Influence of pH on Terminal Carbon Metabolism in Anoxic Sediments from a Mildly Acidic Lake

The carbon and electron flow pathways and the bacterial populations responsible for transformation of H₂-CO₂, formate, methanol, methylamine, acetate, glycine, ethanol, and lactate were examined in sediments collected from Knaack Lake, Wis. The sediments were 60% organic matter (pH 6.2) and did not display detectable sulfate-reducing activity, but they contained the following average concentration (in micromoles per liter of sediment) of metabolites and end products: sulfide, 10; methane, 1,540; CO₂, 3,950; formate, 25; acetate, 157; ethanol, 174; and lactate, 138. Methane was produced predominately from acetate, and only 4% of the total CH₄ was derived from CO₂. Methanogenesis was limited by low environmental temperature and sulfide levels and more importantly by low pH. Increasing in vitro pH to neutral values enhanced total methane production rates and the percentage of CO₂ transformed to methane but did not alter the amount of ¹⁴CO₂ produced from [2-¹⁴C]acetate (~24%). Analysis of both carbon transformation parameters with ¹⁴C-labeled tracers and bacterial trophic group enumerations indicated that methanogenesis from acetate and both heterolactic- and acetic acid-producing fermentations were important to the anaerobic digestion process.     

Partitioning Effects during Terminal Carbon and Electron Flow in Sediments of a Low-Salinity Meltwater Pond near Bratina Island, McMurdo Ice Shelf, Antarctica

A study of anaerobic sediments below cyanobacterial mats of a low-salinity meltwater pond called Orange Pond on the McMurdo Ice Shelf at temperatures simulating those in the summer season (<5°C) revealed that both sulfate reduction and methane production were important terminal anaerobic processes. Addition of [2-¹⁴C]acetate to sediment samples resulted in the passage of label mainly to CO₂. Acetate addition (0 to 27 mM) had little effect on methanogenesis (a 1.1-fold increase), and while the rate of acetate dissimilation was greater than the rate of methane production (6.4 nmol cm⁻³ h⁻¹ compared to 2.5 to 6 nmol cm⁻³ h⁻¹), the portion of methane production attributed to acetate cleavage was <2%. Substantial increases in the methane production rate were observed with H₂ (2.4-fold), and H₂ uptake was totally accounted for by methane production under physiological conditions. Formate also stimulated methane production (twofold), presumably through H₂ release mediated through hydrogen lyase. Addition of sulfate up to 50-fold the natural levels in the sediment (interstitial concentration, ~0.3 mM) did not substantially inhibit methanogenesis, but the process was inhibited by 50-fold chloride (36 mM). No net rate of methane oxidation was observed when sediments were incubated anaerobically, and denitrification rates were substantially lower than rates for sulfate reduction and methanogenesis. The results indicate that carbon flow from acetate is coupled mainly to sulfate reduction and that methane is largely generated from H₂ and CO₂ where chloride, but not sulfate, has a modulating role. Rates of methanogenesis at in situ temperatures were four- to fivefold less than maximal rates found at 20°C.     

Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. III. Experiments with 14C-labeled substrates

An ecological substrate relationship between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten has been studied in experiments using ¹⁴C-labeled acetate and lactate as substrates. Fluoroacetate strongly inhibited the formation of ¹⁴CO₂ from [U-¹⁴C]-acetate and β-fluorolactate gave an inhibition of similar magnitude of the breakdown of [U-¹⁴C]-l-lactate to ¹⁴CO₂ thus confirming earlier results on the specific action of these inhibitors. The turnover-rate constant of l-lactate was 2.37 hr^-1 and the average l-lactate pool size was 12.2 μg per gram of wet mud, giving a turnover rate of 28.9 μg of lactate/gram of mud per hr. The turnover-rate constant of acetate was 0.35 hr^-1 and the average pool size was 5.7 μg per gram of wet mud, giving a rate of disappearance of 1.99 μg of acetate/gram of mud per hr. Estimations of the acetate turnover rate based upon the formation of ¹⁴CO₂ from [U-¹⁴C]-acetate or [1-¹⁴C]-acetate yielded figures of the same magnitude (range 0.45 to 1.74). These and other results suggest that only a portion of the lactate dissimilated is turned over through the acetate pool. The ratio of ¹⁴CO₂/¹⁴CH₄ produced from [U-¹⁴C]-acetate by mud was 1.32; indicating that 0.862 moles of CH₄ and 1.138 moles of CO₂ are formed per mole of acetate. From the rate of disappearance of acetate (0.027 μmoles/gram wet mud per hr) and the rate of methane production (0.034 μmoles/gram wet mud per hr), it may be concluded that acetate is an important precursor of methanogenesis in mud (approximately 70%). A substrate relationship between the two groups of bacteria is likely since ¹⁴CH₄ was formed from [U-¹⁴C]-l-lactate.     

