Organic Marine Geochemistry - American Chemical Society

19 Biogeochemical Cycling of Sulfur Thiols in Coastal Marine Sediments

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on August 26, 2015 | http://pubs.acs.org Publication Date: April 21, 1986 | doi: 10.1021/bk-1986-0305.ch019

Kenneth Mopper and Barrie F. Taylor Division of Marine and Atmospheric Chemistry, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, F L 33149-1098

Thiols are major intermediates in the microbial cycling of sulfur and, because of their high reactivity, they may also play important roles in geochemical processes. Preliminary studies using a new, highly sensitive HPLC assay revealed that thiols are present at concentrations up to 100μΜ in intertidal marine sediments from Biscayne Bay (FL). Methanethiol (MeS) and 3-mercaptopropionate (MP) were the major thiols found. The presence of the latter compound suggests that, in addition to protein degradation, anaerobic decomposition of dimethylpropiothetin (DMPT), a major sulfur compound of marine algae and higher plants, may be an important source of thiols and a significant degradation pathway for DMPT in the environment. Acrylic acid produced from this pathway readily adds HS across the double bond, via Michael addition, to form 3-mercaptopro pionate. Alternatively, this thiol may be formed directly from DMPT by successive anaerobic demethylations; however the biochemical feasibility of this pathway is presently not known. Addition of a specific disulfide cleaving reagent to sediments revealed that thiols are dominantly present in bound forms. Binding of thiols to sediment particles may be an important mechanism for the incorporation of organic sulfur into geopolymers.

Diagenesis o f o r g a n i c matter i n t h e water column and sediments results i n the production o f a wide v a r i e t y o f organosulfur compounds. Most s t u d i e s i n v o l v i n g these compounds i n the marine e n v i r o n m e n t have f o c u s s e d on gaseous and h y d r o p h o b i c s p e c i e s (1 ). In contrast, information regarding reduced, h y d r o p h i l i c s u l f u r o r g a n i c s , i n p a r t i c u l a r the t h i o l s ( g e n e r a l f o r m u l a R-SH, where R i s an o r g a n i c g r o u p ) , i s almost n o n e x i s t e n t . 0097-6156/86/0305-0324$06.00/0 © 1986 American Chemical Society

In Organic Marine Geochemistry; Sohn, M.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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Thiols in Coastal Marine Sediments

325

T h i o l s , or s u l f h y d r y l compounds, p l a y important biochemical r o l e s i n m a i n t a i n i n g macromolecular s t r u c t u r e s , b i n d i n g m e t a l s at a c t i v e s i t e s of enzymes and e l e c t r o n t r a n s p o r t components, c a p t u r i n g and d e t o x i f y i n g m e t a l s , and s e r v i n g as c o e n z y m e s . In aquatic sediments these compounds a r i s e d u r i n g m i c r o b i a l p r o c e s s e s of s u l f a t e r e d u c t i o n and l i t h o t r o p h i c o x i d a t i o n ( o x i c and a n o x i c ) , and f r o m biodégradation of o r g a n i c m a t t e r ( 2 , 3 ) . A b i o t i c s o u r c e s of t h i o l s i n c l u d e r e a c t i o n s of d i s s o l v e d o r g a n i c m a t t e r w i t h H^S and e l e m e n t a l s u l f u r present i n pore water and on p a r t i c l e s u r f a c e s (4,5,6). Functional group, spectrophotometry, electrochemical and elemental analyses have shown the presence of significant c o n c e n t r a t i o n s of t h i o l s and other organo s u l f u r compounds i n a n o x i c m a r i n e waters and sediments (5,7-10). For example, using p o l a r o g r a p h i c t e c h n i q u e s , L u t h e r et a l . (10) r e p o r t e d t h a t t h i o l s were the major reduced s u l f u r s p e c i e s , e i t h e r i n o r g a n i c or o r g a n i c , i n the porewaters of s a l t m a r s h sediments (Great Marsh, Lewes, DE) w i t h t o t a l c o n c e n t r a t i o n s up t o 2.4 mM. The h i g h c o n c e n t r a t i o n s and t h e h i g h c h e m i c a l r e a c t i v i t y of t h i o l s s t r o n g l y suggest t h a t these compounds p l a y a major r o l e i n the e a r l y d i a g e n e s i s of o r g a n i c m a t t e r i n s e d i m e n t s , as w e l l as i n the i n c o r p o r a t i o n of s u l f u r i n t o o r g a n i c geopolymers. For example, t h i o l s r e a d i l y r e a c t t o form d i s u l f i d e and p o l y s u l f i d e bridges ( 5 ) , w h i c h may enhance the c r o s s l i n k i n g and, hence, the m o l e c u l a r weight of o r g a n i c m a t t e r i n sediments. I n a d d i t i o n , t h i o l s form s t r o n g complexes w i t h metal i o n s , e s p e c i a l l y t r a n s i t i o n metals ( 5 ) , and may promote the m o b i l i z a t i o n o f m e t a l s , e.g. a r s e n i c ( 1 1 ) . I t i s l i k e l y that t h r o u g h c o m p l e x a t i o n w i t h m e t a l s on p a r t i c l e s u r f a c e s t h a t t h i o l s a l s o become s t r o n g l y bound. D e s p i t e the b i o g e o c h e m i c a l s i g n i f i c a n c e of t h i o l s , r e l a t i v e l y l i t t l e i s known about the nature and d i s t r i b u t i o n of these s p e c i e s i n sediments. T h e r e f o r e , w i t h the a i d of a newly d e v e l o p e d a n a l y t i c a l method, a study was i n i t i a t e d t o e x p l o r e the r o l e t h a t t h i o l s p l a y i n the marine sedimentary s u l f u r c y c l e . Some of the q u e s t i o n s addressed were: (1) What t h i o l s are present i n sediments? ( 2 ) What are the p o s s i b l e f o r m a t i o n pathways; t h a t i s , what i s the relative importance of biological (e.g. microbial) versus n o n b i o l o g i c a l (e.g. chemical r e a c t i o n s ) sources f o r t h i o l s i n sediments? (3) Are t h i o l s bound t o sediment p a r t i c l e s and (4) What i s the nature of the b i n d i n g ? The major f i n d i n g s and c o n c l u s i o n s of t h i s i n i t i a l study are p r e s e n t e d i n t h i s r e p o r t . Experimental T h i o l s , as w e l l as s u l f i d e and s u l f i t e , were determined i n porewater samples by r e v e r s e d phase h i g h performance l i q u i d chromatography (HPLC). The t e c h n i q u e i s based on precolumn d e r i v a t i z a t i o n w i t h an _o_ p h t h a l a l d e h y d e / a m i n e reagent ( F i g u r e 1) f o l l o w e d by HPLC and f l u o r o m e t r i c d e t e c t i o n . D e r i v a t i z e d porewater samples were i n j e c t e d d i r e c t l y i n t o the HPLC system; the d e t e c t i o n l i m i t i s 0.1 nM ( f o r 100 u l i n j e c t i o n ) . D e t a i l s of the method are g i v e n i n Mopper and Delmas (_12). I n t e r t i d a l B i s c a y n e Bay (FL) sediments were periodically c o l l e c t e d ( u s i n g g l a s s j a r s ) d u r i n g June through September of 1984. A t o t a l o f 27 samples were taken d u r i n g t h i s p e r i o d . S l u r r i e s were


