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[J. Ferment. Technol., Vol. 65, No. 6, 651-658. 1987]

I m m o b i l iz a t i o n o f M i c r o o r g a n i s m s w i t h P V A H a r d e n e db y I t e r a t i v e F r e e z i n g a n d T h a w i n g

OSAMU ARIGA, H IROMI TAKAGI, HIRONORI NISHIZAW A, an d YOSHIKI SANODepartment o f F ine Mater ials Engineering, Facul ty of Text i le Science and Technology,

Shinshu Univers ity , Ueda, Nagan o 386, Japan

In order to utilize polyvinyl alcohol (PVA) as a gel matrix for the immobilizationof microorganisms, PVA w as subjected to iterative freezing-thawing. Th e effects ofthe procedure on the mechanical characteristics of the PVA hydrogel and the stabilityof the immobil ized microorganisms were invest igated. PVA sho w ed rubber-likeelasticity after iterative freezing-thawing . Ge l strength increased with the iterationnu m ber of freezing-thawlng until seven iterations. Although the activities of both thefree and imm obilized cells decreased during the iteration of freezing-thawing, addition o fcryoprotectants such as glycerol and skim m ilk was effective for preventing the decreasein activity.

M a n y w o r k e r s h a v e i n v e s t i g a t e d i m m o b i -l iz e d m i c r o o r g a n i s m s , e s p e c i a l ly t h e p r o -d u c t i o n o f m e t a b o l i t e s , l - 6 ) m e t h o d s o fi m m o b i li z a ti o n 6 - S ~ a n d t h e i r a p p l i c a t i o n tow a s t e w a t e r t re a tm e n t .9 - 1 2 ~ A g a r , p o l y -a e r y l a m i d e , a l g i n a t e o r k a p p a - c a r r a g e e n a nh a v e b e e n u s e d fo r t h e e n t r a p m e n t o fm i c r o o r g a n is m s . H o w e v e r , t h e m e c h a n i c a ls t r e n g t h o f a g a r is r a t h e r w e a k . A c r y l a m i d em o n o m e r is p o is o n o us , s o i m m o b i l i z a ti o n w i tha c r y l a m i d e is o f c o u rs e d e l e t er i o u s f o r t h eo r g a n i s m s . K u u e t a l . 13) p r o p o s e d a g e lp h a s e p o l y m e r i z a t i o n t e c h n i q u e . T h i s f o r m sa re i n f o r c e d g e l m a t r i x h a r d e n e d b y t h ec ro s s- li n ki n g a c t io n o f a c r y l a m i d e m o n o m e rw h i c h h a s p e n e t r a t e d i n t o t h e g e l m a t r i x .H o w e v e r , t h i s t e c h n i q u e n e e d s a n a e r o b i o s i st o r e n h a n c e m e n t o f t h e c r o s s - li n k in g r e a c t io n .T h u s , t h r o u g h o u t t h e p r o c e d u r e o x y g e n - f r e ec o n d i t io n s m u s t b e m a i n t a i n e d . A l g i n a t e g e lr e q u i r e s t h e a d d i t i o n o f C a Z+ a n d A I~ + i o n sf o r s t a b i li z a t io n o f t h e g el . T h i s p r o c e d u r eh a s a d r a w b a c k b e c a u s e t h es e io n s f o r m ap r e c i p i t a t e w i t h p h o s p h a t e i n n o r m a l m e d i -u m . O n th e o t h e r h a n d , K C 1 is r e q u i r e d f o rt h e i m m o b i l iz a t i o n o f k a p p a - c a r r a g e e n a n ,b u t t h i s h a s th e e c o n o m i c a l d i s a d v a n t a g e o fa h i g h r e m o v a l c os t f o r l a m b d a - c a r r a g e e n a n ,

c a u s i n g t h e g el t o b e c o m e w e a k . C o n s e -q u e n t l y , i t m a y b e i m p r a c t i c a l t o a p p l y t h e sep o l y m e r i c m a t e r i a l s t o w a s t e w a t e r t r e a t m e n ta s i m m o b i l i z a t i o n c a r r i e r s .

