1986 emt disruption of microbial cells for intracellular products

7/30/2019 1986 EMT Disruption of Microbial Cells for Intracellular Products

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Disrupt ion o f microbia l c e l l s forintrace l lu lar produc t sY U S U F C H I ST I a nd M U R R A Y M O O - Y O U N G *D e p a r t m e n t o f C h e m i c a l E n g i n e e ri n g , U n i v e r s it y o fW a t e r l o o , W a t e r l o o , O n t a r io , C a n a d a N 2 L 3 G IS u m m a r y . Dis in t eg rat ion o f m icrobia l cel ls i s a neces saryf i r s t s te p f o r t h e p r o d u c t i o n o f i n t r a c el l u la r e n z y m e s a n dorganel I es . W i th increas ing use o f i n t racel lu lar microbia lm a t e r i a l i n i n d u s t r y a n d m e d i c i n e , t h e c e l l d i s r u p t i o n u n i to p e r a t i o n i s g a i n i n g in i m p o r t a n c e .

T h i s r e v i e w e x a m i n e s t h e s t a t e o f t h e a r t o f t h e l ar ge -s c a le c e l l d i s r u p t i o n t e c h n o l o g y a n d d i s r u p t i o n m e t h o d s o fp o t e n t i a l c o m m e r c i a l v a lu e .Keywords.. Dis r u p t io n o f m ic r o o r g a n i sm s ; c e l l d i s i n t e g r a t i o n ;i n t r a c e l l u l a r e n z y m e s

I n t r o d u c t i o nTh e i m p o r t a n c e o f m i c r o o r g an i s m s a s a s o u r c e o f c o m m e r -c ia l ly usefu l chemica l s , an t ib io t i cs and enzymes has beenrecognized for a very long t ime. Near ly a l l chemica l s ofm i c r o b i a l o r i g in p r o d u c e d i n d u s t ri a l ly t o d a y a re o f t h ee x t r a c e l l u l a r t y p e . Th a t i s , t h e y a r e p r o d u c e d wi t h i n t h em i c r o b i a l c e ll , b u t a r e t h e n e x c r e t e d i n t o t h e s u r r o u n d i n ge n v i r o n m e n t . A m u c h l a rg e r p r o p o r t i o n o f t h e p o t e n t i a l l yusefu l microbia l product s i s r e t a ined wi th in the ce l l s . A vas tm a j o r i t y o f t h e e n z y m e s k n o wn , f o r e x a m p l e , a r e i n t r a -c e l lu l a r. 1 Ev e n g r e a t e r u se o f m i c r o b i a l p r o d u c t s , m a n y o fwh i c h wi l l b e i n t r a c e l l u l a r , c a n b e e x p e c t e d f r o m t h ep r e d i c t e d s u r g e i n b i o t e c h n o l o g y . 2

The i so la t ion of in t race l lu la r mater i a l r equi res tha t the

* T o w h o m c o r r e s p o n d e n c e s h o u l d b e a d d re s s e d

ce l l e i ther be gene t i ca l ly engineered so tha t what wouldn o r m a l l y b e a n i n t r a c e l l u l a r p r o d u c t i s e x c r e t e d i n t o t h ee n v i r o n m e n t , o r i t m u s t b e d i s i n t e g r a t e d b y p h y s i c a l ,chemica l or enzymat ic means to re lease i t s content s in tot h e s u r r o u n d i n g m e d i u m . Th e g e n e t ic m a n i p u l a t i o n o fmicrobia l ce l l s to make them leaky i s l imi ted in scope .Making the ce i l fu l ly permeable to any s igni f i cant f rac t iono f t h e i n t ra c e l l u la r p r o d u c t s a n d e n z y m e s wo u l d n o t o n l ybe d i f f i cu l t , but a l so wi l l imply d i scont inued exi s t ence ofthe ce l l . I t i s in th i s context tha t the uni t opera t ion ofmicrobia l ce l l d i s rupt ion for in t race l lu la r product i so la t ionwi ll b e c o m e o f i n c r e as i n g im p o r t a n c e .P r o b a b l y b e c a u s e o f t h e h i g h c a p it a l a n d o p e r a t i n g co s t sof p i lo t p lant s for l a rge-sca le i so la t ion o f in t race l lu la rprod uct s and the req ui rem ent of si zeable t eam s of sc ient i s tsa n d t e c h n i c a l s t a f f t o o b t a i n m e a n i n g f u l b i o c h e m i c a lengineer ing des ign da ta , f ew s tudies have been publ i shedon the subjec t . 3 Thi s review examines the cur ren t s t a t e ofmicrobia l ce l l d i s rupt ion t echnology f rom an indus t r i a la p p l i c a t i o n s p o i n t o f v i e w. Th e d i s r u p t i o n t e c h n i q u e s o fpote nt i a l indus t r i a l use a re a l so d i scussed .In the l as t f ew years severa l in t race l lu la r enzy me s haveb e g u n t o b e p r o d u c e d i n d u s t r ia l l y : f o r e x a m p l e , g l u c os eoxidase for food preserva t ion , penic i l l in acylase for an t i -b io t i c c onve rs ion , and asparaginase for poss ib le cancert h e r a p y . 4 O t h e r e x a m p l e s o f i n tr a c e l lu l a r m i c r o b i a l e n z y m e s

p r o d u c e d c o m m e r c i a l ly a r e g i v en i n T a b l e l .The necess i ty of harves t ing the producing ce l l s , ino r d e r s u b s e q u e n t l y t o e x t r a c t a n i n t e r n a l c o n s t i t u t e n t ,i s a major economic d i sadvantage and, in par t , r esu l t s int h e p r e s e n t p r e o c c u p a t i o n wi t h t h e m a n u f a c t u r e o f p r o -duc t s of very h igh va lue , s Poss ible cos t r ed uc t ion s , how-e v e r , m a y b e a c h i e v e d b y s i m u l t a n e o u s i s o l a t i o n o f an u m b e r o f i n t r a ce l l u la r p r o d u c t s f o l lo wi n g c e ll d i s ru p t io n . 6Th i s wo u l d b e e v e n m o r e s o i f e x t ra c e l h i la r p r o d u c t i s ol a-t i o n p r e c e d e d t h e i n t ra c e l l u la r p r o d u c t i s o l a t io n f r o m t h es am e f e r m e n t a t i o n b a t c h .

T a b l e 1 S o m e e x a m p l e s o f i n t r a c e l lu l a r m i c r o b ia l e n z y m e s p ro d u c e d c o m m e r c i a l l y . [ R e p r o d u c e d f ro m L i l l y , M . D . i nApp l ied B iochem is t ryand Bioengineering ( W i n g a r d J r , L . 8 . , Katchalski .Katzir , E . a n d G o l d s t e i n , L . , ed s ) A c a d e m i c P r es s, N e w Y o r k , 1979, v o [ . 2 , p . 1 b y p e r m i s s i o no f A c a d e m i c P re ss ]E n z y m e S o u r ce E x a m p l e s o f u s eL-AsparagJnase (EC 3 .5 .1 .1 ) Erw/nia carotovora T r e a t m e n t o f a c u t e l y m p h a t i c l e u k a e m i aEscher ich ia co i lC a t a l a s e ( E C 1 . 1 1 . 1 . 6 ) Aspergil lus nigerC h o l e s t e r o l o x i d a s e ( E C 1 . 1 . 3 . 6 ) Nocardia rhodochrous/ 3 -Ga lac tos idase (EC 3 ,2 .1 .23 ) Kluyveromyces fragil isSaccharomyces lactisG l u c o s e i s o m e r a s e ( E C 5 . 3 . 1 . 5 ) Bacillus coagulans

Streptomyces sp .G l u c o s e o x i d a s e ( E C 1 . 1 . 3 . 4 ) Aspergil/us niger

Penici l l ium notatumG l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e ( E C 1 . 1 . 1 . 4 9 ) Y e a s tI n v e r ta s e ( E C 3 . 2 . 1 . 2 6 ) Saccharomyces cerevisiaeP e n i c i l l i n a c y l a s e ( E C 3 . 5 . 1 . 1 1 ) Escher ich ia c oi l

R e m o v a l o f H = O : a f t e r m i l k s t e r i l i z a t i o nS e r u m c h o l e s t e r o l a n a l y s isH y d r o l y s i s o f l a c t o s e i n m i l k / w h e yP r o d u c t i o n o f h i g h - f r u c to s e g l u c o s e s y r u p sS e r u m g l u c o s e a n a l y s i sR e m o v a l o f o x y g e n f r o m f o o d sC l i n i c a l a n a l y s i sC o n f e c t i o n e r yD 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 li n

0 1 4 1 - - 0 2 2 9 / 8 6 / 0 4 0 1 9 4 - - 1 1 $ 0 3 . 0 01 9 4 E n z y m e M i c r o b . T e c h n o l . 1 9 8 6 , v o l . 8 , A p r i l 1 9 8 6 B u t t e r w o r t h & C o . ( Pu b lis h er s) L t d


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C e ll d i s r u p t i o n m e t h o d sM i c r o o r ga n i s m s a r e m o r e r o b u s t t h a n i s g e n e r al l y b e l i e v e d .Th e r e s is t a n ce t o d i s r u p t i o n o f m i c r o o r g a n is m s h a s b e e nr e f e r r e d t o b y W i m p e n n y . 7 He p o i n t s o u t t h a t t h e i n t e rn a lpressure due to osmos i s ins ide an organi sm such as Micro-coccus lysodeikt icus or Sarcina lutea i s a b o u t 2 0 a t m o s -pheres and tha t the s t ruc tures respons ib le for res i s t ing th i spressure a re about as s t rong, weight for weight , asr e i n f o r c e d c o n c r e t e . A v a r i e t y o f d i s r u p t i o n m e t h o d s a r eava i l ab le to d i s in tegra te these s t rong ce l lu la r wal l s andm e m b r a n e s a n d l i b e r a t e t h e c e l l c o n t e n t s .A u s e f u l c l a s s i f i c a t i o n o f t h e c e l l d i s r u p t i o n m e t h o d sis given in Figure 1. On l y s o m e , m a i n l y m e c h a n i c a l , c e l ld i s r u p t i o n m e t h o d s h a v e f o u n d i n d u s t r i a l a p p l i c a t i o n .These wi l l be descr ibed in more de ta i l in the l a t t e r par t s ofth i s r ev iew. Smal l- sca le t echn iques u sed p r imar i ly in micro-b i o l o g y a n d b i o c h e m i s t r y l a b o r a t o r y w o r k h a v e b e e nr e v i e we d p r e v i o u s l y ] 1 So m e o f t h e e a r li e r wo r k h a s b e e nreviewed b y A iba and assoc ia tes , l l

