Effects of Metals on Methanogenesis, Sulfate Reduction, Carbon ...

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Dec 6, 1982 - State University of New York, Stony Brook. (e.g., runoff) ... ed from Flax Pond, a relatively unimpacted salt marsh on the north ..... New Zealand.
Vol. 45, No. 5

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1983, p. 1586-1591

0099-2240/83/051586-06$02.00/0 Copyright © 1983, American Society for Microbiology

Effects of Metals on Methanogenesis, Sulfate Reduction, Carbon Dioxide Evolution, and Microbial Biomass in Anoxic Salt Marsh Sedimentst DOUGLAS G. CAPONE,* DWIGHT D. REESE, AND RONALD P. KIENE Marine Sciences Research Center, State University of New York, Stony Brook, New York 11794 Received 6 December 1982/Accepted 3 March 1983

The effects of several metals on microbial methane, carbon dioxide, and sulfide production and microbial ATP were examined in sediments from Spartina alterniflora communities. Anaerobically homogenized sediments were amended with 1,000 ppm (ratio of weight of metal to dry weight of sediment) of various metals. Time courses in controls were similar for CH4, H2S, and C02, with short initial lags (0 to 4 h) followed by periods of constant gas production (1 to 2 days) and declining rates thereafter. Comparisons were made between control and experimental assays with respect to initial rates of production (after lag) and overall production. Methane evolution was inhibited both initially and overall by CH3HgCl, HgS, and NaAsO2. A period of initial inhibition was followed by a period of overall stimulation with Hg, Pb, Ni, Cd, and Cu, all as chlorides, and with ZnSO4, K2CrO4, and K2Cr2O7. Production of CO2 was generally less affected by the addition of metals. Inhibition was noted with NaAsO2, CH3HgCl, and Na2MoO4. Minor stimulation of CO2 production occurred over the long term with chlorides of Hg, Pb, and Fe. Sulfate reduction was inhibited in the short term by all metals tested and over the long term by all but FeCl2 and NiCl2. Microbial biomass was decreased by FeCl2, K2Cr2O7, ZnSO4, CdCl2, and CuCl2 but remained generally unaffected by PbCl2, HgCl2, and NiCl2. Although the majority of metals produced an immediate inhibition of methanogenesis, for several metals this was only a transient phenomenon followed by an overall stimulation. The initial suppression of methanogenesis may be relieved by precipitation, complexation, or transformation of the metal (possibly by methylation), with the subsequent stimulation resulting from a sustained inhibition of competing organisms (e.g., sulfate-reducing bacteria). For several environmentally significant metals, severe metal pollution may substantially alter the flow of carbon in sediments.

Marine sediments play a vital role in the ecology of coastal areas. The benthic microflora are intimately involved in the cycling of carbon and the regeneration of inorganic nutrients (4, 9). Coincidently, sediments act as a sink for many anthropogenic pollutants (6, 24). Accumulations of such pollutants may affect microbiological activities in the sediments. Changes induced by environmental contaminants in the predominating pathways of terminal carbon flow, nutrient regeneration, and nutrient transformations would have serious consequences for the organisms living within the sediments and in the water column. Heavy metals are an important class of pollutants and derive from both point (e.g., sludge dumping and industrial effluents) and diffuse t Contribution no. 347, Marine Sciences Research Center, State University of New York, Stony Brook.

(e.g., runoff) sources. Metals such as mercury, lead, cadmium, copper, chromium, and zinc have been shown to occur at significantly elevated levels in many nearshore environments, often in the parts-per-thousand (ratio of weight of metal to dry weight of sediment) range (6, 24). Little is known about the effects of such accumulations on the sediment microflora. We have therefore undertaken a study of the effects of several environmentally significant heavy metals on ATP biomass, methanogenesis, sulfate reduction, and CO2 production in anoxic salt marsh sediments. MATERIALS AND METHODS Experimental setup. Sediment samples were collected from Flax Pond, a relatively unimpacted salt marsh on the north shore of Long Island, N.Y. Freshly collected sediments were homogenized at low speed with filtered seawater in a 1:2 ratio of sediment volume

