Anaerobic oxidation of hydrocarbons by Desulfovibrio desulfuricans

Chemical Geology - Elsevier Publishing Company, Amsterdam Printed in The Netherlands

ANAEROBIC OXIDATION OF HYDROCARBONS BY DESULFOVIBRIO

DES ULF URICANS J.B. DAVIS and H.F. YARBROUGH Field Research Laboratory, Socony Mobil Oil Company, Inc., Dallas, Texas (U.S.A.) (Received November 23, 1965)

SUMMARY Data a r e p r e s e n t e d which i n d i c a t e a slow oxidation of r a d i o a c t i v e methane, ethane and n - o c t a d e c a n e by Desulfo~ibrio desulfuricans i s o l a t e d f r o m a f r e s h w a t e r o i l - b e a r i n g aquifer, the C a r r i z o (Eocene) F o r m a t i o n . Oxidation of n - o c t a d e c a n e by s u l f a t e - r e d u c e r b a c t e r i a l c e l l s was i n d i c a t e d a l s o by m e t h y l e n e blue r e d u c t i o n in c o n t r o l l e d e x p e r i m e n t s . The b a c t e r i a may gene r a t e the hydrogen sulfide in the f o r m a t i o n by coupling the oxidation of h y d r o c a r b o n s to s u l f a t e - i o n reduction.

INTRODUCTION

Desulfovibrio desulfuricans is a n o n - s p o r u l a t i n g , m o t i l e , G r a m - n e g a t i v e b a c t e r i u m which o r d i n a r i l y couples r e d u c t i o n of the sulfate ion with i t s ana e r o b i c growth p r o c e s s e s . C e r t a i n s t r a i n s of the b a c t e r i u m have been shown to grow to s o m e extent without sulfate p r e s e n t p r o v i d e d p y r u v a t e is the p r i n cipal s u b s t r a t e ( P o s t g a t e , 1952). S p o r e - f o r m i n g , s u l f a t e - r e d u c i n g b a c t e r i a a l s o have been i d e n t i f i e d including Desulfovibrio orientis (Adams and P o s t g a t e , 1959), the t h e r m o p h i l i c Clostridium nigrificans ( C a m p b e l l et al., 1957) and a r e l a t e d m e s o p h i l i c b a c t e r i u m ( P o s t g a t e and C a m p b e l l , 1963). The c l o s t r i d i a have been shown by P o s t g a t e (1963) to grow on p y r u v a t e in the a b s e n c e of d e t e c t a b l e sulfate ion. Desulfovibrio orientis a p p a r e n t l y does not grow on p y r u v a t e in s u l f a t e - f r e e m e d i a ( A d a m s and P o s t g a t e , 1959). S u l f a t e - r e d u c i n g b a c t e r i a or " s u l f a t e - r e d u c e r s " a r e u s u a l l y c o n s i d e r e d synonymous with the n o n - s p o r e f o r m e r , Desulfovibrio desulfuricans. T h e s e b a c t e r i a have been r e f e r r e d to, p a r t i c u l a r l y by R u s s i a n w o r k e r s , as Microspira desulfuricans ( T a u s s o n and A l e s h i n a , 1932; T a u s s o n and Veselov, 1934). The s a l t t o l e r a n t f o r m has been r e f e r r e d to as Microspira aestzaarii or Desulfovibrio aestuarii. D. desulfuricans was f i r s t i s o l a t e d by B e i j e r i n c k (1895) f r o m mud of the Delft, Holland, s e w e r s . He r e f e r r e d to it as Spirilltmz

desulfuricans. S u l f a t e - r e d u c i n g b a c t e r i a a r e r e m a r k a b l y v e r s a t i l e and ubiquitous, being found in m a r i n e s e d i m e n t s , s o i l s , s u r f a c e and s u b s u r f a c e w a t e r s , p i p e l i n e s , sewage, and p e t r o l e u m r e s e r v o i r s . They a r e e s p e c i a l l y i m p o r t a n t in the s u l f u r cycle of n a t u r e (Butlin, 1953) and t h e i r r o l e in the c o r r o s i o n of i r o n is well Chem. Geol., 1 (1966) 137-144