M e c h a n i c a l c e l l disruptionBoth so l id shear (e .g . bead mi l l ) and l iquid shear (e .g . h igh-p r e s s u r e h o m o g e n i z e r ) b a s e d m e t h o d s o f c e l l d i s r u p t i o nhave proven success fu l on a l a rge sca le . The so l id shearmethods may involve e i ther a gr inding ac t ion as in a ba l lm i l l o r m a y i n v o lv e e x t r u s i o n o f f r o z e n c e l ls , e i t h e r a lo n eo r a s a c e l l - i c e ( o r o t h e r a b r a s i v e ) m i x t u r e , t h r o u g h n a r r o wgaps or or i f i ces under h igh pressure .12M o s t o f t h e c e il d i s ru p t i o n e q u i p m e n t i n c u r r e n t u s e wasor ig ina l ly des igned for the homogeniza t ion and s i ze reduc-t i o n o f v e r y d i f f e r e n t c o m m e r c i a l p r o d u c t s s u c h a s m i l k a n dp a i n t . Fo l l o wi n g D u n n i l l, 6 t h e a p p l i c a ti o n o f t h is h a r d wa r eto ce l l d i s rupt ion has been very success fu l .L i q u i d s h e ar m e t h o d sTh e h i g h - p r e s s u r e h o m o g e n i z e r . Am o n g t h e l i q u i d s h e a rd i s r u p t i o n d e v i c e s , t h e h i g h - p r e s s u r e M a n t o n - Ga u l i n APVt y p e h o m o g e n i z e r i s p r o b a b l y t h e m o s t wi d e l y u s e d . Th etechnica l f eas ib i l i ty of th i s sys tem for ce l l d i s rupt ionwa s d e m o n s t r a t e d b y Du n n i l l a n d L i l l y a n d t h e i r c o -wo r k e r s 13 a n d t h e u s e o f t h is e q u i p m e n t h a s b e e n f u r t h e rrepo r ted . 1 ,4 ,6 ,14-26 Th e h igh-pressure hom oge nizer con-s is ts o f a p o s i ti v e d i s p l a c e m e n t p i s to n p u m p wi t h o n e o rmore p lungers . The ce l l suspens ion i s drawn through a

Disrup t ion o f m icrob ia l ce l ls : Y . Ch is t i and M. M oo- Youngc h e c k v a l v e i n t o t h e p u m p c y l i n d e r a n d , o n t h e p r e s s u r es t roke , i s forced through an adjus table d i scharge va lve(Figure 2) with res t r i c t ed or i f i ce .4Th e d i s r u p t io n o f b a k e r s ' y e a s t , Saccharomyces cerevis-iae, i n a n A P V M a n t o n - G a u l i n h o m o g e n i z e r w a s e x a m i n e db y He t h e r i n g t o n ]3 a n d c o wo r k e r s . Th e ' k n i f e e d g e ' v al vesea t (Figure 3) was found to g ive h igher d i s rupt ion thant h e ' f l a t ' t y p e u n i t a t t h e sa m e o p e r a t i n g p r e s su r e (Figure4 ) . Th e d i s r u p t i o n f o l l o we d f i r s t - o r d e r k i n e t i c s a n d c o u l db e d e s c r i b e d b y :

lo g [ R m / ( R m - - R ) ] = k - / V Pa (1)wh e r e R m i s t h e m a x i m u m o b t a i n a b l e p r o t e i n r e le a s e a f t e rN passes thro ugh the va lve , k i s a d imens iona l (PE a) d i s rup-t i o n r a t e c o n s t a n t . Th e e x p o n e n t , a , o n t h e o p e r a t i n gp r e s s u r e , P , h a d a v a l u e o f 2 . 9 . Th e f o r m o f e q u a t i o n ( 1 )wa s c o n f i r m e d f o r d i s r u p t i o n o f Candida uti l is b y En g l e rand Ro binson 27 in a s l ight ly d i f fe r ent h igh-pressure f lowd e v i c e. Th e v a l ue o f t h e e x p o n e n t , a , f o r a n o r g a n is m i s a

F i g u r e 2 H i gh -p res s u re hom ogen i z e r d i s c ha rge v a l v e un i t . Thed i s c ha rge p ress u re is c on t r o l l ed b y a handwh ee l as s emb l y , A , wh i c h ,t h rou gh a s p r i ng l oaded v a l v e rod , 8 , pos i t i ons t he v a l v e , C , inre l a t i on t o t he v a l v e s ea t , D . Du r i ng d i s c ha rge , t he m a te r i a l p a s s e sb e t w e e n t he v a l ve and i ts s ea t and i mp i nges on an i mpac t r i ng , E .[ R e d r a w n f r o m H e t h e r i n g t o n , P. J , , F o l lo w s , M . , D u n n i l l , P . a n dU t L , M . D . Trans. Ins t . Chem. Eng. 1 9 7 1 , 4 9 , 1 4 2 b y p e rm i s si o n o fThe I ns t i t u t i on o f Chemi c a l E ng i nee rs ]

FN o n - mechanicalIi ILysis Dessication(various)

1E n z y m a t i c:hemical(d e te rg e n ts , s o l v e n ts ,a n t i b i o t i c s )

Physical (osmotic shock,p re s s u re )F i g u r e 1 C e l l d i s r u p t i o n m e t h o d s

Cell d i s r u p t i o nI

IS o l i d s h e a rI

X - pre~,Hughespress,o th e rs

IBead mill

'1M e c h a n i c a l[ 1L i q u i d s h e a r

~ ! i g h -p re s s u reh o m o g e n i z e r ,French pressUl trasonics

Enzym e Microb. 3-echnol. 1986, vol. 8, Ap ril 195


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i M 'q : : : :a bF i g u r e 3 D e t a i l s o f t h e v a l v e s e a t s . (a ) ' F l a t ' u n i t . ( b ) ' K n i f e - e d g e 'u n i t . [ R e d r a w n f r o m H e t h e r i n g t o n , P . J . , F o l lo w s , M ., D u n n il l , P .a n d L i l l y , M . D . Trans. Inst . Chem. E ng. 1971, 49 , 142 , by permis-s ion o f The Inst i tu t ion o f Chemica l Eng ineers ]

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I I l I I0 I 2 3 4 5

NFigure 4 Pro te in re lease wi t h d i f fe ren t va lve sea ts : o , f la t ; o , kn i fee d ge . [Re d r a wn f r o m He th e r i n g to n , P . J . , Fo l l o ws , M . ,D u n n i l I , P .a n d L i l l y , M . D . Trans. Inst. Chem. Eng. 1 9 7 1 , 4 9 , 1 4 2 b y p e rm i s -s i o n o f Th e In s t i t u t i o n o f Ch e m i ca l En gi ne e rs ]

m easu re o f i t s r e s i st an ce to d i s ru p t io n an d is d i f f e r en tfo r d i f f e r en t o rg an i sm s . T h u s , fo r t h e d i s ru p t io n of Escheri-chia coli g ro w n o n a s im p le sy n th e t i c m ed iu m a v a lu eof 2 .2 has b een rep or te d . ~6 For a g iven o rgan ism , a hasb een fo u n d to b e d ep en d e n t o n g ro w th h i s to ry . ~ 6'2 ~'2 8F o r ex am p le , a s sh o w n in Figure 5, E. coli cel ls g rown onco m p lex m ed ia a r e m o re ro b u s t t h an th e ce l l s g ro w n o n as im p le sy n th e t i c m ed iu m ) 6 T h e y eas t d i s ru p t io n p ro cess ~3w as in d ep en d en t o f ce l l co n ce n t r a t i o n o v e r t h e co n cen t r a -t i o n r an g e o f 4 5 0 -7 5 0 k g y eas t m -a o f su sp en s io n w h i l et h e c o n s t a n t , k , w a s t e m p e r a t u r e d e p e n d e n t a n d in c r e as e dw i th i t . T h u s , d i s ru p t io n r a t e i n c reased ~3 b y a b o u t 1 . 5 - fo ldfo r a ch an g e in o p e ra t in g t em p era tu re f ro m 5 to 3 0 C .D i s ru p t io n a t h ig h e r t em p e ra tu res , h o w ev er , w o u ld b eu se fu l o n ly i f t h e d es i r ed p ro d u c t i s n o t h ea t l ab i l e . A to p e ra t in g t em p era tu res h ig h e r t h an 4 0 C , p ro t e in d en a tu ra -t ion dur ing d isrup t ion has b een repo r ted . 13 S ince thet em p era tu re r i s e ac ro ss a G au l in h o m o g en ize r d u e toad iabat ic com pressio n 2s is abou t 2C per 10 MPa, inade-q u a te p reco o l in g , o r f a i l u re t o co o l b e tw een m u l t ip l e

p asses , can r e su l t i n t em p era tu res o f ab o v e 4 0 C an dco n seq u en t d en a tu ra t io n .

E q u a t io n (1 ) i n d i ca t e s a s t ro n g in f lu en ce o f o p e ra t in gp ressu re o n th e d i s ru p t io n p ro cess . B y o p e ra t in g th e h o m o -gen izer a t h igher p ressu re s , i t shou ld be possib le to decreaseth e n u m b er o f p asses o f t h e ce l l s l u r ry th ro u g h th e h o m o -gen izer fo r a g iven degree o f d isrup t ion . S ince the overal lt h ro u g h p u t r a t e ( q C/N, where q is the vo lum e f low rateth ro u g h th e h o m o g en ize r (m 3 h -1 ) , C i s t h e co n cen t r a t i o nof ye ast s lu rry (kg ye ast n f 3 suspe nsion) , and N is then u m b e r o f p a s s e s ) o f t h e h o m o g e n i z e r i s i n v er s e ly d e p e n d e n to n th e n u m b er o f p asses , r ed u ced p asses w o u ld a l l o win c reased th ro u g h p u t . A l so , t h e sev e re d o w n s t r eam c l a r i f i -ca t io n p ro b lem s , t3 d u e to t h e f i n e ce ll d eb ri s r e su l ti n g f ro mfu r th e r d i s in t eg ra t io n o f a l r ead y d i s ru p ted cel ls o n r ep ea t edp assag e th ro u g h th e m ach in e , w o u ld b e r ed u ced . N o te t h a tH e th e r in g to n ' s r e l a t i o n sh ip ( eq u a t io n 1 ) i s v a lid o v e r ap res su re r an g e o f 1 9 . 6 -5 8 . 8 M P a fo r y eas t cel l s l u rr i e s w i th< 7 5 0 k g y eas t m -3 su sp en s io n . F o r m o re co n cen t r a t edsu sp en s ion s (7 5 0 k g y eas t m -3 su sp en s io n ) , t h is eq u a t io nb reak s d o w n fo r o p e ra t in g p res su res g rea t e r t h an ab o u t29 .4 MPa. 13 An op t im al ch o ice o f o perat in g p ressu re isa l s o i m p o r t a n t b e c a u s e t h e p o w e r c o n s u m p t i o n d u r i n gd i s ru p t io n i s a li n ea r fu n c t io n o f t h e o p e ra t in g p res su re .T h e p o w e r c o n s u m p t i o n c o r r e s p o nd s t o a b o u t 3 . 5 k W p e r100 MPa o f operat ing p ressu re .?6

T h e r e l ease o f ab o u t 3 0 % o f t h e av a i lab l e p ro t e in f ro mCandida lipolytica w as ach iev ed b y W h i tw o r th 18 in aM a n t o n - G a u l i n h o m o g e n i z e r a f t e r 6 p a ss e s a t 5 5 M P a .P ro t e in r e l ease d id n o t o b ey th e f i rs t o rd e r k in e t i c s r ep o r t edby o thers . 13'27 Th is d iscre pancy w as ap pare n t ly due to theh e t e r o g e n e o u s m o r p h o l o g y o f C. [ipotytica w h ich co n s i s t edo f a m ix tu re o f f i l am en to u s an d e lo n g a ted -o v o id ce l ls . 18E v en a f t e r r ep ea t ed p asses t h ro u g h th e h o m o g en ize r , n oev id en ce o f p ro t e in d en a tu ra t io n w as fo u n d . A p ilo t -sca l eco n t in u o u s i so l a t i o n o f t h e i n t r ace l lu l a r p ro t e in an d /3-g a l ac to s id ase f ro m E. coli b y d i s ru p t io n in a h ig h -p res su reh o m o g e n i z e r (Figure 6 ) , w i th f l o w ra t e s o f u p to 0 . 2 8 m 3h -1 a t an opera t ing p ress u re o f 54 MPa, was r ep or te d ) 6,21Firs t -o rder d isrup t ion k inet ics were again observed . Usingth e sam e eq u ip m e n t , s ev en in t r ace l lul a r en zy m es o f b ak e r s '

