Methylmercury decomposition in sediments and bacterial cultures: involvement of methanogens and sulfate reducers in oxidative demethylation.

Demethylation of monomethylmercury in freshwater and estuarine sediments and in bacterial cultures was investigated with CH(3)HgI. Under anaerobiosis, results with inhibitors indicated partial involvement of both sulfate reducers and methanogens, the former dominating estuarine sediments, while both were active in freshwaters. Aerobes were the most significant demethylators in estuarine sediments, but were unimportant in freshwater sediments. Products of anaerobic demethylation were mainly CO(2) as well as lesser amounts of CH(4). Acetogenic activity resulted in fixation of some CO(2) produced from CH(3)HgI into acetate. Aerobic demethylation in estuarine sediments produced only CH(4), while aerobic demethylation in freshwater sediments produced small amounts of both CH(4) and CO(2). Two species of Desulfovibrio produced only traces of CH(4) from CH(3)HgI, while a culture of a methylotrophic methanogen formed traces of CO(2) and CH(4) when grown on trimethylamine in the presence of the CH(3)HgI. These results indicate that both aerobes and anaerobes demethylate mercury in sediments, but that either group may dominate in a particular sediment type. Aerobic demethylation in the estuarine sediments appeared to proceed by the previously characterized organomercurial-lyase pathway, because methane was the sole product. However, aerobic demethylation in freshwater sediments as well as anaerobic demethylation in all sediments studied produced primarily carbon dioxide. This indicates the presence of an oxidative pathway, possibly one in which methylmercury serves as an analog of one-carbon substrates.

Effects of acidification on mercury methylation, demethylation, and volatilization in sediments from an acid-susceptible lake.

The effect of experimental acidification on mercury methylation, demethylation, and volatilization was examined in surficial sediment samples from a weakly buffered northern Wisconsin lake. All mercury transformations were measured with radioisotopic tracers. Acidification of sediment pH with H2SO4, HCl, or HNO3 significantly decreased 203Hg(II) methylation. Acidification of pH 6.1 (ambient) sediments to pH 4.5 with either H2SO4 or HCl inhibited methylation by over 65%. The decreased methylation was due to the increased hydrogen ion concentration because methylation was not affected by concentrations of Na2SO4 or NaCl equimolar to the amount of acid added. Inhibition of methylation was observed even after prolonged acidification of sediments to pH 5.0 for up to 74 days. Acidification of sediments to pH 5.5, 4.5, and 3.5 with HNO3 resulted in a near complete inhibition of methylation at each pH. Similarly, the addition of equimolar amounts of NaNO3 resulted in a near complete inhibition of methylation, indicating that the inhibition was due to the nitrate ion rather than to the acidity. Demethylation of methyl mercury was not affected by pHs between 8.0 and 4.4, but sharply decreased below pH 4.4. Volatilization of 203Hg(II) from surface sediments was less than 2% of methylation activity and was not significantly different from that in killed sediments. This study indicated that acidification of sediments inhibits mercury methylation and that the observed increase in the mercury burdens in fish from low pH lakes is not due to increased production of methylmercury in sediments.

Seasonal and spatial variations in mercury methylation and demethylation in an oligotrophic lake.

Microbial mercury methylation and methylmercury decomposition were examined in Lake Clara, an oligotrophic northern Wisconsin seepage lake, using radioisotopic tracers. Methylation activity was near background in the water column, was greatest in the profundal surficial sediments, and decreased with depth in sediment cores. Active demethylation occurred in the water column but was variable. Demethylation was greatest in the surficial sediments and decreased slightly with sediment depth. The methylation/demethylation ratio (M/D) was >1 in the water column, exhibited a sharp peak in surface sediments, and decreased in deeper sediments. Methylation and demethylation activity varied in surface sediments collected along a lake transect. The M/D ratio in surface sediments ranged from 1.4 to 5.8. Methylation in attached microbial communities was near background, while demethylation was high. The M/D ratios in the attached communities were all <0.20. Methylation activity in surface sediments incubated at in situ temperature increased from spring to late summer and decreased in the fall. Demethylation increased from early to midsummer and then declined. The M/D ratio in surface sediments increased from mid- to late summer, and decreased in the fall. These results indicate that the greatest potential for methylation in Lake Clara occurs in the surficial sediments and that methylation in surficial sediments is greatest from mid-July through September. In addition, the net rate of methylmercury production may be significantly affected by demethylation.