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completion of an experiment, sediments were dried to constant weight at 105°C. ATP assays. The effect of the addition of metals on microbial ATP was determined for several metals. ATP was extracted from portions of the sediment slurry with boiling citrate-phosphate buffer (2) and quantified with the luciferin-luciferase reaction. ATP samples were routinely run with and without internal standards to correct for substances in the sample extracts which might affect the activity of the luciferinluciferase preparation (10). Sulfate reduction. For most experiments after 7 October 1981, sulfate reduction was determined by calculating the reduction of 35So4-2 to H235S (8), as well as by gas-chromatographic analysis (see below). For assays of sulfate reduction, 5 to 10 ,uCi of 35S04-2 was added to experimental and control flasks before metals were added. Portions (0.5 ml) were obtained with a syringe through the stopper, placed in a reaction vial, and acidified. The H235S evolved was trapped in 2 ml of 2% CdCI2 solution, combined with 6 ml of Scintiverse, and counted as a gel by liquid scintillation methods with an external standard/channels ratio method of quench correction. Experimental flasks were compared with control flasks in terms of distintegrations per minute per flask. Gas-chromatographic determinations. Methane in the gas phase was determined by flame ionization gas chromatography after separation on a column of Porapak R (2 m, 80/100 mesh) that was held at 60°C and that had an N2 carrier flow rate of 30 ml min-1. Carbon dioxide and H2S were separated under the same conditions except for the use of a He carrier with a flow rate of 30 ml * min-' and determination with a thermal conductivity detector. The solubility of each gas was taken into account when total gas production was computed. Metal determinations. Concentrations of copper, lead, chromium, and nickel were determined on a Perkin-Elmer model 403 atomic absorption spectrophotometer. Cores from the upper 10 cm of the study site were divided into 2-cm segments, oven dried at 105°C, and extracted with concentrated HCl-HNO3 (1:1) for 4 h at 95°C. Concentrations in the upper 10 cm were averaged and expressed in parts per million (ratio of weight of metal to dry weight of sediment). -

TIME (HRS)

FIG. 1. Time course of CH4 and CO2 production in salt marsh sediments. All incubations were run under an N2 atmosphere, with the exception of one incubation (O) which was begun under fully aerobic conditions. Symbols: 0, control; 0, autoclaved sediment; A, 100 ppm of HgCl2; A, 1,000 ppm of HgCl2. Error bars indicate ±SE. to seawater, strained through

a 2-mm mesh to remove material, dispensed in 50-ml portions to 125ml flasks with wide-bore volumetric pipettes, and sealed with black rubber stoppers. All operations were performed while the samples were gassed with N2. Concentrated solutions of each metal were added through the stoppers to yield a final weight of metal proportional to the estimated dry weight of sediment. Solution volumes were typically 0.5 ml. Flasks were incubated at 26 to 27°C with gentle shaking. For most experiments, each metal was run in triplicate. Since there was very low variance among replicates, this was reduced to duplicates in later experiments. The specific chemical compounds used included the chlorides of mercury, monomethyl mercury, lead, nickel, cadmium, iron, and copper; zinc sulfate; sulfides of lead and mercury; sodium molybdate and arsenite; and potassium mono- and dichromate. Samples were taken over the course of several days for various analyses. Experimental flasks were compared with control flasks, which received equal volumes of the carrier solutions lacking any metal. At the

any root

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RESULTS Metal concentrations in the upper 10 cm were relatively low at our study site. Total copper averaged 71 ± 6 ppm (+ standard error [SE] for five samples), lead averaged 47 + 2 ppm, chromium averaged 30 + 2 ppm, and nickel averaged 31 + 5 ppm. Precisions on determinations (as coefficients of variation) were 2% for copper, lead, and nickel and 5% for chromium. Methanogenesis in control flasks exhibited a short lag followed by a period of constant production and, finally, declining rates (Fig. 1). Carbon dioxide production in controls was usually linear from the outset. For controls, initial rates of CH4 production among the experiments varied over about a 14-fold range (0.2 to 2.7 nmol * g of dry sediment-' h-1), and overall

