i s f r e e f r o m c e ll s.

F i g u r e 1 B . $ubt i l is c e ll s i n a n a q u e o u s t w o - p h a s e s y s t e m c o m p o s e do f P E G 3 3 5 0 , 9 % ( w / w ) a n d D e x t r a n T 5 0 0 , 2 % ( w / w ) . C e l ls a c c u -m u l a t e i n t h e b o t t o m p h as e d r o p le t s

334 Enzyme a n d M i c r o b . Technol . , 1985, vo l . 7 , Ju ly

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a-Am ylase p ro du c t io n in aqueous two-phase sys tems: E. Andersson e t aLP a r t i t i o n o r e - a m y l a s e a n d s t a rc h

The p a r t i t i on o f a -am y lase w as s tud ied i n t he aq ueoust w o - p h a s e s y s t e m P E G 3 3 5 0 , 9 % ( w / w ) / D e x t r a n T 5 0 0 ,2% (w /w ) w i t h i nc reas ing p o tass ium p hosp ha te concen tra -t i ons (F ig u re 2 ) . An increase of the potass ium phosphateconcen t ra t i on f r o m 0 t o 0 .3 M inc reased K f r om 0 .33 t o 3 .64 .Bac i l lus sub t i l is does g row in m ed ia con ta in ing up t o0 .2 M p o tassium p hosp ha te . I n an a t t em p t t o e xp lo re t hepos sib i l i ty o f us ing a phase system wi th 0 .2 M potass iump hosp ha te fo r t he p rodu c t i on o f a -am y lase , t h i s system w asst i r red (3 00 rev ra in -1) and aerated (1 vvm) in the fe rmente r .Th i s r esu l ted i n g e l l ing o f t he dex t r an 9 and f l occu la t i onoccu r red i n t he fe rm en te r . I ns tead , t he p hosp ha te concen-t r a t i on i n t he reg u la r m ed ium , co r resp ond ing t o 0 .04 M ,w as us ed i n t he f o l low i ng expe r i m en t s .The p a r t i t i on coe f f i c i en t o f a - am yl a se cou l d be i nc r ea sedus i ng a m i x t u r e o f PEG 60 0 and PEG 3350 a s t op - phas ec o m p o n e n t s (Figure 3) . A phas e s ys t em com pos ed o fP E G 6 0 0 , 8 % ( w / w ) , P E G 3 3 5 0 , 5 % ( w / w ) a n d D e x t r a nT 5 00 , 2% ( w / w ) gave a pa r t i t i on coe f f i c i en t f o r a -am yl a s e

0E0I

0 I I I0 0 . I 0 . 2 0 3P o t a s s i u m p h o s p h a t e c o n c n ( M )

F igure 2 Par t i t io n co eff ic ie n t , K , fo r (z -amylase in a phase systemo f PEG 3350, 9% (w/w), Dextran T 500, 2% (w/w), pH 7 .5, withvar io u s p o tass iu m p h o sp h a te co n cen t r a t i o n s . [ ], V a l u e f r o m r e f . 8

0.5

~0.4

0 2 4 6 8 I0 12PEG 600 (%, w/w)I I I I I I I9 8 7 6 5 4 5PEG 5350 (%,w/w)

Figure 3 Partition coefficient, K, fo r o r -amylase in a phase systemo f Dextran T 500, 2% (w/w) and different concentrations of PEG6 0 0 a n d P E G 3 3 5 0 , p H 7 . 0

F igure 4 Precip i tate d starch par t ic les in the bottom-phase droplets.Aqueous two -p h ase sys tem as in Figure Iof 0 .5 . The vo l um e ra t i o [ vo l um e ( t op phas e ) / vo l um e( bo t t om phas e ) ] o f t h i s phas e s ys t em w as 9 . Theo r e t i c a l l y ,82% o f t he a - am yl a s e i s pa r t i t i oned t o t he t op phas e i ns uch a s y s t em com par ed w i t h 73% i n t he phas e s ys t emP E G 3 3 5 0 , 9 % ( w / w ) / D e x t r a n T 5 0 0 , 2 % ( w / w ).