Anaerobic Degradation of Lactate by Syntrophic Associations of Methanosarcina barkeri and Desulfovibrio Species and Effect of H2 on Acetate Degradation

When grown in the absence of added sulfate, cocultures of Desulfovibrio desulfuricans or Desulfovibrio vulgaris with Methanobrevibacter smithii (Methanobacterium ruminantium), which uses H₂ and CO₂ for methanogenesis, degraded lactate, with the production of acetate and CH₄. When D. desulfuricans or D. vulgaris was grown in the absence of added sulfate in coculture with Methanosarcina barkeri (type strain), which uses both H₂-CO₂ and acetate for methanogenesis, lactate was stoichiometrically degraded to CH₄ and presumably to CO₂. During the first 12 days of incubation of the D. desulfuricans-M. barkeri coculture, lactate was completely degraded, with almost stoichiometric production of acetate and CH₄. Later, acetate was degraded to CH₄ and presumably to CO₂. In experiments in which 20 mM acetate and 0 to 20 mM lactate were added to D. desulfuricans-M. barkeri cocultures, no detectable degradation of acetate occurred until the lactate was catabolized. The ultimate rate of acetate utilization for methanogenesis was greater for those cocultures receiving the highest levels of lactate. A small amount of H₂ was detected in cocultures which contained D. desulfuricans and M. barkeri until after all lactate was degraded. The addition of H₂, but not of lactate, to the growth medium inhibited acetate degradation by pure cultures of M. barkeri. Pure cultures of M. barkeri produced CH₄ from acetate at a rate equivalent to that observed for cocultures containing M. barkeri. Inocula of M. barkeri grown with H₂-CO₂ as the methanogenic substrate produced CH₄ from acetate at a rate equivalent to that observed for acetate-grown inocula when grown in a rumen fluid-vitamin-based medium but not when grown in a yeast extract-based medium. The results suggest that H₂ produced by the Desulfovibrio species during growth with lactate inhibited acetate degradation by M. barkeri.     

Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO₂ and either ethanol or lactate but not H₂ gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO₂ reduction, with a formate production rate and methanogenic turnover constant of 660 μM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 μM) than digestor contents, and the free flora was the primary site for formate cleavage to CO₂ and H₂ (90 μM formate per h). Inhibition of methanogenesis by CHCl₃ resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H₂ gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H₂ transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.     

Growth and Methanogenesis by Methanosarcina Strain 227 on Acetate and Methanol

Methanosarcina strain 227 exhibited exponential growth on sodium acetate in the absence of added H₂. Under these conditions, rates of methanogenesis were limited by concentrations of acetate below 0.05 M. One mole of methane was formed per mole of acetate consumed. Additional evidence from radioactive labeling studies indicated that sufficient energy for growth was obtained by the decarboxylation of acetate. Diauxic growth and sequential methanogenesis from methanol followed by acetate occurred in the presence of mixtures of methanol and acetate. Detailed studies showed that methanol-grown cells did not metabolize acetate in the presence of methanol, although acetate-grown cells did metabolize methanol and acetate simultaneously before shifting to methanol. Acetate catabolism appeared to be regulated in response to the presence of better metabolizable substrates such as methanol or H₂-CO₂ by a mechanism resembling catabolite repression. Inhibition of methanogenesis from acetate by 2-bromoethanesulfonate, an analog of coenzyme M, was reversed by addition of coenzyme M. Labeling studies also showed that methanol may lie on the acetate pathway. These results suggested that methanogenesis from acetate, methanol, and H₂-CO₂ may have some steps in common, as originally proposed by Barker. Studies with various inhibitors, together with molar growth yield data, suggest a role for electron transport mechanisms in energy metabolism during methanogenesis from methanol, acetate, and H₂-CO₂.     