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TABLE 1. Summary of experiments, initial rates of production, and maximum production of CH4 and CO2 in control flask sediment slurries from Spartina communities' CH4 production

Date (mo/day/yr)

Metal(s)

Initial'

Maximum'

CO2 production Maximum' Initiald

2.3 134 ± 10 NDf ND Hg 11/20/80 Pb 7.5 470 ± 132 0.51 21 ± 4.5 4/08/819 1.6 88 ± 3 0.23 16 ± 0.3 Hg 5/05/81 1.4 318 ± 8 ND ND Hg and Pb 6/17/81 1.6 530 ± 19 0.59 43 ± 0.3 6/24/81 Hg and Pb 1.7 144 ± 8 0.20 21 ± 0.9 Hg and Pb 7/21/81 2.7 325 ± 20 0.20 32 ± 1.8 8/18/81 Hg and Pb 1.8 66 ± 15 0.30 19 ± 0.5 Mo, Ni, and As 9/06/81 1.0 39 ± 0.3 0.27 19 ± 1.4 Cr(2),h Cd, and Fe 9/16/81 2.4 250 ± 2 0.24 26 ± 0.5 Hg, HgS, and CH3Hg 10/07/81 2.3 123 ± 26 0.42 29 ± 1.4 Pb and Hg 10/20/81 0.4 25 ± 14 ND ND Hg, HgS, and CH3Hg 10/23/81 1.6 349 ± 229 0.87 50 ± 8.0 Mo, As, Ni, Cr, Cd, and Fe 10/26/81 34 ± 5.2 0.42 38 ± 3.1 0.7 11/17/81 Ni, Cu, and Zn 571 0.17 0.2 10 11/29/81 Hg, Pb, Cr, Cd, Fe, and Ni 30 ± 12 0.74 39 ± 5.5 0.8 1/04/82 Ni, Cu, and Zn 0.8 23 ± 2.0 ND ND Cr and Cr(2) 2/09/82 95 ± 37 0.26 26 ± 2.2 1.4 2/22/82 Ni, Cd, Fe, and Zn 0.25 11 ± 0.1 103 ± 1.6 1.6 11/10/82 Hg, Cu, and Zn a Results are expressed as the mean ± SE for three replicates. b Expressed in nanomoles per gram of dry sediment per hour. c Expressed in nanomoles per gram of dry sediment. d Expressed in micromoles per gram of dry sediment per hour. Expressed in micromoles per gram of dry sediment. f ND, Not done. g The sediment core for this date was collected some distance from the normal site and was somewhat more sandy than the typical cores used. h Cr(2), Dichromate.

production by 6 days varied about 25-fold (23 to 570 nmol g of dry sediment-1) (Table 1). For CO2, both initial rates of production (0.17 to 0.88 ,umol * g of dry sediment-' h-1) and overall rates of production (10 to 50 ,umol * g of dry sediment-') varied about fivefold among experiments (Table 1). For controls, CO2 production had usually ceased by 7 days, and CH4 production had usually ceased by 4 days. Low variance (indicated as SEs in Fig. 1) was associated with replicate assays of both CH4 and CO2 production. In flasks in which assays were begun with oxic atmospheres, severly reduced CH4 production and higher CO2 evolution were apparent (Fig. 1). For all activity parameters, experimental flasks were compared with control flasks with respect to the linear rate (after any lag in controls and over the same time period) and the maximum concentration by the end of the experiment. Mercuric chloride at 10 (data not shown) and 100 ppm (Fig. 1) had little effect on either CH4 or CO2 production. For HgCl2, PbCl2, K2Cr2O7, KCrO4, ZnSO4, NiCl2, CdCl2, FeCl2, and CuCl2 at 1,000 ppm, a period of initial inhibition was followed by stimulation (Fig. 1) (Table 2). In the cases of Ni, Cd, and Fe, this stimulation was not -