P o l y v i n y l a lc o h o l ( P V A ) is a r a w m a t e r i a lo f v i n y l o n a n d c a n b e p r o d u c e d i n d u s tr i al lyr a t h e r c h e a p ly . F u r t h e r m o r e , i m m o b i l i-z a t io n w i t h P V A h a v e b e e n f r e q u e n t lyi n v e s t i g a t e d b e c a u s e P V A i s n o n - t o x i c t oo r g a n i s m s . H a s h i m o t o e t a l . s t u d i e d w a s t e -w a t e r t r e a t m e n t u s i n g a c t i v a t e d s lu d g ee n t r a p p e d i n P V A r e i n f o r c e d b y b o r i c a c i dt r e a t m e n t TM o r f re ez in g .1 5 ) T h u s , P V A m a yb e t h e m o s t e c o n o m i c a l p o l y m e r i c su p p o r t .I f it c a n a t t a i n s u ff ic ie n t m e c h a n i c a l s t r e n g t h ,t h is p o l y m e r i c s u p p o r t w i l l b e u s e f u l a s a g e lm a t r i x fo r i m m o b i l i z a t i o n . R e c e n t l y , t h eh a r d e n i n g o f P V A g e l b y i t e r a ti v e f r e ez i n g -t h a w i n g w a s d e v e l o p e d b y N a m b u .1 6 ~ T h i sm e t h o d n e e d s n o c h e m i c a l r e a g e n t s a n d t h u st h e r e s u l ti n g g e l c a n m a i n t a i n h i g h w a t e rc o n t e n t ( th i s g e l is c a l l e d h y d r o g e l ) . I f t h ei t e r a t i v e f r e e z i n g - t h a w i n g m e t h o d c a n b ea p p l i e d t o P V A g e l f o r h a r d e n i n g , a n e c o -n o m i c a l im m o b i l i z a t io n m e t h o d m a y b er e a l i z e d .

I n t h e p r e s e n t w o r k , u t il i za t io n o f P V A a sa g e l m a t r i x f o r i m m o b i l i z a t i o n w a s i n v es ti -

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Vol. 65, 1987] Immobilization with PVA 653Table 2. Compositionof L-broth.

ConcentrationComponent [g/l]Y e a s t e x t r a c t 5P e p t o n e 1MgSO4.7H20 1.23NaC1 5

curve at 5 ppm was defined as the oxygen consumptionrate, i . e . microbial respiratory activity.R e s u l t s a n d D i s c u s s i o n

H a r d e n i n g o f P V A g e l b y fr e e z in g -t h a w i n g Polyvinyl alcohol as a gelmatrix for enzymes or microorganisms hasbeen investigated. Recently, Imai et al . 8 ,17)entrap ped enzymes within a polyvinyl alcoholmemb ran e cross-linked by means of ultra-violet ray irradiation. However, in thisprocedure several hours are necessary forirradiation which ma y lead to death of theorganisms. Hashimoto e t a l . TM investigatedwastewater treatment using activated sludgeimmobilized with PVA. The y used boricacid in order to hard en the PV A gel. How-ever, there is concern about unfavorableeffects on the microorganisms because boricacid is generally used as a sterilizer, eventhough they insisted that high resistance bythe activated sludge to a severe environment,as well as stabilization effects of the polymericsupport were observed. Although manyattempts to utilize PVA as a polymericsupport have been carried out, no effectiveimmobilization methods with PVA have beenreported.

Recently, it was reported that a PVAsolution became gelatinous by freezing andthat the gel strength increased duringitera tions of freezing and thawing.10~ Withthis treatment, a rubber-like hydrogel can beobtained from a PVA solution without usingany chemical reagents. In the present work,the strength of PVA gel was measured andthe effects of several factors on gel strengthwere investigated.

Figure 2 shows the effects of a nu mbe r of

E

o t

I I I I

I I I I1 2 3 4L [ k g l c m 2 1Fig. 2. Effects of freezing-thawing on gel strength.(5% PVA) . , 2/0 agar; Iteration number (n):0, 1; O, 2; , 5; [3, 7; , , 10.

freezing- thawing cycles (n) on gel strengthfor 5% PVA gel. The ordin ate (X) repre-sents the pene trat ion dept h of the test needleas the strain generated on the gel surfacewhen the stress (L) was applied. As shownin Fig. 2, gel strength increased with thenum ber of iterations until a maxi mum ofseven times. Similarly, Fig. 3 shows theresults of 10% PV A gel. Ten percent PV Agel was more rigid t han 5 % PVA gel and thehardness did not increase after seven iter-ations, similar to that for 5% PVA gel. Theeffects of PVA concentration on gel strengthare shown in Fig. 4. Gel strength increasedwith an increase in the PVA concentration.The results in Figs. 2, 3 and 4 indicate thatgel strength is significantly affected by theiteration n umbe r of the freezing-thawing

6 I _

L [ k g l c r n z ]F i g . 3. E f f e ct s o f f r e e z i n g - t h a w i n g o n g e l s t r e n g t h .

