Adsorcion de Metales Pesados Poer Bacterias

7/28/2019 Adsorcion de Metales Pesados Poer Bacterias

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V o l . 5 9 , N o . 5P P L I E D AND ENVIRONMENTAL M I C R O B I O L O G Y , May 1 9 9 3 , p . 1 2 7 4 - 1 2 8 20 0 9 9 - 2 2 4 0 / 9 3 / 0 5 1 2 7 4 - 0 9 $ 0 2 . 0 0 / 0C o p y r i g h t 1 9 9 3 , A m e r i c a n S o c i e t y f o r M i c r o b i o l o g y

S o r p t i o n o f Heavy M e t a l s t o t h e F i l a m e n t o u s B a c t e r i u mT h i o t h r i x S t r a i n AlKAY L . SHUFTLEWORTHt AND RICHARD F . UNZ*

L a b o r a t o r y o f E n v i r o n m e n t a l M i c r o b i o l o g y , I n t e r c o l l e g i a t e P r o g r a m i n E c o l o g y a n d D e p a r t m e n t o f C i v i l a n dE n v i r o n m e n t a l E n g i n e e r i n g , T h e P e n n s y l v a n i a S t a t e U n i v e r s i t y , U n i v e r s i t y P a r k , P e n n s y l v a n i a 1 6 8 0 2R e c e i v e d 2 3 O c t o b e r 1 9 9 2 / A c c e p t e d 1 0 F e b r u a r y 1 9 9 3

A s t u d y w a s u n d e r t a k e n t o d e t e r m i n e t h e a b i l i t y o f t h e f i l a m e n t o u s b a c t e r i u m T h i o t h r i x s t r a i n Al t o s o r bh e a v y m e t a l s f r o m s o l u t i o n . C e l l s o f T h i o t h r i x s t r a i n Al w e r e h a r v e s t e d , w a s h e d , a n d s u s p e n d e d i n s o l u t i o n so f m e t a l s . A f t e r a n e q u i l i b r a t i o n p e r i o d , b i o m a s s w a s s e p a r a t e d f r o m s o l u t i o n a n d t h e m e t a l c o n t e n t i na c i d - d i g e s t e d c e l l s a n d / o r f i l t r a t e s w a s d e t e r m i n e d b y a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y . S o r p t i o n o f n i c k e la n d z i n c w a s v e r y r a p i d ; m o s t o f t h e s o r b ed m et a l w a s b o u n d i n l e s s t h a n 1 0 m i n. T h e s o r p t i o n d a t a f o r c o p p e rf i t t h e F r e u n d l i c h i s o t h e r m , a n d n i c k e l a n d z i n c d a t a f i t b i p h a s i c F r e u n d l i c h i s o t h e r m s . S o r p t i o n o f b o t h n i c k e la n d z i n c w a s d e p e n d e n t o n c e l l a g e . C e l l s h a r v e s t e d 2 4 h a f t e r i n o c u l a t i o n s o r b e d a p p r o x i m a t e l y o n e - h a l f o f t h ea m o u n t o f m e t a l p e r g r a m c e l l p r o t e i n t h a n d i d c e l l s h a r v e s t e d a f t e r 4 8 , 7 2 , o r 9 6 h . C a l c i u m a n d m a g n e s i u me f f e c t i v e l y c o m p e t e d w i t h z i n c f o r b i n d i n g s i t e s , w h e r e a s p o t a s s i u m h a d o n l y a s l i g h t e f f e c t o n t h e c a p a c i t y o fc e l l s t o s o r b z i n c . The p r i m a r y m e c h a n i s m o f m e t a l s o r p t i o n a p p a r e n t l y w a s i o n e x c h a n g e , b e c a u s e 6 6 t o 75%o f n i c k e l o r z i n c c o u l d b e d e s o r b e d b y p l a c i n g m e t a l - l a d e n c e l l s i n a s o l u t i o n o f 5 mM C a C l 2 . A c o m p e t i t i o ne x p e r i m e n t w i t h n i c k e l a n d z i n c i n d i c a t e d t h a t b o t h m e t a l s o c c u p i e d t h e s a m e s o r p t i o n s i t e s . T h e s t r o n gc h e l a t i n g a g e n t s E D TA a n d N T A e f f e c t i v e l y p r e v e n t e d m e t a l u p t a k e , b u t l a c t a t e e n h a n c e d t h e u p t a k e o f n i c k e l .T h i o t h r i x s t r a i n Al g r o w n i n n i c k e l - c o n t a i n i n g medium h a d a r e l a t i v e l y l o w u p t a k e o f n i c k e l c o m p a r e d w i t hu p t a k e b y r e s t i n g c e l l s s u s p e n d e d i n a s i m p l e b u f f e r s o l u t i o n .F i l a m e n t o u s s u l f u r b a c t e r i a , i n c l u d i n g T h i o t h r i x s p p . , o c -c u r i n n a t u r a l a q u a t i c e n v i r o n m e n t s ( 1 4 ) a n d i n b i o l o g i c a lw a s t e w a t e r t r e at m en t s y s t em s , p a r t i c u l a r l y t h o s e w h i c h a r eo x y g e n d e f i c i e n t o r s e p t i c ( 3 1 , 3 6 ) . When c o n d i t i o n s a r ef a v o r a b l e f o r t h e i r g r o w t h , T h i o t h r i x s p p . m ay b e c o m ed o m i n a n t i n t h e m i c r o b i a l f l o c s o f t h e a c t i v a t e d - s l u d g ep r o c e s s o f w a s t e w a t e r t r e a t m e n t a n d c o n t r i b u t e t o t h e p h e -nomenon o f f i l a m e n t o u s s l u d g e b u l k i n g , w h i c h i s c h a r a c t e r -i z e d b y p o o r s e t t l i n g a n d l i m i t e d c o m p a c t i o n o f t h e s l u d g e

b i o m a s s .Heavy m e t a l s o c c u r b o t h i n d o m e s t i c a n d i n d u s t r i a l w a s t e -w a t e r s ( 2 4 ) , a n d t h e U . S . E n v i r o n m e n t a l P r o t e c t i o n A g e n c y ,t h r o u g h t h e 1 9 8 7 W a t e r Q u a l i t y A c t , s e e k s t o s t r i n g e n t l yl i m i t t h e d i s c h a r g e o f b i o a v a i l a b l e h e a v y m e t a l s t o t h ee n v i r o n m e n t ( 1 0 ) . I t i s p a r t i c u l a r l y i m p o r t a n t , t h e r e f o r e , t ou n d e r s t a n d t h e e x t e n t t o w h i c h m i c r o o r g a n i s m s i n d i g e n o u st o b i o l o g i c a l t r e a t m e n t s y s t e m s m ay m o b i l i z e o r i m m o b i l i z em e t a l s p r e s e n t i n w a s t e w a t e r s . One i m p o r t a n t f a c t o r i n m e t a ls o r p t i o n i s t h e a v a i l a b l e s u r f a c e a r e a . A c c o r d i n g t o t h eh y p o t h e s i s o f P i p e s ( 2 5 ) , a f i l a m e n t p o s s e s s e s a g r e a t e rs u r f a c e a r e a - t o - v o l u m e r a t i o t h a n a c l u m p o f c e l l s d o e s ;c o n s e q u e n t l y , f l o c s i n b u l k i n g s l u d g e s may h a v e a l a r g e rs u r f a c e a r e a t h a n t h o s e i n n o n b u l k i n g s l u d g e s a n d mayt h e r e f o r e e x h i b i t a g r e a t e r p o t e n t i a l f o r m e t a l u p t a k e . L a w -s o n e t a l . ( 1 5 ) w e r e u n a b l e t o e s t a b l i s h d e f i n i t i v e p r o o f o fe n h a n c e d m e t a l u p t a k e b y b u l k i n g s l u d g e s , h o w e v e r , a n ds t u d i e s o f m e t a l i n t e r a c t i o n s w i t h a x e n i c c u l t u r e s o f w a s t e -w a t e r f i l a m e n t o u s b a c t e r i a h a v e b e e n l i m i t e d t o S p h a e r o t i l u ss p p . ( 3 , 1 7 ) .S h u t t l e w o r t h a n d Unz ( 2 6 ) r e p o r t e d t h a t s e v e r a l h e a v ym e t a l s h a d t o x i c e f f e c t s o n T h i o t h r i c s t r a i n A l , w h i c h w a s a n

* C o r r e s p o n d i n g a u t h o r .t P r e s e n t a dd r es s : D i v is i o n o f M i c r o b i o l o g y , N a t i o n a l C e n t e r f o rT o x i c o l o g i c a l R e s e a r c h , J e f f e r s o n , AR 7 2 0 7 9 .

i s o l a t e f r o m b u l k i n g a c t i v a t e d s l u d g e ( 3 6 ) . I t was e n v i s i o n e dt h a t t h i s o r g a n i s m m ay d i f f e r f r o m u n i c e l l u l a r b a c t e r i a i n i t su p t a k e o f h e a v y m e t a l s a s a r e s u l t o f i t s f i l a m e n t o u s g r o w t hh a b i t a n d t h e u t i l i z a t i o n o f r e d u c e d s u l f u r f o r e n e r g y ( 3 7 ) . I nt h i s r e p o r t , t h e p e r f o r m a n c e o f T h i o t h r i x s t r a i n Al i n t h er e m o v a l o f h e a v y m e t a l s f r o m s o l u t i o n i s d e s c r i b e d a n d t h ei m p l i c a t i o n s o f m e t a l s o r p t i o n o n m e t a l t o x i c i t y a r e d i s -c u s s e d .MATERUILS AN D M E T H O D S