significant when compared with controls. A general inhibition of methanogenesis was noted with HgS, CH3HgCl, and NaAsO2, whereas PbS caused only an initial inhibition (Table 2). Methanogenesis was stimulated from the outset by Na2MoO4. Although methanogenesis generally leveled off in experimental flasks within 4 days, for ZnCl2 and CuCl2 a prolonged period of inhibition was followed by an extended period of enhanced methanogenesis. In general, CO2 production was not severely affected by the addition of metals. A slight initial inhibition occurred with Na2MoO4, ZnCl2, and CdCl2, whereas substantial inhibition occurred with K2Cr2O7 and NaAsO2. Inhibition over 6 days was noted with Na2MoO4, K2Cr2O7, ZnCl2, CH3HgCl, and NaAsO2. A slight stimulation of overall CO2 production occurred with HgCI2, PbC12, NiC12, and FeCl2. Initial rates of H2S production on the seven dates studied ranged from 0.02 to 0.24 ,umol * g of dry sediment-1 h-1, whereas overall production ranged from 4 to 42 ,mol * g of dry sediment-1. Evaluation of the effects of metals was generally confined to the radioisotopic assays to avoid underestimating SO2-2 reduction in metalenriched flasks because of possible metal precip-

EFFECTS OF METALS ON MARINE SEDIMENT MICROBIOTA

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TABLE 2. Effect of metals at 1,000 ppm on methanogenesis and CO2 production in salt marsh sedimentsa H235S production CO2 production CH4 production Metal Initial

Maximum

Initial

Maximum

Initial

Maximum

0.01 0 0.77 ± 0.15 0.76 ± 0.19 (2) 23 ± 6.9 6.8 ± 1.7 (2) 0.82 1.26 ± 0.08" 0.39 1.03 ± 0.05 (6) 1.48 ± 0.06b 0.75 ± 0.12 (9)" 0.54 0.59 1.20 ± 0.12 0.93 ± 0.15 (3) 2.81 ± 0.39b 0.60 ± 0.16 (4)b 0.01 0.03 0.61 ± 0.18 0.16 ± 0.11 (2)" 3.0 ± 0.18" 0.19 ± 0.06 (3)b K2Cr2O7 ND ND ND NDc 2.4 0.16 (1) K2CrO4 0.05 0 0.97 ± 0.15 4.43 ± 2.26 0.% ± 0.17 (4) 0.15 ± 0.01 (4)b ZnSO4 1.03 0.2 1.13 ± 0.16 1.06 ± 0.14 (6) 1.37 ± 0.44 0.51 ± 0.07 (5)b NiCl2 0.03 0.08 1.05 ± 0.20 0.81 ± 0.23 (4) 2.14 ± 1.15 0.18 ± 0.06 (4)b CdCl2 1.33 0.68 1.65 ± 0.27 1.25 ± 0.27 (3) 1.18 ± 0.47 0.83 ± 0.24 (3) FeCl2 0.05 0.0 1.18 ± 0.28 0.92 ± 0.01 (3)" 4.84 ± 1.78 0.10 ± 0.05 (3)" CuCl2 0.93 0.61 1.07 ± 0.01b 0.98 ± 0.07 (2) 1.04 ± 0.16 PbS 0.66 ± 0.04 (2)b 0.65 0.50 1.07 ± 0.00 1.00 ± 0.19 (2) 0.68 ± 0.26 0.58 ± 0.06 (3)b HgS 0.63 0.42 0.69 ± 0.11 1.08 ± 0.17 (2) 0.54 ± 0.27 (2) 0.07 ± 0.01b CH3HgCl 0.05 0.14 0.68 ± 0.07 0.38 ± 0.24 (2) 0.01 ± 0.09b 0.02 ± 0.01 (2)b NaAs02 a Values are expressed as the averages from several experiments and represent the ratios between results for experimental and control flasks ± SE. Numbers in parentheses represent the number of experiments. b Significant (P < 0.05) stimulation or inhibition as determined by a one-tailed Student's t test. c ND, Not done.