137

known (Von Wolzogen-Kuhr and Van der Vlugt, 1934; Starkey, 1958; Horvath, 1961). A variety of organic compounds are utilized by the b a c t e r i a under anaerobic conditions in the presence of sulfate ion. Rosenfeld (1947), for example, reported the utilization of hydrocarbons by sulfate-reducing bacteria. The cultures, however, were not pure. Hydrocarbons are not included as utilizable compounds in the official description of the species p r e p a r e d by C.E. Zobell (In: Breed et al., 1957, pp.248-249). On the other hand, autotrophic growth by the bacteria is known (Butlin and Adams, 1947) and is r e f e r r e d to in Zobell's description. In the work reported here, experiments were designed to demonstrate, if possible, the oxidation of hydrocarbons by sulfate, reducing bacteria. Such b a c t e r i a were isolated from an oil-bearing f r e s h water aquifer, the highly permeable C a r r i z o Formation of the Wilcox Group (Eocene), Atascosa County Texas. Hydrogen sulfide is also present in the formation. These observations suggested a possible relationship between the bacteria and hydrocarbon utilization coupled with hydrogen sulfide generation. However, before such a relationship in nature could be considered seriously, it was n e c e s s a r y to determine an experimental relationship. METHODS AND RESULTS

Sampling Samples of water were taken from artesian wells to a depth of 4,200 ft. flowing from the C a r r i z o Formation in southern Texas. This oil-bearing aquifer produces fresh water which is used extensively for irrigation purposes in the region. Methane (up to 62%) and ethane (up to 2%) along with t r a c e s of hydrogen sulfide were in the gas collected over water samples. The water contains sulfate ion, 12-52 p.p.m., and sulfide ion up to 22 p.p.m. Sulfate-reducing bacteria, while not detected in all water samples, were found in numbers up to 10/ml.

Methylene blue reduction The strain of Desulfovibrio desulfuricans used in a study of methylene blue reduction as an index of hydrocarbon oxidation was No.8326 (Postgate), cultured in a liquid medium to obtain cells for experimentation. The medium employed was essentially medium C of Butlin et al. (1949), modified by Postgate (1961). However, 0.5% phosphate salts were used instead of 0.05% (Table I). The b a c t e r i a were grown for 48 h in a liter of modified medium C, centrifuged, and washed twice with M/15 phosphate buffer at pH 7. The yield of cells was 1.0 g (wet weight). These were r e - s u s p e n d e d in M/15 phosphate buffer. Activity of the cells and their adaptability to glucose oxidation were determined by adding a 2-ml suspension containing 5 mg (dry weight) of sulfate r e d u c e r s to each of two Thunberg tubes. 10 mg of glucose in 3 ml of M/15 phosphate buffer was added to tube A; 3 ml of M/15 phosphate buffer only was added to tube B (control). Methylene blue (10 ~g) was added initially 138

Chem. Geol.. 1 (1966) 137--144

TABLE I Modified medium C 1 Components

% in distilled water

NaC1 NH4C1 Na2SO 4 CaC12.2H20 MgSO4.7H20 Sodium lactate Yeast extract

1.0 0.1 0.26 0.01 0.2 0.6 (as 70% w/w sol.) 0.1

1 The medium above was s t e r i l i z e d and 10 p.p.m, of Fe(NH4)2(SO4)2 added from a sterile 1% solution. This was followed by a sterile stock solution of phosphate salts to yield a final medium concentration of 0.3% Na2HPO 4 and 0.2% KH2PO4. TABLE II Activity and adaptability of sulfate-reducing bacteria Decoloration of methylene blue (MB) in min 0 h {room temp.) 3 h added additional MB, 10 # g 3.5 h added additional MB, 10 /zg

tube A {glucose)

tube B {control)