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p r e p a r e d u s i n g two p a r t s sediment and one p a r t d e a e r a t e d seawater (V/V). The s l u r r i e s (~ 500ml) were s t o r e d a t 25°C under argon i n glass j a r s . Background d a t a f o r t y p i c a l sediment samples a r e g i v e n i n Table I .


T a b l e I . T y p i c a l Background Data on I n t e r t i d a l B i s c a y n e Bay Sediments

Date

Porewater pH

Aug. 1, 1984

Aug. 8, 1984

Aug. 24, 1984

7.4

7.4

7.5

Size F r a c t i o n urn

% Dry CaC0~ Sediment

Organic C

φ > 300 300 < φ 63 < φ

Ν.A.

63

72.3 20.1 7.6

64.8

>

39.9

1.53

φ > 300 300 < φ 63 < φ

N.A.

63

74.1 16.1 9.8

64.1

>

37.7

1.34

φ > 300 300 < φ 63 < φ

N.A.

63

69.7 22.3 8.0

58.1

>

41.7

1.76

N.A. - n o t a n a l y z e d ; φ = medium g r a i n s i z e .

P r i o r t o t h i o l a n a l y s i s , s l u r r i e s were a l l o w e d t o s e t t l e and a 2 ml a l i q u o t of t h e s u p e r n a t a n t was f i l t e r e d (0.2 pm, Nucleopore) and d e r i v a t i z e d . Some sediment s l u r r i e s were t r e a t e d w i t h a s p e c i f i c S-S c l e a v i n g r e a g e n t , t r i b u t y l p h o s p h i n e ( 1 3 ) , i n o r d e r t o e v a l u a t e t h e d e g r e e t o which t h i o l s were bound v i a d i s u l f i d e l i n k a g e s . The r e a g e n t was added t o a f i n a l c o n c e n t r a t i o n o f 0.5-1.0 ml p e r l i t e r slurry. R e s u l t s and D i s c u s s i o n I d e n t i f i c a t i o n o f T h i o l s i n Marine Sediment P o r e w a t e r s . T h i o l s were p r e s e n t a t s i g n i f i c a n t l e v e l s ( u p t o a b o u t 100 uM) i n a n o x i c , i n t e r t i d a l B i s c a y n e Bay sediments d u r i n g t h e e n t i r e s a m p l i n g p e r i o d . The c o n c e n t r a t i o n s found were s i m i l a r t o those r e p o r t e d f o r o t h e r low m o l e c u l a r weight o r g a n i c s i n sediment porewaters ( T a b l e I I ) . More t h a n 30 i n d i v i d u a l t h i o l s w e r e d e t e c t e d o f w h i c h 13 were p o s i t i v e l y or tentatively i d e n t i f i e d ( F i g u r e 2 and T a b l e I I I ) . Peaks were i d e n t i f i e d by c o - i n j e c t i o n w i t h a u t h e n t i c compounds under d i f f e r e n t chromatographic and d e r i v a t i z a t i o n c o n d i t i o n s , as o u t l i n e d i n F i g u r e 3. F o r example, a l t e r i n g t h e pH o f the m o b i l e phase was particularly effective f o r t h e i d e n t i f i c a t i o n of c a r b o x y l a t e d t h i o l s , such as 3-mercaptopropionate, because p r o t o n a t i o n o f t h e carboxy group s e l e c t i v e l y enhanced t h e r e t e n t i o n o f these compounds.