Na2MoO4 HgC12 PbCl2

itation of S-2. 35S04-2 reduction was severely inhibited, both initially and finally, in the presence of all metals tested, with the exception of FeCl2, HgCl2, and NiCl2 (Table 2). Similar results were obtained for total H2S production in metal-enriched flasks, as compared with controls. ATP concentrations in control flasks on 5 May 1981, 24 June 1981, 21 July 1981, and 4 January 1982 were quite similar, averaging 4.3 + 0.33 ,ug g of dry sediment-' (±SE) (Table 3). For PbCl2 and HgCl2 at various levels, no significant decrease in ATP pools was noted. In fact, when each was added at a low level (10 ppm), a slight increase in ATP levels may have occurred. NiCl2 also had little effect on ATP. Within individual experiments, significant (P < 0.05) decreases in ATP, relative to controls, were noted with copper, chromium, cadmium, and zinc. In order of increasing severity, ZnCl2, CdCl2, K2Cr2O7, and CuCl2 all substantially reduced sediment ATP levels, and the severity of inhibition generally increased with time (Table 3). -

DISCUSSION In general, S04-2 reduction is the predominating pathway of terminal carbon oxidation in anoxic marine sediments since S04 2 reducers effectively outcompete methanogens for common substrates (1, 13, 25) and have a higher

theoretical growth yield (22). Methanogenesis does occur concomitantly with S04-2 reduction (17, 20), but it is usually of only minor significance until S04-2 levels are depleted to a point at which S04-2 respiration is limited, as occurs

in the deeper portions of the sediment column (14). In freshwater sediments, low concentrations of S04-2 allow methanogenesis to predominate as the main terminal oxidative process (25), although recent evidence indicates that substantial amounts of S04-2 reduction may also occur in some freshwater systems (7). Differential inhibition of SO4-2-respiring bacteria has been shown to result in higher methanogenic activities (16). Besides interspecific competition, other factors may influence the relative proportions of methanogenesis and S042 reduction in a particular system. Sulfate (25) and sulfide (3, 12) have both been implicated as direct inhibitors of methanogenesis. A variety of metals were shown to be highly and specifically inhibitory to SO42-respiring bacteria (21). Furthermore, Pederson and Sayler (18) found little evidence of the inhibition of methanogenesis in lake sediments amended with mercury. The recognized ability of methanogens to methylate a variety of metals (19) may contribute to a relatively lower sensitivity to those metals. It should be noted, however, that the addition of high levels of CH3HgCl (1,000 ppm) to our assay system was very inhibitory to methanogenesis (Table 2). The levels of metals used in our experiments may seem exceptionally high. However, concentrations of Cr, Cu, Hg, Pb, and Zn in excess of 1,000 ppm have been noted in coastal sediments subject to heavy pollutant loading (6, 24). With regard to effects on microbiological processes, the availability or activity of each metal is a more important factor. However, the actual speciation of individual metals in anoxic marine sediments is not well understood. Whereas the

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TABLE 3. Effect of metals on extractable ATP in salt marsh sedimentsa ATP Concn 0h

4h

1.1 ± 0.1 (2) 0.9 ± 0.1 (2) 0.8 ± 0.2 (5)

1.0 ± 0.1 (2)

1.4 ± 0.4 (2)

1.4 (1)

100 1,000

0.9 (1) 0.7 ± 0.1 (4)

1.0 ± 0.1 (2) 0.9 ± 0.1 (4)

1.3 (1) 0.9 ± 0.1 (2)

PbCl2

10 1,000

1.0 (1) 0.9 ± 0.1 (2)

1.1 (1) 1.0 ± 0.4 (2)

1.2 (1) 1.3 ± 0.4 (2)

1.4 (1) 0.8 (2)

NiCI2

1,000

1.1 (1)

0.7 (1)

1.2 (1)

1.1 (1)

FeCl2

1,000

0.8 (1)

0.9 (1)

0.6 (1)

K2Cr2O7

1,000

0.9 (1)

0.2 (1)

0.3 (1)

CdCl2

1,000

1.2 (1)

