515

Photosynthesis Research 10." 551-518 (1986) © Martinus N i j h o f f Publishers, Dordrecht - Printed in the Netherlands

ACCUMULATION OF SILVER SILv~kTHIOSULFATE

BY ChromatiumvinosuuFROM

SOLUTIONS

CONTAINIWG

MASA0 KITAJIMA Research Laboratories, S a i t a m a - k e n 351, Japan Key

words:Chromatium bacteria, s i l v e r

Asaka,

Fuji

Photo

Film

vinosum, photosynthetic r e s i s t a n t bacteria, s i l v e r

Co.,

Ltd.,

Asaka-shi,

sulfur bacteria, sulfur a c c u m u l a t i o n by bacteria.

1. A B S T R A C T

The p h o t o s y n t h e t i c s u l f u r bacterium, C h r o m a t i u m vinosum, was c u l t u r e d in i n o r g a n i c p h o t o g r a p h i c processing solutions containing silver thiosulfate complex salt (AgNa3(S203)2) under light. It w a s f o u n d t h a t Chromatium w a s r e s i s t a n t to A g a n d a c c u m u l a t e d granular silver in t h e membrane during growth. The amount of Ag a c c u m u l a t e d in the cel is depended on the initial c o n c e n t r a t i o n s of the A g s a l t in the c u l t u r e solution. When the c o n c e n t r a t i o n of Ag was 300 mg/l, the bacteria a c c u m u l a t e d Ag as high as 30% of t h e d r y c e l l w e i g h t . T h e s i z e of t h e g r a n u l e s w a s 0.1 to 0 . 3 ~ m . R e s u l t s from X-ray m i c r o a n a l y s i s indicated that these granules c o n s i s t e d m o s t l y of Ag ° with s m a l l fractions of Ag2S and AgCl. 2. r t q T R O ~ I O N Ag is one of the most toxic h e a v y m e t a l s to bacteria. Its inhibitory a c t i o n is s t r o n g e r t h a n t h a t of Cd, Zn a n d Hg. A n t i m i c r o b i a l agents such as s i l v e r s u l f a d i a z i n e were w i d e l y used as drugs for t r e a t m e n t of infected burns a n d wounds. S e v e r a l bacteria which are resistant to s i l v e r s u l f a diazine were isolated from burns and their properties w e r e s t u d i e d [I]. The m e t a b o l i s m of Ag in P s e u d o m o n a s stutzeri i s o l a t e d from a s i l v e r mine was studied. The Ag a c c u m u l a t i o n was c l o s e l y c o r r e l a t e d with the presence of a p l a s m i d [2]. We e a r l i e r reported that Thiobacillus novellus and Thiobacillus thioparus, as w e l l as C h r o m a t i u m vinosum, were grown in used p h o t o g r a p h i c processing solutions containing silver thiosulfate complex salt (AgNa3(S203) 2) a n d suggested that these bacteria c o u l d be u s e d for t r e a t i n g p h o t o g r a p h i c waste water [3,4]. S i l v e r resistance of other m i c r o o r g a n i s m s in p h o t o g r a p h i c industrial water was also reported [5]. A c o m m u n i t y of bacteria, c o n s i s t i n g of Pseudomonas m a l t o ~ h i l i a , S t a p h y l o c o c c u s aureus and a coryneform organism, was isolated from soil near a silver mine and was found to be resistant to A g c o n c e n t r a t i o n up to 100 mM; moreover, these Ag adapted bacteria accumulated A g [6]. Thiobacillus thiooxidans and Thiobacillus ferrooxidans were also reported to a c c u m u l a t e small Ag g r a n u l e s c o n s i s t i n g of Ag2S on the surface of c e l l m e m b r a n e s [7]. I now report that a photosynthetic sulfur bacterium, Chromatium vinosum, c u l t u r e d in the presence of s i l v e r t h i o s u l f a t e c o m p l e x s a l t accumulates Ag ° granules in t h e m e m b r a n e . Conditions and some suggested m e c h a n i s m s for the a c c u m u l a t i o n w i l l be discussed. 3. M E T H O D S