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327

S-R


O-Phthalaldehyde

Thiol

Isoindole

F i g u r e 1. F l u o r e s c e n c e d e r i v a t i z a t i o n of t h i o l s ; the e x c i t a t i o n and e m i s s i o n maxima of the f l u o r e s c e n t i s o i n d o l e p r o d u c t a r e a t 340 nm and 450 nm r e s p e c t i v e l y ( 1 2 ) .

STANDARD

. 6

Figure 2. Upper; G r a d i e n t s e p a r a t i o n of ο-phthalaldehyde d e r i v a t i v e s of 11 t h i o l s and sodium s u l f i t e a c c o r d i n g t o Mopper and Delmas ( 1 2 ) . Peaks: (1) sodium s u l f i t e (100 pmol); (2) glutathione (7 pmol); (3) t h i o g l y c o l l a t e (200 pmol); (4) Ν-acetylcysteine (7 pmol); (5) 2-mereaptoethanesulfonate (Co-M) (10 pmol); (6) 3-mercaptopropionate (10 pmol); (8) m o n o t h i o g l y c e r o l (10 pmol); (9) 2 m e r c a p t o e t h a n o l (10 pmol); ( 1 0 ) m e t h a n e t h i o l (15 pmol); (11) e t h a n e t h i o l (10 pmol); (12) 2-propanethiol (15 pmol); (13) 1 p r o p a n e t h i o l (15 pmol). M i d d l e : T h i o l s i n porewater i n r e d u c i n g sediment s l u r r y from B i s c a y n e Bay. Porewater water was f i l t e r - s t e r i l i z e d p r i o r t o d e r i v a t i z a t i o n . Peak 7: s u l f i d e (Note: response f a c t o r i s about 200 t i m e s l o w e r t h a n f o r t h i o l s ) . Lower : r e a g e n t b l a n k i n porewater m a t r i x .


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T a b l e I I . C o n c e n t r a t i o n Range of T h i o l s i n Comparison t o Other Low M o l e c u l a r Weight O r g a n i c s i n Sediment Porewaters Compounds

Typical Concentrations

Reference

Thiols

0.1 - 100 μΜ; 0.1 - 2.4 mM

T h i s work and 10, respectively

Sugars

0.2 - 2 uM

33_, 34

Amino A c i d s

1 - 200 μΜ *

35, 36

^LMW C a r b o x y l i e Acids

1 - 30 μΜ

37, 38

LMW

0.1 - 5 μΜ

Mopper (unpublished)

Carbonyls

* Note: The h i g h e s t v a l u e was measured i n T h i o p l o c a mats u n d e r l y i n g t h e Peru u p w e l l i n g . Maximum amino a c i d c o n c e n t r a t i o n s found i n t y p i c a l marine sediments a r e 10 - 20 uM. f LMW = Low m o l e c u l a r w e i g h t .

Table I I I .

T h i o l s I d e n t i f i e d i n S l u r r i e s of Marine

Sediments

Major T h i o l s (0.5 - 20 μΜ) 3-Mercaptopropionate (dominant) M e t h a n e t h i o l (dominant) Ethanethiol Monothioglycerol 2 -Mercaptoethanol 2 -Mercaptopyruvate M i n o r T h i o l s ( C H S C H 3

DMPT

DMS

3

+ CH =CHC00H 2

acrylic

acid

DMS i s a l s o generated d u r i n g a n a e r o b i c f e r m e n t a t i o n o f d i m e t h y l p r o p i o t h e t i n (24) and was d e t e c t e d as t h e p r i n c i p a l v o l a t i l e s u l f u r compound e v o l v e d from s a l t marsh f l a t s ( 2 5 ) . T h e r e f o r e , i t i s r e a s o n a b l e t o h y p o t h e s i z e t h a t DMPT, i s a l s o a major p r e c u r s o r f o r other o r g a n o s u l f u r compounds, e.g. t h i o l s , i n anoxic marine sediments. However, no s t u d i e s have been r e p o r t e d on t h e m i c r o b i a l p r o d u c t i o n o f t h i o l s from t h i s compound. Hypothetical formation pathways f o r m e t h a n e t h i o l and 3-mercaptopropionate from DMPT a r e g i v e n i n F i g u r e 4. Two s u c c e s s i v e d e m e t h y l a t i o n s o f DMPT would y i e l d 3-methiolpropionate and 3-mercaptopropionate. The f i r s t demethylation of d i m e t h y l p r o p i o t h e t i n i s biochemically f e a s i b l e ; f o r e x a m p l e , homocysteine can accept a methyl-group from DMPT, i n a r e a c t i o n analogous t o t h a t i n v o l v i n g d i m e t h y l t h e t i n ( 2 6 ) , t o y i e l d m e t h i o n i n e and 3 - m e t h i o l p r o p i o n a t e ( 2 7 ) . The second d e m e t h y l a t i o n , t h a t o f 3 - m e t h i o l p r o p i o n a t e , has n o t been demonstrated but i t might be c a t a l y z e d by methanogenic and/or a c e t o g e n i c b a c t e r i a . Methyl t r a n s f e r r e a c t i o n s from m e t h y l a t e d s u l f u r s u b s t r a t e s c o u l d o p e r a t e and i n v o l v e c o b a l a m i n e - c o n t a i n i n g enzymes as shown f o r methanol (28) and p o s t u l a t e d f o r methylamines and methoxy-aromatic molecules (29,30). F i n a l l y , 3 - m e t h i o l p r o p i o n a t e i s a known p r e c u r s o r o f methanethiol (18).