0.6 (1)

0.3 (1)

CUC12

1,000

0.5 (1)

0.1 (1)

0.2 (1)

HgCl2

(ppm) 10

0.3 (1)

24 h

Final

1.3 (1) 1.0 (1) 0.7 (1) 0.4 (1) ZnSO4 1,000 a Results are expressed as the ratios of means of results for two to three experimental flasks to the means of results for two to three controls flasks in each experiment. Numbers in parentheses represent the number of experiments. When more than one experiment was run, the ratios were reported as the mean ± SE.

low Eh of anoxic sediments is thought to promote mobilization of metals, the production of S-2 by S04-2 reduction should remove available metals through precipitation of insoluble metal sulfides (5, 11). Complexation of metals with organic substances adds another dimension of complexity to metal speciation. Khalid et al. (11) reported an increase in the dissolved concentrations of several metals in estuarine sediments experimentally subjected to low 02. Similar phenomena have been found in soils and freshwater sediments (5). The effect of the addition of heavy metals on both methanogens and S04-2-respiring bacteria should be mitigated to some extent in marine sediments by sulfide precipitation. Severe metal loading beyond this capacity appears, in several cases, to inhibit S04-2 reduction selectively, thereby favoring methanogenesis. Mountfort and Asher (15) recently reported that heavy organic pollution of intertidal sediments produced a shift from S04-2 reduction to methanogenesis. They attributed this observation to the rapid depletion of interstitial S04-2 through intense S04-2 reduction which was fueled by organic loading. Many sources of organic pollution, such as sewage sludge, also contain high levels of heavy metals (24), and such factors may additionally have contributed to the phenomena observed by Mountfort and Asher (15). Methanogenesis was stimulated to various degrees by the majority of the metals tested. Oremland and Taylor (16) attributed the stimulation of methanogenesis by Na2MoO4 to differen-

tial inhibition of S04-2 respirers. Although no other metal elicited as great a response in our system as Na2MoO4, we suspect a similar explanation may account for our observations. In fact, our evidence indicates a sustained inhibition of S04-2 reduction by most of the metals which stimulated CH4 production. Selection and enrichment of metal-resistant strains of methanogens is another possibility. Such phenomena have been noted in bacterial populations of marine sediments subject to chronic metal pollution (23). Further work is being undertaken to investigate the nature of the observed stimulation. For several environmentally significant metals, chronic pollution may shift the natural preponderance of the terminal flow of carbon in the sediments from S04-2 reduction to methanogenesis, possibly lowering the catabolic efficiency of the sediment system so affected. A further implication of such a shift is the potential ability of methanogens to aggravate metal toxicity at higher trophic levels through methylation. ACKNOWLEDGMENTS We thank Jennifer Jesty, Edward Carpenter, Mary Scranton, Glenn Lopez, Ted Jereb, and Jim Bauer for their comments or assistance or both on various aspects of this study. Thanks are particularly extended to Dave Hirschberg for metal analysis. Financial support was provided by the National Oceanic and Atmospheric Administration through the Office of Marine Pollution Assessment (grant NA-80-RAD-00057), by the Environmental Protection Agency (grant R-809475-01-0), and by the National Science Foundation (grant OCE-82-00157).