Substrates for sulfate reduction and methane production in intertidal sediments.

The activity of and potential substrates for methane-producing bacteria and sulfate-reducing bacteria were examined in marsh, estuary, and beach intertidal sediments. Slow rates of methane production were detected in all sediments, although rates of sulfate reduction were 100- to 1,000-fold higher. After sulfate was depleted in sediments, the rates of methane production sharply increased. The addition of methylamine stimulated methanogenesis in the presence of sulfate, and [C]methylamine was rapidly converted to CH(4) and CO(2) in freshly collected marsh sediment. Acetate, hydrogen, or methionine additions did not stimulate methanogenesis. [methyl-C]methionine and [2-C]acetate were converted to CO(2) and not to CH(4) in fresh sediment. No reduction of CO(2) to CH(4) occurred in fresh sediment. Molybdate, an inhibitor of sulfate reduction, inhibited [2-C]acetate metabolism by 98.5%. Fluoracetate, an inhibitor of acetate metabolism, inhibited sulfate reduction by 61%. These results suggest that acetate is a major electron donor for sulfate reduction in marine sediments. In the presence of high concentrations of sulfate, methane may be derived from novel substrates such as methylamine.

Microbial methanogenesis and acetate metabolism in a meromictic lake.

Methanogenesis and the anaerobic metabolism of acetate were examined in the sediment and water column of Knaack Lake, a small biogenic meromictic lake located in central Wisconsin. The lake was sharply stratified during the summer and was anaerobic below a depth of 3 m. Large concentrations (4,000 mumol/liter) of dissolved methane were detected in the bottom waters. A methane concentration maximum occurred at 4 m above the sediment. The production of (14)CH(4) from (14)C-labeled HCOOH, HCO(3) (-), and CH(3)OH and [2-(14)C]acetate demonstrated microbial methanogenesis in the water column of the lake. The maximum rate of methanogenesis calculated from reduction of H(14)CO(3) (-) by endogenous electron donors in the surface sediment (depth, 22 m) was 7.6 nmol/h per 10 ml and in the water column (depth, 21 m) was 0.6 nmol/h per 10 ml. The methyl group of acetate was simultaneously metabolized to CH(4) and CO(2) in the anaerobic portions of the lake. Acetate oxidation was greatest in surface waters and decreased with water depth. Acetate was metabolized primarily to methane in the sediments and water immediately above the sediment. Sulfide inhibition studies and temperature activity profiles demonstrated that acetate metabolism was performed by several microbial populations. Sulfide additions (less than 5 mug/ml) to water from 21.5 m stimulated methanogenesis from acetate, but inhibited CO(2) production. Sulfate addition (1 mM) had no significant effect on acetate metabolism in water from 21.5 m, whereas nitrate additions (10 to 14,000 mug/liter) completely inhibited methanogenesis and stimulated CO(2) formation.

Anaerobic metabolism of immediate methane precursors in Lake Mendota.

Lake Mendota sediments and the immediate overlying water column were studied to better understand the metabolism of the methanogenic precursors H2/CO2 and acetate in nature. The pool size of acetate (3.5 microns M) was very small, and the acetate turnover time (0.22h) was very rapid. The dissolved inorganic carbon pool was shown to be large (6.4 to 8.3 mM), and the turnover time was slow (111 H.). CO2 was shown to account for 41 +/- 5.5% of the methane produced in sediment. Acetate and H2/CO2 were simultaneously converted to CH4. The addition of H2 to sediments resulted in an increase specific activity of CH4 from H(14)CO3- and a decrease in specific activity of CH4 from [2-14C]acetate. Acetate addition resulted in a decrease in specific activity of CH4 from H(14)CO3-. The metabolism of H(14)CO3- or [2-14C]acetate to 14CH4 was not inhibited by addition of acetate or H2. After greater than 99% of added [2-14C]acetate had been turned over, 42% of the label was recovered as 14CH4 20% was recovered as 14CO2 and 38% was incorporated into sediment. Inhibitor studies of [2-14C]acetate metabolism in sediments demonstrated that CHCl3 completely inhibited CH4 formation, but not CO2 production. Air and nitrate addition inhibited CH4 formation and stimulated CO2 production, whereas fluoroacetate addition totally inhibited acetate metabolism. The oxidation of [2-14C]acetate to 14CO2 was shown to decrease with time when sediment was incubated before the addition of label, suggesting depletion of low levels of an endogenous sediment electron acceptor. Acetate metabolism varied seasonally and was related to the concentration of sulfate in the lake and interstitial water. Methanogenesis occurred in the sediment and in the water immediately overlying the sediment during period of lake stratification and several centimeters below the sediment-water interface during lake turnovers. These data indicate that methanogenesis in Lake Mendota sediments was limited by "immediate" methane precursor availability (i.e., acetate and H2), by competition for these substrates by nonmethanogens, and by seasonal variations which altered sediment and water chemistry.