(10% PV A) Iteration number (n): O, 1;O, 3; A, 5; , 7; V, 10.

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654 AR1OA e l a l . [ , J . F e r m e n t . T e c h n o l . ,

6

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O0 5 10L I k g / c r n ~ I

F i g . 4 . E f fe c ts o f P V A c o n c e n t r a t i o n o n ge l s t r e n g t h .( n = l ) P V A c o n c e n t r a ti o n ( % ) : O , 7 .5 ; A ,10 ; A , 12 ; I , 15 .

c y c l e , a s w e l l a s t h e P V A c o n c e n t r a t i o n .T h e s e r e s u l t s a r e i n a g r e e m e n t w i t h t h o s e o fN a m b u l e , x s,19 ) a n d W a t a s e , 20) a l t h o u g h t h et y p e o f P V A u s e d i n t h is s t u d y i s d i f fe r e n tf r o m t h e i r ' s .

T o c o m p a r e t h e s t re n g t h o f P V A g e l w i t ht h a t o f o t h e r s, t h e s t r e n g t h o f 2 % a g a r w a se x a m i n e d . A s c a n b e s e e n i n F ig . 2, t h es tr e n g th o f t h e 2 % a g a r w a s g r e a t e r t h a n t h a to f t h e 5 % P V A g e l. H o w e v e r , b r e a k a g e o ft h e g e l w a s o b s e r v e d a t 2 k g / c m 2 u n d e r t h ec o n d i t io n s o f t h e p r e s e n t s tu d y . T a k a t ae t a l . TM m e a s u r e d g e l s t r e n g t h u s i n g ar h e o m e t e r a n d r e p o r t e d t h a t 2 . 8 - 4 . 0 %c a r r a g e e n a n g e l b r o k e a t 0 . 5 5 - 1 . 2 k g / e m 2.O n t h e o t h e r h a n d , n o b r e a k a g e w a s ob s e rv e d ,e v e n o v e r 3 .5 k g / c m 2, f o r 5 % P V A g e l . T h er e s u l t i n g h y d r o g e l w h i c h e x p e r i e n c e d t h ef r e e z i n g - t h a w i n g p r o c e s s p o s s e s s e d a r u b b e r -l ik e e l a s ti c i ty . E v e n w i t h v i o l e n t a g i t a t i o n ,b r e a k a g e o f t h e P V A g e l p a rt i c le s w a s n o to b s e rv e d , a l t h o u g h t h e a g a r , c a r r a g e e n a n a n da l g i n a t e g e l s w e r e e a s i ly d e s t r o y e d . S u c hr u b b e r - l i k e e l a s t i c it y is a g r e a t a d v a n t a g e f o ra p p l i c a t i o n o f t h e g e l t o a f l u i d i z e d - b e dr e a c t o r i n w h i c h t h e r e m a y b e e x p o s u re t oh i g h s h ea r s t r e s s .

O c h i a i e t a l . ~ 2) u s e d P V A a s a p o l y m e r i cs u p p o r t fo r c h l o r o p l a s t p h o t o s y st e m s . T h e yp r e p a r e d i m m o b i l i z e d c h l o r o p l a s t f i l m b yd r y i n g l i q u i d f i l m s p r e a d o n a g l a s s p l a t e .T h i s t e c h n i q u e is a ls o a p p l i c a b l e t o t h e o t h e rp o l y m e rs . I n o u r p r e l i m i n a r y e x p e r i m e n t ,

t h e fi lm w a s to o w e a k t o t r e a t. H o w e v e r , i tw a s c o n f i r m e d t h a t i t e ra t i v e f re e z i n g - t h a w i n gw a s a l so e f f e c t iv e f o r t h e h a r d e n i n g o f P V Af i l m p r e p a r e d i n t h i s w a y .