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LITERATURE CITED 1. Abram, J. W., and D. B. Nedwell. 1978. Inhibition of methanogenesis by sulphate reducing bacteria competing for transferred hydrogen. Arch. Microbiol. 117:89-92. 2. Bulied, N. C. 1978. An improved method for the extraction of adenosine triphosphate from marine sediments and seawater. Limnol. Oceanogr. 23:174-178. 3. Cappenburg, T. E. 1974. Interrelationships between sulfate-reducing and methane-producing bacteria in bottom sediments of a fresh water lake II. Inhibition experiments. Antonie van Leeuwenhoek J. Microbiol. Serol. 40:297306. 4. Fencdel, T., and T. H. Blackburn. 1979. Bacteria and mineral cycling. Academic Press, Inc., New York. 5. Gadd, G. M., and A. J. Grifiths. 1978. Microorganisms and heavy metal toxicity. Microb. Ecol. 4:303-317. 6. Grieg, R., and R. McGrath. 1977. Trace metals in sediments of Raritan Bay. Mar. Pollut. Bull. 8:188-192. 7. Iagvorsen, K., J. G. Zedkus, and T. Brock. 1981. Dynamics of bacterial sulfate reduction in a eutrophic lake. Appl. Environ. Microbiol. 42:1029-1036. 8. Jorgensen, B. B. 1978. A comparison of methods for the quantification of bacterial sulfate reduction in coastal marine sediments. I. Measurement with radiotracer techniques. Geomicrobiol. J. 1:11-27. 9. Jorgensen, B. B. 1982. Mineralization of organic matter in the sea bed-the role of sulphate reduction. Nature (London) 296:643-645. 10. Karl, D. M., and P. A. LaRock. 1975. Adenosine triphosphate measurements in soil and marine sediments. J. Fish. Res. Board Can. 32:599-667. 11. Khald, R. A., W. H. Patrick, Jr., and R. P. Gambrill. 1978. Effect of dissolved oxygen on chemical transformations of heavy metals, phosphorus and nitrogen in an estuarine sediment. Estuarine Coastal Mar. Sci. 6:21-35. 12. Khan, A., and T. Trottler. 1978. Effect of sulfur-containing compounds on anaerobic degradation of cellulose to methane by mixed cultures obtained from sewage sludge. AppI. Environ. Microbiol. 35:1027-1034. 13. Lovely, D. R., D. F. Dwyer, and M. J. King. 1982. Kinetic analysis of competition between sulfate reducers and methanogens for hydrogen in sediments. Appl. Environ. Microbiol. 43:1373-1379.

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14. Marte, C. S., and R. A. Berner. 1974. Methane production in the interstitial waters of sulfate-depleted marine sediments. Science 185:1167-1169. 15. Mountrort, D. O., and R. A. Asher. 1981. Role of sulfate reduction versus methanogenesis in terminal carbon flow in polluted intertidal sediments of Waimea Inlet, Nelson, New Zealand. Appl. Environ. Microbiol. 42:252-258. 16. Oremland, R., and B. Taylor. 1978. Sulfate reduction and methanogenesis in marine sediments. Geochim. Cosmochim. Acta 42:209-214. 17. Oremland, R. S., L. M. Marsh, and S. Polin. 1982. Methane production and simultaneous sulphate reduction in anoxic saltmarsh sediments. Nature (London) 296:143145. 18. Pedeson, D., and G. Sayler. 1981. Methanogenesis in fresh water sediments: inherent variability and effects of environmental contaminants. Can. J. Microbiol. 27:198205. 19. RIkey, W. P., L. J. Dizkes, and J. M. Wood. 1977. Biomethylation of toxic elements in the environment. Science 197:329-332. 20. Senior, E., E. B. Lindstrom, I. M. Banat, and D. B. Nedwell. 1982. Sulfate reduction and methanogenesis on the east coast of the United Kingdom. Appl. Environ. Microbiol. 43:987-996. 21. Taylor, B., sad R. Oremband. 1979. Depletion of ATP in DesuUfovibrio by oxyanions of group VI elements. Curr. Microbiol. 3:101-103. 22. Thauw, R. K., K. Jungerman, and K. Decker. 1981. Energy conservation in chemotrophic anaerobic bacteria. Bacteriol. Rev. 41:100-180. 23. Timoney, J. F., J. Port, J. GUes, and J. Spanler. 1978. Heavy-metal and antibiotic resistance in the bacterial flora of sediments of New York Bight. Appl. Environ. Microbiol. 36:465-472. 24. Williams, S. C., H. J. Simpson, C. R. Olsen, and R. F. Bopp. 1978. Sources of heavy metals in sediments of the Hudson River estuary. Mar. Chem. 6:195-213. 25. Winfrey, M. R., and J. G. Zelksu. 1977. Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments. Appl. Environ. Microbiol. 33:275-281.