AND MATERIALS

3.1. C u l t u r e conditions. C h r o m a t i u m v i n o s u m was grown in a standard m e d i u m c o n t a i n i n g p e r l i t e r : N a C I , 3.0g; N H 4 C I , 1.0g; C a C I 2 , 0.05g; K H 2 P O 4 , 0.5g;

[369]

516

[3vo]

FeCI 3 6H20, 0.05g; K 2 H P O 4 , 0.5g; a n d N a H C 0 3 , 2.0g. To t h i s s o l u t i o n w e r e added 2g of N a 2 S 2 0 3 . 5 H 2 0 for c o n t r o l e x p e r i m e n t s or v a r i o u s c o n c e n t r a t i o n s of A g N a 3 ( S 2 0 3 ) 2 p r e p a r e d from photographic fixing solutions used for p r o c e s s i n g p h o t o g r a p h i c film. The pH of the final s o l u t i o n was 5.8 to 6.0. Narrow-neck, 125 ml g l a s s b o t t l e s were f i l l e d with the m e d i u m c o n t a i n i n g a b o u t 1 0 0 m g d r y c e l l s p e r 1 a n d w e r e s e a l e d w i t h wax. O x y g e n in t h e s o l u t i o n s w a s c o n s u m e d f o r o x i d a t i o n of S 2- or $ 2 0 ~,-~ a n d t h u s a n a e r o b i c c o n d i t i o n s were m a i n t a i n e d d u r i n g the incubation. The b o t t l e s were kept at o . . . . 30 C and were 1 1 1 u m l n a t e d wlth a set of i n c a n d e s c e n t lamps w h i l e stirring. T h e l i g h t i n t e n s i t y at t h e s u r f a c e of t h e b o t t l e w a s 2 m W / c m 2. T h e c e l l s were c o l l e c t e d on m e m b r a n e filters, and dry c e l l w e i g h t was m e a s u r e d after d r y i n g the f i l t e r s in an o v e n at 110°C for I hr. 3.2. ~ u a n t i t a t i v e analysis of A@. T h e a m o u n t of A g a c c u m u l a t e d in t h e cells was determined by two different procedures: d e c r e a s e of A g in t h e c u l t u r e s o l u t i o n s and direct m e a s u r e m e n t of Ag in the cells. Ag c o n c e n t r a tions in c u l t u r e s o l u t i o n s were m e a s u r e d with an atomic a b s o r p t i o n spectrophotometer (Hitachi Z-7000) before inoculation a n d a f t e r r e m o v a l of t h e c u l t u r e d c e l l s f o l l o w i n g v a r i o u s i n c u b a t i o n times. The amount of Ag accum u l a t e d in the c e l l s was d e t e r m i n e d d i r e c t l y by the f o l l o w i n g procedure. A l l c e l l s in a b o t t l e were c o l l e c t e d by c e n t r i f u g a t i o n and were r e d i s p e r s e d in 5ml of 5% g e l a t i n solution. The w h o l e amount of the s o l u t i o n was then coated homogeneously on a 5 0 c m 2 g l a s s p l a t e a n d dried. T h e a m o u n t of Ag p e r u n i t a r e a of t h e p l a t e w a s m e a s u r e d w i t h an X - r a y f l u o r o p h o t o m e t e r ( P h i l i p s P W 1410). The results were corrected with a calibration curve prepared by using solutions containing predetermined concentrations of AgNO 3 • X-ray m i c r o a n a l y s i s of each c e l l was p e r f o r m e d w i t h a S h i m a d z u E P M 810 X-ray microanalyzer. One drop of the c e l l c u l t u r e from a high c o n c e n t r a tion t h i o s u l f a t e s o l u t i o n was s p o t t e d on a g l a s s plate, dried a n d p l a c e d on the m i c r o a n a l y z e r . An e l e c t r o n p h o t o m i c r o g r a p h of one c e l l was taken, and then the X-ray diffraction p a t t e r n of a g r a n u l e in t h e m e m b r a n e w a s measured. 4. R E S U L T S A N D D I S C U S S I O N C h r o m a t i u m v i n o s u m w a s f o u n d to be r e s i s t a n t to s i l v e r t h i o s u l f a t e c o m p l e x s a l t (AgNa3(S203) 2. It grew c o n t i n u o u s l y in a m e d i u m c o n t a i n i n g Ag at c o n c e n t r a t i o n s lower than 10mg/l. The c u l t u r e r e a c h e d a steady state at 350 to 6 0 0 m g of dry c e l l s per 125mi in 2 to 3 days. When the Ag c o n c e n t r a t i o n w a s h i g h e r , t h e c o l o r of t h e c e l l s c h a n g e d f r o m r e d to d a r k r e d a n d finally black. T h e c e l l s u s p e n s i o n w a s s t a b l e at t h e b e g i n n i n g of t h e incubation, but w h e n the b a c t e r i a a c c u m u l a t e d a s u b s t a n t i a l amount of Ag, t h e y s e d i m e n t e d e a s i l y upon t e r m i n a t i o n of stirring. As C h r o m a t i u m a c c u m u l a t e d A g from the medium, Ag c o n c e n t r a t i o n s in the m e d i u m were reduced. W h e n the i n i t i a l Ag c o n c e n t r a i o n was 4.6mg/l, a l m o s t a l l A g w a s r e m o v e d f r o m t h e m e d i u m in 18 hr. W h e n t h e i n i t i a l A g c o n c e n t r a t i o n was 76mg/l, 43% was r e m o v e d in 18 hr a n d 98% in 62 hr. The amount of Ag a c c u m u l a t e d in the c e l l s were d e t e r m i n e d d i r e c t l y by an X - r a y f l u o r o p h o t o m e t e r . It was found that the a c c u m u l a t i o n of Ag in the c e l l s d e p e n d e d on the i n i t i a l Ag c o n c e n t r a t i o n s in the c u l t u r e medium. The results a r e s h o w n in T a b l e I. When the initial Ag concentration was 45mg/l, the cells accumulated A g u p to 10% of t h e d r y c e l l w e i g h t . When the i n i t i a l c o n c e n t r a t i o n was 266mg/I, 29% of the dry c e l l w e i g h t was Ag. In o r d e r to i d e n t i f y t h e s i t e of A g a c c u m u l a t i o n , a single cell was observed and analyzed w i t h an X - r a y m i c r o a n a l y z e r . Figure 1 is an e l e c t r o n p h o t o m i c r o g r a p h of C h r o m a t i u m c u l t u r e d in a m e d i u m w i t h high Ag