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Number of passes ( N )Figure 5 Release of/3-galactosidase fro m E. c o l i M L 3 0 8 , g r o w n o nd e f in e d a n d c o m p l e x m e d i a, b y d i s r u p t io n i n a M a n t o n - G a u l i nAP V h o m o g e n i ze r a t ~ 2 0 M Pa . [Re d r a wn f r o m G r a y , P . P . , Du n n i l l ,P . a n d L i l l y , M . D . i n Fe rme n ta t io n Te ch n o lo g y To d a y ( Te r u i , G . ,e d . ) 1 9 7 2 , p . 3 4 7 b y p e r m i ss i o n o f Th e So c i e ty o f Fe r m e n ta t i o nTe ch n o l o g y , Ja pa n ]

196 Enzyme Mic rob . Techno l . 1986, vo l. 8 , Apr i l


4/11

y eas t w ere r e l eased . ~4 T h e r e l ease o f an en zy m e r e l a t iv e t oth e o v e ra l l p ro t e in r e l ease w as i n d ep e n d en t o f t he d i s ru p -t i o n p res su re , t h e t em p era tu re a n d th e i n it i a l y eas t co n cen -t r a t i o n . A t d i s ru p t io n t em p era tu res o f 3 0 C o r l e s s , n o l os so f en zy m e ac t i v i t y o ccu r r ed . D ep en d in g o n t h e l o ca t i o n o fth e en zy m e ac t i v i t y i n t h e ce l l , t h e e n zy m e re l ease w asfas t e r t h an , eq u a l t o , o r s l o w er t h an , t h e o v e ra l l p ro t e inrelease (Figure 7).

T h e co n t in u o u s i so l a t i o n o f an i n t r ace l l u la r a l i p h a ti cam id ase f ro m Pseudomonas aeruginosa b y ce l l d i s ru p t io nin a h ig h -p res su re h o m o g en iz e r w as a l so d esc r ib ed . 1 A l is to f 2 1 l a rge - scal e en zy m e p u r i f i ca t i o n p ro ced u res 29 m en t io n sp ro cesses w h ich u t i l i ze h ig h -p res su re M an to n -G au l inh o m o g en ize r s t o r e l ease en zy m es f ro m th e so u rce ce l l .T h e d i s ru p t io n o f ce ll s to a s t a t e w h ere m e m b r an e -b o u n den zy m es a r e r e l eased h as b een r e p o r t ed 2 t o b e m o red i f fi c u lt . F o r e x a m p l e , m e m b r a n e - b o u n d e n z y m e c y t o -ch ro m e o x id ase f ro m P. aeruginosa r eq u i r ed t h ree p as ses a t5 5 M P a in a h ig h -p res su re h o m o g en ize r w h ereas u n b o u n din t r ace l l u la r en zy m es co u ld n o rm a l ly b e r e l eased b y a s in gl ep ass a t t h e sam e p res su re . 2 E x p er i en ce g a in ed f ro m ce l ld i s ru p t io n i n h ig h -p res su re h o m o g en ize r s fo r i so l a t i o n o fin t r ace l l u la r m a te r i a l , an d o th e r ca re fu l w o rk 3 '3 a sh o w th a tm o s t f r ee en zy m es a r e n o t su scep t ib l e t o sh ea r d am ag e toan y s ig n if i can t d eg ree in t h e ab sen ce o f g as - l i q u id i n t e r -f aces . T h u s , l o s ses d u r in g n o rm a l ce l l d i s ru p t io n m a y b ereg a rd ed a s n eg l ig ib l e , z3 a l t h o u g h i t m u s t b e ap p rec i a t edth a t o n ce t h e i n t eg r i t y o f t h e ce l l en v i ro n m en t i s d es t ro y ed ,th e en zy m e m ay b e su scep t ib l e t o d eg rad a t io n o r i n ac t i v a -t i o n b y a n u m b er o f o th e r f ac to r s su ch a s p ro t eases . 2~M e m b r a n e - b o u n d e n z y m e s a n d m u l t i e n z y m e c o m p l e x e s ,o n t h e o th e r h an d , m ay b e m o re sh ea r s en s i t i v e an d l e s ssu i tab le fo r i so la t ion in a h igh-p ressu re f low dev ice. Fore x a m p l e , i s o la t i on o f a l k an e h y d r o x y l a s e , a m u l t i e n z y m em e m b r a n e - a s s o c i a t e d c o m p l e x , b y h o m o g e n i z a t i o n o fPseu d o mo n a s p u t i d a i s r ep o r t ed t o h av e b ad ly d am ag edth e en zy m e . 32A l th o u g h , i n ad d i t i o n t o y e as t s an d b ac t e r i a l ce l l d i s ru p-t i o n , t h e h ig h -pres su re h o m o g en ize r h as b een u sed fo r t h ed i s in t eg ra t io n o f m y ce l i a l o rg an i sm s su ch a s Aspergillusnig er , 4,26'33 i t h as n o t b een fo u n d v e ry su i t ab l e fo r th i sp u rp o se . P ro b l em s w ere en co u n te red 26 w i th Aspergillus sp .

A B C D E F

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- t ]Figure 6 F l o w - s h e e t f o r t h e c o n t i n u o u s i s o l a t i o n o f ~ < j a l a c t o -s i d a s e f r o m E . coli M L 3 O 8 . A , C o n t i n u o u s f l o w m e d i a s t e ri li z e r;B , l m 3 f e r m e n t e r ; C a n d F , h e a t e x c h a n g e r s ; D , c e n t r i f u g e ; E ,M a n t o n - - G a u l i n A P V K R 3 h o m o g e n i z e r ; G a nd I , m i x i n g c h a m b e r s ;H , r o t a r y v a c u u m f i l t e r ; J , c e n tr i fu g e . [ R e d r a w n f r o m G r a y , P . P . ,D u n n i l l , P . a n d L i l l y , M . D . in Fermentation Technology TodayT e r u i , G . , e d .) 1 9 7 2 , p . 3 4 7 b y p e r m i s si o n o f T h e S o c i e t y o f F e r -m e n t a t i o n T e c h n o l o g y , J a p an ]

Disru pt ion o f m icrob ia l ce ll s : Y . Ch is t i and M, M oo- Young

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F i g u r e 7 Enzyme re lease vs. prote in re lease for : A , acid phos-phatase; B, inver tase; C, G6PDH and 6PGDH; D, ADH; E, a lka l inephospha tase ; F , fumarase . [Redrawn f r om Fo l lows, M. , He the r ing ton ,P . J ., D unn i l l , P . and L i l l y , M . D . Biotechnol. Bioen9. 1971, 13,549 by permission of John Wiley & S ons Inc. ]

due to the b lockage o f the homogen izing valve. S imilarp ro b lem s h av e b een r ep o r t ed b y Z e t e l ak i 33 w i th A. nigerw h ere , d esp i t e h ig h ly d i lu t ed ce l l su spen s io n s , sm a l l m y ce l i a lpel le ts gave r ise to c logg ing o f the homogen izer valve. Inad d i t i o n , i n su f f i c i en t ly h ig h o p e ra t i n g p res su res h av e b eenfoun d to b e avai lab le ~6 fo r s ing le-pass d isrup t io n o f cer ta inf i l am en to u s fu n g i , a l t h o u g h m u l t i p l e p as ses a t l o w er p res -su res g en e ra l l y g av e sa t i s f ac to ry r e su l ts .

L a r ge M a n t o n - G a u l i n A P V h o m o g e n i z e rs c a p a b l e o fp rocess ing up to 53 m 3 h - t a t 55 .2 M Pa are avai lab le . 26O th e r h ig h sh ea r ce l l d i s ru p t io n eq u ip m en t su ch a s t h eex t r em e p res su re p u m p ~ an d th e F ren ch p res s , 3 s w h icho p era t e o n a s im i l a r p r in c ipl e as t h e M a n to n -G au l i n A P Vh o m o g en ize r h av e b een d esc r ib ed , b u t h av e n o t g a in edin d u s t r i a l accep t an ce .T h e m ech an i sm o f ce l l d i s ru p t io n i n h ig h -p res su re f l o wd ev ices , su ch a s t h e A P V M an to n -G au l in h o m o g en ize r ,has be en the sub jec t o f mu ch at te n t ion . ~v 'lg,27'34,3sA ca re fu l s t u d y iv o f p r e s su re v a r i a t i o n an d v a lv e m o v e -

m e n t i n a M a n t o n - G a u l i n A P V h o m o g e n i z e r , u s e d f o rd i s r up t i on o f b a k e r s ' y e a s t , w as c o n d u c t e d b y B r o o k m a nw i th t h e a im o f e s t ab l ish in g a m ech a n i sm fo r ce ll d i sru p -t i o n . T u rb u len ce , a s a p o ss ib l e cau se o f d i s ru p t io n , w asd i sco u n ted s in ce v a r i a t i o n s i n ce l l co n cen t r a t i o n (1 0 -8 0 %w /v ) w ere fo u n d to h av e n o s ig ni f i can t e f f ec t o n d i s ru p t io nra t e a n d , a l so , b ecau se t h e m e an su sp en s io n v e lo c i t yth ro u g h th e v a lv e r em a in ed co n s t an t o v e r a w id e p res su rer a ng e ( 3 5 - 1 0 0 M P a ) . I n c o n t r a s t t o H e t h e r i n g t o n' s e q u a t i o n(equa t ion 1 ) , the p ressu re d rop across the valve, no t theo p e ra t i n g p res su re , w as su g g es t ed a s t h e m a in f ac to r i n ce l ld i s ru p t io n . ~7 F u r th e r ex p e r im e n t s 3 s su p p o r t e d t h i s co n c lu -s ion , and the in f luence on cel l d isrup t ion o f the ra te a tw h ich t h e p res su re d ro p o ccu r s w as r ev ea l ed . 3 s T h u s ,m ea su rem en t s m ad e w i th t h e v a lve r ep l aced b y v a r io u sl en g th s o f h y p o d erm ic n eed l es sh o w ed th a t , a t co n s t an to p e ra t i o n a l p r e s su re (1 2 4 M P a) , p ro t e in r e l ease d ec l i n edw i th i n c reas in g n eed l e l en g th (Figure 8). The m o r e r a p i dp res su re d ro p m a y a l so b e t h e ex p la n a t io n fo r t h e o b se rv ed ~3

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Tube length (ram)Figu re 8 P lo t o f pro te in re lease vs . tube len gth a t con s tan t pressu rep e a k w i t h 6 0 % w / w y e a s t s u s p e ns i on a t p H 7 . 4 . [ R e d r a w n f r o mB r o o k m a n , J . S . G . Bio t echno l . B ioen9 . 1 9 7 5 , 1 7 , 4 6 5 b y p e r m is -s i on o f J ohn W i l ey & S ons I nc . ]b e t t e r p e r fo rm an ce o f t h e ' k n i f e - ed g e ' v a lve sea t re l a t i ve t othe f la t type seat in cel l d isrup t ion .