V o l u m e t r i c c h a n g e s i n P V A g e l i nf r e e z l n g - t h a w i n g c y c l e s D u r i n g i t e r -a t io n s o f fr e e z in g - t h a w i n g , v o l u m e t r i c c h a n g e si n P V A g e l w e r e o b s e r v e d . T h i s r e s u lt s i na s u b s e q u e n t i n c r e a s e i n t h e P V A c o n c e n -t r a t i o n i n t h e g e l. I n c r e a s i n g P V A c o n c e n -t r a t i o n m a y r e s u l t i n e n h a n c e m e n t o f t h e g e ls t re n g t h . T h e r e f o r e , v o l u m e t r i c c h a n g e s i nt h e P V A g e l d u r i n g i t e r a ti v e f re e z i n g - t h a w i n gw e r e i n v e s t i g a te d f o r c h a r a c t e r i z a t i o n o f th eP V A g el .

F i g u r e 5 s h o w s t y p i c a l v o l u m e t r i c c h a n g e si n t h e b l oc k s a n d b e a d s o f P V A g e ls d u r i n gf r e e z i n g - t h a w i n g i t e r a t io n s . F i g u r e 5 ( a )s ho w s t h e f r e e z i n g - t h a w i n g p r o c e d u r e c a r r i e do u t i n a p l a s ti c c o n t a i n e r . F i g u r e 5 ( b )s h o w s t h e v o l u m e t r i c c h a n g e s i n g e l b e a d s .T h e y w e r e f i rs t f r o z e n i n l i q u i d p a r a f f i n a n dt h e n i n d i s t i l l e d w a t e r t b r s u b s e q u e n t i t e r -a t i o n s . I n F i g . 5 ( b ) , t h e v o l u m e r a t i o isb a s e d o n t h a t o f l i q u id d r o p s w i t h o u t f r e e z i n g .I n a n y c a se , t h e v o l u m e s o f th e g e ls d e c r e a s e dw i t h i t e r a t i o n s a n d t h e l o w e r P V A c o n c e n -t r a t i o n r e s u lt e d i n l a r g e r v o l u m e t r i c c h a n g e s.S y n e r e s i s w a s o b s e r v e d d u r i n g f r e e z i n g -t h a w i n g o f t h e g e l b lo c k s. T h i s p h e n o m e n o nm a y c a u s e th e v o l u m e t r i c c h a n g e s . A te v e r y P V A c o n c e n t r a ti o n , t h e v o l u m e r a t i or e a c h e d a p l a t e a u a f t e r s e v e n i t e r a ti o n s .T h e s e p l a t e a u s w e r e s i m i l a r t o t h o s e o f t h e

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bI I I I2 4 6 8n { - }

V o l u m e t r i c c h a n g e s i n P V A g e l .( a ) g e l b l o c k f r o z e n i n a p l a s ti c c o n t a i n e r . ( b ) g e lb e a d s f r o z en i n l i q u i d p a r a f f i n. P V A c o n c e n -t r a t i o n ( % ) : Q , 7 . 5 ; O , 1 0 ; A , 1 2 . 5 ; & , 1 5 .

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Vol. 65, 1987] Imm obilization with PVA 655g e l s t r e n g t h m e a s u r e m e n t s .