So l ub l e st a r ch w as u s ed f o r t he enz ym e p r odu c t i onexpe r i m en t s . The pa r t i t i on coe f f i c i en t o f s t a r ch i n t hephas e s ys t em PEG 3350 , 9% ( w / w ) / D ex t r an T 500 , 2%( w / w ) w as 0 .55 and i n t he phas e s ys t em PEG 600 , 8%( w / w ) , PE G 335 0 , 5% ( w / w ) / D ex t r an T 500 , 2% ( w / w ) i twas 0 .8 0. T hese f igures refer only to so luble s tarch , becauses o l ub le s t a r ch p r ec i p i ta t e s i n t he phas e s ys t em and accum u-l a t e s i n t he bo t t om phas e (Figure 4). I t is observed as am i l ky p r ec i p i t a t e . H ow eve r , du r i ng t he f e r m en t a t i ons t hea - am yl a se p r o duced deg r ades t he s t a r ch and t he p r ec i p i ta t edi sappears (Figure 5).P r o d u c t i o n o f a - a m y l a s e in a q u e o u s t w o - p h a s es y s t e m

Bacillus sub tilis was f i r s t cul t iva ted in a normal growthm e d i u m (Figure 6). A620 reached 23 af ter 112 h , 0 .5 8 Um l - ] a - am yl a s e w as p r oduce d and 38 g 1 ~ ou t o f 50 g 1 ~s t a rch w as cons um ed . The ove r a ll p r oduc t i v i t y w as 5 .2 x10 -3 U m1-1 h -1 and dur ing the f i r s t 60 h o f the cu l t iva t ioni t was 8 .4 10 -3 U m1-1 h-LThe p r oduc t i on o f a - am yl a s e w as t hen s t ud i ed ba t ch -w i s e i n t he aqueous t w o- phas e s ys t em PEG 3350 , 9%( w / w ) / D ex t r an T 500 , 2% ( w / w ) (Figure 7). This phases ys t em w as chos en becaus e t he com pos i t i on o f t op and21bo t t om phas es , r e s pec t ive l y , i s know n . Th i s m akes i tpos s ib l e t o add f r e s h t op phas e w i t h t he com pos i t i on ,w h i ch i s nece s s a r y t o m a i n t a in t he phas e s ys t em w hen t hetop phase i s changed.

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a f t e r 9 0 h . T h e a - a m y l a s e c o n c e n t r a t i o n i n t h e t o p p h a s ew a s 0 .3 5 U m l - ~, w h i c h c o r r e s p o n d s t o a t o t a l p r o d u c t i o no f 0 .4 8 U m 1- 1. T h i s p r o d u c t i o n i s 8 3 % o f t h e p r o d u c t i o ni n t h e n o r m a l g r o w t h m e d i u m (F igure 6 ) . The overa l lp r o d u c t i v i t y w a s 5 .3 x 10 -3 U m1-1 h -1.

I n o r d e r t o i m p r o v e t h e p r o d u c t i o n o f a - a m y l a s e , ap h a s e s y s t e m w i t h a h ig h e r p a r t i t i o n c o e f f i c i e n t w a s s t u d ie d[ P E G 6 0 0 , 8 % ( w / w ) , P E G 3 3 5 0 , 5 % ( w / w ) / D e x t r a n , 2 %( w / w ) ] o v e r t w o r e p e a t e d b a t c h e x p e r i m e n t s . F o r t h isp h a se s y s t e m t h e c o m p o s i t i o n o f t o p a n d b o t t o m p h a s ew a s n o t p r e v i o u sl y k n o w n . T h e c o m p o s i t i o n o f t h e t o pp h a s e w a s d e t e r m i n e d t o b e P E G 6 0 0 , 8 .4 % ( w / w ) , P E G3 3 5 0 , 5 .2 % ( w / w ) / D e x t r a n T 5 0 0 , 0 .8 % ( w / w ) a c c o r d i n gt o t h e p r o c e d u r e g i v e n i n M a t e ri a ls a n d m e t h o d s .

A f t e r r e m o v a l o f t h e t o p p h a s e i t w a s a s s u m e d t h a t t h ec o n c e n t r a t i o n s o f s t a rc h a n d o t h e r n u t r i e n t s r e m a i n i n g i nt h e b o t t o m p h a s e w e r e h i g h e n o u g h t o s u p p o r t g r o w t ha n d a - a m y l a s e p r o d u c t i o n w i t h B. subti l is . C o n s e q u e n t l y ,t h e c o n c e n t r a t i o n s o f st a r c h a n d o t h e r n u t r i e n t s i n t h ef r e s h l y a d d e d t o p p h a s e w e r e t h e s a m e as i n t h e g r o w t hm e d i u m .

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