A t t em p t s h av e b een m ad e to ex p la in ce l l d i s ru p t io n o nth e b as i s o f th e h y d ro d y n am ics o f f l o w in t h e v a lv e , 9F ro m a co n s id e ra t i o n o f K o lm o g o ro fP s t h eo ry , 36 D o u lahan d co w o rk e r s 19 d e r iv ed t h e t h eo re t i ca l ex p res s io n :Sp = 1 - - exp { - - [ (P - -P 0 ] /c~] ~ } (2 )

wher e P o , c~ and /3 are cons tan ts , P i s the opera t ing p ressu re ,and Sp is the so lub le p r o tei n re lease . The values o f t3 werefo u n d to d ep en d o n cel l co n ce n t r a t i o n o f S . c e r e v i s i a e ,t h u s co n t r ad i c t i n g sev e ra l p r ev io u s r ep o r t s ( r e f e r en ces 1 3 ,1 4 , 1 7 an d 2 7 fo r ex am p le ) t h a t ce l l d i s ru p t io n w as i n d e -p en d e n t o f co n cen t r a t i o n . V ar i a t i o n o f su sp en s io n v i sco s i t yw i th ce l l co n cen t r a t i o n an d co n seq u en t v a r i a t i o n i n t u rb u -l en ce i n t en s i t y i s t h e ex p lan a t io n t h ey ad v an c ed fo r t h e i ro b se rv ed co n ce n t r a t i o n d e p en d en ce o f d is ru p t io n .

In an o th e r s t u d y o f th e m ech a n i sm o f cel l d i s ru p t io nin h igh-p ressu re f low dev ices ,z7 ex p e r im en t s w ere co n d u c-t ed i n a h ig h p res su re i n t en s i fi e r cap ab le o f p ro d u c in gp res su res o f u p t o 3 0 0 M P a w h ich co u ld b e eq u ip p ed w i thv a r io u s o u t l e t n o zz l e s . T h e eq u ip m en t h an d led sm a l lv o lu m es (1 5 0 m l ) o f ce ll su sp en s ion i n b a t ch es . A n 8 0 # mi . d . o r i f i ce , w i th an d w i th o u t an im p in g em en t p l a t e , w asu sed to s tu d y th e e f f ec t o f im p in g e m en t o n ce ll r u p tu re ,w h i l e t h e e f f e c t o f sh ea r o n d i s ru p t io n w as i n v es t i g a t edb y r ep l ac in g t h e o r i fi ce w i th cap i l la r i e s o f 1 0 2 o r 1 5 2 /~ mi . d . w i th l en g th s v a ry in g f ro m 0 . 0 2 6 to 0 . 1 5 0 m . C a n d i d aut i l i s ce ll s g ro w n o n a fu l l y d e f in ed m ed iu m w ere u sed i n a l lt h e ex p e r im en t s . S u sp en s io n co n cen t r a t i o n s r an g ed f ro m 9 3to 1 4 8 k g d ry w t n 3 an d co n cen t r a t i o n w as fo u n d to h av en o s ig n i f i can t e f f ec t o n d i s ru p t io n . T h e i n v es t i g a to r sc o n c l u d e d t h a t : ' I m p i n g e m e n t o f a h i g h ve l o c i ty j e t o fsu sp en d ed ce i l s o n a s t a t i o n a ry su r f ace i s n ecessa ry fo reffect ive d isrup t ion o f cel l wal ls in h igh-p ressu re f low

dev ices ' , and that : 27 'Th e rap id re lease o f p ressu re i s no t ,b y i t s e l f , an e f f ec t i v e m ean s o f d i s ru p t in g t h e m ic ro b i a lcel l wal ls ' . I t shou ld be recogn ized , however , that in ad e vi ce s u c h a s th e M a n t o n - G a u l i n A P V h o m o g e n i z e r t h eim p in g em en t s t r e s s i s p ro b ab ly d i r ec t l y p ro p o r t i o n a l t oth e o p e ra t i n g p res su re .U l t r a so n ica t i o n . U l t r a so n ica t i o n i s an o th e r l i q u id - sh ea r

m e th o d o f d i s ru p t io n w h ich h as r ece iv ed so m e a t t en t i o n i nth e l i t e r a tu re , s7-~9 U l t r a so u n d , so u n d o f f r eq u e n cy h ig h e rth an 1 5 -2 0 k H z w h ich i s i n aud ib l e t o t h e h u m an ea r , 7i s k n o w n to cau se b o th i n ac t i v a t i o n 38 an d , a t h ig h e r aco u s t i cp o w e r i n p u t s , d i s ru p t io n o f m ic ro b i a l ce ll s i n su sp en s ion .

T h e p ro t e in r e l ease co n s t an t fo r b r ew er s ' y eas t so n i ca t eda t p o w er s u p t o 2 0 0 aco u s t i c w a t t s a t 2 0 k H z h as b eensh o w n to b e i n d ep en d en t o f ce l l co n cen t r a t i o n u p t o v a lu eso f 6 0 0 k g w e t w t m 3 an d ah n o s t p r o p o r t i o n a l t o t h e i n p u ta c o u s ti c p o w e r f r o m 6 0 - 1 9 5 a c o u s ti c w a t ts . 4 T h e m e c h a n -i sm o f ce l l d i s ru p t io n b y u l t r a so u n d i s p ro b ab ly a s so c i a t edw i th t h e cav i t a t i o n p h e n o m en a a s so c i a t ed w i th u l t r a -so n i ca t i o n . A t su f f i c i en t ly h ig h aco u s t i c p o w er i n p u t s ,m ic ro b u b b les fo rm a t v a r io u s n u c l ea t i o n s i t e s i n t h e f l u id .T h ese b u b b les g ro w d u r in g t h e r a r e f ac t i o n p h ase o f t h eso u n d w av e , an d th en , i n t h e co m p ress io n p h ase , th e b u b b leco n te n t i s co m p re ssed t o a m in im u m rad ius w h ere t h eb u b b le co l l ap ses , r e l eas in g a v io l en t sh o ck w av e w h ichp ro p ag a t e s t h ro u g h th e m ed iu m . T h i s fo rm a t io n , g ro w than d co l l ap se o f g as an d v a p o u r b u b b les o w in g to t h e ac t i o no f i n t en se so u n d w av es is t e rm ed cav i t a t i o n . In th e co l l ap sep h ase o f cav i t a t i o n o f b u b b les , a l a rge q u an t i t y o f so n i cen e rg y i s co n v e r t ed t o m ech an ica l en e rg y in t h e fo rm o fe l a s t i c w av es . T h e d i s s ip a t i o n o f t h i s m ech an ica l en e rg yin t h e f l u id su sp en d ing th e ce l l h as b een ex p la in ed b yD o u lah 39 u s in g K o lm o g o ro f f ' s3 6 t h eo ry o f l o ca l i so t ro p i ctu rbu lence. Accord ing to Dou lah the f lu id edd ies smal lerth an t h e d im en s io n s o f a ce ll w il l im p a r t m o t io n s o f v a r io u sin t en s it i e s t o i t , an d w h en th e k in e t i c en e rg y co n ten t o f acel l exceeds the wal l s t reng th , the cel l d is in teg rates . Forth e d i s ru p t io n o f b rew er s ' y eas t ce l ls , th e t h eo re t i ca lanalysis s9 leads to th e express ion :

1 - - S p = ex p - - (k t ) ( 3 )fo r a g iv en aco u s t i c p o w er i n p u t , w h ere S p i s t h e p ro t e inre l ease , k i s t h e p ro t e in r e l ease co n s t an t (m in -~ ) , an d ti s the e xposure t ime ( ra in ) . Th is express ion is iden t ical tot h a t d e t e r m i n e d e x p e r i m e n t a l l y b y a n i n d e p e n d e n t g r o u p 4 fo r p ro t e in r e l ease f ro m a b rew er s ' y eas t su sp ens io n (2 0 0 k gw e t w t n f 3 o f su sp en s io n ) u s in g a s e l f - t un in g 2 0 k H zg en era to r w i th a cap ac i ty o f 1 9 0 aco u s t i c w a t t s . T h ed ep en d en ce o f t h e p ro t e in r e l ease co n s t an t k o n aco u s t i cp o w er i n p u t w as e s t ab l i sh ed s9 t o b e an ex p o n e n t i a l o n e ,k = b ( p - p o ) -9 ( 4 )whe re b i s a con stan t , p i s the pow er inpu t (J kg ~ s ~ ) andp o i s t h e cav i t a t i o n t h re sh o ld p o w er . 39

T h e ab so rp t io n o f so n ic en e rg y b y a f l u id is d ep en d en to n th e p rev a il i n g pres su re , an d an i n c rease in t h e am b ien tp res su re u p t o a ce r t a in l im i t i n c reases t h e co n v er s io n o fso u n d en e rg y to sh o ck w av e en e rg y . 41 T h e co n s t an t , k ,i s t h u s p res su re d ep en d en t . T h i s i s co n f i rm ed b y th e o b se r -vat ion th at an increase in the a mb ien t p ressu re on cel l sus-p en s io n s d o es i n d eed l ead t o e n h an ced ce ll d i s in t eg ra ti o n , s7A l th o u g h u l t r a so n ic d i s in t eg ra to rs cap ab le o f h an d l in gu p to ab o u t 1 0 1 h - ' o f ce ll su spen s io n w ere av a i lab l e a s f a rba ck as 1 967 , 4z and large-scale u l t rason ic homo gen iz ers arein u se i n t h e ch em ica l in d u s t ry fo r co n t in u o u s h o m o g en iza -

198 Enzyme Microb, Technol. 1986, vol. 8, Apri l


6/11

t i o n o f l i q u id - l i q u id d i sp e r sio n s an d fo r b reak in g d o w nag g lo m era t e s o f p o w d ered p a r t i c l e s , 43 th e u se o f t h i st ech n iq u e fo r i n du s t r ia l - sca le ce l l d i s ru p t io n i s n o t p rac t i sedto our knowledge. There are several reasons fo r th is . Mosto f t h e u l t r a so n ica t io n en e rg y ab so rb ed in to su sp en s io n su l t im a te ly ap p ea r s a s h ea t , an d g o o d t em p era tu re co n t ro l i sn ecessa ry . A l so , t h e p ro cess o f u l t r a so n ica t io n i s k n o w n tog iv e r i s e t o ch em ica l e f f ec t s 39 su ch a s t h e fo rm a t io n o f f r eerad i ca ls . T h e l a t t e r , o r u l t r a so u n d i t s e l f , m ay h av e d am ag in gin f lu en ce o n so m e o f t h e m o lecu les o f i n t e re s t . P ro d u c t io no f v e ry f i n e ce l l d eb r is 4 o cc u r s an d can l ead to su b seq u e n tp ro cess in g p ro b lem s . T h e d i f f i cu l ty o f t r an sm i t t i n g su f f i c i en tp o w e r t o l a rg e v o lu m es o f ce l l m a te r i a l h as b een m en -t io n ed ; 26 in ad d i t i o n , t h i s m e th o d o f ce l l d i s ru p tio n seem sless effect ive fo r fungal o rgan isms such as A. niger. 33S o l i d s h ea r m e t h o d s