Hydrogen metabolism during methanogenesis from acetate by Methanosarcina barkeri

A tritium exchange assay and a sensitive gas chromatographic technique were used to demonstrate that hydrogenase was active and that hydrogen was produced by Methanosarcina barkeri strain MS grown on acetate. Both methane and hydrogen production rates were dependent on the concentration of acetate in the medium. H₂ was produced at 0.5–2% of the rate of CH₄ formation. Chloroform and potassium cyanide, inhibitors of methanogenesis from acetate, inhibited H₂ production but not hydrogenase activity. The addition of hydrogen gas to cell suspensions did not inhibit CH₄ or carbon dioxide production from the methyl group of acetate. H₂ production appears to be linked to several intracellular redox processes which follow the cleavage of acetate.     

Growth of Desulfovibrio in Lactate or Ethanol Media Low in Sulfate in Association with H2-Utilizing Methanogenic Bacteria

In the analysis of an ethanol-CO₂ enrichment of bacteria from an anaerobic sewage digestor, a strain tentatively identified as Desulfovibrio vulgaris and an H₂-utilizing methanogen resembling Methanobacterium formicicum were isolated, and they were shown to represent a synergistic association of two bacterial species similar to that previously found between S organism and Methanobacterium strain MOH isolated from Methanobacillus omelianskii. In lowsulfate media, the desulfovibrio produced acetate and H₂ from ethanol and acetate, H₂, and, presumably, CO₂ from lactate; but growth was slight and little of the energy source was catabolized unless the organism was combined with an H₂-utilizing methanogenic bacterium. The type strains of D. vulgaris and Desulfovibrio desulfuricans carried out the same type of synergistic growth with methanogens. In mixtures of desulfovibrio and strain MOH growing on ethanol, lactate, or pyruvate, diminution of methane produced was stoichiometric with the moles of sulfate added, and the desulfovibrios grew better with sulfate addition. The energetics of the synergistic associations and of the competition between the methanogenic system and sulfate-reducing system as sinks for electrons generated in the oxidation of organic materials such as ethanol, lactate, and acetate are discussed. It is suggested that lack of availability of H₂ for growth of methanogens is a major factor in suppression of methanogenesis by sulfate in natural ecosystems. The results with these known mixtures of bacteria suggest that hydrogenase-forming, sulfate-reducing bacteria could be active in some methanogenic ecosystems that are low in sulfate.     

Sulfate Reduction Relative to Methane Production in High-Rate Anaerobic Digestion: Technical Aspects

The effect of different substrates and different levels of sulfate and sulfide on methane production relative to sulfate reduction in high-rate anaerobic digestion was evaluated. Reactors could be acclimated so that sulfate up to a concentration of 5 g of sulfate S per liter did not significantly affect methanogenesis. Higher levels gave inhibition because of salt toxicity. Sulfate reduction was optimal at a relatively low level of sulfate, i.e., 0.5 g of sulfate S per liter, but was also not significantly affected by higher levels. Both acetoclastic and hydrogenotrophic methane-producing bacteria adapted to much higher levels of free H₂S than the values reported in the literature (50% inhibition occurred only at free H₂S levels of more than 1,000 mg/liter). High levels of free H₂S affected the sulfate-reducing bacteria only slightly. Formate and acetate supported the sulfate-reducing bacteria very poorly. In the high-rate reactors studied, intensive H₂S formation occurred only when H₂ gas or an H₂ precursor such as ethanol was supplied.     