R e c e n t l y , m a n y p o l y m e r s c i e n t i s t s h a v eb e c o m e i n t e r e st e d i n t h e g e l a t i o n o f P V Ad u r i n g f r e e z in g - t h a w i n g . T h e e x a c t m e c h a -n i s m o f P V A g e l a t i o n i s s ti ll n o t c l e a r , b u t t h ef o ll o w in g s h a v e b e e n e l u c i d a t e d : P V A h a sm a n y h y d r o x y l g r o u p s i n t h e s ke l e to n a n d t h eh y d r o g e n b o n d s b e t w e e n t h e h y d r o x y l g r o u p so f t h e P V A m o l e c u l e , a n d t h e H 2 0 a d j a c e n tt o t h e P V A s k e l e t o n c a u s e s a l a r g e r a t t r a c t i v ef o r ce a t t h e e n t a n g l e m e n t p o i n t. A p o r t i o no f t h e w a t e r m o l e c u le s e x is t a s b o u n d w a t e r ,a n d t h e o t h e r as f r ee w a t e r . D u r i n g f r e e z in go f t h e P V A s o l u ti o n , f re e H ~ O m o l e c u l e s a rer e p e l l e d a w a y f r o m t h e e n t a n g l e m e n t p o i n t sa n d s t r o n g e r h y d r o g e n b o n d s a r e f o r m e db e t w e e n t h e h y d r o x y l g ro u p s o f t h e P V As k e l e t o n s . 23> O n t h e o t h e r h an d , W a t a s ee t a l . 2 4) a n d N a g u r a e t a l . TM h a v e i n s i s t e dt h a t t h e h y d r o g e n b o n d s b e t w e e n t h e h y d r o x y lg r o u p s o f t h e P V A s k el e to n s a n d t h e w a t e rm o l e c u l e s a r e t h e m a j o r r e a s o n f o r g e l a t i o n .N a m b u s tu d i ed t h e r h e o lo g y o f P V A a n dd i s cu s s e d t h e e f f ec t s o f th e s u b s t i t u e n t g r o u p so n s te r ic h i n d r a n c e i n t h e r e s u l t in g p o l y m e r s .F r o m t h e r es ul ts o f l a C - N M R a n d 1 H - N M Ra n a l y s e s a n d X - r a y d i f f r a c t i o n , h e p r e s u m e dt h a t g e l a t i o n o f t h e P V A s o l u ti o n a n d t h er e i n f o r c e m e n t o f t h e r u b b e r - l i k e e l a s t i c i tym i g h t b e a t tr i b u t a b l e t o th e t b r m a t i o n o fm i c r o c r y s t a ls a t t h e e n t a n g l e m e n t p o i n t o f t h ep o l y m e r a n d t h e g r o w t h o f a c r o s s- li n k in gs t ru c t u r e . T h e r e f o r e , t h e p l a t e a u s o b s e r v e di n t h e p r e s e n t w o r k m a y c o r r e s p o n d t oa l i m i t o f f o r m a t i o n o f m i c r o c ry s t a ls a n d t h eg r o w t h o f c r o s s- li n k in g . C o n t r a r y t o t h eo b s e r v a t i o n s i n F i g . 5 ( a ) , t h e g e l b e a d sp r e p a r e d i n l i q u i d p a r a f f i n s w e l l e d i n i t i a l l y ,a n d v o l u m e t r i c i n c r e a s e s w e r e o b s e r v e d a f t e rt h e b e a d s w e r e w a s h e d w i t h d i s t i l l e d w a t e r .T h e r e h a v e b e e n p a p e r s o n f r e e z i n g - t h a w i n gp r o c e d u r e s i n l i q u i d p a r a f f in a s y e t . W eb e l ie v e t h a t t h is d i f fe r e n c e m a y b e d u e t od i f f e re n c e s i n t h e i n t e r f a c e s ( a i r - w a t e r o rw a t e r - o r g a n i c s o lv e n t) o n f o r m a t i o n o f t h e g e lp a r t i c l e s a n d t h e f r e e z i n g o r t h a w i n g r a t e s .W a t a s e e t a l . 2 6 ) r e p o r t e d c h a n g e s i n t h er h e o l o g i ca l p r o p e r t i e s o f t h e g e l d e p e n d i n g o nt h e p r e p a r a t i o n c o n d i t io n s a n d c o n c l u d e d

t h a t t h e f r e e z in g o r t h a w i n g t e m p e r a t u r e a la dt h e t h a w i n g r a t e s i g n i f i c a n tl y a f f e c t e d t h er h e o l o g ic a l p r o p e r t ie s o f t h e P V A g el . W h e nf r e e z i n g w a s p e r f o r m e d i n l iq u i d p a r a f f i n ,a l a r g e v o l u m e o f l i q u id p a r a f f i n m i g h tp r e v e n t ra p i d f r e e z i n g o r th a w i n g w h i c h m a yl e a d t o i n s u ff ic i e n t e n t a n g l e m e n t o f P V Ap o l y m e r s .