B a c t e r i a . T h i o t h r i x s t r a i n Al i s a member o f o u r c u l t u r ec o l l e c t i o n . T h e b a c t e r i u m was m a i n t a i n e d a n d g r o w n o nl a c t a t e - t h i o s u l f a t e - N - 2 - h y d r o x y e t h y l p i p e r a z i n e - N ' - 2 - e t h a n e -s u l f o n i c a c i d ( H E P E S ) ( L T H ) m e d i u m , d e s c r i b e d e l s e w h e r e( 2 6 ) .S o r p t i o n e x p e r i m e n t s . LTH medium was i n o c u l a t e d w i t hg r o w i n g b r o t h c u l t u r e ( l o g p h a s e ) o f T h i o t h r i x s t r a i n Al t op r o d u c e a n o p t i c a l d e n s i t y a t 4 0 0 nm ( O D 4 0 0 ) o f 0 . 0 0 7 5 .I n c u b a t i o n was c a r r i e d o u t a t 2 5 C o n a n o r b i t a l s h a k e r ( 1 1 0r p m ) f o r 2 . 5 t o 3 d a y s u n l e s s o t h e r w i s e s t a t e d . B i o m a s s wash a r v e s t e d b y c e n t r i f u g a t i o n ( 7 , 0 0 0 x g a t 2 5 C f o r 1 5 m i n ) ,w a s h e d t w i c e , a n d r e s u s p e n d e d i n 2 0 mM HEPES b u f f e r ( p H7 . 2 0 ) . C o n c e n t r a t e d c e l l s u s p e n s i o n s w e r e k e p t a e r o b i c b yb u b b l i n g w i t h a i r u n t i l n e e d e d . T h e c e l l d e n s i t y w a s a p p r o x -i m a t e d b y m e a s u r i n g t h e O D 4 0 0 . S u b s a m p l e s o f c e l l s w e r ef r o z e n a t - 2 0 C f o r s u b s e q u e n t p r o t e i n a n a l y s i s .T h e s t a n d a r d r e a c t o r c o n d i t i o n s f o r s o r p t i o n e x p e r i m e n t s ,u n l e s s o t h e r w i s e n o t e d , c o ns i s t ed o f 2 0 m l o f c e l l s ( O D 4 0 0 o f0 . 3 f o r Cu a n d 1 . 6 f o r Zn a n d N i ) s u s p e n d e d i n 2 0 mMH EP ES b u f f e r ( p H 7 . 2 0 ) i n 1 2 5 - m l h i g h - d e n s i t y p o l y e t h y l e n eb o t t l e s . P r e l i m i n a r y e x p e r i m e n t s r e v e a l e d t h a t n o n s p e c i f i cm e t a l s o r p t i o n was l o w e r w i t h h i g h - d e n s i t y p o l y e t h y l e n et h a n p o l y p r o p y l e n e b o t t l e s . A l l m e t a l s w e r e a d d e d a s c h l o -r i d e s a l t s a t a p p r o p r i a t e c o n c e n t r a t i o n s . T h e c e l l - m e t a l m i x -t u r e s w e r e i n c u b a t e d f o r 6 h a t 2 5 C a n d 1 1 0 r p m . B a c t e r i a1 2 7 4


2/9

SORPTION O F HEAVY METALS T O TH IOTH RIX STRAIN Al 1 2 7 5were a l w a y s t h e l a s t component t o b e a d d e d t o t h e r e ac t io nm i x t u r e s , a n d a l l e x p e r i m e n t s were p e r f o r m e d i n d u p l i c a t e .At i n t e r v a l s , c e l l s were r e c o v e r e d by f i l t e r i n g 1 5 m l o fs a m p l e t h r o u g h 0 . 2 - , u m - p o r e - s i z e f i l t e r s ( d i a m e t e r , 2 5 m m ) .P o l y s u l f o n e f i l t e r s ( G e l m a n S c i e n c e s , Ann A r b o r , M i c h . )were u s e d f o r e x p e r i m e n t s i n v o l v i n g n i c k e l o r z i n c . P o l y c a r -b o n a t e f i l t e r s were r e q u i r e d f o r t h e c o ppe r s t u d y t o m i n i m i z en o n b i o l o g i c a l m e t a l s o r p t i o n . F o r a l l m e t a l s , t h e u s e o fp o l y s u l f o n e f i l t e r s u p p o r t u n i t s ( G e l m a n S c i e n c e s ) was nec-essary t o p r e v e n t s o r p t i o n o f m e t a l s t o t h e f i l t e r a p p a r a t u s .C e l l s were r i n s e d t w i c e i n s i t u w i t h 2 . 5 ml o f u l t r a p u r e waterand t h e n q u a n t i t a t i v e l y removed f r o m t h e f i l t e r w i t h appro x-i m a t e l y 7 ml o f u l t r a p u r e w a t e r . Then 1 m l o f c o n c e n t r a t e dH N O3 was a d d e d t o e a c h s a m p l e , a n d t h e volume wasr e d u c e d t o 5 ml b y g e n t l e h e a t i n g . C e l l s were d i g e s t e d bya dd it i o n o f 6 ml o f c o n c e n t r a t e d H N O3 a n d e v a p o r a t i o n t od r y n e s s a t j u s t below t h e b o i l i n g p o i n t . D i g e s t e d s a m p l e swere r e s u s p e n d e d i n 1 5 m l o f 1% H N O3 i n p r e p a r a t i o n f o ra n a l y s i s b y f l a m e a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y( A A S ) o r i n 0.2% H N O 3 f o r a n a l y s i s t o b e c o m p l e t e d byg r a p h i t e f u r n a c e AAS. I n p r e l i m i n a r y e x p e r i m e n t s , f i l t r a t e swere a l s o k e p t f o r m e t a l a n a l y s i s . M e t a l r e c o v e r y ( s u m o fc e l l s an d f i l t r a t e s ) was b a s e d on t h e measured t o t a l m e t a lad d ed and was between 9 2 and 1 0 3 % , w i t h an average o f9 8 . 8 % . Th e d i f f e r e n c e i n r e s u l t s between d u p l i c a t e s wast y p i c a l l y l e s s t h a n 4%. For o n e s t u d y done i n t r i p l i c a t e , t h erange f o r t h e s t a n d a r d d e v i a t i o n was 0 . 3 t o 2.4% o f t h e meanand t h e average s t a n d a r d d e v i a t i o n was 1 . 4 % o f t h e mean.S o r p t i o n under a n o x i c c o n d i t i o n s . C e l l s c o n t a i n i n g anabundance o f s u l f u r g r a n u l e s ( 2 0 h o l d ) a n d c e l l s v i r t u a l l yd e v o i d o f s u l f u r g r a n u l e s ( 2 . 5 d a y s o l d ) were h a r v e s t e d . Thec e l l s were washe d an d r e s u s p e n d e d , and o x i c a n d a n o x i cc o n d i t i o n s were c r e a t e d by b u b b l i n g h u m i d i f i e d a i r o r n i t r o -g e n , r e s p e c t i v e l y , t h r o u g h t h e s o l u t i o n s f o r 3 0 m i n . Thenm e t a l c h l o r i d e was added t o g i v e a f i n a l c o n c e n t r at i o n o f 5 0F L M . Th e c e l l s w e r e bubbled f o r a n o t h e r 3 h and t h e ns e p a r a t e d from t h e b u l k s o l u t i o n b y f i l t r a t i o n . M e t a l a n a l y s e swere p e r f o r m e d o n l y o n t h e f i l t r a t e s b e c a u s e m e t a l s u l f i d ep r e c i p i t a t e s m i g h t n o t be q u a n t i t a t i v e l y r e c o v e r e d from t h ef i l t e r a l o n g w i t h t h e c e l l f r a c t i o n .M e t a l u p t a k e d ur i ng g r o w t h . LTH m e d i u m was s u p p l e -mented w i t h N i C l 2 an d 6 3 N i C 1 2 ( s p e c i f i c a c t i v i t y , 5 2 . 7 MBq/, m o l ; Amersham, A r l i n g t o n H e i g h t s , I l l . ) t o p r o d u c e t h ed e s i r e d n i c k e l c o n c e n t r a t i o n an d an a c t i v i t y o f 2 0 0 B q / m l . As u f f i c i e n t inoculum o f washe d T h i o t h r i x s t r a i n Al c e l l s wasadded t o p r o d u c e an i n i t i a l O D 4 . o f 0 . 0 1 , an d t h e c u l t u r e swere i n c u b a t e d a t 2 5 C on an o r b i t a l s h a k e r ( 1 1 0 r p m ) . Ati n t e r v a l s , a l i q u o t s were re mov e d from t h e c u l t u r e s f o r mea-surement o f O D 4 . , p H , an d c e l l - b o u n d n i c k e l . T h e OD o fs o n i c a t e d s a m p l e s was used t o q u a n t i f y c e l l g r o w t h ( 2 6 ) .C e l l - b o u n d n i c k e l was determined by f i l t e r i n g s a m p l e st h r o u g h a 0 . 2 - , u m f i l t e r , r i n s i n g t w i c e with 5 m l o f u l t r a p u r ew a t e r , an d a i r d r y i n g . Th e c e l l s were p a r t i a l l y d i g e s t e d b yb e i n g i n c u b a t e d w i t h 2 m l o f c o n c e n t r a t e d HCI f o r 5 h . Each2 - m l H Cl s a m p l e was m i xed w i t h 1 5 m l o f S c i n t i v e r s e I Ic o c k t a i l ( F i s h e r S c i e n t i f i c , P i t t s b u r g h , P a . ) , an d c e l l - b o u n dn i c k e l was determined fr o m r a d i o a c t i v e c o u n t s o b t a i n e dw i t h a l i q u i d s c i n t i l l a t i o n counter ( m o d e l 1 2 1 7 R a c k b e t a ;LKB I ns t r um en t s, G ai t h er s b ur g , M d . ) . Th e e x t e r n a l s t a n -d a r d s r a t i o method was used f o r q u e n c h c o r r e c t i o n s .Metal u p t a k e i n t h e presence o f l i g a n d s . Th e e f f e c t o fv a r i o u s l i g a n d s on m e t a l s p e c i a t i o n was p r e d i c t e d by u s i n gt h e c o m p u t e r m o d e l MINEQL ( 3 5 ) as d e s c r i b e d p r e v i o u s l y( 2 6 ) . E q u i l i b r i u m c o n s t a n t s wh ich were s u b s t i t u t e d f o r v a l -u e s i n t h e MINEQL d a t a b as e a nd c o n s t a n t s w hic h were n o tp a r t o f t h e o r i g i n a l d a t a base ar e g i v e n i n T a b l e 1 . A l l

TABLE 1 . A d d i t i o n s t o a n d c h a n g e s i n t h e t h e r m o d y n a m i cc o n s t a n t s u s e d i n t h e MINEQL p r o g r a m '

Compound ( I D n o . ) b Lo g K C o m p o n e n t s t o i c h i o m e t r y T y p es p e c i e s dN i a c e t a t e ( 6 9 1 0 ) 1 . 4 3 5 N i ( 1 ) a c e t a t e ( 1 ) I IZn a c e t a t e ( 6 1 4 0 ) 1 . 5 8 Zn ( 1 ) a c e t a t e ( 1 ) I IH T r i s ( 1 3 5 9 0 ) 8 . 2 8 ' H ( 1 ) T r i s ( 1 ) I IN i T r i s ( 7 5 7 0 ) 3 . 0 3 e N i ( 1 ) T r i s ( 1 ) I IN i T r i s ( 7 5 8 0 ) 5 . 1 2 ' N i ( 1 ) T r i s ( 2 ) I IZn T r i s 2 . 6 7 T Zn ( 1 ) T r i s ( 1 ) I IC u ( O H ) 2 ( 2 0 5 0 0 ) - 8 . 6 8 Cu ( 1 ) H ( - 2 ) VZ n 3 ( P O 4 ) 2 ( 2 0 6 5 0 ) 3 5 . 3 Zn ( 3 ) P 0 4 ( 2 ) VH g l y c o l a t e 3 . 8 3 H ( 1 ) g l y c o l a t e ( 1 ) I ICu g l y c o l a t e 2 . 9 0 Cu ( 1 ) g l y c o l a t e ( 1 ) I IN i g l y c o l a t e 2 . 2 6 N i ( 1 ) g l y c o l a t e ( 1 ) I IZn g l y c o l a t e 2 . 3 8 Zn ( 1 ) g l y c o l a t e ( 1 ) I IH l a c t a t e 3 . 8 6 H ( 1 ) l a c t a t e ( 1 ) I ICa l a c t a t e 1 . 4 2 Ca ( 1 ) l a c t a t e ( 1 ) I IM g l a c t a t e 1 . 3 7 M g ( 1 ) l a c t a t e ( 1 ) I ICu l a c t a t e 3 . 0 2 Cu ( 1 ) l a c t a t e ( 1 ) I ICu l a c t a t e 4 . 8 4 Cu ( 1 ) l a c t a t e ( 2 ) I IN i l a c t a t e 2 . 2 2 N i ( 1 ) l a c t a t e ( 1 ) I IN i l a c t a t e 3 . 6 2 N i ( 1 ) l a c t a t e ( 2 ) I IZn l a c t a t e 2 . 2 2 Zn ( 1 ) l a c t a t e ( 1 ) I IZn l a c t a t e 3 . 7 5 Zn ( 1 ) l a c t a t e ( 2 ) I I

a A l l o t h e r t h e r m o d y n a m i c c o n s t a n t s u s e d w e r e t h o s e c o n t a i n e d i n t h e d a t ab a s e ( t h e r m . d a t ) s u p p l i e d w i t h t h e p r o g r a m . T h e MINEQL i o n c . d a t d a t a b a s ew a s m o d i f i e d b y a d d i t i o n o f t he i on c h a r g e v a l u e s ( - 1 ) f o r g l y c o l a t e a n dl a c t a t e . T h e i o n c . d a t c h a n g e s w e r e n e c e s s a r y f o r t h e p r o g r a m t o p r o p e r l yc a l c u l a t e t h e c h a n g e s i n l a c t a t e a n d g l y c o l a t e K v a l u e s a s a f u n c t i o n o f i o n i cs t r e n g t h .b I D n o . i s t h e n u m b e r i n t h e t h e r m . d a t d a t a b a s e u s e d t o i d e n t i f y t h ec o m p o u n d . O n l y t h e n u m b e r s t h a t w e r e i n c l u d e d i n t h e o r i g i n a l d a t a b a s e a r eg i v e n h e r e .c T h e l o g K v a l u e s a r e t h e t h e r m o d y n a m i c c o n s t a n t s a n d a r e g i v e n i n t h ec o n v e n t i o n u s e d i n t h e MINEQL p r o g r a m .d T h e t y p e s p e c i e s a r e l i s t e d a c c o r d i n g t o t h e MINEQL f o r m a t .' T h e l i t e r a t u r e v a l u e s f o r t h e s e c o m p o u n d s w e r e c o n c e n t r a t i o n e q u i l i b r i u mc o n s t a n t s ( K I ) . T h e e x t e n d e d D e b y e- H u i c k el e q u at i o n was u s e d t o d e t e r m i n et h e a c t i v i t y c o e f f i c i e n t s n e c e s s a r y t o c a l c u l a t e t h e K v a l u e s f r o m t h e K v a l u e s( 3 2 ) .

s u b s t i t u t e d v a l u e s , e x c e p t t h o s e f o r z i n c p h o s p h a t e , w e r eo b t a i n e d f r o m t a b l e s o f c r i t i c a l s t a b i l i t y c o n s t a n t s ( 1 8 , 1 9 ,2 8 - 3 0 ) ; t h e z i n c p h o s p h a t e c o n s t a n t was o b t a i n e d f r o mL i n d s a y ( 1 6 ) .D e s o r p t i o n o f m e t a l s . B a t c h e s o f c e l l s were i n c u b a t e d f o r 8h a t 2 5 C a n d 1 1 0 rpm i n 2 1 . 5 , u M C u , 6 3 . 3 , u M N i , o r 5 7 . 0,M Z n . A l i q u o t s ( 2 0 m l ) o f c e l l s were r e m o v e d , f i l t e r e d , a n dr i n s e d t w i c e w i t h 2 . 5 m l o f u l t r a p u r e w a t e r . T h e c e l l s w e r eq u a n t i t a t i v e l y r e m o v e d f r o m t h e f i l t e r b y b e i n g w a s h e d o f fw i t h e x a c t l y 2 0 m l o f t h e d e si re d d e s o r p t i o n s o l u t i o n . T h ed e s o r p t i o n s o l u t i o n s were 6 mM K C l , 5 mM C a C l 2 , a n d 5mM EDTA; e a c h c o m p o u n d was d i s s o l v e d e i t h e r i n u l t r a -p u r e w a t e r ( p H u n a d j u s t e d ) o r i n 2 0 mM HEPES ( p H 7 . 2 0 ) .T h e c e l l s , t h u s r e s u s p e n d e d i n t h e v a r i o u s d e s o r p t i o n s o l u -t i o n s , were a g a i n i n c u b a t e d f o r 8 h . A l i q u o t s o f c e l l s ( 1 5 m l )w e r e t h e n p r o c e s s e d f o r m e t a l a n a l y s i s a s s t a t e d a b o v e .I s o t h e r m m o d e l s . T h e s o r p t i o n d a t a w e r e f i t t e d t o F r e u n -d l i c h a n d L a n g m u i r i s o t h e r m m o d e l s ( 3 4 ) . The l i n e a r i z e df o r m o f t h e F r e u n d l i c h e q u a t i o n i s a s f o l l o w s : l o g q e = ( l o gK f ) + [ ( 1 / n ) ( l o g C ) ] , w h e r e C i s t h e c o n c e n t r a t i o n i n s o l u t i o na t e q u i l i b r i u m , q e i s t h e m o l e s o f s o l u t e s o r b e d p e r u n i tw e i g h t a t c o n c e n t r a t i o n C , a n d K f a n d n a r e c o n s t a nt s .T h e L a n g m u i r e q u a t i o n was l i n e a r i z e d b y t h e m e t h o d o fD o w d a n d R i g g s ( 4 ) a n d i s a s f o l l o w s : q e = Q - [ ( 1 I b ) ( q , /C ) ] , w h e r e q e a n d C a r e t h e same a s f o r t h e F r e u n d l i c he q u a t i o n , Q i s t h e m o l e s o f s o l u t e s o r b e d p e r u n i t w e i g h t o f

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F I G . 1 . F r e u n d l i c h s o r p t i o n i s o t h e r m s f o r T h i o t h r i x s t r a i n A l . Ci s t h e c o n c e n t r a t i o n o f m et a l i n s o l u t i o n a t e q u i l i b r i u m , a n d Kf a n dn a r e c o n s t a n t s . T h e r a n g e s o f t o t a l m e t al c o nc en t ra t i o ns t e s t e dw e r e 2 . 5 t o 1 0 0 , u M C u , 0 . 5 t o 5 0 0 , u M N i , a n d 2 . 5 t o 1 0 0 0 , u M Z n .T h e c o n c e n t r a t i o n s o f c e l l p r o t e i n w e r e 3 9 . 5 , u g / m l ( C u ) , 1 6 8 p g / m l( N i ) , a n d 1 8 2 , u g / m l ( Z n ) .

a d s o r b e n t t o f o r m a c o m p l e t e m o n o l a y e r , a n d b i s a c o n -s t a n t .AAS. AAS a n a l y s e s w e r e c o m p l e t e d w i t h a P e r k i n - E l m e rm o d e l 3 0 3 0 B a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t e re q u i p p e d w i t h a m o d e l HGA-600 f u r n a c e . A l l z i n c a n dh i g h - c o n c e n t r a t i o n c o p p e r s a m p l e s w e r e a n a l y z e d b y a i r -a c e t y l e n e f l a m e AAS u n d e r t h e c o n d i t i o n s r e c o m m e n d e d b yt h e i n s t r u m e n t m a n u f a c t u r e r . L o w - c o n c e n t r a t i o n c o p p e rs a m p l e s a n d a l l n i c k e l s a m p l e s w e r e a n a l y z e d b y t h e g r a p h i t ef u r n a c e t e c h n i q u e . No m a t r i x i n t e r f e r e n c e s w e r e f o u n d .O t h e r p r o c e d u r e s . C e l l p r o t e i n was d e t e r m i n e d b y t h eLowry m e t h o d w i t h t h e d i g e s t i o n p r o c e d u r e m o d i f i e d t op r e v e n t p o t e n t i a l i n t e r f e r e n c e f r o m s u l f u r g r a n u l e s ( 3 7 ) .U l t r a p u r e w a t e r was o b t a i n e d f r o m a M i l l i - Q f o u r - b o w ls y s t e m ( M i l l i p o r e C o r p . , B e d f o r d , M a s s . ) . G l a s s w a r e a n dp l a s t i c w a r e w e r e w a s h e d i n L i n b r o 7x d e t e r g e n t , r i n s e d ,s o a k e d f o r a minimum o f 4 h i n 2 0 % n i t r i c a c i d , a n d r i n s e df o u r t i m e s w i t h u l t r a p u r e w a t e r . C h e m i c a l s w e r e r e a g e n tg r a d e a n d c o m m e r c i a l l y a v a i l a b l e .

RESULTST h e c a p a c i t y o f T h i o t h r i x s t r a i n Al c e l l s t o s o r b m et a l swas a f u n c t i o n o f t h e m e t a l t y p e a n d m e t a l c o n c e n t r a t i o n( F i g . 1 ) . W i t h i n t h e e q u i l i b r i u m c o n c e n t r a t i o n ( C ) r an g e o f0 . 1 7 8 t o 3 3 , u M Cu ( 2 . 5 t o 1 0 0 , u M t o t a l c o p p e r ) , c o p p e rs o r p t i o n d a t a f i t t h e F r e u n d l i c h i s o t h e r m . T h e s o r p t i o n o fn i c k e l a n d z i n c b e t w e e n C r a n g e s o f 0 . 0 8 t o 4 5 0 , u M a n d 0 . 2t o 9 3 0 , u M , r e s p e c t i v e l y , d i d n o t f i t e i t h e r t h e F r e u n d l i c hi s o t h e r m o r t h e L a n g m u i r i s o t h e r m . H o w e v e r , b o t h n i c k e la n d z i n c s o r p t i o n c o u l d b e d e s c r i b e d b y b i p h a s i c F r e u n d l i c hi s o t h e r m s . T h e e q u a t i o n was a p p l i e d o n c e f o r C c 4 . 8 , u M

a n d a s e c o n d t i m e f o r C 2 4 . 8 , u M ( F i g . 1 ) . A t t h e l o w e rm e t a l c o n c e n t r a t i o n s t h e c e l l s h a d a g r ea te r a f f i n i t y f o r z i n ct h a n f o r n i c k e l , b u t a t C 2 4 . 8 , u M t h e r e w e r e n o d i f f e r e n c e si n t h e s o r p t i o n o f n i c k e l a n d z i n c ( F i g . 1 ) . T h e K a n d n v a l u e sf o r t h e F r e u n d l i c h i s o t h e r m s a r e g i v e n i n T a b l e 2 .T h e s o r p t i o n o f N i a n d Zn was v e r y f a s t . M o s t o f t h e m e t a li o n s w h i c h w e r e t a k e n u p f r o m s o l u t i o n w e r e b o u n d w i t h i n1 0 m i n , a n d a l m o s t n o i n c r e a s e i n t h e l e v e l o f b o u n d m e t a lo c c u r r e d a f t e r 1 h ( F i g . 2 ) . M e t a l u p t a k e was a l s o a f u n c t i o no f c e l l a g e a n d was n o t l i n e a r w i t h c e l l p r o t e i n c o n c e n t r a t i o n( F i g . 3 ) . T h e r e was v i r t u a l l y n o d i f f e r e n c e i n m e t a l u p t a k e

T A B L E 2 . C o n s t a n t s f o r F r e u n d l i c h i s o t h e r m s a , bM e t a l l o g K f 1 / n r 2

C o p p e r 2 . 2 2 0 . 6 5 0 0 . 9 9 9N i c k e lC < 4 . 8 1 . 3 4 0 . 8 8 5 0 . 9 9 2C > 4 . 8 1 . 7 9 0 . 2 5 0 0 . 9 9 6Z i n cC c 4 . 9 1 . 5 3 0 . 5 9 8 0 . 9 8 8C > 4 . 9 1 . 8 0 0 . 2 3 6 0 . 9 5 4a T h e l o g K f i s t h e s o r p t i o n c a p a c i t y f o r C = 1 , a n d t h e s l o p e i n d i c a t e ss o r p t i o n i n t e n s i t y .b C e l l p r o t e i n c o n c e n t r a t i o n s f o r c o p p e r , n i c k e l , a n d z i n c were 3 9 . 5 , 1 6 8 ,a n d 1 8 2 , u g / m l , r e s p e c t i v e l y .

b e t w e e n c e l l s w h i c h w e r e 2 , 3 , 4 , o r 5 d a y s o l d , b u t c e l l sw h i c h w e r e 1 d a y o l d s o r b e d s u b s t a n t i a l l y l e s s m e t a l t h a no l d e r c e l l s d i d . O n e - d a y - o l d c e l l s were i n t h e e a r l y t o m i d - l o gp h a s e a n d were p a c k e d w i t h s u l f u r g r a n u l e s ( F i g . 4 A ) . Byd a y 2 , c e l l s w e r e i n t h e l a t e l o g t o e a r l y s t a t i o n a r y p h a s e a n dw e r e v i r t u a l l y d e v o i d o f s u l f u r g r a n u l e s ( F i g . 4 B ) .T h e s o r p t i o n o f z i n c was i n h i b i t e d b y t h e p r e s e n c e o f o t h e rd i v a l e n t c a t i o n s , a n d c a l c i u m was a s o m e w h a t m ore e f f e c t i v ei n h i b i t o r t h a n was m a g n e s i u m ( F i g . 5 ) . C e l l s i n z i n c s o l u t i o nw i t h n o a d d i t i o n a l c a t i o n s s o r b e d 57% o f t h e m e t a l ; h o w -e v e r , i n t h e p r e s e n c e o f c a l c i u m o r m a g n e s i u m a t 1 0 t i m e st h e z i n c c o n c e n t r a t i o n , 2 3 a n d 3 0 % , r e s p e c t i v e l y , o f t h e z i n cwas s o r b e d . A t e a c h c o n c e n t r a t i o n , t h e r e was a s t a t i s t i c a l l ys i g n i f i c a n t d i f f e r e n c e ( P < 0 . 0 2 ; S t u d e n t ' s t t e s t ) b e t w e e n t h ee f f e c t s o f c a l c i u m a n d o f m a g n e s i u m on z i n c u p t a k e . T h ep r e s e n c e o f u p t o 2 . 0 mM KCI h a d v i r t u a l l y n o e f f e c t o n z i n cs o r p t i o n ( F i g . 5 ) . Z i n c a n d n i c k e l i n c o m b i n a t i o n a l s o a p -p e a r e d t o m u t u a l l y c o m p e t e f o r s o r p t i o n s i t e s ( F i g . 6 ) . T h ea m o u n t o f z i n c s o r b e d was d e c r e a s e d b y t h e a d d i t i o n o fn i c k e l ( F i g . 6 B ) , a n d , c o n v e r s e l y , t h e a m o u n t o f n i c k e ls o r b e d was l o w e r e d b y t h e p r e s e n c e o f z i n c ( F i g . 6 A ) . F o re x a m p l e , 1 5 4 , u m o l o f z i n c p e r g p r o t e i n was s o r b e d f r o m a5 0 , u M s o l u t i o n b u t o n l y 1 1 2 , u m o l o f z i n c p e r g o f p r o t e i nwas s o r b e d f r o m a 1 0 0 , u M s o l u t i o n w h i c h was 50 % z i n c a n d5 0 % n i c k e l . H o w e v e r , 2 1 9 , u m o l o f z i nc p e r g o f p r o t e i n wass o r b e d f r o m a 1 0 0 , u M z i n c s o l u t i o n . S i m i l a r l y , 1 2 8 , u m o l o fn i c k e l p e r g o f p r o t e i n was s o r b e d f r o m 5 0 , u M n i c k e l ; 9 1 . 6, u m o l o f n i c k e l p e r g o f p r o t e i n was s o r b e d f r o m a 1 0 0 , u Ms o l u t i o n o f 50 % n i c k e l a n d 50 % z i n c , b u t 1 6 6 , u m o l o f n i c k e lp e r g o f p r o t e i n was s o r b e d f r o m 1 0 0 , u M n i c k e l . T h e

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F I G . 2 . K i n e t i c s o f n i c k e l a n d z i n c b i n d i n g t o T h i o t h r i x s t r a i nA l . M e t a l c o n c e n t r a t i o n s were 5 3 . 2 F . M n i c k e l a n d 4 5 . 0 , u M z i n c .The p r o t e i n c o n c e n t r a t i o n was 1 9 7 , u g / m l f o r b o t h n i c k e l a n d z i n c .

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100 200 300 400

P r o t e i n c o n c e n t r a t i o n ( m g / L )F I G . 3 . E ff e ct o f c e l l ag e a n d c e l l c o n c e n t r a t i o n o n t h e s o r p t i o no f n i c k e l ( A) a n d z i n c ( B ) t o T h i o t h r i x s t r a i n A l . Th e n i c k e l a n d z i n cc o n c e n t r a t i o n s were 5 2 . 4 a n d 4 8 . 9 , u M , r e s p e c t i v e l y . S y m b o l s : 0, 1d a y o l d ; A , 2 d a y s o l d ; * , 3 d a y s o l d ; E, 4 d a y s o l d ; V, 5 d a y s o l d .

c o m p a r a t i v e a b i l i t y o f n i c k e l a n d z i n c t o compete f o r sorp-t i o n s i t e s was a n a l y z e d b y l i n e a r r e g r e s s i o n . I f n i c k e l a n dz i n c c o m p e t e d e q u a l l y w e l l f o r t h e same s i t e s , a p l o t o f t h er a t i o s o f n i c k e l s o r b e d / t o t a l m e t a l s o r b e d v e r s u s n i c k e la d d e d / t o t a l m e t a l a d d e d s h o u l d p r o d u c e a s l o p e o f 1 . I fn i c k e l was n o t bound as e f f e c t i v e l y as z i n c , t h e s l o p e s h o u l db e < 1 , a nd , c o n v e rs e l y , i f n i c k e l bound more r e a d i l y t h a nz i n c , t h e s l o p e s h o u l d b e > 1 . The s l o p e was 0 . 9 1 a n d wass i g n i f i c a n t l y d i f f e r e n t f r o m 1 . 0 0 a t P < 0 . 0 0 5 , i n d i c a t i n g t h a tt h e r e was a s l i g h t s e l e c t i v i t y f o r z i n c o v e r n i c k e l .The b i n d i n g o f n i c k e l a n d z i n c was a l so a f f e c te d b y t h epre s ence o f v a r i o u s o r g a n i c l i g a n d s ( F i g . 7 ) . I t was assumedt h a t o n l y f r e e m e t a l i o n s would b e a v a i l a b l e f o r b i n d i n g t o t h ec e l l s , a n d , as e x p e c t e d , t h e c h e l a t e d m e t a l s p e c i e s ( N i -EDTA, N i - n i t r i l o t r i a c e t i c a c i d [ N T A ] , Zn-EDTA, and Z n -NTA) d i d n o t b i n d t o t h e c e l l s . S i m i l a r l y , t h e presence o fa c e t a t e , g l y c o l a t e , T r i s , o r c i t r a t e i n h i b i t e d t h e s o r p t i o n o fn i c k e l a n d z i n c t o b i o m a s s . H o w e v e r , a c e t a t e , g l y c o l a t e , andT r i s ar e r e l a t i v e l y weak l i g a n d s f o r n i c k e l a n d z i n c ; t h e r e -f o r e , h i g h c o n c e n t r a t i o n s o f t h e s e l i g a n d s were needed t oa c h i e v e a 3 0 t o 50 % r e d u c t i o n i n t h e p r e d i c t e d m e t a l s p e c i e sa va il a b l e f o r s o r p t i o n . The i o n i c s t r e n g t h o f t h e s o l u t i o n swas g r e a t l y i n c r e a s e d i n t h e t r i a l s c o n t a i n i n g s u c h h i g hc o n c e n t r a t i o n s o f l i g a n d , and c o n t r o l s w i t h 3 0 mM KCIshowed t h a t h i g h i o n i c s t r e n g t h a l o n e s u b s t a n t i a l l y reducedt h e s o r p t i o n o f n i c k e l and z i n c ( F i g . 7 ) . I t was n o t e w o r t h yt h at l a ct at e d i d n o t c a u s e a d e c r e a s e i n t h e s o r p t i o n o f n i c k e l .The d e s o r p t i o n o f m e t a l s b y w a t e r , HEPES, K C I , C a C l 2 ,a n d EDTA i s s h ow n i n F i g . 8 . Th e c h e l a t i n g a g e n t EDTAwas t h e m o s t e f f i c i e n t d e s o r p t i o n a g e n t t e s t e d , w i t h HEPES-

b u f f e r e d 5 mM EDTA s o l u t i o n r e m o v i n g 8 0 t o 83% o f t h ei n i t i a l m e t a l s o r b e d . C a l c i u m c h l o r i d e was t h e s e c o n d b e s tm e t a l d e s o r p t i o n a g e n t , a n d i t r e m o v e d n i c k e l a n d z i n c moree f f e c t i v e l y t h a n i t r e m o v e d c o p p e r ( F i g . 8 ) . More m e t a l wasr e m o v e d b y C a C 1 2 i n u n b u f f e r e d s o l u t i o n ( p H a p p r o x i m a t e l y5 . 6 t o 6 . 0 ) t h a n i n H E P E S - b u f f e r e d s o l u t i o n ( p H 7 . 2 ) , event h o u g h t h e p r e s e n c e o f 2 0 mM HEPES s u b s t a n t i a l l y i n -c r e a s e d t h e i o n i c s t r e n g t h o f t h e s o l u t i o n . T h e p r e s e n c e o fKCl h a d v i r t u a l l y no i m p a c t o n t h e d e s o r p t i o n o f c o p p e r , b u tKCI h a d a p r o n o u n c e d e f f e c t on t h e r e t e n t i o n o f n i c k e l a n dz i n c b y c e l l s i n u n b u f f e r e d u l t r a p u r e w a t e r . T h e r e was nod e s o r p t i o n o f z i n c b y u l t r a p u r e w a t e r , a l t h o u g h 21% o f t h es o r b e d c o p p e r was r e m o v e d b y w a t e r a l o n e .I n p r e v i o u s s t u d i e s , o b s er v a t i o n s o f h y d r o g e n s u l f i d e o d o ra n d b l a c k i s h g r e y d i s c o l o r a t i o n i n s o m e u n a e r a t e d T h i o t h r i xc u l t u r e s were t a k e n a s e v i d e n c e o f p o s s i b l e s u l f u r r e d u c t i o n .I t was b e l i e v e d t h a t u n d e r a n o x i c c o n d i t i o n s t h e b a c t e r i u mm i g h t c a u s e m e t a l s t o p r e c i p i t a t e a s s u l f i d e s . H o w e v e r , t h e r ewas no e v i d e n c e o f m e t a l s u l f i d e p r e c i p i t a t i o n when c e l l s ,e i t h e r w i t h o r w i t h o u t s u l f u r g r a n u l e s , were s u b j e c t e d t o an i t r o g e n a t m o s p h e r e .G r o w i n g c u l t u r e s o f T h i o t h r i x s t r a i n Al were c a p a b l e o ft a k i n g u p n i c k e l f r o m t h e g r o w t h medium ( F i g . 9 ) ; h o w e v e r ,t h e amount o f c e l l - a s s o c i a t e d n i c k e l was m uch s m a l l e r t h a nw o u l d b e p r e d i c t e d f r o m t h e i n f o r m a t i o n o b t a i n e d on u p t a k eu n d e r n o n g r o w t h c o n d i t i o n s . O n l y 17% o f 1 . 1 9 , u M n i c k e lwas r e m o v e d d u r i n g g r o w t h i n n i c k e l - c o n t a i n i n g m e d i u m ,w h e r e a s 79 % o f 1 , u M n i c k e l was s o r b e d t o c e l l s r e s u s p e n d e da t a c o m p a r a b l e c e l l d e n s i t y i n HEPES b u f f e r . C e l l g r o w t h ,c e l l - a s s o c i a t e d n i c k e l l e v e l , a n d pH a l l i n c r e a s e d c o n c o m i -t a n t l y . A t t e m p t s t o p r e v e n t pH c h a n g e s d u r i n g g r o w t h wereu n s u c c e s s f u l b e c a u s e c e l l g r o w t h was i n h i b i t e d b y h i g h e rc o nc en t r at i o n s o f HEPES b u f f e r . T h e c e l l - a s s o c i a t e d n i c k e lwas n o t r e a d i l y d e s o r b e d . No m e t a l was r e m o v e d f r o m3 - d a y - o l d c e l l s ( g r o w n i n 1 . 1 9 p , M N i ) b y i n c u b a t i n g h a r -v e s t e d c e l l s f o r 4 h i n 1 0 mM HEPES b u f f e r ( p H 5 , 6 , 7 , o r8 ) o r i n H E P E S - b u f f e r e d 0 . 5 mM C a C l 2 ( p H 7 . 5 ) . F u r t h e r -m o r e o n l y 6% o f t h e c e l l - a s s o c i a t e d n i c k e l was r e m o v e d b y0 . 1 mM EDTA i n 1 0 mM HEPES b u f f e r ( p H 7 . 5 ) .

DISCUSSIONT h e F r e u n d l i c h a n d L a n g m u i r i s o t h e r m m o d e l s a r e c o m -m o n l y u s e d t o d e s c r i b e t h e s o r p t i o n o f m e t a l s o n t o m i c r o b i a ls u r f a c e s a n d a r e u s u a l l y i n a g r e e m e n t over m o d e r a t e r a n g e so f t h e e q u i l i b r i u m s o r b a t e c o n c e n t r a t i o n ( 3 4 ) . I n t h e p r e s e n ts t u d y , s o r p t i o n d a t a b e s t f i t t h e F r e u n d l i c h i s o t h e r m , a l -t h o u g h t h e c u r v e s were b i p h a s i c f o r n i c k e l a n d z i n c . B e c a u s et h e c u r v e s a r e b i p h a s i c , t h e r e a r e p r o b a b l y a t l e a s t tw o t y p e so f b i n d i n g s i t e s f o r n i c k e l a n d z i n c ( 6 ) . T h e c h e m i s t r y o f c e l ls u r f a c e s i s c o m p l e x , a n d t h e p o t e n t i a l t h e r e f o r e e x i s t s f o rm e t a l s t o s o r b a t a v a r i e t y o f s i t e s ( 1 , 2 0 ) . A d d i t i o n a l l y ,m e t a l s m ay n o t o n l y b e s o r b e d t o t h e s u r f a c e b u t m ay a l s o b et a k e n u p i n t o t h e i n t e r i o r o f t h e c e l l s .M e t a l s o r p t i o n can v a r y a c c o r d i n g t o pH a n d i o n i cs t r e n g t h . In t h i s s t u d y , t h e pH w as m a i n t a i n e d a t 7 . 2 0b e c a u s e ( i ) T h i o t h r i x s t r a i n A l i s pH s e n s i t i v e a n d d o e s n o tg r o w w e l l b e l o w pH 7 ( p e r s o n a l o b s e r v a t i o n ) a n d ( i i ) o r g a n i cw a s t e w a t e r s t y p i c a l l y h a v e a c i r c u m n e u t r a l p H . M a i n t a i n i n gt h i s p H n e c e s s i t a t e d u s i n g an e f f e c t i v e b u f f e r ( H E P E S ) ,w h i c h u n d o u b t e d l y a f f e c t e d t h e r e s u l t s o f m e t a l u p t a k ee x p e r i m e n t s . A l t h o u g h t h e m e t a l - b i n d i n g c a p a c i t y o fHEPES h a s b e e n r e p o r t e d t o b e " n e g l i g i b l e " ( 9 ) , m e t a lc o m p l e x a t i o n b y t h e b u f f e r m ay n e v e r t h e l e s s o c c u r ( 1 2 ) .M e t a l b i n d i n g b y b a c t e r i a h a s b e e n s h o w n t o b e p r o -f o u n d l y i n f l u e n c e d b y p H , b u f f e r t y p e , i o n i c s t r e n g t h , i n c u -

V O L . 5 9 , 1 9 9 3 1 2 7 7


5/9

1 2 7 8 SHUTT L EWO R T H AND UNZ A P P L . E N V I R O N . M I C R O B I O L .


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F I G . 5 . E f f e c t o f c a t i o n s on t h e s o r p t i o n o f z i n c t o T h i o t h r i xs t r a i n A l . Th e z i n c c o nc e nt r a ti o n was 5 0 , u M , a n d t h e p r o t e i nc o n c e n t r a t i o n was 1 7 3 , u g / m l .

b a t i o n t i m e , m e t a l c o n c e n t r a t i o n , a n d o t h e r p a r a m e t e r s ( 2 0 ) .I n a d d i t i o n , u n d e r i d e n t i c a l c o n d i t i o n s m e t a l u p t a k e h a s b e e ns h o w n t o b e d e p e n d e n t on t h e t e s t o r g a n i s m ( 2 1 ) . I t i st h e r e f o r e n o t s u r p r i s i n g t h a t r e p o r t s on t h e e x t e n t o f m et a la c c u m u l a t i o n i n b a c t e r i a l c e l l s g i v e w i d e l y d i f f e r e n t r e s u l t s ;t h e h i g h e s t r e p o r t e d m e t a l c o n c e n t r a t i o n i n c e l l s i s 44% o fc e l l d r y w e i g h t ( 8 ) . In t h e c u r r e n t s t u d y , n i c k e l o r z i n cc o n t r i b u t e d 0 . 7 t o 0 . 8 % o f T h i o t h r i x s t r a i n Al c e l l d r y w e i g h twhen t h e b a c t e r i u m was e x p o s e d t o a m e t a l c o n ce n t r at i o n o f1 0 0 , u M . U n d e r i d e n t i c a l c o n d i t i o n s , c o p p e r a m o u n t e d t o6 . 3 % o f c e l l d r y w e i g h t . T h e s e c o n c e n t r a t i o n s ar e n o t

.co L00.

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F I G . 6 . C o m p e t i t i o n between n i c k e l and z i n c f o r b i n d i n g s i t e s onT h i o t h r i x s t r a i n A l . ( A ) E f f e c t o f z i nc on t h e s o r p t i o n o f n i c k e l ; ( B )e f f e c t o f n i c k e l on t h e s o r p t i o n o f z i n c . Th e p r o t e i n c o n c e n t r a t i o nwas 1 7 3 p . g / m l .

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co cetato w a 1 6 gm.Abeiton:1,1 u eaO m i uctaeLatE0mimlcat;Gy,1 Msdu0~~~~~~~~~~~~~~~~i1.0500 . 0 1 0 20 KCI M C e t Lact G lyc TRIS c i t r E D T A NTA

FIG. 7. E f f e c t of ligands onthe s or ption of nickel and zinc toT h i othr ix s t r a i n Al. T h e total metal concentration was0.40 pmol (2 0c p M ) f o r a l l cas e s e x c e p t the 10.M controls. Complexed metals p e c i e s were c o n s i d e r e d tobe unavailable for sorption. The p rote i nconcentration was 168 p.g/ml. Abbreviations: 10 , 10 p.M metalc o ntr o l; 2 0, 20 p.M metal c o ntr o l ; KCI, 30mMCI; Acet, 30mMsodium acetate; Lact, 10mMsodium lactate; G l y c , 10mMsodiumglycolate; TRIS, 20mMr i s ; Citr, 20 p.M sodium citrate; EDTA, 10p.M sodium EDTA; and NTA, 10 p.M sodium NTA.

unusual among bacteria ( 8 ) ; hence the filamentous morphol-ogy of the Thiothrix strain did not appear to enhance thec a p a c i ty for metal adsorption.The a f f i n i t y of the cells for metals testedwasCu >Zn1000-o

40000c , , L

Cu N i Z nF I G . 8 . D e s o r p t i o n o f m e t a l s f r o m m e t a l - l a d e n T h i o t h r i x c s t r a i nAl c e l l s . T h e i n i t i a l amount o f m et a l s s o r b e d was 4 3 1 p m o l o f Cu p e rg o f c e l l p r o t e i n , 1 6 0 p L m o l o f N i p e r g o f c e l l p r o t e i n , o r 1 7 4 , u m o l o fZn p e r g o f c e l l p r o t e i n . Th e d e s o r p t i o n medium c o ns i s te d o f 6 mMK C I , 5 mM C a C 1 2 , o r 5 mMEDTA made e i t h e r i n u l t r a p u r e w a t e r o ri n 2 0 mM HEPES b u f f e r a t pH 7 . 2 0 . S y m b o l s : _, w a t e r ; =,HEPES b u f f e r ; E D 1 , K C I ; K3 KCI i n HEPES b u f f e r ; G M Z , C a C l 2 ;1, C a C 1 2 i n HEPES b u f f e r ; M , EDTA i n HEPES b u f f e r . * , nod e s o r p t i o n .

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T i m e ( d a y s )FIG. 9 . U p t a k e o f n i c k e l b y T h i o t h r i x s t r a i n Al d u ri ng g r o w t h .T h e i n i t i a l n i c k e l c o n c e n t r a t i o n was 1 . 1 9 , u M .

Ni . I n e x p e r i m e n t s with mixtures o f m e t a l s , t h e r e a p p e a r e dt o b e l i t t l e s e l e c t i v i t y betw e e n n i c k e l and z i n c . T h e r e g r e s -s i o n o f t h e r a t i o s m e t h o d u s e d t o determine s e l e c t i v i t y ofn i c k e l an d z i n c was u n i q u e , an d i t i s s u g g e s t e d t h a t t h i sm e t h o d b e use d i n t h e f u t u r e t o p r o v i d e a q u a n t i t a t i v eassessment of t h e c o m p e t i t i o n o f c a t i o n s f o r b i n d i n g s i t e s .A l t h o u g h others h av e s h o w n c o m p e t i t i o n between m e t a l s f o rb i n d i n g s i t e s and metal s e l e c t i v i t y ( 5 , 7 , 2 1 ) , t h e p o t e n t i a l f o rp r e f e r e n t i a l u p t a k e of metals a t s p e c i f i c s i t e s on m i c r o b i a lsurfaces merits f u r t h e r s t u d y because of i t s i m p o r t a n c e t ob i o s o r p t i o n t e c h n o l o g y .T h e s o r p t i o n o f metal i o n s t o r e s t i n g c e l l s o f T h i o t h r i xs t r a i n Al a p p e a r e d t o i n v o l v e p r i m a r i l y an i o n - e x c h a n g et y p e of r e a c t i o n . This contention i s s u p p o r t e d b y t h e d a t aobtained i n t h e k i n e t i c s , c a t i o n c o m p e t i t i o n , and d e s o r p t i o ne x p e r i m e n t s . I t i s well s u b s t a n t i a t e d t h a t m e t a b o l i c a l l y i n d e -p e n d e n t u p t a k e o f metals i s u s u a l l y v e r y r a p i d ( 8 ) , an d metalu p t a k e b y T h i o t h r i c s t r a i n Al was v e r y r a p i d . I n a d d i t i o n ,metal d e s o r p t i o n f r o m T h i o t h r i x s t r a i n Al was enhanced b yi n c r e a s i n g t h e i o n i c s t r e n g t h an d b y a d d i n g c o m p e t i n g c a t -ions an d c h e l a t i n g a g e n t s ; t h e r e f o r e , m o st o f t h e m e t a l wasrather l o o s e l y associated with t h e c e l l s . H o w e v e r , a p p r o x i -m a t e l y 20% of t h e b o u n d metal i o n s were n o t r e m o v e d fromT h i o t h r i x s t r a i n Al b y t h e s t r o n g c h e l a t i n g a g e n t EDTA; t h i snonextractable m e ta l c o ul d have d i f f u s e d i n t o t h e i n t e r i o r o fth e c e l l s . Xue e t a l . ( 3 8 ) r e p o r t e d t h a t Cu u p t a k e i n t h e a l g aC h l a m y d o m o n a s r h e i n h a r d i i occurred i n tw o s t e p s an dp r e s e n t e d ev idence t h a t t h e second s t e p was a r e s u l t o f t h ed i f f u s i o n of metals i n t o t h e c e l l s . A l t e r n a t i v e e x p l a n a t i o n s o fth e o b s e r v e d phenomenon i n T h i o t h r i x s t r a i n Al i n c l u d e ( i )c o m p l e x a t i o n of metals a t s i t e s with h i g h e r s t a b i l i t y c o n-s t a n t s than EDTA and ( i i ) t h e p r e s e n c e o f m e t a l - b i n d i n g s i t e sw h i c h ar e i n a c c e s s i b l e t o EDTA. I t seems u n l i k e l y , h ow-e v e r , t h a t there w e r e s u r f a c e - b i n d i n g s i t e s with h i g h e r a f f i n -i t i e s f o r metals than EDTA, because t h e r e was no e v i d e n c eo f a n y b a c t e r i a l metal u p t a k e f ro m p r e f o r m e d metal-EDTAc o m p l e x e s .T h i o t h r i x s t r a i n Al has an u n u s u a l m o d e o f m e t a b o l i s m i nt h a t i t a p p e a r s t o b e an o b l i g a t e m i x o t r o p h an d r e q u i r e s ar e d uc e d i n o r g a n i c s u l f u r s o u r c e f o r g r o w t h ( 3 7 ) . I n a g r o w t hmedium of a c e t a t e and t h i o s u l f a t e , m o s t o f t h e t h i o s u l f a t e i su t i l i z e d i n t h e e a r l y l o g p h a s e o f g r o w t h an d s u l f u r g r a n u l e sar e f o r m e d d u r i n g t h i s p e r i o d o f t h i o s u l f a t e u t i l i z a t i o n ( 3 3 ) .As t h e c u l t u r e a g e s , s t o r e d s u l f u r d i s a p p e a r s . T h e r e f o r e ,c e l l s i n e a r l y l o g p h a s e d i f f e r m e t a b o l i c a l l y from t h o s e i n l a t el o g p h a s e , an d metabolic d i f f e r e n c e s may h a v e c o n t r i b u t e dto the observed e f f e c t o f c e l l a g e on n i c k e l and z i n c u p t a k e .H o w e v e r , i t i s a l s o p o s s i b l e t h a t o t h e r t i m e - d e p e n d e n tc h a n g e s accounted f o r t h e o b s e r v e d v a r i a t i o n s , b e c a u s e

r e s e a r c h e r s w o r k i n g w i t h a d i s s i m i l a r t y p e o f b a c t e r i u m ,Z o o g l o e a r a m i g e r a , a l s o f o u n d t h a t m e t a l u p t a k e was c u l -t u r e a g e d e p e n d e n t ( 2 3 ) .The p r e s e n c e o f o r g a n i c l i g a n d s i s u s u a l l y a s s u m e d t or e d u c e t h e s o r p t i o n o f m e t a l s t o b a c t e r i a b e c a u s e o n l y f r e em e t a l i o n s a r e b i o a v a i l a b l e . T h i s i s t r u e p r o v i d e d t h a t ( i )l i g a n d s a r e n o t d e g r a d e d b y t h e b a c t e r i a a n d ( i i ) s t a b i l i t yc o n s t a n t s f o r t h e o r g a n i c l i g a n d s a r e g r e a t e r t h a n c o n s t a n t sf o r t h e s i t e s on t h e b a c t e r i a l s u r f a c e . I n s t u d i e s on l i g a n de f f e c t s , most r e s e a r c h e r s h a v e u s e d v e r y s t r o n g l i g a n d sw h i c h a r e g e n e r a l l y n o t d e g r a d e d . I n o u r work w e n o t o n l ye x a m i n e d t h e a b i l i t y o f weak l i g a n d s t o i n f l u e n c e b a c t e r i a lm e t a l s o r p t i o n b u t a l s o u s e d a c o m p u t e r m o d e l o f t h et h e o r e t i c a l m e t a l s p e c i a t i o n t o a s s i s t i n t h e i n t e r p r e t a t i o n o ft h e r e s u l t s . T h e o b s e r v e d e f f e c t s o f t h e s t r o n g c h e l a t i n ga g e n t s E D T A a n d NTA on n i c k e l a n d z i n c s o r p t i o n t oT h i o t h r i x s t r a i n Al were a c c u r a t e l y p r e d i c t e d f r o m t h e s et h e o r e t i c a l c o n s i d e r a t i o n s . T h e EDTA a n d NTA s p e c i e sw e r e e x a c t l y 50 % o f t h e 0 . 4 p u m o l o f t o t a l m e t a l ; t h e r e f o r e ,0 . 2 , u m o l was p r e d i c t e d t o b e a v a i l a b l e f o r b i o s o r p t i o n . T h ea m o u n t s o f n i c k e l a n d z i n c w h i c h were s o r b e d f r o m t h eE D T A a n d NTA s o l u t i o n s were n e a r l y i d e n t i c a l t o t h ea m o u n t s s o r b e d f r o m t h e c o n t r o l s , w h i c h c o n t a i n e d 0 . 2 , u m o lo f t o t a l m e t a l .T h e e f f e c t s o f a c e t a t e , l a c t a t e , g l y c o l a t e , a n d T r i s o n m e t a ls o r p t i o n w e r e m o r e d i f f i c u l t t o i n t e r p r e t . B e c a u s e t h e s t a b i l -i t y c o n s t a n t s o f t h e s e c o m p o u n d s a r e r e l a t i v e l y l o w , h i g hc o n c e n t r a t i o n s w e r e r e q u i r e d t o s i g n i f i c a n t l y a l t e r m e t a ls p e c i a t i o n . T h e h i g h c o n c e n t r a t i o n o f l i g a n d s e l e v a t e d t h ei o n i c s t r e n g t h o f t h e r e a c t i o n m e d i u m , a n d i n t h e s e c a s e s , i twas n o t p o s s i b l e t o s e p a ra t e t h e i o n i c s t r e n g t h e f f e c t s f r o mt h e l i g a n d e f f e c t s , e v e n t h o u g h a c o nt r o l f o r i o n i c s t r e n g t hwas i n c l u d e d i n t h e e x p e r i m e n t s . O f t h e l i g a n d s u s e d , o n l ya c e t a t e a n d l a c t a t e a r e d e g r a d a b l e b y T h i o t h r i x s t r a i n A l( 3 7 ) . D e g r a d a t i o n o f t h e s e l i g a n d s was e x p e c t e d t o c a u s e a ni n c r e a s e i n t h e a m o u n t o f m e t al s o r b ed o v e r t h e amountp r e d i c t e d t o b e a v a i l a b l e . T h e r e was n o e v i d e n c e f o r i n -c r e a s e d s o r p t i o n w i t h a c e t a t e , p o s s i b l y b e c a u s e t h e l a c t a t e -g r o w n c e l l s d i d n o t d e g r a d e e n o u g h a c e t a t e t o a f f e c t m e t a ls p e c i a t i o n . I n c o n t r a s t , n i c k e l s o r p t i o n i n t h e p r e s e n c e o fl a c t a t e was n o t o n l y h i g h e r t h a n p r e d i c t e d i n t h e a b s e n c e o fl a c t a t e d e g r a d a t i o n b u t a l s o h i g h e r t h an t h a t o f t h e c o n t r o l( n o l a c t a t e p r e s e n t ) . T h i s r e s u l t was r e p r o d u c i b l e .T h e t r i a l s w i t h c i t r a t e w e r e t h e m o s t d i f f i c u l t t o i n t e r p r e t ,a n d i t i s p o s s i b l e t h a t t h e p r e d i c t e d s p e c i a t i o n w a s n o ta c c u r a t e . I t i s w e l l known t h a t t h e r m o d y n a m i c c o n s t a n t sm ay b e i n a c c u r a t e b e c a u s e o f a n a l y t i c a l l i m i t a t i o n s ( 3 2 ) , a n di n many i n s t a n c e s t h e e q u i l i b r i u m c o n s t a n t s i n t h e MINEQLc o m p u t e r p r o g r a m w e r e s u b s t a n t i a l l y d i f f e r e n t f r o m c o n -s t a n t s f o u n d i n t h e l i t e r a t u r e ( 1 8 , 1 9 , 2 8 - 3 0 ) . H o w e v e r , i t wasn o t p o s s i b l e t o r e p l a c e t h e c i t r a t e t h e r m o d y n a m i c d a t a i n t h eMINEQL p r o g r a m , b e c a u s e some o f t h e n e e d e d c r i t i c a ls t a b i l i t y c o n s t a n t s w e r e n o t a v a i l a b l e .T h e e f f e c t ( s ) o f l i g a n d s o n t h e s o rp t io n o f m et a l s h a s a l s ob e e n n o t e d i n o t h e r s t u d i e s . F o r i n s t a n c e , S i n g h a n d Y a d a v a( 2 7 ) d e m o n s t r a t e d t h a t c i t r a t e s t i m u l a t e d cadmium u p t a k e i nt h e c y a n o b a c t e r i u m A n a c y s t i s n i d u l a n s w h e r e a s E D T A i n -h i b i t e d c a d m i u m u p t a k e . T h e s e w o r k e r s a t t r i b u t e d t h e c i t -r a t e - e n h a n c e d u p t a k e t o t h e a v a i l a b i l i t y o f c i t r a t e a s a c a r b o ns o u r c e f o r t h e o r g a n i s m . S i m i l a r l y , J a r d i m a n d P e a r s o n ( 1 3 )f o u n d t h a t c i t r a t e d e g r a d a t i o n d u r i n g g r o w t h o f P l e c t o n e m ab o r y a n u m c o u l d c a u s e a n i n c r e a s e i n t h e a m o u n t o f f r e ei o n i c c o p p e r i n t h e g r o w t h m e d i u m .P r e d i c t i n g a n d i n t e r p r e t i n g m e t a l u p t a k e b y g r o w i n g c u l -t u r e s o f b a c t e r i a i s c o m p l e x b e c a u s e m e d i a c o n t a i n many

A P P L . E N V I R O N . M I C R O B I O L .


8/9

SORPTION O F HEAVY METALS TO T H I O T H R I X STRA IN Al 1 2 8 1c o m p o n e n t s w h i c h c a n i n f l u e n c e m e t a l t o x i c i t y a n d / o r m e t a ls p e c i a t i o n . In t h i s s t u d y t h e me dium c o n t a i n e d s u b s t a n t i a la m o u n t s o f c a l c i u m a nd m a g n e s i u m , b o t h o f w h i c h w e r eshown t o i n h i b i t t h e s o r p t i o n o f z i n c i n s h o r t - t e r m e x p e r i -m e n t s . T h e l a c t a t e i n t h e m e d i u m m u s t h a v e f o r m e d c o m -p l e x e s w i t h t h e m e t a l s , b u t t h e s e c o m p l e x e d m e t a l i o n s w e r ep r o b a b l y made a v a i l a b l e by m i c r o b i a l d e g r a d a t i o n o f t h el a c t a t e . F u r t h e r m o r e , t h e p H o f t h e LTH m e d i u m r o s es i g n i f i c a n t l y d u r i n g g r o w t h o f T h i o t h r i x s t r a i n A l , a n d t h es o l u t i o n p H c a n a f f e c t t h e e x t e n t o f m e t a l b i n d i n g . Ther e m o v a l o f n i c k e l f r o m t h e g r o w t h me dium d i d n o t , h o w e v e r ,a p p e a r t o b e a r e s u l t o f p H - r e l a t e d s o r p t i o n , s i n c e t h ec e l l - a s s o c i a t e d n i c k e l was n o t d e s o r b e d by l o w e r i n g t h e p H .I n f a c t , t h e l i m i t e d r e m o v a l o f c el l - a ss o ci a t ed n i ck el byEDTA s u g g e s t s t h a t t h e m e t a l u p t a k e d u r i n g g r o w t h was n o ta n i o n - e x c h a ng e p h e n o m e n o n ; t h e r e f o r e , t h e m e c h a n i s m f o rm e t a l u p t a k e i n g r o w i n g c e l l s o f T h i o t h r i x s t r a i n Al m ayd i f f e r f r o m t h e m e c h a n i s m f o r u p t a k e i n r e s t i n g c e l l s . Thea v a i l a b i l i t y o f a n e n e r g y s o u r c e ( s ) m ay c a u s e a c t i v e l y g r o w -i n g c u l t u r e s t o r e s p o n d d i f f e r e n t l y f r o m r e s t i n g c e l l s , b e c a u s ee n e r g y - d e p e n d e n t m e t a l e f f l u x a n d e n er g y - d e p e n d en t m et a lu p t a k e s y s t e m s h a v e b o t h b e e n r e p o r t e d ( 2 , 2 2 ) . F u r t h e r -m o r e , m i c r o o r g a ni s m s h a v e b e e n shown t o a l t e r t h e i r g r o w t hmedium i n w a y s w h i c h c a n i n h i b i t m et a l u p t a k e ( 1 1 , 1 2 ) .S h u t t l e w o r t h a n d Un z ( 2 6 ) d e t e r m i n e d t h a t c o p p e r wasm o r e t o x i c t h a n e i t h e r n i c k e l o r z i n c t o T h i o t h r i x s t r a i n A l ,a n d r e s u l t s f r o m t h e p r e s e n t s t u d y r e v e a l e d t h a t T h i o t h r i xs t r a i n Al h a d t h e h i g h e s t s o r p t i o n c a p a c i t y f o r c o p p e r ,a l t h o u g h t h e h i gh es t a d s o r p t i o n i n t e n s i t y o c c u r r e d a t l o wc o n c e n t r a t i o n s o f n i c k e l . C a l c i u m a n d m a g n e s i u m w e r ef o u n d t o r e d u c e t h e t o x i c i t y o f h e a v y m e t a l s ( 2 6 ) a n d t or e d u c e t h e s o r p t i o n o f z i n c t o t h e b a c t e r i u m . S i m i l a r l y ,n i c k e l t o x i c i t y w a s d i m i n i s h e d b y t h e p r e s e n c e o f z i n c , a n dt h e a b i l i t y o f z i n c a n d n i c k e l t o c o m p e t e f o r s o r p t i o n s i t e swas c o n f i r m e d . T h e c h e l a t i n g a g e n t EDTA e f f e c t i v e l y p r e -v e n t e d n i c k e l a n d z i n c s o r p t i o n , a s w e l l a s d e c r e a s i n g t h et o x i c i t y o f t h e s e m e t a l s t o t h e b a c t e r i u m . T h u s , a l l o f t h et o x i c e f f e c t s o f m e t a l s o n T h i o t h r i x s t r a i n A l , e x c e p t t h es y n e r g i s t i c t o x i c i t y o f c o p p e r - n i c k e l a n d c o p p e r - z i n c m i x -t u r e s , c a n b e a cc o u n t e d f o r b y m e t a l s o r p t i o n .

ACKNOWLEDGMENTST h i s s t u d y was f u n d e d , i n p a r t , b y NSF g r a n t E CE 8 4 1 7 6 6 3 a n d b yt h e E n v i r o n m e n t a l R e s o u r c e s R e s e a r c h I n s t i t u t e , C o l l e g e o f E n g i -n e e r i n g , a n d D e p a r t m e n t o f C i v i l E n g i n e e r i n g , a l l o f T h e P e n n s y l -v a n i a S t a t e U n i v e r s i t y .We t h a n k C h r i s Co x f o r h i s i n s t r u c t i o n o n t h e MINEQL p r o g r a ma n d W i l l i a m T a r u t i s f o r h i s h e l p w i t h t h e s t a t i s t i c a l a n a l y s e s .

REFERENCES1 . B e v e r i d g e , T . J . 1 9 8 9 . R o l e o f c e l l u l a r d e s i g n i n b a c t e r i a l m e t a la c c u m u l a t i o n a n d m i n e r a l i z a t i o n . A n n u . R e v . M i c r o b i o l . 4 3 :1 4 7 - 1 7 1 .2 . B u c h e d e r , F . , a n d E . B r o d a . 1 9 7 4 . E n e r g y - d e p e n d e n t z i n ct r a n s p o r t b y E s c h e r i c h i a c o l i . E u r . J . B i o c h e m . 4 5 : 5 5 5 - 5 5 9 .3 . C h a n g , Y . , J . T . P f e f f e r , a n d E . S . K . C h i a n . 1 9 8 0 . D i s t r i b u t i o no f i r o n i n S p h a e r o t i l u s a n d t h e a s s o c i a t e d i n h i b i t i o n . A p p l .E n v i r o n . M i c r o b i o l . 4 0 : 1 0 4 9 - 1 0 5 2 .4 . D o w d , J . E . , a n d D . S . R i g g s . 1 9 6 5 . A c o m p a r i s o n o f e s t i m a t e so f M i c h a e l i s - M e n t e n k i n e t i c c o n s t a n t s f r o m v a r i o u s l i n e a r t r a n s -f o r m a t i o n s . J . B i o l . C h e m . 2 4 0 : 8 6 3 - 8 6 9 .5 . D o y l e , R . J . , T . H . M a t t h e w s , a n d U . N . S t r e i p s . 1 9 8 0 . C h e m i c a lb a s i s f o r s e l e c t i v i t y o f m e t a l i o n s b y t h e B a c i l l u s s u b t i l i s c e l lw a l l . J . B a c t e r i o l . 1 4 3 : 4 7 1 - 4 8 0 .6 . E l r a s h i d i , M. A . , a n d G . A . O ' C o n n o r . 1 9 8 2 . I n f l u e n c e o fs o l u t i o n c o m p o s i t i o n o n s o r p t i o n o f z i nc b y s o i l s . S o i l S c i . S o c .Am . J . 4 6 : 1 1 5 3 - 1 1 5 8 .7 . F a i s o n , B . D . , C . A . C a n c e l , S . N . L e w i s , a n d H . I . A d l e r . 1 9 9 0 .

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V O L . 5 9 , 1 9 9 3


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1 2 8 2 SHUTT L EWO R T H AND UNZ2 n d e d . , p . 8 - 1 2 0 . J o h n W i l e y & S o n s , I n c . , Ne w Y o r k .3 3 . U n z , R . F . , a n d T . M. W i l l i a m s . 1 9 8 9 . S u b s t r a t e u t i l i z a t i o n b yf i l a m e n t o u s s u l f u r b a c t e r i a o f a c t i v a t e d s l u d g e , p . 4 1 2 - 4 1 6 . I n T .H a t t o r i , Y . I s h i d a , Y . M a r u y a m a , R . Y . M o r i t a , a n d A . U c h i d a( e d . ) , R e c e n t a d v a n c e s i n m i c r o b i a l e c o l o g y . J a p a n S c i e n t i f i cS o c i e t i e s P r e s s , T o k y o .3 4 . W e b e r , W. J . , J r . 1 9 7 2 . P h y s i c o c h e m i c a l p r o c e s s e s f o r w a t e rq u a l i t y c o n t r o l , p . 1 9 9 - 2 6 0 . J o h n W i l e y & S o n s , I n c . , Ne wY o r k .3 5 . W e s t a l l , J . C . , J . L . Z a c h ar y , a n d F . M. M. M o r e l . 1 9 8 6 .MINEQL: a c o m p u t er p r o g r am f o r t h e c a l c u l a t i o n o f t h e

A P P L . E N V I R O N . M I C R O B I O L .c h e m i c a l e q u i l i b r i u m c o m p o s i t i o n o f a q u e o u s s y s t e m s . O r e g o nS t a t e U n i v e r s i t y r e p o r t n o . 8 6 - 0 1 . V e r s i o n 1 . O r e g o n S t a t eU n i v e r s i t y , C o r v a l l i s .3 6 . W i l l i a m s , T . M . , a n d R . F . U n z . 1 9 8 5 . I s o l a t i o n a n d c h a r a c t e r -i z a t i o n o f f i l a m e n t o u s b a c t e r i a p r e s e n t i n b u l k i n g a c t i v a t e ds l u d g e . A p p l . M i c r o b i o l . B i o t e c h n o l . 2 2 : 2 7 3 - 2 8 2 .3 7 . W i l l i a m s , T . M . , a n d R . F . U n z . 1 9 8 9 . T h e n u t r i t i o n o f T h i o t h r n x ,t y p e 0 2 1 N , B e g g i a t o a , a n d L e u c o t h r i x s t r a i n s . W a t e r R e s .2 3 : 1 5 - 2 2 .3 8 . X u e , H . - B . , W. S t u m m , a n d L . S i g g . 1 9 8 8 . T h e b i n d i n g o f h e a v ym e t a l s t o a l g a l s u r f a c e s . W a t e r R e s . 2 2 : 9 1 7 - 9 2 6 .

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