Stable Carbon Isotope Fractionation by Methanosarcina barkeri during Methanogenesis from Acetate, Methanol, or Carbon Dioxide-Hydrogen

Methanosarcina barkeri was cultured on methanol, H₂-CO₂, and acetate, and the ¹³C/¹²C ratios of the substrates and the methane produced from them were determined. The discrimination against ¹³C in methane relative to substrate decreased in the order methanol > CO₂ > acetate. The isotopic fractionation for methane derived from acetate was only one-third of that observed with methanol as the substrate. The data presented indicate that the last enzyme of methanogenesis, methylreductase, is not the primary site of isotopic discrimination during methanogenesis from methanol or CO₂. These results also support biogeochemical interpretations that gas produced in environments in which acetate is the primary methane precursor will have higher ¹³C/¹²C ratios than those from environments where other substrates predominate.     

Trophic links between fermenters and methanogens in a moderately acidic fen soil

Trophic links between fermentation and methanogenesis of soil derived from a methane‐emitting, moderately acidic temperate fen (pH 4.5) were investigated. Initial CO₂:CH₄ production ratios in anoxic microcosms indicated that methanogenesis was concomitant to other terminal anaerobic processes. Methane production in anoxic microcosms at in situ pH was stimulated by supplemental H₂–CO₂, formate or methanol; supplemental acetate did not stimulate methanogenesis. Supplemental H₂–CO₂, formate or methanol also stimulated the formation of acetate, indicating that the fen harbours moderately acid‐tolerant acetogens. Supplemental monosaccharides (glucose, N‐acetylglucosamine and xylose) stimulated the production of CO₂, H₂, acetate and other fermentation products when methanogenesis was inhibited with 2‐bromoethane sulfonate 20 mM. Glucose stimulated methanogenesis in the absence of BES. Upper soil depths yielded higher anaerobic activities and also higher numbers of cells. Detected archaeal 16S rRNA genes were indicative of H₂–CO₂‐ and formate‐consuming methanogens (Methanomicrobiaceae), obligate acetoclastic methanogens (Methanosaetaceae) and crenarchaeotes (groups I.1a, I.1c and I.3). Molecular analyses of partial sequences of 16S rRNA genes revealed the presence of Acidobacteria, Nitrospirales, Clamydiales, Clostridiales, Alpha‐, Gamma‐, Deltaproteobacteria and Cyanobacteria. These collective results suggest that this moderately acidic fen harbours phylogenetically diverse, moderately acid tolerant fermenters (both facultative aerobes and obligate anaerobes) that are trophically linked to methanogenesis.     

Mesophilic syntrophic acetate oxidation during methane formation by a triculture at high ammonium concentration

In a mesophilic (37°C) triculture at a high ammonium concentration and pH8, methanogenesis from acetate occurred via syntrophic acetate oxidation. Studies with ¹⁴C-labelled substrates showed that the amount of labelled methane formed from 1-¹⁴C-labelled acetate was equal to that formed from 2-¹⁴C-labelled acetate. Labelled methane was also formed from H¹⁴CO₃ ^-. These results clearly showed that both the methyl and carboxyl groups of acetate were oxidized to CO₂ and that CO₂ was reduced to CH₄ through hydrogenotrophic methanogenesis. During growth of the triculture, a significant isotopic exchange between the carboxyl group of acetate and bicarbonate occurred. As a result, there was a decrease in the specific activity of 1-¹⁴C-acetate, and the production of ¹⁴CO₂ was slightly higher from 1-¹⁴C- than from 2-¹⁴C-acetate. For each mole acetate degraded, 0.94 mol methane was formed; 9.2 mmol acetate was metabolized during the 294 days of incubation.     

Fate of Immediate Methane Precursors in Low-Sulfate, Hot-Spring Algal-Bacterial Mats