Association of hydrogen metabolism with methanogenesis in Lake Mendota sediments.

Lake Mendota sediments were studied to determine the role of H2 in sediment methanogenesis. H2 was generally not detectable in sediment. The addition of H2 to sediment significantly increased methanogenensis. The amount of methane produced was proportional to the concentration of hydrogen added. H2 addition stimulated the reduction of CO2 to methane, but did not significantly stimulate the conversion of methanol or the methyl position of acetate to methane. Various organic compounds also stimulated sediment methanogenesis. Formate, ethanol, and glucose were shown to serve as electron donors for CO2 reduction to methane. The addition of formate to sediment resulted in H2 evolution. H2 was not deith the phenomenon of interspecies hydrogen transfer. The results indicate that hydrogen is an important intermediate and a rate-limiting factor in sediment methanogenesis.

Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments.

The effect of sulfate on methane production in Lake Mendota sediments was investigated to clarify the mechanism of sulfate inhibition of methanogenesis. Methanogenesis was shown to be inhibited by the addition of as little as 0.2 mM sulfate. Sulfate inhibition was reversed by the addition of either H2 or acetate. Methane evolved when inhibition was reversed by H2 additions was derived from 14CO2. Conversely, when acetate was added to overcome sulfate inhibition, the evolved methane was derived from [2-14C]acetate. A competition for available H2 and acetate was proposed as the mechanism by which sulfate inhibited methanogenesis. Acetate was shown to be metabolized even in the absence of methanogenic activity. In the presence of sulfate, the methyl position of acetate was converted to CO2. The addition of sulfate to sediments did not result in the accumulation of significant amounts of sulfide in the pore water. Sulfate additions did not inhibit methanogenesis unless greater than 100 mug of free sulfide per ml was present in the pore water. These results indicate that carbon and electron flow are altered when sulfate is added to sediments. Sulfate-reducing organisms appear to assume the role of methanogenic bacteria in sulfate-containing sediments by utilizing methanogenic precursors.

Temperature limitation of methanogenesis in aquatic sediments.

Microbial methanogenesis was examined in sediments collected from Lake Mendota, Wisconsin, at water depths of 5, 10, and 18 m. The rate of sediment methanogenesis was shown to vary with respect to sediment site and depth, sampling date, in situ temperature, and number of methanogens. Increased numbers of methanogenic bacteria and rates of methanogenesis correlated with increased sediment temperature during seasonal change. The greatest methanogenic activity was observed for 18-m sediments throughout the sampling year. As compared with shallower sediments, 18-m sediment was removed from oxygenation effects and contained higher amounts of ammonia, carbonate, and methanogenic bacteria, and the population density of methanogens fluctuated less during seasonal change. Rates of methanogenesis in 18-m sediment cores decreased with increasing sediment depth. The optimum temperature, 35 to 42 C, for sediment methanogenesis was considerably higher than the maximum observed in situ temperature of 23 C. The conversion of H2 and [14C]carbonate to [14C]methane displayed the same temperature optimum when these substrates were added to sediments. The predominant methanogenic population had simple nutritional requirements and were metabolically active at 4 to 45 C. Hydrogen oxidizers were the major nutritional type of sediment methanogens; formate and methanol fermentors were present, but acetate fermentors were not observed. Methanobacterium species were most abundant in sediments although Methanosarcina, Methanococcus, and Methanospirillum species were observed in enrichment cultures. A chemolithotropic species of Methanosarcina and Methanobacterium was isolated in pure culture that displayed temperature optima above 30 C and had simple nutritional requirements.