E f f e c t s o f c r y o p r o t e c t a n t s o n m i c r o -b i a l a c t i v i t y d u r i n g f r e e z l n g - t h a w i n gG e n e r a l l y s p e a k i n g , t h e r e s p i r a t o r y a c t i v i t yo r v i a b i l i t y o f m i c r o o r g a n i s m s f a ll s d u r i n gf r ee z i n g a n d th a w i n g . W h e n a m i c r o o r g a n -i s m is p r e s e r v e d b y a f r e e z i n g m e t h o d , ac r y o p r o t e c t a n t is u s u a l ly a d d e d t o p r e v e n ta l o w e r i n g o f m i c r o b i a l a c ti v i ty . I f m i c r o b i a la c t i v i t y w a s m a i n t a i n e d a t a f i x e d l e v e l b y t h ea d d i t io n o f a c r y o p r o t e c t a n t , P V A m a y b eu s e fu l f o r t h e i m m o b i l i z a t i o n w h i c h a c c o m p a -n i e s i t e r a t i v e f r e e z i n g - t h a w i n g t r e a t m e n t .I n t h e p r e s e n t w o r k , c h a n g e s i n a c t i v i t y i n t h em i c r o o r g a n i s m s w e r e m e a s u r e d d u r i n gi t e r a t i v e f r e e z i n g - t h a w i n g a n d p o s s i b l e e f f e c -

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a c t i v a t e d s l u d g e d u r i n g f r e e z i n g - t h a w i n g . ( f r e ec e l l ) S y m b o l s i n th e u p p e r f i g u r e c o r r e s p o n d t ot h e g l y c e r o l c o n c e n t r a t i o n i n t h e l o w e r f ig u r e .

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656 ARIOA t a l . [J. Ferment. Technol.,Table 3. Effects of various substances on cell viabi lity duringfreezing-thawlng.

n [--]Substance . . . . . . . . . . .0 1 3 5no addition I. 0 0. 61 0. 39 0. 37100 ppm Tween 80 1.0 0. 57 0.30 0. 2450 ppm Tween 80 1.0 0. 53 0. 39 0. 27saline 1.0 0. 45 0. 26 0. 17no addit ion 1.0 0.80 0. 56 0. 361% glucose I. 0 0. 50 0. 17 0.073% glucose 1.0 0. 54 0. 19 0.065% glucose 1.0 0. 65 0. 20 0. 091% lactose 1.0 0. 83 O. 56 0. 283% lactose I. 0 0.82 0. 60 0. 325% lactose 1.0 0. 69 0. 67 0. 40no addition 1.0 0.87 0. 52 0. 450.5% maltose 1.0 0. 82 0. 35 0. 201% maltose 1.0 0. 88 0. 37 0. 232% maltose 1.0 0. 82 0. 36 0. 231% oleic acid I. 0 0. 62 0. 30 0.073% oleic acid 1.0 0.50 0. 16 0. 035% oleic acid 1.0 0.38 0. 13 0.03

tive cryoprotectants were investigated.The effects of various substances on therespiratory act ivi ty of act ivated sludge duri ngiterat ive freezing-thawing are shown inTab le 3. Rela tive activit y was defined as therat io of the oxygen cons umpt ion rate to that | .0when i terat ion number (n) is zero, i . e . , thefirst freezing. This relative value is expressed q"as the act ivi ty rat io. As shown in Tab le 3, othe ac tivity of the ac tivate d sludge decreasedsignificantly dur ing freezing- thawing, al- L.though some scattering due to fluctuation of ~>'0.5>the organis ms in the activa ted sludge was :~observed. Comp ari ng the act ivi ty rat ios .~und er condit ions with no addit ive to thosewith an addit ive, all of the comp ounds inTa bl e 3 seemed to be ineffective or rath erharmful as a cryoprotectant . On the otherhand, as shown in Figs. 6 and 7, the additionof small amou nt s of glycerol or skim milk was Fig. 7.effective. It seemed that these substancessuppressed the lowering of the act ivi ty duri ngiterative freezing- thawing. In Fig. 7, the

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n l - !Effects of skim milk on the activity ratio ofactivated sludge during freezing-thawing. (freecell) Skim milk concentration (%): O, 0;O, 3; A, 5; A, 10.