19.

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331

A b i o t i c Production of Thiols. W h i l e t h e f o r m a t i o n pathway o f 3 - m e r c a p t o p r o p i o n a t e from s u c c e s s i v e d e m e t h y l a t i o n s o f DMPT remains to be proven, DMPT may n o n e t h e l e s s be an i m p o r t a n t p r e c u r s o r o f t h i s t h i o l by a l e s s s p e c u l a t i v e pathway i n v o l v i n g e s t a b l i s h e d r e a c t i o n s . The f i r s t r e a c t i o n i s the a n a e r o b i c c l e a v a g e o f DMPT t o DMS and a c r y l i c a c i d ( r e a c t i o n V I I , Figure 4 ) . I f t h i s r e a c t i o n occurs i n the s u l f a t e r e d u c i n g zone, a c r y l i c a c i d w i l l then r a p i d l y r e a c t w i t h HS ( a p o w e r f u l n u c l e o p h i l e ) by a d d i n g i t a c r o s s the double bond v i a t h e w e l l known M i c h a e l a d d i t i o n r e a c t i o n (31) ( F i g u r e 4 ) . I n t h e p a s t , t h i s r e a c t i o n has been s t u d i e d a t h i g h c o n c e n t r a t i o n s of r e a c t a n t s and i n t h e a b s e n c e o f w a t e r ( 3 2 ) . Therefore, the environmental relevance of t h i s r e a c t i o n i s questionable. S e v e r a l e x p e r i m e n t s were performed t o examine t h i s p o s s i b l e r e a c t i o n . A c r y l i c a c i d and sodium s u l f i d e were added t o d e a e r a t e d , aged G u l f Stream seawater t o f i n a l c o n c e n t r a t i o n s o f 0.1 mM and 1.0 mM, r e s p e c t i v e l y . C o n t r o l s c o n s i s t e d o f seawater a l o n e , seawater p l u s a c r y l i c a c i d , and seawater p l u s sodium s u l f i d e . The r e a c t i o n was r u n a t pH 8.2-8.4 under argon a t 25°C and 60°C. A f t e r two h o u r s , a l i q u o t s were removed f o r t h i o l a n a l y s i s by HPLC. Only two t h i o l s were d e t e c t e d , o f which 3-mercaptopropionate, t h e e x p e c t e d p r o d u c t , was one. The i d e n t i t y o f t h e o t h e r t h i o l has n o t been established. C o n t r o l s showed n e g l i g i b l e t h i o l p r o d u c t i o n ( F i g u r e 5). The apparent y i e l d o f the r e a c t i o n (% a c r y l i c a c i d c o n v e r t e d t o 3-mercaptopropionate) was about 1-2% a t 60"C and 0.3-0.4% a t 25"C. A d d i t i o n o f t r i b u t y l p h o s p h i n e , a d i s u l f i d e c l e a v i n g r e a g e n t , t o the r e a c t i o n m i x t u r e e i t h e r b e f o r e o r a f t e r t h e 2 hour i n c u b a t i o n , i n c r e a s e d t h e t h i o l y i e l d by about a f a c t o r o f two. These r e s u l t s s u g g e s t t h a t a b i o t i c r e a c t i o n s may i n d e e d be r e s p o n s i b l e f o r the f o r m a t i o n o f some t h i o l s i n t h e environment and t h a t t h i o l s o x i d i z e r a p i d l y t o form d i s u l f i d e compounds even under r e d u c i n g c o n d i t i o n s . A k i n e t i c study o f t h e M i c h a e l r e a c t i o n i n seawater i s c u r r e n t l y being undertaken. In order t o p r o v i d e a d d i t i o n a l evidence i n support of the p r o p o s e d M i c h a e l a d d i t i o n r e a c t i o n , a s e d i m e n t s t u d y was a l s o performed. A c r y l i c a c i d was added d i r e c t l y t o s l u r r i e s o f r e d u c i n g sediments from B i s c a y n e Bay and t h e f o r m a t i o n of 3-mercaptopro p i o n a t e i n t h e p o r e w a t e r , r e l a t i v e t o u n s p i k e d c o n t r o l s , was m o n i t e r e d by HPLC. The c o n c e n t r a t i o n o f added a c r y l i c a c i d was 0.1 mmol p e r l i t e r s l u r r y ( 0.2 mM i n t h e porewater) and t h e s l u r r i e s w e r e i n c u b a t e d u n d e r a r g o n a t 37°C f o r 2 h o u r s p r i o r t o t h i o l analysis. F i g u r e 6 c l e a r l y shows t h a t a d d i t i o n o f a c r y l i c a c i d t o r e d u c i n g sediment g i v e s r i s e t o 3-mercaptopropionate, t h e main product expected from the M i c h a e l a d d i t i o n o f HS t o a c r y l i c a c i d . The a d d i t i o n o f h^S, t r a c e s o f w h i c h w i l l be p r e s e n t a t t h e pH of t h e porewater ( T a b l e I ) , t o a c r y l i c a c i d p r o b a b l y f o l l o w s t h e Markownikoff a d d i t i o n r u l e t o y i e l d 2-mercaptopropionate:

SH I n f a c t , 2-mercaptopropionate was t e n t a t i v e l y i d e n t i f i e d ( a t t r a c e c o n c e n t r a t i o n s ) i n some B i s c a y n e Bay sediments ( T a b l e I I I ) . I t i s t e m p t i n g t o conclude from these s t u d i e s t h a t a b i o t i c r e a c t i o n s p l a y a major r o l e i n the f o r m a t i o n o f t h i o l s i n s e d i m e n t s .




332

SEAWATER ALONE

Figure 5. Upper: A b i o t i c production of 3-mercaptopropionate (MP) and an unknown t h i o l (?) i n deaerated seawater from reaction of a c r y l i c acid (0.1 mM) with sodium s u l f i d e (1.0 mM) at 60°C f o r 2 hours under argon; detection by fluorescence d e r i v a t i z a t i o n and HPLC. Middle: C o n t r o l = sodium s u l f i d e (1.0 mM) alone i n seawater under i d e n t i c a l reaction conditions as above. Lower: Control = seawater alone under i d e n t i c a l reaction conditions as above.


M O P P E R A N D TAYLOR


333

MP


THIOLS

IN

I

INCUBATED HS"

SEDIMENT

SLURRY WITH

SLURRY

SPIKED

ACRYLIC

AC ID

F i g u r e 6. Upper: 3-mercaptopropionate (MP) and hydrogen s u l f i d e (HS ) p r e s e n t i n aqueous phase o f B i s c a y n e Bay sediment i n c u b a t e d a t 37 C f o r 2 hours under argon; d e t e c t i o n by f l u o r e s c e n c e d e r i v a t i z a t i o n and HPLC; the l a r g e i n i t i a l peaks are p r o b a b l y humic s u b s t a n c e s . Lower : A l i q u o t of same sediment s l u r r y sample i n c u b a t e d under the same c o n d i t i o n s as above but w i t h added a c r y l i c a c i d (10 pmole p e r 100 ml s l u r r y ) ; n o t e i n c r e a s e i n 3-mercaptopropionate and d e c r e a s e i n HS .



334

W h i l e t h i s may be t r u e f o r 3-mercaptopropionate, t h e a c t u a l i m p o r t a n c e of these r e a c t i o n s r e l a t i v e t o m i c r o b i a l p r o d u c t i o n o f t h i o l s can o n l y be p r o p e r l y a s s e s s e d w i t h t r a c e r experiments u s i n g r a d i o l a b e l l e d s u b s t r a t e s and r e a c t a n t s .


R e l e a s e o f Bound T h i o l s . Tributylphosphine q u a n t i t a t i v e l y and r a p i d l y reduces d i s u l f i d e s t o t h i o l s and, by m a i n t a i n i n g reducing c o n d i t i o n s , prevents t h e i r r e o x i d a t i o n (13): R'-S-S-R + ( C H ) P + H 0 4

9

3

2

> R'SH + RSH + ( C ^ ^ P ^ O

A l t h o u g h t h i s reagent had o n l y been used i n the p a s t t o p r e s e r v e s t a n d a r d t h i o l s o l u t i o n s , i t was f e l t t h a t i t c o u l d a l s o be employed to q u a n t i f y t h e r e l a t i v e amounts o f -S-S- bound t h i o l s i n s e d i m e n t s . However, s i n c e t r i b u t y l p h o s p h i n e had n o t been p r e v i o u s l y used i n s e a w a t e r media, a p r e l i m i n a r y t e s t was performed t o e v a l u a t e i t s c h e m i c a l b e h a v i o r under these c o n d i t i o n s . D i m e t h y l d i s u l f i d e and o x i d i z e d g l u t a t h i o n e were added t o seawater t o a f i n a l c o n c e n t r a t i o n o f 1 μΜ e a c h . T r i b u t y l p h o s p h i n e was a d d e d ( 0 . 5 m l p e r l i t e r s e a w a t e r ) and t h e samples were i n c u b a t e d a t 25 C i n t h e presence o f air. A l i q u o t s were p e r i o d i c a l l y removed f o r t h i o l a n a l y s i s . A f t e r 20 m i n , 95% of t h e d i s u l f i d e s were reduced t o t h i o l s ( F i g u r e 7 ) . The t h i o l s were s t a b l e f o r t h r e e days even i n t h e presence of a i r . A f t e r t h i s p e r i o d , r e o x i d a t i o n o c c u r r e d p r o b a b l y due t o l o s s o f the excess and p r o t e c t i v e phosphine by r e a c t i o n w i t h oxygen. From these t e s t s i t was concluded t h a t t h e phosphine reagent c o u l d be used t o s t u d y t h e r e l a t i v e abundance o f f r e e v e r s u s -S-S- bound t h i o l s i n reducing sediments. Sediment s l u r r i e s were i n c u b a t e d a t 30°C (approximate i n s i t u t e m p e r a t u r e ) w i t h and w i t h o u t t r i b u t y l p h o s p h i n e . A l i q u o t s of p o r e w a t e r were p e r i o d i c a l l y removed f o r t h i o l a n a l y s i s over the f o l l o w i n g 2-4 days. D u r i n g t h e course of t h i s s t u d y , a t o t a l o f 7 s u c h i n c u b a t i o n s were performed on f r e s h l y c o l l e c t e d sediment. R e s u l t s were s i m i l a r i n a l l cases and t y p i c a l l y showed that t r i b u t y l p h o s p h i n e induced a d r a m a t i c and r a p i d r e l e a s e o f bound ( o r o x i d i z e d ) t h i o l s ( T a b l e IV and F i g u r e 8 ) . Bound t h i o l s were p r e s e n t a t a p p r o x i m a t e l y 20 times g r e a t e r c o n c e n t r a t i o n s than f r e e t h i o l s ( i . e . , ^ 9 5 % o f a l l t h i o l s r e l e a s e d from sediment were i n i t i a l l y bound). I f a i r i s not e x c l u d e d d u r i n g the i n c u b a t i o n , r e l e a s e d t h i o l s become r e o x i d i z e d a f t e r s e v e r a l days ( F i g u r e 8) p r o b a b l y due t o t h e o x i d a t i o n o f t h e p r o t e c t i v e phosphine. Addition of fresh t r i b u t y l p h o s p h i n e regenerated the t h i o l s . A d d i t i o n o f t r i b u t y l p h o s p h i n e t o e x t r a c t e d porewater ( p a r t i c l e f r e e ) r e s u l t e d i n o n l y a minor i n c r e a s e i n t h i o l c o n c e n t r a t i o n s . This r e s u l t i n d i c a t e s that the dramatic increases obtained with s l u r r i e s (Table IV and F i g u r e 8) a r e p r o b a b l y due t o r e l e a s e o f t h i o l s bound t o p a r t i c l e s u r f a c e s , a s o p p o s e d t o r e l e a s e f r o m d i s u l f i d e s d i s s o l v e d i n the i n t e r s t i a l water. S u r f a c e b i n d i n g i s most l i k e l y t h r o u g h -S-6- l i n k a g e s , but i t i s a l s o c o n c e i v a b l e t h a t some f r a c t i o n o f t h e r e l e a s e d t h i o l s may be due t o d i s p l a c e m e n t of t h i o l s from m e t a l complexes on p a r t i c l e s u r f a c e s by t h e phosphine n u c l e o p h i l e . However, when an e q u i v a l e n t amount o f a s t r o n g m e t a l complexing agent (EDTA) was s u b s t i t u t e d f o r the phosphine, o n l y n e g l i g i b l e r e l e a s e s o f t h i o l s were o b s e r v e d , suggesting t h a t , f o r t h e sediments s t u d i e d , b i n d i n g by m e t a l


19.

772/o/5 in Coastal Marine Sediments


1 uM GLUTATHIONE ( O X , )

RED.

GLUT.

I

1 μΜ GLUTATHIONE ( O X . )


I r

TRIBUTYLPHOSPHINE

1 M—

^u__j

RED.

^

GLUT.

2 μΜ GLUTATHIONE ( R E D . )

r

K

J

/ V L _

U^-Lr

F i g u r e 7 . C l e a v a g e o f o x i d i z e d g l u t a t h i o n e i n s e a w a t e r by t r i b u t y l p h o s p h i n e t o y i e l d reduced g l u t a t h i o n e ( r e d . G l u t . ) ; d e t e c t i o n by f l u o r e s c e n c e d e r i v a t i z a t i o n a n d HPLC. U p p e r : O x i d i z e d g l u t a t h i o n e (luM) alone. Middle: Oxidized glutathione ( l u M ) p l u s t r i b u t y l p h o s p h i n e (50 μΙ/lOOml seawater) r e a c t e d f o r 20 min a t 25 C; Lower : Reduced g l u t a t h i o n e s t a n d a r d (2μΜ) i n seawater.

I n c u b a t i o n time ( h r )

F i g u r e 8. R e l e a s e o f t h i o _ l s ( 3 - m e r c a p t o p r o p i o n a t e - MP a n d m e t h a n e t h i o l - MeS) and HS upon a d d i t i o n o f t r i b u t y l p h o s p h i n e (50μ1 p e r 100 ml s l u r r y ) t o a B i s c a y n e Bay sediment s l u r r y ; incubated at 25 C; control slurries received no tributylphosphine·


335

336


complexation may not be as important as b i n d i n g by -S-S- bond formation. Specific methods for releasing metal bound t h i o l s are being explored.

Table IV. Release of Bound Thiols from Sediment


Incubated Sediment*

3-Mercaptopropionate (μΜ)

Methanethiol

HS~

SO^

(μΜ)

(mM)

(mM)

Control Slurry*

0.19

0.05

4.24 0.071

Slurry with Tributylphosphine*

6.53

1.31

6.16 0.033

% T h i o l Bound*

97%

96%

31%

0%

* 8/24/84; Biscayne Bay i n t e r t i d a l sediment incubated f o r 23h at 25 C; t h i o l s detected i n the "control s l u r r y " (no tributylphosphine added) are interpreted as being i n the unbound (or free) state; t h i o l s detected i n "slurry with tributylphosphine" are interpreted as free plus bound species; the "% t h i o l bound" was calculated from: [(thiols i n tributylphosphine treated s l u r r y ) - ( t h i o l s i n control)] χ 100%/(thiols i n tributylphosphine treated s l u r r y ) .

Summary and Conclusions Thiols are present at s i g n i f i c a n t levels i n reducing, i n t e r t i d a l sediments and apparently arise as a result of interacting b i o t i c (microbial) processes and a b i o t i c reactions (Figure 9). Over 30 t h i o l s were detected, of which methanethiol and 3-mercaptopropionate were present i n the highest concentrations throughout the entire 4 month sampling period. Methanethiol can readily arise through a number of known anaerobic pathways; however, no such pathways have been reported for the formation of 3-mercaptopropionate. I t can be speculated that this compound i s produced by successive anaerobic demethylations of dimethylpropiothetin (DMPT), a major sulfur compound of marine algae and plants. On the other hand, the known anaerobic breakdown pathway of DMPT i s v i a enzymatic cleavage to y i e l d dimethylsulfide and a c r y l i c acid. A c r y l i c acid i s a highly reactive species and, i n zones of active sulfate reduction, i t w i l l readily undergo a Michael addition with HS to yield 3-mercaptopropionate (Figure 6). I f this reaction i s i n fact o c c u r r i n g i n sediments and i n the water column of anoxic basins, a number of important geochemical implications can be inferred. For every mole of DMPT hydrolyzed, up to two moles of organosulfur compounds (dimethylsulfide and 3-mercaptopropionate) are produced. If direct b i o l o g i c a l sources are indeed negligible for the l a t t e r compound, then i t s c o n c e n t r a t i o n and turnover may be used to estimate that of a c r y l i c acid and i n d i r e c t l y that of DMPT hydrolysis







338

ORGANIC MARINE GEOCHEMISTRY

i n the environment. More generally, the results imply that a major chemical pathway f o r the i n c o r p o r a t i o n of s u l f u r i n t o o r g a n i c geopolymers i s by reaction of HS with reactive s i t e s , e.g. o l e f i n i c double bonds, displaceable halogens (39), within sedimentary organic matter. The Michael addition reaction of HS to a c r y l i c acid may be used as a model case of such interactions. Addition of a specific -S-Scleaving reagent, tributylphosphine, to reducing marine sediments resulted i n a dramatic and r a p i d r e l e a s e of t h i o l s i n t o the porewater. The r e s u l t s showed that In the sediments s t u d i e d bound t h i o l s a r e present i n at least 20 times greater concentration than freely dissolved t h i o l s or d i s u l f i d e s . These results imply that another important route f o r the incorporation of sulfur into organic geopolymers may be binding of t h i o l s to reactive s i t e s (e.g., R-SH groups and metal ions) on p a r t i c l e s . The results of this i n i t i a l study indicate that t h i o l s play an a c t i v e r o l e i n the biogeochemical c y c l i n g of s u l f u r i n marine sediments (Figure 9). Many questions remain to be addressed. In p a r t i c u l a r , how fast i s the turnover of t h i o l s i n sediments and what organisms are involved? What f r a c t i o n of sedimentary sulfur passes through the t h i o l pool? What are the precursors of the thiols? How do thiol-metal interactions affect the geochemistry (e.g. migration and binding) of heavy metals and t h i o l s i n sediments? How i s b i o t i c and abiotic production of t h i o l s i n porewaters related to the sulfur content and sulfur speciation within organic geopolymers? Acknowledgments We would l i k e to thank R. Cuhel, G.R. Harvey, and G.E. Luther f o r valuable input regarding possible formation routes f o r t h i o l s . F i n a n c i a l support was provided i n part by a grant from the National Institutes of Health (Biomedical Research Support Grant to K.M.) and from the National Science Foundation (OCE-8516020). Literature Cited

1. Balzer, W. In "Marine Organic Chemistry"; Duursma, E.K.; Dawson, R., Eds.; Elsevier: Amsterdam, 1981; Chap. 13. 2. Trudinger, P.A. Phil. Trans. Roy. Soc. 1982, B298, 563-581. 3. Zinder, S.H.; Doemel, W.N; Brock, T.D. Appl. Environ. Microbiol. 1977, 34, 859-860. 4. Altschuler, Z.S.; Schnepfe, M.M.; Silber, C.C.; Simon, F.O. Science 1983, 221, 221-227. 5. Boulegue, J.; Lord, C.J.; Church, T.M. Geochim. Cosmochim. Acta 1982, 46, 453-464. 6. Martin, T.H.; Hodgson, G.W. Chem. Geol. 1973, 12, 189-208. 7. Adams, D.D.; Richards, F.A. Deep-Sea Res. 1968, 15, 471-481. 8. Nissenbaum, Α.; Kaplan, I.R. Limnol. Oceanogr. 1972, 17, 570-582. 9. Dyrssen D.; Haraldsson, C.; Westerlund, S.; Aren, K. Mar. Chem., submitted. 10. Luther, G.W.; Church, T.M.; Giblin, A.E.; Howarth, R.W. In "Organic Marine Geochemistry"; Sohn, M., Ed.; ACS Symposium Series No. American Chemical Society: Washington, D.C., 1986; in press.



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M O P P E R AND T A Y L O R


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11. Cullen, W.R.; McBride, B.C.; Reglinski, J. J. Inorg. Biochem. 1984, 21, 174-194. 12. Mopper, K.; Delmas, D. Anal. Chem. 1984, 56, 2558-2560. 13. Humphrey, R.E.; Potter, J.L. Anal. Chem. 1965, 37, 164-165. 14. Bird, R.P.; Moir, R.J. Aust. J. Biol. Sci. 1972, 25, 835-848. 15. Salsbury, R.L.; Merricks, D.L. Plant Soil 1975, 43, 191-209. 16. Zikakis, J.P.; Salsbury, R.L. J. Dairy Sci. 1969, 52, 2014-2019. 17. Zinder, S.H.; Brock, T.D. Appl. Environ. Microbiol. 1978, 35, 344-352. 18. Cooper, A.J.L. Ann. Rev. Biochem. 1983, 52, 187-222. 19. Bremner, J.M.; Steele, C.G. Adv. Microbial Ecol. 1978, 2, 155-201. 20. White, R.H. J. Mar Res. 1982, 40, 529-536. 21. Vairavamurthy, Α.; Andreae, M.O.; Iverson, R.L. Limnol. Oceanogr. 1985, 30, 59-70. 22. Cantoni, G.L.; Anderson, D.G. J . Biol. Chem. 1956, 222, 171-177. 23. Kadota, H.; Ishida, Y. Ann. Rev. Microbiol. 1972, 26, 127-138. 24. Wagner, C; Stadtman, E.R. Arch. Biochem. Biophys. 1962, 98, 331-336. 25. Aneja, V.P.; Overton, J.H., Jr.; Cupitt, J.T.; Durham, J.L.: Wilson, W.E. Tellus 1979, 31, 174-178. 26. Lehninger, A.L. "Biochemistry"; Worth: New York, 1975; 2nd Edition. 27. Maw, G.A.; du Vigneaud, V. J. Biol. Chem. 1948, 176, 1037-1045. 28. Wood, J.M.: Moura, I.; Moura, J.G.G.; Santos, M.H.; Xavier, Α.V.; LeGall, J.; Scandellari, M. Science 1982, 216, 303-305. 29. Naumann, E.; Fahlsbuch, K.; Gottschalk, G. Arch. Microbiol. 1984, 138, 79-83. 30. Tschech, Α.; Pfennig, N. Arch. Microbiol. 1984, 137, 163-167. 31. Noller, C.R. In "Textbook of Organic Chemistry"; W.B. Saunders: Philadelphia, 1966; 3rd Edition; p. 619. 32. Dahlbom, R. Acta. Chem. Scand. 1951, 5, 690-698. 33. Mopper, K.; Dawson, R.; Liebezeit, G.; Ittekkot, V. Mar. Chem. 1980, 10, 55-66. 34. King, G.M.; Klug, M.J. Appl. Environ. Microbiol. 1982, 44, 1308-1317. 35. Henrichs, S.M.; Farrington, J.W.; Lee, C. Limnol. Oceanogr. 1984, 29, 20-34. 36. Jørgensen, N.O.G.; Lindroth, P.; Mopper, K. Oceanolog. Acta 1981, 4, 465-474. 37. Hordijk, K.A.; Cappenberg, T.E. Appl. Environ. Microbiol. 1983, 46, 361-369. 38. Ansbaek, J.; Blackburn, T.H. Microb. Ecol. 1980, 5, 253-264. 39. Schwarzenbach, R.P.; Giger, W.; Schaffner, C.; Wanner, 0. Environ. Sci. Technol. 1985, 19, 322-327. RECEIVED

September 16, 1985


Organic Marine Geochemistry - American Chemical Society

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