The bead mil l . Cel l d isrup t ion in bead mil ls i s regarded aso n e o f t h e m o s t e f f i c i en t t ech n iq u es fo r p h y s i ca l ce ll d i s-rup t io n .23 Vario us designs o f b ead mil ls have bee n used fo rm ic ro b ia l ce l l d i s ru p t io n , l s ' 3 3 ' ~ - s s T h ese m i l l s co n s is t o fe i t h e r a v e r t i ca l o r a h o r i zo n ta l cy l in d r i ca l ch am b er w i th am o to r -d r iv en cen t r a l sh a f t su p p o r t in g a co l l ec t io n o f o f f -cen t r ed d i scs o r o th e r ag i t a t i n g e l em en t (Figures 9 an d 10) .The chamber is f i l led to the desi red level wi th s teel o rbal lo t in i g lass beads which p rov ide the g r ind ing act ion . Thech a rg e o f g r in d in g b ead s i s r e t a in ed in t h e ch am b er b y as i ev e -p l a t e co v e r in g th e b o t to m in l e t i n v e r t i ca l m ach in es(Figure 9), w h i l e i n h o r i zo n ta l u n i t s t h e f l u id en t ry i s ab o v eth e l ev e l o f t h e b ead s i n t h e ch am b er an d n o r e t en t io nm ech an i sm i s r eq u i r ed (Figure 10). A t th e f l u id ex i t p o r t ,t h ree d i f f e r en t t y p e s o f b ead r e t en t io n sy s t em s h av e b eenem p lo y ed : a s i ev e -pl a t e , a d i sc ro t a t i n g in v e ry c lo se p ro x -im i ty t o a p l a t e w i th a cen t r a l ex i t p o r t i n i t (Figure 10) san d a v ib ra t in g s lo t . s3 's6 T h e l a t t e r tw o ty p es o f b eadreten t ion dev ices are said to reduce fou l ing p rob le ms . Is 's3T h e h o r i zo n ta l co n f ig u ra t io n o f t h e m i l l is k n o w n to g iv e ab e t t e r e f f i c i en cy o f d is ru p t io n r e l a t i v e t o t h e v e r t i ca l o n e . ~T h i s i s b ecau se th e u p w ard f lu id f l o w in v e r t i ca l m ach in es

B

FG

GH

I

F i g u r e 9 S i m p l i f i e d d r a w i n g o f t h e N e t z s c h - - M o l i n e x K E 5 a g i t a t o rm i l l s y s t e m : A , d r i v e m o t o r ; B , v a r ia b l e V - b e l t d r i v e ; C , b e a r in g s ;D , a g i t a t o r c o u p l i n g ; E , c y l i n d r i c a l s i e v e p l a t e ; F , a g i t a t o r d i s c s; G ,t e m p e r a t u r e j a c k e t i n l e t a n d o u t l e t ; H , b o t t o m s i e ve p l a te ; I , t e m -p e ra t u re m e a s u r i n g p o c k e t ; J , r e c i r c u l a t i n g p u m p . [ R e d r a w n f r o mC u r r i e , J . A . , D u n n i l l , P . a n d L i l l y , M . D . Biotechnol. Bioeng. 1 9 7 2 ,1 4 , 7 2 5 b y p e r m i s s i o n o f J o h n W i l e y & S o n s I n c . ]

Disrup t ion o f m icrob ia l ce l ls : Y . Ch is t i and M. M oo- Young

.__~11~____~ B ea rin g eals ] [ - - ~ J - Co o l an t

F i g u r e 1 0 Th e D yn e M i l l . Th e f e e d i n l e t a n d o u t l e t s a re a b o vet a n k l e ve l a n d t h e ro t a ~ i n g d isc i s u se d t o re t a i n b e a d s . [ R e d ra w nf ro m W a n g , D . I . C . , C o o n e y , C . L . , D e m a i n , A . L . , D u n n i l l , P . ,H u m p h r e y , A . E . a n d L i l l y , M . D . Fermentation and EnzymeTechnology J o h n W i l ey , N e w Y o r k , 1 9 7 9 , p p. 2 3 8 - 3 1 0 b y p e r-m i s s io n o f J o h n W i l ey & S o ns I n c . ]t en d s t o f l u idi ze t h e g r in d in g b ead s t o so m e d eg ree , t h e reb yred u c in g g r in d in g e f f i c i en cy .

S ch em es fo r i so l a t i o n an d p u r i f i ca t io n o f t h e i n t r a -ce l lu l a r fo rm a te d eh y d ro g en ase f ro m C. boidinii usingd isrup t io n o f the ceils in bea d mil ls have been pub l ished , s~a n d a c o n t i n u o u s f o o d - f e e d p r o t e i n p r o d u c t i o n p r o c e s sin v o lvin g d is ru p t io n o f 1 0% d ry w e ig h t b ak e r s ' y eas t an db rew e rs ' y eas t s lu rr i e s i n a 5 l i tr e n o m in a l cap a c i ty b eadm i l l h as b een r ep o r t ed . 49 O th e r s im i l a r p ro cesses h av e b eend iscussed in the l i tera tu re .48 'sT h e k in e t i c s o f p ro t e in r e l ease f ro m b ak e r s ' y eas t b y

d i s ru p t io n in a h ig h - sp eed b a l l m i l l h av e b een in v es t ig a t edb y D u n n i l l an d L i l ly i s an d d ep en d o n th e co n s t ru c t io n o fth e m i l l . F i rs t -o rd e r d i s ru p t io n k in e t i c s h av e b een o b se rv edin m ach in es w i th p red o m in an t p lu g f lo w , w h ereas i nm ach in es i n w h ich th e ro to r d es ig n p e rm i t s s ig n i f i can tb ack m ix in g th e d i s ru p t io n d ev ia t e s f ro m th e f i r s t -o rd e rb eh av io u r , i s A n u m b er o f o th e r au th o r s h av e a lso r ep o r t e df i r s t -o rd e r d i s ru p t io n in b ead mills , sl"s2's4 F o r e x a m p l e , at eam o f i n v es t ig a to rs s4 e x am in e d b a t c h an d co n t in u o u sd i s ru p t io n o f b ak e r s ' y eas t i n l ab o ra to ry an d p i lo t s ca l em i l l s (0 . 6 an d 5 l i t re d i s ru p t io n c h am b er v o lu m es )a n do b se rv ed f i r s t -o rd e r k in e t i c s i n t h e b a t ch o p e ra t io n , w h ereth e r a t e o f p ro t e in r e l ease w as d i r ec t ly p ro p o r t io n a l t oth e am o u n t o f u n re leased p ro t e in :

d R / d t = k ( R m - - R ) ( 5)w h ere R i s t h e w e ig h t o f p ro t e in r e l eased p e r u n i t w e ig h t o fp a c k e d y e a s t , R m is t h e m a x i m u m m e a s u r e d p r o t e i n r e l ea s e(0 . 1 0 0 + 0 . 0 0 5 k g p ro t e in r e l eased k g 1 p ack ed y eas t ) .In t eg ra t io n o f eq u a t io n (5 ) fo r t = 0 an d t = t (b a t ch t im e)y ields:

in [ R m / ( R m - - R ) ] = l n D = k t (6 )w h ere D i s t h e r ec ip ro ca l o f t h e f r ac t io n o f u n re l easedp ro t e in .F o r co n t in u o u s d i s ru p t io n , f i r s t -o rd e r k in e t i c s h e ld , an dD co u ld b e r e l a t ed to t h e n a tu re o f m ix in g in t h e m i l lex p ressed in t e rm s o f a co n t in u o u s s t i r r ed t an k r eac to r(CST R) in -ser ies mode l , s4 thus:

D = R m / ( R m - - R ) = [1 + (k 'c /] ) ] (7 )

Enzyme Microb. Technol. 1986, vol. 8, Apri l 199


7/11

Revieww h ere r i s t h e m e an r e s id en ce t im e in t h e m iU ( to t a l v o lu m eo f t h e m i l l , V , d iv ided b y t h e t o t a l t h ro u g h p u t q ) an d j ist h e n u m b er o f C S T R s in se r i e s ; t h i s m ay in c lu d e f r ac t i o n so f C S TR s .S 4 T h e v a lu es o f / ' w ere o b t a in e d ex p e r im en ta l l yf ro m res iden ce t im e d i s t r i b u t io n s tu d i e s .T h e d i s ru p t io n r a t e co n s t an t , k , i s k n o w n to b e a fu n c -t i o n o f s ev e ra l p a ram e te r s : t em p era tu re , im p e l l e r ro t a t i o n a lspee d , l s ' s l -ss bead load ing , XS,s3,ss bead s ize l s ' s l ' ss and cel lconcentration.XS'SSThere i s ev idence s4 tha t , wi th in l im i ts , the d isru p t ionra t e co n s t an t , k , i n c reases w i th t h e ag i t a to r t i p sp eed , U t .T h i s v a r i a t i o n h as b een d esc r ib ed s4 b y :

k = K U (8 )(w h ere K = 0 . 0 0 3 6 m -1 fo r a 0 . 6 1 i t r e m i l l ) . A n o th e rr ep o r t s s n o t ed t h a t p ro t e in r e l ease f ro m b ak e r s ' y eas ti n c reased w i th ag i t a to r t i p sp eed to a l im i t (Figure 11)b ey o n d w h ich n o fu r th e r i n c rease o ccu r r ed . E x cess iv e lyh ig h ag i t a to r sp eeds can l ead t o s ign i f i cant f r ag m en ta t i o n o fg la ss b ead s . 4 S u ch h ig h ag i t a to r sp eed s h av e n o t , h o w ev e r ,b een fo u n d n ecessa ry . A l th o u g h in c reased ag i t a to r sp eed sin c rease d i s ru p t io n , t h e am o u n t o f h ea t g en e ra t ed an d th ep o w er co n su m ed a l so i n c rease .T h e d i s ru p t io n e f f i c i en cy , E , d e f in ed s4 a s :

E = R C q / P ' (9 )w h ere R i s t h e a m o u n t o f p ro t e in r e l eased p e r kg p ack e dy eas t , q t h e t h ro u g h p u t , C th e y eas t co n ce n t r a t i o n , an dP ' t h e p o w er co n su m p t io n fo r d is ru p t io n i n t h e 5 l i t re m i l l,i s shown in Figure 12 . F r o m t h is p l o t a n o p t i m u m e f f i c ie n c ycan b e seen t o ex i s t f o r a g iv en th ro u g h p u t an d p ro t e inre l ease . A t a co n s t an t im p e l l e r t i p sp eed , t h e e f f i c i en cyd ec l in es w i th t h ro u g h p u t . T h i s can b e ex p la in ed in t h efo l l o w in g w ay : an i n c rease i n t h e l o n g i tu d in a l d i sp e r s io no r b ack f io w , an d a co n seq u en t d ec rease i n t h e n u m b ero f e f f ec t i v e C S T R s in t h e m i l l , w i l l ( eq u a t io n 7 ) lead t o ad ec rease i n t h e y i e ld o f t h e m i l l an d , h en ce , a d ec l i n e i ni t s e f f i c i en cy , s4 T h u s , w h ereas t h e d i s ru p t io n r a t e co n s t an tt en d s t o a m ax i m u m v a lu e w i th i n c reas in g ag i t a to r t i psp eed ( eq u a t io n 8 ) , t h e d eg ree o f d i sp e r s io n a l so i n c reases .T h e tw o co n f l i c t i n g e f f ec t s r e su l t i n t h e ex i s t en ce o f ano p t im u m d i s ru p t io n e f f i c i en cy . E q u a t io n (7 ) a l so sh o w s th ein f lu en ce o f m ean r e s iden ce t im e o f t h e ce l l s i n t h e g r in d ingch am b er o n d i s ru p t io n . D i s ru p t io n d ec l in es w i th i n c reasedf lo w ra t e o f t h e ce l l s l u r ry t h ro u g h th e m ach in e , an o b se r -vat ion a lso re por t ed by several o thers . 46 ,s l 's3 ,ss T he in f lu -