517

[3711 Table I. A m o u n t of s i l v e r a c c u m u l a t e d by Chromatium vinosum from s o l u t i o n s c o n t a i n i n g v a r i o u s concentrations of s i l v e r thiosulfate. Initial Ag Conc. (mg/l)

0 3.0 45.2 226

Dry cell w e i g h t (mg/125ml)

342 541 430 561

Ag found in cells (mg/125ml) 0 3.0 43.3 135

Ag content (%)

0 0.6 10 29

concentration. It m a y be s e e n f r o m t h e f i g u r e t h a t t h e A g g r a n u l e s (electron dense spots) are located m o s t l y in the c e l l membrane. The number of t h e g r a n u l e s is n o t v e r y l a r g e b u t t h e y l o o k to a l m o s t fill the membrane. The size of the g r a n u l e s varies but many of them are as large as 0.1 to 0.3 ~ m . F i g u r e 2 is a r e s u l t of an X - r a y d i f f r a c t i o n p a t t e r n of o n e c e l l . A d i s t i n c t i v e l y high peak a s s i g n e d as A g ° and s m a l l peaks assigned as Ag2S and AgCI are observed. This i n v e s t i g a t i o n indicates that Ag is a c c u m u l a t e d b y C h r o m a t i u m m a i n l y in m e t a l l i c form, r a t h e r t h a n A g 2 S as r e p o r t e d b y P o o l e y [7] with T h i o b a c i l l u s . Some experiments were carried o u t to u n d e r s t a n d more about the mechanisms of A g a c c u m u l a t i o n by Chromatium. Chromatium vinosum is a f a c u l t a t i v e autotrophic bacterium. It u t i l i z e s reducing e n e r g y of ~ 2 - or S20 in s t r i c t l y inorganic medium. B u t w h e n it is c u l t u r e d in t h e presence of organic proton acceptor such as malate, it u t i l i z e s the organic reductant preferably to t h e i n o r g a n i c r e d u c t a n t s . In o r d e r to c o n f i r m whether the Ag accumulation p r o c e s s is c o r r e l a t e d to an e n e r g y u p t a k e m e c h a n i s m of the bacterium, the effect of m a l a t e on the Ag a c c u m u l a t i o n was studied. It was found that Chromatitun grew more r a p i d l y in the presence of malate but accumulation of A g d i m i n i s h e d markedly as c o m p a r e d with