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& , s a l i n e . G l y c e r o l c o n c e n t r a t i o n ( % ) : Q , 0 ;O , 1 ; A , 3 ; D , 5 ; L 1 0 .

a d d i t i o n o f 3 - 5 % g l y c e r o l i n d u c e d a c t i v i tyr a t i o s l a r g e r t h a n u n i t y , b u t t h i s m a y h a v eb e e n d u e t o e x p e r i m e n t a l e r r o r.S i m i l a r l y , a s s h o w n i n F i g . 8 , g l y c e r o l w a sa l so e f f e c t iv e f o r th e i t e r a t i v e f r e e z i n g -t h a w i n g p r o c e d u r e w i t h E . c o l i , a l t h o u g h t h i sw a s m o r e s e n s it iv e t h a n t h e a c t i v a t e d s l u d g et o f r e e z i n g - t h a w i n g .I m m o b i l iz a t io n o f m i c r o o r g a n i s m sw i t h P V A I m m o b i l iz a t io n o f a c t i v a te ds lu d g e w i t h v a r i o u s c o n c e n t r a t i o n s o f p o l y - 1 .5v i n y l a l c o h o l w a s c a r r i e d o u t a n d t h ea c t iv i ti e s o f o x y g e n c o n s u m p t i o n a f t e r i te r a -t iv e f r e e z i n g - t h a w i n g w e r e m e a s u r e d . I nF i g . 9, t h e a c t i v i t y r a t i o s b a s e d o n t h e a c t i v i t ya t s e c o n d c y c l e o f f r e e z i n g - th a w i n g w e r e ~ 1 . 0p l o t t e d a g a i n s t t h e i t e r a t i o n n u m b e r b e c a u s e 25 % P V A g e l b e a d s a t in i t ia l f r e e z in g - t h a w i n gw e r e t o o f r a g i l e f o r m e a s u r i n g r e s p i r a t i o na c t i v i t y .A l t h o u g h a n e q u a l m a s s o f c e ll s w a s 0 .5i m m o b i l i z e d , t h e a c t i v i ty r a ti o s v a r i e d w i t ht h e P V A c o n c e n t r a t io n . F r o m th e r e su l ts i n F i g . 10.F i g . 5 , t h e v o l u m e s o f t h e g e l b e a d s w e r ed e c r e a s e d b y f r e e z in g - t h a w i n g . H o w e v e r ,a s a m p l e o f t h e 5 m l P V A g e l w a s t a k e n i n

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F i g . 9 . E f fe ct s o f P V A c o n c e n t r a t i o n o n t h e a c t i v i tyr a t i o o f a c t i v a t e d s l u d g e d u r i n g f r e e z i n g - t h a w l n g .( i m m o b i l i z e d c e l l) P V A c o n c e n t r a t i o n ( % ) " O , 5 ;A, 7 .5 ; @, 10; A , 12; 0 , 15 .

t h i s e x p e r i m e n t , t h e r e f o r e , t h e a p p a r e n t c e l ld e n s i t y i n t h e g e l b e a d s w a s i n c r e a s e d . I f t h ec e ll s i m m o b i l i z e d i n t h e s u p p o r t a r e q u i t es t a b l e d u r i n g f r e e z i n g - t h a w i n g , t h e a p p a r e n ta c t i v i t y r a t i o s o u g h t t o i n c r e a s e w i t h t h ei t e ra t i o n s. S i n c e th e v o l u m e t r i c c h a n g e sw e r e l a r g e r f o r t h e l o w e r P V A c o n c e n -t r a t i o n s , t h e r e s u l t s s h o w n i n F i g . 9 s u g g e s tt h a t h i g h e r c o n c e n t r a t io n s o f P V A c o n t r ib u t et o t h e s t a b i l i z a t io n o f c e l l a c t i v i t y d u r i n gf r e e z i n g - t h a w i n g , s i m i l a r t o t h a t w h e n p o l y -v i n y l p y r r o l i d o n e o r d e x t r a n a r e u s e d a sc o m m o n p o l y m e r i c c ry o p r o t e c ta n t s .

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T h e a c t i v i t y r a t i o s fo r a c t iv a t e d s l u d g ei m m o b i l i z e d w i t h 1 0 % P V A a r e sh o w n inF i g . 1 0 a n d t h o s e f o r E . c o l i i n F i g . 1 1. T h ee f fe c ts o f p r o t e c t i o n a g a i n s t a c t i v i t y lo w e r i n gw e r e c l e a r l y s h o w n i n t h e c a s es w h e n g l y c e r o la n d s k i m m i l k w e r e u s e d a s c r y o p r o t e c t a n t s .M o r e o v e r , t h e s e c o m p o u n d s d i d n o t a f fe c tt h e g e l s t r e n g t h ( d a t a n o t s h o w n ) . T h e r e f o r e ,u t i l i z a t i o n o f P V A h a r d e n e d b y i t e ra t iv ef r e e z i n g - th a w i n g a s a n i m m o b i l i z a t i o ns u p p o r t m a y b e p o s s ib l e i f g l y c e r o l o r s k i mm i l k is a d d e d a s a c r y o p r o t e c t a n t .