E 20

~05 6 7 8 9 IO

Tip speed (m s I)F i gu re 11 E f f ec t o f ag i t a t o r s peed on p ro te i n re l eas e . The ra teo f p ro te i n rel eas e a t d i f f e r en t ag i t a t o r t i p s peeds a t bead loadv o l u m e : ~ , 7 0 % ; ~ , 8 0 % ; o , 8 5 % . C o n s t a n t p a r a m e t e r s: b e ad d i a m e t e r ,0 . 55 - -0 .85 m m; f l ow ra te o f c e l l s l u r r y , 100 I h - I ; c e l l s us pens i on ,4 0 % ( w / v ) . [ R e d r a w n f r o m S c h d t t e , H . , K r o n e r , K . H ., H u s te d t , H .a n d K u l a , M . - R , Enzyme Microb. Technol . 1 9 8 3 , 5 , 1 4 3 ]

~c

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0.04 0.06 0.08 0.10/~ ( kg protein rele ese d/kg pocked yeast)

F i g u r e 12 E f f i c i enc y o f a bead m i l l a t v a r i ous f l ow ra tes andi mpe l l e r t i p s peeds . F l ow ra te (m 3 s 1 ) : m ,20 X 10 -6 ;A ,5 0 X 10 -6 ;o , 1 00 X 1 0 6 . Im p e l le r t ip s p ee d : 1 , 8 m s - ~ ; 2 , 1 0 m s - 1 ; 3 , 1 5 ms - l ; 4 , 2 0 m s 2 . [ R e d r a w n f r o m L i m o n - L a s o n , J. , H o a r e , M . ,O r s b o r n , C . B . , D o y l e , D . J . a n d D u n n i l l , P . Biotechnol. Bioeng.1 9 7 9 , 2 1 , 7 4 5 b y p e r m i ss i o n o f J o h n W i l e y & S o n s I n c . ]

ence o f f lo w rate , q (m 3 h 1 ) , on the degree o f d is in teg ra-t ion , x (% ), has bee n desc r ibed s2 accor d ing to :

l o g [1 0 0 / (1 0 0 - x ) ] = k /q (1 0 )where k i s a f i rs t -o rder d isrup t ion ra te constan t . The f ind ingth a t i n c reased f l o w ra t e o f t h e ce ll su sp en s io n t h ro u g h th em i l l r ed u ces t h e d eg ree o f d i s in t eg ra t i o n w as co n f i rm e db y o th e r ex p e r im en te r s , s3 F o r 1 5% ce l l s lu r ri e s ab o u t 9 0 %d i s ru p t io n o f S. cerevisiae h as b een r ep o r t ed s3 a t a r e s id en cet im e o f 2 . 5 m in i n a 2 0 li t r e n o m in a l ca p ac i ty m i l l . U n d erth e sam e co n d i t i o n s , 8 4 % o f C. utilis co u ld b e b ro k en .T h i s d i f f e r en ce i n t h e d eg ree o f d i s ru p t io n o f th e tw oo rg an i sm s is p a r t l y d u e t o t h e i r d i f f e r en t ce l l s i zes , b u tm a in ly b ecau se o f t h e h ig h e r m ech an ica l s t r en g th o f th ecel l wal l o f C. utilis. In a s im i l a r w ay , i n an o th e r r ep o r t s2C. utilis w as fo u n d to b e m o re r e s i s t an t t o d i s ru p t io nc o m p a r e d t o S. cerevisiae.

In co n t r a s t t o ce l l d i s ru p t io n i n th e h ig h -p res su re h o m o -g en ize r , D u n n il l an d L i l ly i s fo u n d th a t t h e d i s ru p t io n r a t ein h ig h - sp eed b a l l m i ll s i s d ep en d en t o n y eas t co n cen t r a t i o nb e tw e en 3 0 an d 6 0 % p ack ed w e ig h t p e r v o lu m e an d d e -c reases w i th i n c reas in g ce l l co n cen t r a t i o n . C o n t r ad i c t i n gth i s a r e o th e r o b se rv a t io n s s2 w h ich i n d i ca t e n o i n fu e n ceo f y e a s t c e ll c o n c e n t r a ti o n ( 4 - 2 0 % d r y w e i g h t r a n ge ) o nd i s ru p t io n r a t e .

The bead d iameter and bead load ing in the g r ind ingch am b er o f t h e m i l l a r e o th e r f ac to r s w h ich a f f ec t d i s ru p -t i o n r a t e c o n s t an t . I n c reased b ead lo ad in g en h an ces t h e r a t eo f d i s ru p t io n , l s 's3 's s In a 2 0 l i t r e N e tzsch L M E 2 0 in d u s t r ia lb ead m i l l , u sed s s fo r t h e r e l ease o f p ro t e in an d so m ee n z y m e s ( a g i ta t o r t i p s p e e d o f 5 . 2 - 1 0 . 6 m s - ~ , s lu r r yf lo w ra t e s o f 5 0 -5 0 0 1 h 1 , ce l l co n cen t r a t i o n s o f 3 0 -5 0 % w / v , 7 0 - 9 0 % g la ss b e a d l o a d i n g , a n d 0 . 4 5 - 1 m mg lass b e ad d i am e te r ) , in c reased b ead lo ad in g s w ere fo u n d toen h an ce t h e r a t e o f p ro t e in r e l ease u p t o a b ead lo ad in g o f80%. F ur the r increase in bead load led to a decl ine inp ro t e in r e l ease . T h is l a t t e r o b se rv a t io n , n o t r e p o r t ed b e fo re ,i s p ro b ab ly sy s t em sp ec i f i c an d a r i s e s d u e t o i n e f f i c i en tag i t a t i o n a t h ig h b ead lo ad in g s , p o ss ib ly d u e t o an i n su f f i -c i en t ly p o w er fu l ag i t a to r m o to r . T em p era tu re i n c reases an dp o w er co n su m p t io n a r e a l so s t ro n g ly i n f lu en ced b y t h ebea d load ing ; s3 ,ss th is i s show n in Figures 13 a n d 14 .

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F i g u r e 1 3 Te m p e r a tu r e ri se d u r i n g d i s r u p t i o n a t b e a d l o a d vo l -u m e s : ~ , 7 0 %; z~, 8 0 %; e , 9 0 %. Co n s ta n t p a r a m e te r s : b e a d d i a m e te r ,0 .5 5 - - 0 .B5 m m ; f l o w r a te , 1 0 0 I h -~ ; ye a s t ce ~ l su sp e n s io n , 4 0 % w ]v .[Re d r a wn f r o m Sch L i t t e , H ., Kr o n e r , K . H . , Hu s te d t , H . a n d Ku l a ,M . - R . E n z y m e M i c r o b , T e c h n o l . 1 9 8 3 , 5 , 1 4 3 ]

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T i p s p e e d (ms l)F i g u r e 1 4 Po we r co n su m p t i o n o f t h e Ne tzsch L M E 2 0 m i l l a tva r i o u s t i p sp e e ds a n d b e a d l o a d vo l u m e s : ~ , 7 0 %; A , 7 5 %; o , 8 5 %.Constan t parameters : as in F i g u r e 1 3 . [Redrawn f rom SchL i t te ,H . , K r o n e r , K . H . , Hu s te d t , H . a n d Ku l a , M . - R . E n z y m e M i c r o b .T e c h n o l . 1 9 8 3 , 5 , 1 4 3 ]

t o R eh ac ek an d S ch ae fe r , s3 i s i n d ep en d en t o f v essel an dag i t a to r co n f ig u ra t io n . M o re r ap id d i s ru p t io n i s ach i ev edw i th sm a l l e r b ead s ; a s h o w ev e r , t h e re i s an o b v io u s l o w erl im i t t o t h e b ea d s i ze d i c t a t ed b y th e b ead f lu id i za t io n an ds iev e b l in d in g p ro b lem s en co u n te re d w i th v e ry sm a ll b ead s ) sCon tr ad ict ing th is is the c la im sl tha t increa sing bead d ia-m e te r a t f i r s t i n c reases t h e p ro t e in r e l ease f ro m S. carls-bergens i s , b u t p ro t e in r e l ease d ec lin es s l ig h t ly w i th a fu r th e rin c rease i n b ead s i ze . T h i s o b se rv a t io n r em a in s u n ex p la in ed .F o r en zy m e re l ease , t h e l o ca t io n o f th e d es i r ed en zy m e inth e ce l l i s o f im p o r t an ce to t h e ch o ice o f b ead d i am e te r an db ead lo ad in g , s~ F o r ex am p le , w h i le c y to p lasm ic en zy m e sl ik e o -g lu co se -6 -p h o sp h a te d eh y d ro g en ase f ro m y eas t ce ll sw ere so lu b i l i zed b es t w i th 0 . 5 5 - -0 . 8 5 m m g las s b ead s , ce l ld i s in t eg ra t io n fo r a -D -g lu co sid ase i so l a ti o n w as b e t t e ra c c o m p l i s h e d w i t h s o m e w h a t l a r g e r b e a d s o f 1 m m d i a m e t e r .T h i s o b se rv a t io n can b e r e l a t ed to t h e f ac t t h a t a - o - g l u c o -s id ase i s l o ca t ed p red o m in an t ly i n t h e p e r ip l a sm ic sp ace

Disrupt ion of m icrobia l cel ls : Y. Chist i and M. Moo-Youngan d c o m p le t e d i s in t eg ra t io n o f t h e ce ll s is n o t n ecessa ry toso lub i l ize th is en zym e . ss