F I G U R E 1. E l e c t r o n p h o t o m i c r o g r a p h granules in the membrane.

of C h r o m a t i u m v i n o s u m c o n t a i n i n g

silver

518

[372]

F I G ~ R E 2. X - r a y d i f f r a c t i o n p a t t e r n of a s i l v e r brane m e a s u r e d by an X - r a y m i c r o a n a l y z e r .

granule

in t h e c e l l

mem-

e x p e r i m e n t s done in the inorganic medium. Effect of i l l u m i n a t i o n on the Ag a c c u m u l a t i o n was a l s o investigated. It was found that the b a c t e r i u m accum u l a t e d Ag under dark conditions, a l t h o u g h it grew m u c h s l o w e r than w i t h illumination. T h e e f f e c t of s o d i u m p e n t a c h l o r o p h e n o l a t e , a strong u n c o u p l e r of p h o s p h o r y l a t i o n [8], w a s a l s o e x a m i n e d . The bacterium was found to a c c u m u l a t e Ag in the p r e s e n c e of the uncoupler. The amount of the a c c u m u l a t e d Ag was about 2/3 of the control. AqNa3(S203) 2 is s t a b l e in s o l u t i o n and its s o l u b i l i t y is quite high. C h r o m a t i u m m a y n o t d i s t i n g u i s h ~ N a 2 S 2 0 3 a n d A g N a 3 ( S 2 0 3 ) 2 at t h e m e m b r a n e ht u t i l i z e s $ 2 0 ~- as e n e r g y s o u r c e a n d m a y l e a v e A g in t h e intramembrane structure. As t h e i n t r a m e m b r a n e r e g i o n is m a i n t a i n e d in reductive atmosphere, A g + is r e d u c e d a n d m a y d e p o s i t as A g ° a n d f o r m granules. P r e l i m i n a r y studies do not p r o v i d e m u c h d e t a i l about the accumul a t i o n mechanism, and m o r e w o r k is n e c e s s a r y to u n d e r s t a n d it fully. S

T h i s s t u d y w a s s t a r t e d at t h e U n i v e r s i t y of C a l i f o r n i a , San Diego u n d e r t h e a u s p i c e s of t h e l a t e P r o f e s s o r W a r r e n L. B u t l e r . The author w i s h e s to t h a n k h i m f o r h i s s u p p o r t a n d s u g g e s t i o n s . Chromatium vinosum was o b t a i n e d t h r o u g h the courtesy of P r o f e s s o r Shigehiro M o r i t a at Tokyo U n i v e r s i t y of A g r i c u l t u r e and Technology. REFERENCES

I. R e s e n k r a n z HS, C o w a r d JE, W l o o d k o w s k i TJ and Carr NS (1974) A n t i m i c r o b A n t i g e n t s & C h e m o t h e r 5: 199-201 2. H a e f e l i CC, F r a n k l i n C and Hady K (1984) J B a c t e r i o l 1 5 8 : 3 8 9 - 3 9 2 3. K i t a j i m a M, A b e A a n d T o m o t s u T (1976) In: P r o c A n n M e e t i n g P h o t o graphic Soc Japan, pp.I07-I09 4. K i t a j i m a M (1977) U S P a t e n t 4,155,810 5. B e l l y RT and K y d d GC (1982) D e v e l o p Industr M i c r o b i o l 2 3 : 5 6 7 - 5 7 7 6. C h a r l e y RC and B u l l AT (1979) A r c h M i c r o b i o l 123: 239-244 7. P o o l e y FD (1982) Nature 296: 642-643 8. K r o g m a n n DW, J a g e n d o r f AT and A v r o n M (1959) P l a n t P h y s i o l 34:272-275