45 2.5 1.0

50 22.5 21.0

TABLE III Reduction of methylene blue by sulfate reducers in the presence of n-oetadecane or glucose Decoloration of MB 1 {min ) 1st experiment 2nd experiment {after re-aeration and evacuation)

tube A

tube B

tube C

20.5 9.0

15.5 7.0

28:5 14.0

1 Added 40 pg methylene blue. to e a c h t u b e and th e t u b e s e v a c u a t e d and f i l l e d with tank N 2 ( s e e T a b l e II). 10 m g of s o d i u m l a c t a t e ( g r o w t h s u b s t r a t e ) in 3 m l M / 1 5 p h o s p h a t e b u f f e r w a s ad d ed to a f r e s h 2 - m l s u s p e n s i o n of s u l f a t e - r e d u c e r c e l l s in a t h i r d T h u n b e r g tube f o l l o w e d by s t a n d a r d e v a c u a t i o n and addition of N 2. R e d u c t i o n ( d e c o l o r a t i o n ) of m e t h y l e n e blue in t h i s t h i r d tube o c c u r r e d in l e s s than a m i n u t e , a s c o m p a r e d with t h e 45 m i n r e q u i r e d o r i g i n a l l y f o r g l u c o s e in t u b e A. With t h i s b a c k g r o u n d of i n f o r m a t i o n , an e x p e r i m e n t with n - o c t a d e c a n e w a s p e r f o r m e d with the c r o p of c e l l s . A f r e s h 5 - m l s u s p e n s i o n of c e l l s c o n t a i n i n g 40 m g ( d r y w ei g h t ) of s u l f a t e r e d u c e r s w a s a d d e d to e a c h of t h r e e T h u n b e r g t u b e s . 15 m g of n - o c t a d e c a n e a d s o r b e d on 150 m g of L i n d e m o l e c u l a r s i e v e 5A (a z e o l i t e ) w a s ad d ed to tube A, 15 m g of g l u c o s e p l u s 150 m g of m o l e c u l a r s i e v e 5A t o tube B, and 150 m g m o l e c u l a r s i e v e 5A only to tube C. Th e t u b e s w e r e e v a c u a t e d and f i l l e d with N2. T a b l e lII s h o w s the r e s u l t s . T u b e s A, B, and C w e r e o p e n e d and 2.5 m g of FeSO4.7H20 ad d ed to each. Chem. Geol., 1 {1966) 137--144

139

TABLE IV Reduction of methylene blue by Desl~lfovibFio desulfuricans in the presence of n-octadeeane or sodium lactate Test systems 1 n - o c t a d e c a n e , 10 m g s o d i u m l a c t a t e , 10 m g control a control b

control a. 160 mg molecular sieve 5A added control b, 10 mg n-octadeemlc adsorbed on 100 mg molecular sieve added

Decoloration of methylene blue (min.) expt. I

expt. 2

60 26 60 60

56 55 90 100

expt. 3

expt. l a

expt. 2a

expt. 3a

>9(!

135

$5

> 90

45

25

45 ,i5 >120 > 120

1All test systems originally contained suspension of 30 mg (dry weight) bacterial cells. J60 /~g methylene blue and 100 mg molecular sieve 5A. with or without substrate as indicated. M e t h y l e n e blue w a s at t h i s t i m e d e c o l o r i z e d in all t u b e s . In 3 h b l a c k e n i n g ( F e S $ ) had o c c u r r e d at the b o t t o m of t u b e s A ( n - o c t a d e c a n e ) and B ( g l u c o s e ) . T u b e C ( c o n t r o l ) had no b l a c k e n i n g until the f o l l o w i n g day. T h e s e r e s u l t s with r e s t i n g c e l l s s u g g e s t that t h e s u l f a t e ion s e r v e d as a h y d r o g e n a c c e p t o r in the b a c t e r i a l d e h y d r o g e n a t i o n ( o x id a t io n ) of both n - o c t a d e c a n e and g l u c o s e : 9S 2SO 4 + 8H - - ~