In ad d i t i o n to sev e ra l y eas t s (S. cerevisiae, S. carlsber-gens i s , C . bo id in i i, C . u t i l i s ) , a n u m b er o f b ac t e r i a , i n c lu d in gE. co i l , Bac i l lu s sphaer icus , Lac tobac i l lu s con fusus , Brev i -b a c t e r i u m a mm o n i a g e n e s s s a n d B a c i l l u s s u b t i l i s , 46 haveb een d i s ru p ted in h ig h - sp eed b ead m i l l s . T h e d i sin t eg ra t io no f b ac t e r i a i n a b ead m i l l is h am p ere d b y th e sm a l l d im en -s io n s o f su ch ce l ls , s s w h ich a re ap p r o x im a te ly l / 1 0 th e s izeo f a y e as t ce l l. T h u s , w h i le a 2 0 l i tr e b ea d m i l l w as fo u n dcap ab le o f h an d l in g 2 0 0 k g b ak e r s ' y eas t h - ] w i th > 8 5 %d is in teg ra t io n u n d e r o p t i m u m co n d i t i o n s (1 1 0 0 r ev m in - ] ,a t 1 0 0 l h -~ an d ce ll co n cen t r a t i o n o f 4 0 % w /v ) , o n lyb e tw ee n 1 0 an d 2 0 k g h - a o f b ac t e r i a co u ld b e p ro cessed ,d ep en d in g o n th e o rg an i sm . 5sT h e p e r fo rm an ces o f a 0. 6 l i t r e D y n o -M i l l , a 2 0 l i tr e m i l la n d a M a n t o n - G a u l i n h o m o g e n i z e r f o r t h e r e le a s e o fl eu c in e d eh y d ro g en ase f ro m B . s p h a e r i c u s ( F i g u r e 1 5 ) h av eb een co m p ared , s s W h i le t h e l a rg e r rn il l an d th e h o m o g en iz e rg av e s im i l a r p e r fo rm an ces , t h e sm a l l e r m i l l w h ich u sed0 . 2 5 - O . 5 0 m m b e a d s g a ve s i g ni f ic a n t ly b e t t e r e n z y m ere l ease , s s T h e b e t t e r p e r fo rm an ce o f t h e sm a l l e r m i l l isp ro b a b ly d u e to t h e sm a l l e r b ead s ize u sed in i t.The w ork o f Sch~i tte and h is g roup , ss show n in T a b l e 2 ,

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F i g u r e 1 5 D i s r u p t i o n o f B a c i l l u s s p h a e r i cu s using var ious typeso f h o m o g e n i ze r s . Th e r el ea se o f l e u c in e d e h yd r o g e n a se a c t i v i t y wa sfo l l o w e d fo r t h r e e p a sse s i n : ~ , Dyn o - M i l l Typ e K DL ; o , Ne tzschL M E 2 0 m i l l ; ~ , M a n to n - Ga u l i n h i g h p re ssu re h o m o g e n i ze r , Dyn o -M i l l : a g i ta to r t i p sp e e d , 1 0 m s t ; gl a ss b e a d d ia m e te r , 0 .2 5 - - 0 .5 0mm ; f lo w ra te , 5 I h ~ ; bead load vo lum e, 85%; ce l l suspension , 40%( w /v ) . N e tzsch L M E 2 0 m i l l : a g i ta to r t i p sp e e d , 9 .5 m s- Z ; glassb e a d d i a m e te r , 0 ,5 5 - - 0 .8 5 m m ; f l o w r a te , 9 3 I h - t ; b e a d l o a dvo l u m e . 8 0 %; ce ll su spe n s i o n, 4 0 % ( w /v ) . M a n to n - Ga u l i n : p re ssu re58 .8 MPa; f low ra te , 54 I h ~ ;ce l l suspension , 40% (w /v) . [Red raw nf r o m Sch f i t t e , H . , K r o n e r , K . H . , Hu s te d t , H . a n d Ku l a , M . -R .E n z y m e M i c ro b , T e c h n o l, 1 9 8 3 , 5 , 1 4 3 ]

T a b l e 2 Com par ison o f th e d is in tegra t ion o f va r ious yeasts and bacte r ia in an industr ia l bead m i l l ( Netzsch L M E 2 0 ) . [Re p r o d u c e d f r o mSch d t te , H . , K r o n e r , K . H . , Hu s te d t , H . a n d Ku l a , M , - R . E n z y m e M i c r o b . T e c h n o l. 1 9 8 3 , 5 , 1 4 3 ]

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S a c c h a r o my c e s c a r l s b e r g e n s i s D- Gl u co se - 6 -p h o sp h a te d e h yd r o g e n a se 8 6S a c c h a r o my c e s c e r e v i s i a e D- Gl u co se - 6 -p h o sp h a te d e h yd r o g e n a se 1 0 0C a n d i d a b o i d i n i i Fo r m a te d e h yd r o g e n a se 9 5E s c h e r i c h ia c o i l M R E 6 0 0 I s o l e u c y l - t R N A s y n th e t as e 9 5B r e v i b a c t e ri u m a m m o n i a g e n e s Fumarase 85B a c i l l u s s p h a e r i c u s L e u c i n e d e h yd r o g e n a se 8 0L a c t o b a c i l l u s c o n f u s u s L a c ta te d e h yd r o g e n a se 9 2

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Rev i ewc l ea r ly in d i ca t e s t h e n eed fo r m u l t i p l e p as ses t h ro u g h th e2 0 1 i t r e m i l l f o r t h e r e l ease o f b ac t e r i a l en zy m es . T h i sco n s id e rab ly l o w er s t h e e q u ip m en t cap ac i ty fo r b ac t e r i a lce l l d i s ru p t io n s in ce t h e f l o w ra t e m u s t a l so b e r a th e r l o w .O th e r m ic ro o rg an i sm s w h ich h av e b een d i s in t eg ra t edin bead mil ls include a lgae S c e n e d e s r n u s o b l i q u u s an dSp i ru l ina p la tens i s 48 fo r i n t r ace l l u l a r p ro t e in , an d fu n g isuch as Asp ergi l lu s niger. 33 '46 C o m p le t e d i s in t eg ra t i o no f A . n i g e r i n b ead m i l l s h as b een sh o w n to b e p o ss ib le , 46an d th e ag i t a to r m i l l i s s a id t o b e p a r t i cu l a r ly v a lu ab l efo r d i s ru p t io n o f m y ce l i a w h ich can b l in d t h e h ig h-p res su reh o m o g e n i z e r ) O f a n u m b e r o f d i s r up t i on m e t h o d s e x a m -ined by Zetel ak i 3a fo r A. n iger , b ead m i l l d i s t ru p t io n w asfo u n d to b e t h e m o s t e f f ec t i v e .

P r o d u c t io n o f h e a t d u e t o i m p a c t s a n d f r ic t i on b e t w e e nth e g r in d in g e l em en t s i s a g en e ra l p ro b l em in ag i t a t edm i l l s : a In sm a l l m i l l s , a j ack e t ed g r in d in g ch am b er i s g ene r -a l l y su f fi c i en t t o h an d le t h e g en e ra t ed h ea t l o ad . W h en ,h o w ev er , m i l l s o f m o re t h an 1 0 l i tr e cap ac i ty a r e u sed fo rsubstances sensi t ive t o t em p era tu res ab o v e 4 5 C , co o l in gth ro u g h th e v es se l w a l l p o ses se r io u s p ro b l em s . T h i s p ro b -l em h as b een o v e rco m e in a 2 0 l i tr e h o r i zo n ta l i n d u s t r i a lb ead m i l l , t h e N e tzsch L M 2 0 . s3 T h i s p a t e n t ed s s d es ignp ro v id es , i n ad d i t i o n t o a j ack e t ed g r in d in g ch am b er , aco o led ag i t a to r sh a f t an d co o led d i sc s . T h e d i sc s a r e m o u n t edo n th e sh a f t a l t e rn a t e ly , r ad i a l l y an d o b l iq u e ly t o t h e sh a f t( F i g u r e 1 6 ) . T h e r ad i a l ag i t a to r d i sc s im p a r t a r ad i a l m o t io nto the g r ind ing elemen ts , wh i le the o b l ique d iscs g ive r i se toax i a l m o v e m e n t o f a su b s t an t i a l p a r t o f t h e e l em e n t s . T h ecross ing o f the paths thus ach ieved g ives the mi l l a veryh igh eff ic iency .S3

T h erm a l an d sh ea r d en a tu ra t i o n o f p ro t e in s i n b eadmil ls are d is t inct possib i l i t ies . For the re lease o f a num bero f i n t r ace l l u l a r en zy m es f ro m b ak e r s ' y eas t i n a 5 l l t reh o r i zo n ta l b ead m i l l , s2 n o d en a tu ra t i o n w as n o t i ced ,h o w ev e r . T h i s is t r u e , i n g en e ra l , a t l o w o p e ra t i o n t em p era -tu res . I n an i so l a t ed case , so m e en zy m e in ac t i v a t i o n h asb een r ep o r t ed s l ev en a t t em p era tu re s a s lo w as 2 C . Inan o th e r i n v es t i g a t i o n , s4 i t w as fo u n d th a t i n c reasin gt e m p e r a t u r e r e d u c e d t h e m e a s u r e d p r o t e i n re l ea s e (F igure] 7 ), ap p a ren t ly d u e t o t h e rm a l d en a tu ra t i o n a n d /o r m o d i -f i ca t i o n o f p h y s i ca l p ro p e r t i e s su ch a s su sp en sio n v i sco s i t y( r ed u ced v i sco s i t y a t h ig h e r t em p era tu res w o u ld l ead t og r e a t e r b a c k m i x i n g ) .

F i g u r e 1 6 S i m p l i f i e d d ra w i n g o f t h e N e tz s c h m o d e l L M 2 0 m i l l :A , c y l i nd r i c a l g r i nd i ng v es se l w i t h c oo l i ng j ac k e t ; B , ag i t a t o r w i t hc oo l ed s ha f t and d i s cs ; C , annu l a r v i b ra t i ng s l o t s epa ra to r ; D , v a r i -ab l e s peed d r i v e m o to r ; 1 and 2 , p rod uc t i n l e t and ou t l e t ; 3 and 4 ,ag i t a t o r c o o l i ng i n l e t and o u t l e t ; 5 and 6 , v es se l c oo l i ng i n l e t ando u t l e t . [ R e d r a w n f r o m R e h a c e k , J . a n d S c h a e f e r , J . B i o t e c h n o l .Bioen9. 1977 , 19 , 1523 by pe rm i s s i on o f J ohn W i l ey & S ons I nc . ]

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TemperQture (C)E f f ec t o f t empe ra tu re on t he d i s rup t i on k i ne t i c s i n ai gu re 175 l i t re mi l l w i th s ta in less s tee l impe l lers : q = 0 .24 m 3 h - l , U = S ms 1 , C = 0 . 4 5 . [ R e d r a w n f r o m L i m o n - L a s o n , J . , H o a r e , M . , O r s b o r n ,C . B , , Doy l e , D . J . and Dun n i l l , P . Bio t echno l . B ioeng. 1 9 7 9 , 2 1 ,745 by pe rm i s s i on o f J ohn W i l ey & S ons I nc . ]