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p o l y v i n y l a l c o h o l c o u l d b e u s e d a s a n i m -m o b i l i z i n g s u p p o r t f or b o t h i t e r a ti v e f re e z in g -t h a w i n g t r e a t m e n t a n d w h e n u s i n g c r y o -p r o t e c t a n t s s u c h a s g l y c e r o l o r s k i m m i l k .T h i s i m m o b i l i z a t i o n m e t h o d s h o u l d r e su l t i na n e c o n o m i c a l a d v a n t a g e w h e n c o m p a r e d t oo t h e r e n t r a p m e n t s ys te m s . T h i s s y s te m m a yb e a p p l i c a b l e to w a s t e w a t e r t r e a tm e n t .

A c k n o w l e d g m e n t sT he a u tho r s t ha nk K ur a r a y C o . L td . f o r k ind lysupp ly ing the P V A . W e w i sh to t ha nk P r o f . S .M a t s u z a w a , D i s . K . Y a m a u r a a n d M . N a g u r a o f

Shinshu Univ ers i ty for the i r va lu able sugges tions.

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Miorobiol. Biotechnol., 13, 133 (1981).2 ) T ya g [ , R . D . , G hose , T . K . : Biotechnol. Bioeng.,24, 781 (1982).3 ) G h o m m l d h , C . , N a v a rr o , J . M . , D u r a n d , G . :Biotechnol. Bioen g., 24, 605 (1982).

4 ) A t r a t , P ., H u l l e r , E . , H or ho ld , C . : Enr. J . Appl .Microbiol. Biotechnol., 12, 157 (1981).

5 ) W a da , M . , K a to , J . , C h iba t a , I . : Eur. J . Appl .?fficrobiol. Biotechn ol., 10, 275 (1980).6 ) W a d a , M . , K a to , J . , C h iba t a , I . : Eur. J . Appl .Microbiol. B iotechnol., 8, 241 (1979).7 ) K okuf u t a , E . , M a t sum oto , W . , N a k a m ur a , I . :Biotechnol. Bioen g., 24, 1591 (1982).8 ) I m a i , K . , S h iom i , T . , S a to , K . , F u j i sh im a , A . :Biotechnol. Bioen g., 25, 613 (1983).9) Shi ta ra , S . , Watanabe , A. , Suzuki , T . : J . JapanSewage W orks Assoc. , 21, 35 (1984).

10) Shi ta ra , S . , Watanabe , A. , Suzuki , T . : J . JapanSewage W orks Assoc. , 20, 31 (1983).

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12) Ni lson , I . , Ohlson, S . : Eur. J. Appl. Microbiol.Biotechnol., 14, 86 (1982).

13) Ku u, W . Y. , Polack , J . A . : Biotechnol.Bioeng., 25,1995 (1983).14) H a sh im o to , S ., F u r uka w a , K . , H a m a , H . : J .

Japan Sewage Works Assoc., 23, 41 (1986).15) H a sh im o to , S . , F u r uka w a , K . , H a m a , H . : J .Japan Sewage W orks Assoc., 23, 16 (1986).16) Na mb u, IV[ .: .Japanese Pa ten t La id Op en, No.130543-82 (1982).17) I m a i , K . , S h iom i , T . , U c h id a , K . , I V f iya , M . :Biotechnol. Bioeng., 28, 1721 (1983).

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186, 1081 (1985).21) Taka ta , I . , Yarnamoto , K. , Tosa , T . , Chiba ta , I . :Enzyme Microb. Technol., 2, 30 (1980).22) O c h ia i , H . , S h iba t a , H . , M a t suo , T . , H a sh inokuc h i ,

K . , I na m ur a , I . : Ag r ic . B io l. Che m. , 42~ 683(1978).23) 1V[aeda, H. , Ka wa i , T . , K ashiw agi , R . : KobunshiKagaku, 13, 193 (1956).24) W ata se, 1V[.,N i s h i n a r i , K . : Makromol . Chem., 196,1081 (1985).

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(Rece ived Februa ry 28 , 1987)