S2- + F e 2 ' - - ~ . F e S L a t e r , b l a c k e n i n g of the c o n t r o l (tube C) a s w e l l as m e t h y l e n e blue r e d u c t i o n ( T a b l e III) o c c u r r e d due o s t e n s i b l y to d e h y d r o g e n a t i o n of e n d o g e n o u s s u b s t r a t e s The p r e c e d i n g r e s u l t s with m e t h y l e n e blue w e r e c o n f i r m e d by a d d i t i o n a l e x p e r i m e n t s with a n e w b a t c h of s u l f a t e - r e d u c i n g b a c t e r i a l c e l l s . T h e s e w e r e g r o w n and w a s h e d t h r e e t i m e s with M / 1 5 p h o s p h a t e b u f f e r , pH 7; t h e c e n t r i f u g e d y i e l d was 1.3 g/1 (wet weight). T h e c e l l s w e r e r e - s u s p e n d e d in 25 ml p h o s p h a t e b u f f e r and 3 m l of s u s p e n d e d c e l l s w e r e added to eq u al v o l u m e s of p h o s p h a t e b u f f e r in T h u n b e r g t u b e s c o n t a i n i n g : (1) ~'l-o c t a d e c a n e a d s o r b e d on m o l e c u l a r s i e v e 5A; (2) s o d i u m l a c t a t e on m o l e c u l a r s i e v e 5A; (3) m o l e c u l a r s i e v e 5A a l o n e ( d u p l i c a t e c o n t r o l s ) . M e t h y l e n e blue was added to e a c h t ube, the t u b e s w e r e a e r a t e d t h o r o u g h l y by s h a k i n g in a i r , then e v a c u a t e d and f i l l e d with N2. T h e r e s u l t s a r e shown u n d e r e x p e r i m e n t 1 in T a b l e IV. T h e s y s t e m s w e r e then r e f r i g e r a t e d and e x p e r i m e n t 2 and e x p e r i m e n t 3 w e r e p e r f o r m e d the f o l l o w i n g day at 9h00 a . m . and l l h 0 0 a . m . , r e s p e c t i v e l y . Th e r e s u l t s a r e a l s o shown in T a b l e IV. The c o n t r o l s ( T a b l e IV) w e r e t r e a t e d f u r t h e r as f o l l o w s . To c o n t r o l (a) an a d d i t i o n a l 100 m g of m o l e c u l a r s i e v e 5A only w as added. To c o n t r o l (b) wa~ added , - o c t a d e c a n e a d s o r b e d on m o l e c u l a r s i e v e 5A. Th e two s y s t e m s were' a e r a t e d to r e - o x i d i z e the m e t h y l e n e blue, then e v a c u a t e d and N 2 added. No d e c o l o r a t i o n o c c u r r e d in 90 m i n ( e x p e r i m e n t l a ) . Both s y s t e m s w e r e r e f r i g e r a t e d . Th e next day the t u b e s w e r e opened, r e - a e r a t e d , then e v a c u a t e d and N2 added; e x p e r i m e n t 2a w a s p e r f o r m e d at 25°C and e x p e r i m e n t 3a at 30°C ( T a b l e IV). 140

(!hl~m. (;t~o[., 1 (J.t)66) 137--14q

Oxidation of radioactive hydrocarbons Desulfovibrio desulfuricans i s o l a t e d f r o m th e a r t e s i a n w a t e r of the C a r r i z o F o r m a t i o n w e r e c u l t u r e d r e p e a t e d l y in the m e d i u m of M a c P h e r s o n and M i l l e r (1963). T h i s m e d i u m ( T a b l e V) h a s an Eh of a b o u t - 3 0 0 m V and s u p p o r t s good gro wt h . A p p r o x i m a t e l y 0.5 g c e l l s ( d r y w e i g h t ) / l i t e r h a v e b e e n o b t a i n e d f r o m a s m a l l i n o c u l u m a f t e r one w e e k ' s i n c u b a t io n. When h a r v e s t e d , the c e l l s u s p e n s i o n s a r e e s s e n t i a l l y F e S - f r e e b e c a u s e of t h e low f r o n co n t en t of the m e d i u m The s u l f a t e r e d u c e r s w e r e i n o c u l a t e d into the t e s t m e d i u m which w as v a r i e d only with r e s p e c t to l a c t a t e c o n t e n t (Tabl~ V]). R a d i o a c t i v e (14C) m e t h a n e , e t h a n e , and n - o c t a d e c a n e a d s o r b e d on m o l e c u l a r s i e v e 5A, w e r e TABLE V MacPherson and M i l l e r ' s medium Components

g/l

lactic acid KH2PO4 NH4C1 Na2SO4 CaC12.2H20 MgSO4.7H20 FeSO4.7H20 Na2S trace metals (stock sol.)