The fates of acetate and carbon dioxide were examined in several experiments designed to indicate their relative contributions to methane production at various temperatures in two low-sulfate, hot-spring algal-bacterial mats. [2-¹⁴C]acetate was predominantly incorporated into cell material, although some ¹⁴CH₄ and ¹⁴CO₂ was produced. Acetate incorporation was reduced by dark incubation in short-term experiments and severely depressed by a 2-day preincubation in darkness. Autoradiograms showed that acetate was incorporated by long filaments resembling phototrophic microorganisms of the mat communities. [³H]acetate was not converted to C³H₄ in samples from Octopus Spring collected at the optimum temperature for methanogenesis. NaH¹⁴CO₃ was readily converted to ¹⁴CH₄ at temperatures at which methanogenesis was active in both mats. Comparisons of the specific activities of methane and carbon dioxide suggested that of the methane produced, 80 ± 6% in Octopus Spring and 71 ± 21% in Wiegert Channel were derived from carbon dioxide. Addition of acetate to 1 mM did not reduce the relative importance of carbon dioxide as a methane precursor in samples from Octopus Spring. Experiments with pure cultures of Methanobacterium thermoautotrophicum suggested that the measured ratio of specific activities might underestimate the true contribution of carbon dioxide in methanogenesis.     

Hydrogen as a substrate for methanogenesis and sulphate reduction in anaerobic saltmarsh sediment

Hydrogen gas stimulated sulphate reduction in a saltmarsh sediment and the importance of H₂ transferred from organotrophic bacteria to the sulphate-reducers is discussed. -fluorolactate inhibited sulphate reduction whether lactate, ethanol or hydrogen was being used as growth substrate. When added to sediment -fluorolactate inhibited sulphate reduction with a consequent increase in methane production.Addition of H₂ stimulated methanogenesis in sediment and this stimulation was greater if CO₂ was also present. Hydrogen availability was the primary limitation of methanogenesis but the low concentration of dissolved CO₂ in seawater may limit methane production even if H₂ is available.The removal of inhibition of methanogenesis by the use of fluorolactate to suppress sulphate reduction or by the provision of hydrogen indicates competitive inhibition of methanogens by sulphate reducers utilizing transferred hydrogen.Abbreviations HSRB hydrogen utilizing sulphate reducing bacteria - HDO hydrogen donating organism     

Phylogenetic diversity and activity of anaerobic microorganisms of high-temperature horizons of the Dagang oil field (P. R. China)

The number of microorganisms of major metabolic groups and the rates of sulfate reduction and methanogenesis processes in the formation waters of the high-temperature horizons of Dagang oil field have been determined. Using cultural methods, it was shown that the microbial community contained aerobic bacteria oxidizing crude oil, anaerobic fermentative bacteria, sulfate-reducing bacteria, and methanogens. Using cultural methods, the possibility of methane production from a mixture of hydrogen and carbon dioxide (H₂ + CO₂) and from acetate was established, and this result was confirmed by radioisotope methods involving NaH¹⁴CO₃ and ¹⁴CH₃COONa. Analysis of enrichment cultures 16S rDNA of methanogens demonstrated that these microorganisms belong to Methanothermobacter sp. (M. thermautotrophicus), which consumes hydrogen and carbon dioxide as basic substrates. The genes of acetate-utilizing bacteria were not revealed. Phylotypes of the representatives of Thermococcus spp. were found among archaeal 16S rDNA. 16S rRNA genes of bacterial clones belong to the orders Thermoanaerobacteriales (Thermoanaerobacter, Thermovenabulum, Thermacetogenium, and Coprothermobacter spp.), Thermotogales, Nitrospirales (Thermodesulfovibrio sp.) and Planctomycetales. 16S rDNA of a bacterium capable of oxidizing acetate in the course of syntrophic growth with H₂-utilizing methanogens was found in high-temperature petroleum reservoirs for the first time. These results provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs, indicating that syntrophic processes play an important part in acetate degradation accompanied by methane production.     

Methanogenic fermentation of benzoate in an enrichment culture

Enrichment cultures inoculated with black mud fermented benzoate according to the stoichiometric equation: 4 C₆H₅CO₂H+18 H₂O 15 CH₄+13 CO₂.Trans-2-hydroxycyclohexanecarboxylate, 2-oxo-cyclohexanecarboxylate, pimelate, caproate, butyrate, acetate, and molecular hydrogen were shown to be regular components of the culture fluid occurring in low concentrations. Inhibition of methanogenesis by chloroform, 4-chlorobutyrate, or 2-bromooctanoate resulted in a cessation of the benzoate breakdown after all intermediates had accumulated. It is proposed that benzoate is fermented via a direct reductive pathway to butyrate, acetate, H₂, and CO₂, whereafter butyrate is converted to acetate and H₂, and the latter substrates are fermented to CH₄ and CO₂ by methane producers.     