T h e f r eeze -p res s . F reeze -p res s in g o f m ic ro b i a l ce l lsuspensions can be used to d isrup t the cel ls . Exampleso f t h e f r eeze -p res s in g eq u ip m en t i n c lu d e t h e H u g h es p res sin w h ich a f ro zen p as t e o f ce l l s i s fo rced t h ro u g h a n a r ro wsl i t o r o r i f ice , e i the r in the p re sence o f an ab ras ive a tt em p era tu re s j u s t b e lo w ze ro o r w i th o u t t h e ab ras iv e a tt e m p e r a t u r e s o f a b o u t - 2 5 C ] I n t h e l a t t e r c a s e, p h a s ean d co n seq u en t v o lu m e ch an g es o f i ce co n t r i b u t e t o d i s-rup t ion . 7 '12 In add i t ion , so l id shear due to c rysta l l ine ice isim p o r t an t . A cco rd in g to W im p en n y , 7 ce l l b r eak ag e i n th eH u g h es p res s y i e ld s ce l l w a l l m em b ran e p rep a ra t i o n s t h a ta r e r e l a t iv e ly i n t ac t an d m ay b e a g o o d m e th o d fo r i so la t i o no f m em b ran e -as so c i a t ed en zy m es . A sem ico n t in u o u s d es ig no f X -P res s f r eeze -p res s in g eq u ip m en t cap ab le o f h an d l in gab o u t 1 0 k g m a te r i a l ev e ry h o u r h as b een s tu d i ed , s9 F o r as imi lar mach ine, a rep or t 6 suggests that the dev ice can bes c a l e d u p a l m o s t u n l i m i t e d l y . T h e t h e r m o d y n a m i c s o fco m p re ss io n , an d rh eo lo g y in su ch d ev i ces h av e r ece iv edth eo re t i ca l t r ea tm en t i n a n u m b e r o f p ap e r s ( e. g . r e f . 1 2 ).W e a re n o t aw are o f an y in d u s t r ia l f r eeze -p res s in g eq u ip -m e n t co m m erc i a l l y av a i l ab l e o r i n u se . T h e r ead e r i s r e f e r r edto o th e r papers 12'sg'61-6s fo r i n fo rm a t io n o n t h i s t ech n iq u e .O t h e r m e t h o d s o f d i s r u p t i o nM an y o th e r m e th o d s o f m ic ro b i a l ce ll d i s ru p t io n ex i s t , b u th av e fo u n d n o l a rg e - sca l e ap p l i ca t i o n s , O sm o t i c sh o ck , 4enz ym at ic 31 and chem ical ly s is 22'66 are som e exam ples .E n zy m ic ce l l l y s i s, w h ich i s a t t r ac t i v e i n t e rm s o f i tsd e l i cacy an d sp ec i f i c i t y fo r ju s t t h e ce ll w a l l s t ru c tu re , isr e s t r i c t ed b y t h e co s t z s o f t h e en zy m e w h ich i s l o s t i n tot h e e x t r a c t ) T h is c an p r o b a b l y b e o v e r c om e b y t he u s eo f so lu b l e im m o b i l i zed en zy m es i n u l t r a f i l t e r r eac to r s a sd esc r ib ed b y D u n n i l l . 6 E n zy m at i c ce l l ly s i s o f P. pu t idau s in g l y so zy m e h as b een su ccess fu l fo r t h e i so l a t i o n o fa lk an e h y d ro x y las e , an en zy m e d am ag ed sev e re ly d u r in gm ech a n ica l i so l a t i o n . 32 R ed u c t io n i n t h e co s t o f ce l ll y t i c en zy m es m ay a l so b e p o ss ib l e b y i n c reas in g t h e sca l eo f p r o d u c t i o n , f o r e x a m p l e . P r o d u c t io n o f y e a s t @ t i ce n z y m e s f r o m C y t o p h a g a h as b een ca r r i ed o u t o n a p i l o tscale 1 .4 m 3 ferm en te r . 67-69 Cell ly t ic enz ym e p r odu ct io nh as b een ex am in ed b y G a las e t aL ,70 am o n g o th e r s , S en s i -t i v i t y o f m ic ro o rg an i sm s to v a r iou s l y t i c en zy m es v a r i e s

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g r e a t l y wi t h t h e p h a s e o f g r o wt h a n d f e r m e n t a t i o n c o n -d i t i o n s Y I n s o m e c a s e s , a u t o l y s is o f m i c r o b i a l c el ls w i t h o u ta n y f o r e i g n e n z y m e m a y b e p o s s i b l e . Au t o l y s i s o f Bacil lusa my l o l i q u e f a c i e n s h a s b e e n r e p o r t e d f l s Al t h o u g h a u t o l y s i si s , i n g e n e r a l , s l o we r t h a n o t h e r d i s r u p t i o n m e t h o d s , i t i sv o l u m e i n d e p e n d e n t 7s a n d s o 1 0 0 m 3 o f c ei ls m a y b elysed as fas t as 100 cm a .Acids , a lk a l i sy sur fac tant s ?a and so lvent s 66 have beenut i l i zed for ce l l lys i s . Se lec t ive ext rac t ion of choles te ro lo x i d a s e f r o m No c a r d i a r h e d o c r o u s b y c e ll p e r m e a b i l i z a t i o n ,u s i ng t h e s u r f a c t a n t T r i t o n X- 1 0 0 , h a s b e e n r e p o r t e d ] ~This sur fac tant i s qui t e expens ive , however , and l a rge-sca leu s e m a y n o t b e p o s s i b l e . A l s o , t h e c o n t a m i n a t i o n o f t h ep r o d u c t wi t h t h e s u r f a c t a n t i s a n o t h e r d r a wb a c k . Ot h e rchem ica l s , such as ac ids and a lka l is , a re not ve ry se lec t ivea n d t e n d t o d a m a g e s e n s it iv e p r o t e i n s a n d e n z y m e s a l o n gwi th the ce l l wai l s .Co m b i n a t i o n d i s r u p t i o n m e t h o d s m a y p r o v e u s e fu l f o rorgani sms more res i s t an t to d i s rupt ion , where mechanica ld i s i n te g r a t io n a l o n e m a y b e c o s t l y .2 s Fo r e x a m p l e , i m p r o v e dp r o t e i n e x t r a c t a b i l i t y f r o m C. l ipolyt ica wa s o b t a i n e d i n ah i g h - p r e s s u r e h o m o g e n i z e r wh e n t h e c e l l s we r e s u s p e n d e di n a l k a l in e s o l u t io n s f o r a s h o r t p e r i o d p r i o r t o h o m o -gen izat ion .22D o w n s t r e a m p r o ce s s in gA c e l l d i s r u p t io n p r o c e s s c a n n o t b e c o n s i d e r e d i n i s o l a ti o nf r o m f u r t h e r d o wn s t r e a m p r o c e s s i n g . Th i s i s b e c a u s e t h ec e l l d i sr u p t i o n o p e r a t i o n a f f e c t s t h e p h y s i c a l p r o p e r t i e sof the ce l l s lur ry such as v i scos i ty ,24 dens i ty , par t i c l e s i ze ,and se t t l eabi l i ty of su spens ion , 22 which in turn a f fec t subse-q u e n t p r o c e s s in g . Ch a n g e s , in s o m e p r o p e r t i e s o f a 4 5 %p a c k e d we i g h t p e r v o l u m e s u s p e n s i o n o f b a k e r s ' y e a s t o np as sa ge t h r o u g h a M a n t o n - G a u i l n A P V h o m o g e n i z e r at49 MPa a nd 5C 24 are sho wn in Fi g u r e 1 8 . H o m o g e n i z e dce l l suspens ions ha ve been fou nd to be more s t able . 22Se d i m e n t a t i o n o f m e c h a n i c a l l y d i s r u p t e d c e l l s u s p e n s i o n sin indus t r i a l cent r i fuges has bee n s tud ied 24 and a re por t 72on c la r i f i ca t ion of these suspens ions v ia ro ta ry vacuumprecoa t f i l t r a t ion exi s t s . These s tudies indica te the needf o r t h e c h o i c e o f a p p r o p r i a t e c e l l d i s r u p t i o n e q u i p m e n t ,a n d f o r t h e e x t e n t o f t r e a t m e n t i n a n e q u i p m e n t , i n o r d e r

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F i g u r e 18 Changes in the p roper t ies o f yeast suspensions dur ingd i s r u p t i o n , o , Ap p a r e n t v i sco s i t y o f t h e su sp en s io n a t 2 1 7 Hz ; o ,appa ren t v iscos i ty o f suspension superna tan t a t 217 H z; o , packeds o l i d con ten t o f suspension ; ,5, so lub le p ro te in in the suspensionsu p e r n a ta n t . [Re d r a w n f r o m M o sq u e i r a , F . G . . H i g g in s , J , J ., D u n n i l l ,P . a n d L i l l y , M . D . Biotechnol. Bioeng. 1 9 8 1 , 2 3 , 3 3 5 b y p e r m i s s i o no f Jo h n W i l e y & So n s In c . ]

Di s rup t i on o f m i c rob i a l ce l ls : Y . Ch i s t i and M. M oo - Y oungto fac i l i t a t e downs t ream process ing of the d i s ruptedmater i a l .

B i o s a f e t y i n ce l l d i s r u p t i o nI n m o s t t r e a t m e n t s o f b i o p r o c e s s e s, th e p r o c e s s sa f e t y t e n d sto be igno red . T hi s is a l so t rue of the microbia l ce l l d i s rup-t i o n o p e r a t i o n s . So m e s a f e t y c o n s i d e r a t i o n s i n t h e d i s ru p -t i o n o f p a t h o g e n i c o r g a n i s m s i n i n d u s t r i a l e q u i p m e n t s u c ha s t h e M a n t o n - G a u l i n A P V h o m o g e n i z e r a n d i n b a l lmi ll s have been t rea ted in the l i t e ra tur e . 73 The poss ib i l i tyof gaske t f a i lure in h igh-pressure devices and consequentf o r m a t i o n o f a j e t o f p a r t l y d i s r u p t e d c e ll s u sp e n s i o nindica tes the need for ap propr ia te enc losures . 73 The opera-t iona l sa fe ty of some ce l l d i s rupt ion m achines has a lso beendi scussed by Dunn i l l . 3C o n c l u s i o n sFor l a rge-sca le ce l l d i s rupt ion , the mechanica l methods ofd i s r u p t i o n a r e s e e m i n g l y t h e m o s t p o p u l a r . Of t h e s e , t h eh i g h p r e s s u r e h o m o g e n i z e r a n d t h e b e a d m i l l a r e t h e m o s tc o m m o n l y u s e d . T h e h i g h -p r e ss u r e h o m o g e n i z e r i s b e s ts u i t e d f o r s o m e b a c t e r i a a n d y e a s t s , wh il e t h e b e a d m i l lappears m ore su i t ab le for the d i s in tegra t ion o f yeas t cel lsand for mycel i a l organi sms and a lgae . There i s enoughe v i d e n c e t o s h o w t h a t t h e m e c h a n i c a l c e l l d i s r u p t i o nm e t h o d s d o n o t d a m a g e m o s t i n tr a c e l lu l a r e n z y m e s a n dp r o t e i n s ; m e m b r a n e - a s s o c i a t e d e n z y m e s a n d m u l t i e n z y m ec o m p l e x e s m a y b e t h e e x c e p t i o n s .Other t echniques which may have potent i a l l a rge-sca leappl i ca t ions a re u l t rason ica t ion , f reeze-press ing and enzy -m a t i c l y s is . A c o m b i n a t i o n o f t wo o r m o r e d i s r u p t i o nt e c h n i q u e s f o r d i s r u p t io n o f m o r e r e s is t a n t o r g an i s m s m a yh a v e e c o n o m i c a d v a n ta g e s .Th e f i n d in g t h a t f e r m e n t a t i o n c o n d i t i o n s i n fl u e n c e th eres i s t ance of microbia l ce l l s to d i s rupt ion should be fur theri n v e s t ig a t e d a n d c o u l d b e f a v o u r a b l y u s e d t o m a n i p u l a t ethe ease o f d i s rupt ion of a ce l l . S tudies of d i s ru pt ion k ine t i csa n d o f t h e i n f l u e n c e o f c e l l m o r p h o l o g y o n k i n e t i c s o fd i s i n te g r a t io n a r e n e e d e d ; a n d v e r y l it t le i n f o r m a t i o n i sava i l ab le on d i s rupt ion of mycel i a l organi sms .N o m e n c l a t u r ea

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1986 emt disruption of microbial cells for intracellular products

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