9.0 0.35 0.50 7.0 0.80 0.60 0.0007 0.08 10 ml

TABLE VI Oxidation oi hydrocarbons by Radioactive substrate methane, 0.02% (uninoculated control) ethane, 0.02% (uninoeclated eontrol) ~z-oetadecane, 0.01~ (uninoculated control)

Desulfovibrio desulfi~ricans

Lactate

e /m i n

(%)

CO2

0.1 0.01 0 0.1

110 67 19 15

0.1 0.01 6 0.1

44 29 22 9

0.1 0.01 0 0.1

64 42 5 2

cells 10 6 6 6 (no cells) 1341 16 3

0 (no cells)

TABLE VII El'feet of sulfate ion on ethane oxidation by

Desulfovibrio desulfuricans Ethane oxidation, e/min (CO2) SO4 + cells cells, no SO4 control, no cells

117 23 19

Chem. Geol., 1 (1966) 137-144

141

i n t r o d u c e d into s e p a r a t e s y s t e m s . T o t a l a v a i l a b l e r a d i o a c t i v i t y of e a c h s u b s t r a t e with the counting p r o c e d u r e e m p l o y e d w a s about 60,000 c / m i n , b a s e d on c o m p l e t e o x i d a t i o n and r e c o v e r y . F l a s k s w e r e i n c u b a t e d f o r 22 d a y s in o r d e r to a l l o w t h o s e c o n t a i n i n g 0.01% l a c t a t e t i m e to p r o d u c e s a t i s f a c t o r y t u r b i d i t y . I n o c u l a t e d f l a s k s c o n t a i n i n g 0.1% l a c t a t e w e r e q u i t e t u r b i d a f t e r one w e e k , w h i l e no g r o w t h w a s o b v i o u s in t h o s e c o n t a i n i n g no l a c t a t e . U n i n o c u l a t e d c o n t r o l s w e r e e m p l o y e d a s shown in T a b l e VI. At the end of the i n c u b a t i o n p e r i o d , the e x p e r i m e n t a l s y s t e m s w e r e a c i d i f i e d , e v a c u a t e d and the r e s p i r a t o r y c a r b o n d i o x i d e t r a p p e d in a l k a l i . The c a r b o n a t e w a s p r e c i p i t a t e d with BaC12 and the BaCO3 p r e c i p i t a t e s f i l t e r e d onto 1 - i n c h d i a m e t e r f i l t e r p a p e r d i s c s f o r r a d i o a c t i v i t y m e a s u r e m e n t s . The b a c t e r i a l c e l l s f r o m t h e m e t h a n e and e t h a n e ( g a s e o u s ) s y s t e m s w e r e then f i l t e r e d onto f i l t e r p a p e r d i s c s f o r r a d i o a c t i v i t y m e a s u r e m e n t . B a c t e r i a l c e l l s in the n - o c t a d e c a n e s y s t e m s :could not be m e a s u r e d f o r r a d i o a c t i v i t y b e c a u s e of t h e n o n - g a s e o u s r a d i o a c t i v e s u b s t r a t e which o b v i o u s l y c o u l d not be s p a r g e d f r o m the c u l t u r e . The a c e t o n e - d r i e d BaCO3 p r e c i p i t a t e s and b a c t e r i a l c e l l s , r e s p e c t i v e l y , w e r e a n a l y z e d f o r r a d i o a c t i v i t y u s i n g a thin endwindow G e i g e r - M [ i l l e r tube a t t a c h e d to a b a s i c s c a l e r . The d a t a of T a b l e VI i n d i c a t e that the b a c t e r i a l o x i d a t i o n of t h e h y d r o c a r b o n s , while slow, was d e f i n i t e . L a c t a t e a p p a r e n t l y i n c r e a s e d o x i d a t i v e a c t i v i t y of t h e b a c t e r i a l c e l l s , although to a m u c h l e s s e r e x t e n t than e x p e c t e d b a s e d on o b s e r v e d g r o w t h r e s p o n s e . Radioactive ethane oxidation was further tested using a resting cell s u s p e n s i o n of s u l f a t e r e d u c e r s g r o w n on the s a m e m e d i u m . W a s h e d c e l l s , 0.5 m g ( d r y w e i g h t ) / m l , w e r e s u s p e n d e d in M / 1 5 p h o s p h a t e b u f f e r u n d e r 0.02% r a d i o a c t i v e e t h a n e in a i r , with and without N a ~ O 4 (0.7%). The p r e s e n c e of s u l f a t e s t i m u l a t e d e t h a n e o x i d a t i o n by n o n - g r o w i n g c e l l s ( T a b l e VII). DISCUSSION The o x i d a t i o n of p u r e h y d r o c a r b o n s by s u l f a t e - r e d u c i n g b a c t e r i a h a s not b e e n c l e a r l y s u b s t a n t i a t e d in s c i e n t i f i c j o u r n a l s , although g r o w t h in m e d i a cont a i n i n g h y d r o c a r b o n s , and c r u d e oil i t s e l f , h a s b e e n o b s e r v e d often ( B e c k , 1947; K u z n e t s o v , 1950; U p d e g r a f f and W r e n , 1954). V i g o r o u s g r o w t h of s u l f a t e r e d u c i n g b a c t e r i a with a h y d r o c a r b o n a s the s o l e c a r b o n s o u r c e h a s not b e e n d e s c r i b e d to o u r k n o w l e d g e ; i . e . , w h e r e c e l l y i e l d s a r e s u b s t a n t i a l enough to w a r r a n t t h e d e f i n i t e c o n c l u s i o n t h a t the h y d r o c a r b o n w a s u t i l i z e d . E v e n t h e m o s t r e c e n t r e v i e w of t h e m e c h a n i s m s of p e t r o l e u m - h y d r o c a r b o n o x i d a t i o n s (Van d e r L i n d e n and T h i j s s e , 1965) p r e s e n t s no new r e f e r e n c e s c o n c e r n i n g s u l f a t e r e d u c e r s . The c r i t e r i o n f o r g r o w t h and o s t e n s i b l y the c r i t e r i o n f o r u t i l i z a t i o n of the h y d r o c a r b o n s u b s t r a t e h a s been, in m a n y c a s e s , a b l a c k e n i n g of the c u l t u r e m e d i u m , due to F e S f o r m e d when Ht~ g e n e r a t e d a s a r e s u l t of s u l f a t e r e d u c t i o n r e a c t s with i r o n p r e s e n t in the m e d i u m . P r o v o c a t i v e l y , c r u d e c u l t u r e s and even m a r i n e m u d s have b e e n e m p l o y e d a s i n o c u l a in s o m e i n v e s t i g a t i o n s of h y d r o c a r b o n u t i l i z a t i o n by t h e s e b a c t e r i a ( T a u s s o n and A l e s h i n a , 1932; R o s e n f e l d , 1947). It i s r e m a r k a b l e that d e f i n i t e p r o o f of t h e i r a b i l i t y to u t i l i z e h y d r o c a r b o n s a s food h a s not b e e n c l e a r l y d e m o n s t r a t e d by r e s e a r c h e r s in the p a s t , p a r t i c u l a r l y s i n c e a g r e a t d e a l of i m p o r t a n c e h a s b e e n a t t a c h e d to t h e i r a c t i v i t i e s in p e t r o l e u m r e s e r v o i r s ( Z o b e l l , 1946). A l s o , d e s p i t e t h e a l l e g e d a b i l i t y of t h e s e b a c t e r i a to o x i d i z e h y d r o c a r b o n s , 142

Chem. Geol., ] (1966) 137--144

p r e s u m a b l y c o u p l i n g t h i s o x i d a t i o n with the r e d u c t i o n of t h e s u l f a t e ion, no e x p e r i m e n t s h a v e b e e n r e p o r t e d p r e v i o u s l y in w h i c h r a d i o a c t i v e h y d r o c a r b o n s w e r e e m p l o y e d . In v i e w of t h e i m p o r t a n c e a t t a c h e d to t h e a c t i v i t i e s of t h e s e b a c t e r i a in m a n y n a t u r a l e n v i r o n m e n t s , e . g . , r e d u c t i o n of s u l f a t e ion in m a r i n e s e d i m e n t s and s u b s u r f a c e w a t e r s , k n o w l e d g e of t h e i r m e t a b o l i s m i s of i n t e r e s t , p a r t i c u l a r l y a k n o w l e d g e of the s u b s t r a t e s the b a c t e r i a u t i l i z e and the m o d i f i c a t i o n s t h e s e s u b s t r a t e s u n d e r g o . Desulfovibrio desulfuricans i s an i m p o r t a n t g e o l o g i c ( b i o c h e m i c a l ) a g e n t by v i r t u e of i t s r o l e in t h e f o r m a t i o n of huge s u l f u r d e p o s i t s ( T h o d e et a l . , 1954; Ivanov, 1964). T h e p u r p o s e of o u r i n v e s t i g a t i o n d e s c r i b e d a b o v e w a s to r e l a t e , if p o s s i b l e , t h e m e t a b o l i s m of p e t r o l e u m c o n s t i t u e n t s by the b a c t e r i a to s u l f a t e r e d u c t i o n in a n a t u r a l e n v i r o n m e n t , n a m e l y an o i l - b e a r i n g f r e s h water aquifer. In o u r e x p e r i m e n t s the a m o u n t of r a d i o a c t i v e c a r b o n d i o x i d e p r o d u c e d f r o m r a d i o a c t i v e h y d r o c a r b o n s i s s m a l l , but d e f i n i t e . H o w e v e r , v e r y l i t t l e if a n y t h i n g i s knowri about the a c t u a l m e t a b o l i s m of a p u r e h y d r o c a r b o n s u b s t r a t e by s u l f a t e r e d u c e r s ; i . e . , the m e c h a n i s m of o x i d a t i o n o r even a c a r b o n b a l a n c e . T h e r e i s e v i d e n c e of d e h y d r o g e n a t i o n of an a l k a n e s u b s t r a t e s u c h a s n - o c t a d e c a n e , w h e r e i n m e t h y l e n e b l u e can a c t a s the h y d r o g e n a c c e p t o r e x p e r i ~ m e n t a l l y . S u l f a t e ion p r e s u m a b l y i s the h y d r o g e n ( e l e c t r o n ) a c c e p t o r in the o x i d a t i o n of h y d r o c a r b o n s a s it i s in the o x i d a t i o n of l a c t a t e d u r i n g g r o w t h of the b a c t e r i a . But f u r t h e r i n v e s t i g a t i o n i s r e q u i r e d to l e a r n e v e n t h e m o s t r u d i m e n t a r y f a c t s of a n a e r o b i c o x i d a t i o n of h y d r o c a r b o n s by s u l f a t e r e d u c e r s . C e l l s of Desulfovibrio desulfuricans g r o w n in the p r e s e n c e of r a d i o e t h a n e d e f i n i t e l y i n c o r p o r a t e s o m e of the e t h a n e c a r b o n into t h e i r c e l l s . In f a c t , t h e r e w a s , in one i n s t a n c e , a r e l a t i v e l y high r a t i o ( a p p r o x . 3 0 ) of r a d i o c a r b o n i n c o r p o r a t e d into c e l l s to that in r e s p i r a t o r y c a r b o n d i o x i d e . In the o x i d a t i o n of e t h a n e by t h e a e r o b i c m y c o b a c t e r i a t h i s r a t i o a p p r o a c h e s one ( D a v i s et a l . , 1956). E x p e r i m e n t s with r a d i o a c t i v e s u b s t r a t e s h a v e the a d v a n t a g e of y i e l d i n g s p e c i f i c i n f o r m a t i o n . V i g o r o u s b a c t e r i a l g r o w t h o r a c t i v i t y i s not r e q u i r e d to y i e l d p o s i t i v e r e s u l t s . N e g a t i v e r e s u l t s a d m i t t e d l y h a v e d u b i o u s v a l u e s i n c e t h e y m a y be a t t r i b u t e d to t h e p a r t i c u l a r b a c t e r i a l s t r a i n on the c u l t u r a l p r o c e d u r e s . In g e o l o g i c t i m e , s l o w b a c t e r i a l a n a e r o b i c o x i d a t i o n of h y d r o c a r b o n s m a y be a m e a n s of g e n e r a t i n g s i g n i f i c a n t q u a n t i t i e s of h y d r o g e n s u l f i d e in p e t r o l e u m - b e a r i n g f o r m a t i o n s .

ACKNOWLEDGMENT We g r a t e f u l l y a c k n o w l e d g e t h e a s s i s t a n c e of C.H. C a l v e r t and J . P . S t a n l e y in the p e r f o r m a n c e of e x p e r i m e n t s .

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