Pyruvate oxidation by Methanococcus spp.

In the absence of H₂, Methanococcus spp. utilized pyruvate as an electron donor for methanogenesis. For Methanococcus voltae A3, Methanococcus maripaludis JJ1, and Methanococcus vannielii, typical rates of pyruvate-dependent methanogenesis were 3.4, 2.8, and 3.9 nmol min^-1 mg^-1 cell dry wt, respectively. These rates were 1–4% of the rates of H₂-dependent methanogenesis. For M. voltae, the concentration of pyruvate required for one-half the maximum rate of methanogenesis was 7 mM, and pyruvate-dependent methanogenesis was linear for 3 days. Radiolabeled acetate was formed from [3-¹⁴C]pyruvate, and the stoichiometry of pyruvate consumed per acetate produced was 1.12±0.27. The stoichiometry of pyruvate consumed per CH₄ produced was 3.64±0.34. These values are close to the expected values of 1 acetate and 4 CH₄. Although 10–30% of total cell carbon could be obtained from exogenous pyruvate during growth with H₂, pyruvate did not replace the nutritional requirement for acetate in Methanococcus voltae A3 or two acetate auxotrophs of Methanococcus maripaludis, JJ6 and JJ7. These results suggest that pyruvate was not oxidized in the presence of H₂. The inability to oxidize pyruvate during H₂-dependent methanogenesis would prevent a futile cycle of pyruvate oxidation and biosynthesis during autotrophic growth.     

The use of direct-fed microbials for mitigation of ruminant methane emissions: a review

Concerns about the environmental effect and the economic burden of methane (CH₄) emissions from ruminants are driving the search for ways to mitigate rumen methanogenesis. The use of direct-fed microbials (DFM) is one possible option to decrease CH₄ emission from ruminants. Direct-fed microbials are already used in ruminants mainly to increase productivity and to improve health, and are readily accepted by producers and consumers alike. However, studies on the use of DFM as rumen CH₄ mitigants are scarce. A few studies using Saccharomyces cerevisiae have shown a CH₄-decreasing effect but, to date, there has not been a systematic exploration of DFM as modulators of rumen methanogenesis. In this review, we explored biochemical pathways competing with methanogenesis that, potentially, could be modulated by the use of DFM. Pathways involving the redirection of H₂ away from methanogenesis and pathways producing less H₂ during feed fermentation are the preferred options. Propionate formation is an example of the latter option that in addition to decrease CH₄ formation increases the retention of energy from the diet. Homoacetogenesis is a pathway using H₂ to produce acetate, however up to now no acetogen has been shown to efficiently compete with methanogens in the rumen. Nitrate and sulphate reduction are pathways competing with methanogenesis, but the availability of these substances in the rumen is limited. Although there were studies using nitrate and sulphate as chemical additives, use of DFM for improving these processes and decrease the accumulation of toxic metabolites needs to be explored more. There are some other pathways such as methanotrophy and capnophily or modes of action such as inhibition of methanogens that theoretically could be provided by DFM and affect methanogenesis. We conclude that DFM is a promising alternative for rumen methane mitigation that should be further explored for their practical usage.     

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 μmol/liter) of dissolved methane were detected in the bottom waters. A methane concentration maximum occurred at 4 m above the sediment. The production of ¹⁴CH₄ from ¹⁴C-labeled HCOOH, HCO₃⁻, and CH₃OH and [2-¹⁴C]acetate demonstrated microbial methanogenesis in the water column of the lake. The maximum rate of methanogenesis calculated from reduction of H¹⁴CO₃⁻ 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 CH₄ and CO₂ 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 μg/ml) to water from 21.5 m stimulated methanogenesis from acetate, but inhibited CO₂ 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 μg/liter) completely inhibited methanogenesis and stimulated CO₂ formation.     

Methanogenic activity and diversity in the centre of the Amsterdam Mud Volcano, Eastern Mediterranean Sea

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes.