Methanosarcina spp. drive vinyl chloride dechlorination via interspecies hydrogen transfer

Two highly enriched cultures containing Dehalococcoides spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by Methanosaeta, while the other culture was dominated by Methanosarcina, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by Methanosaeta had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by Methanosarcina displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts. Methanosarcina-dominated cultures converted a higher percentage of [2-(14)C]acetate to (14)CO(2) when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H(2)) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H(2) levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H(2) levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H(2) as electron donor, in cultures both with and without BES. These findings all indicate that Methanosarcina (but not Methanosaeta), while cleaving acetate to methane, simultaneously oxidizes acetate to CO(2) plus H(2), driving hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides.     

Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces

Fecal suspensions from humans were incubated with 13CO2 and H2. The suspensions were from subjects who harbored 10(8) and 10(10) methanogens per g (dry weight) of feces, respectively, and from a subject who did not harbor methanogens. Quantitative nuclear magnetic resonance spectroscopy showed that acetate labeled in both the methyl and carboxyl groups was formed by suspensions from the subject without methanogens and the subject with the lower concentrations of methanogens. The amounts of labeled acetate formed were in agreement with the amounts expected based on measurements of H2 utilization. No labeled acetate was formed by suspensions from the subject with the higher concentrations of methanogens, and essentially all of the H2 used was accounted for by CH4 production. Suspensions from the subject with lower concentrations of methanogens produced both methane and acetate from H2 and CO2. The results indicate that reduction of CO2 to acetate may be a major pathway for microbial production of acetate in the human colon except when very high concentrations of methanogens (ca. 10(10) per g [dry weight] of feces) are present. Double-labeled acetate was also formed from H2 and 13CO2 by fecal suspensions from nonmethanogenic and moderately methanogenic rats.     

Acetate production from hydrogen and [13C]carbon dioxide by the microflora of human feces.

Fecal suspensions from humans were incubated with 13CO2 and H2. The suspensions were from subjects who harbored 10(8) and 10(10) methanogens per g (dry weight) of feces, respectively, and from a subject who did not harbor methanogens. Quantitative nuclear magnetic resonance spectroscopy showed that acetate labeled in both the methyl and carboxyl groups was formed by suspensions from the subject without methanogens and the subject with the lower concentrations of methanogens. The amounts of labeled acetate formed were in agreement with the amounts expected based on measurements of H2 utilization. No labeled acetate was formed by suspensions from the subject with the higher concentrations of methanogens, and essentially all of the H2 used was accounted for by CH4 production. Suspensions from the subject with lower concentrations of methanogens produced both methane and acetate from H2 and CO2. The results indicate that reduction of CO2 to acetate may be a major pathway for microbial production of acetate in the human colon except when very high concentrations of methanogens (ca. 10(10) per g [dry weight] of feces) are present. Double-labeled acetate was also formed from H2 and 13CO2 by fecal suspensions from nonmethanogenic and moderately methanogenic rats.     

Thermoanaerobacteriaceae oxidize acetate in methanogenic rice field soil at 50°C

Rice field soils contain a thermophilic microbial community. Incubation of Italian rice field soil at 50°C resulted in transient accumulation of acetate, but the microorganisms responsible for methane production from acetate are unknown. Without addition of exogenous acetate, the δ(13)C of CH(4) and CO(2) indicated that CH(4) was exclusively produced by hydrogenotrophic methanogenesis. When exogenous acetate was added, acetoclastic methanogenesis apparently also operated. Nevertheless, addition of [2-(13)C]acetate (99% (13)C) resulted in the production not only of (13)C-labelled CH(4) but also of CO(2), which contained up to 27% (13)C, demonstrating that the methyl group of acetate was also oxidized. Part of the (13)C-labelled acetate was also converted to propionate which contained up to 14% (13)C. The microorganisms capable of assimilating acetate at 50°C were targeted by stable isotope probing (SIP) of ribosomal RNA and rRNA genes using [U-(13)C] acetate. Using quantitative PCR, (13)C-labelled bacterial ribosomal RNA and DNA was detected after 21 and 32 days of incubation with [U-(13)C]acetate respectively. In the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 140, 120 and 171 bp length predominated. Cloning and sequencing of 16S rRNA showed that these T-RFs were affiliated with the bacterial genera Thermacetogenium and Symbiobacterium and with members of the Thermoanaerobacteriaceae. Similar experiments targeting archaeal RNA and DNA showed that Methanocellales were the dominant methanogens being consistent with the operation of syntrophic bacterial acetate oxidation coupled to hydrogenotrophic methanogenesis. After 17 days, however, Methanosarcinacea increasingly contributed to the synthesis of rRNA from [U-(13)C]acetate indicating that acetoclastic methanogens were also active in methanogenic Italian rice field soil under thermal conditions.     

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.     

Microbial Methane Formation from Hard Coal and Timber in an Abandoned Coal Mine

About 7% of the global annual methane emissions originate from coal mining. Also, mine gas has come into focus of the power industry and is being used increasingly for heat and power production. In many coal deposits worldwide, stable carbon and hydrogen isotopic signatures of methane indicate a mixed thermogenic and biogenic origin. In this study, we have measured in an abandoned coal mine methane fluxes and isotopic signatures of methane and carbon dioxide, and collected samples for microbiological and phylogenetic investigations. Mine timber and hard coal showed an in-situ production of methane with isotopic signatures similar to those of the methane in the mine atmosphere. Enrichment cultures amended with mine timber or hard coal as sole carbon sources formed methane over a period of nine months. Predominantly, acetoclastic methanogenesis was stimulated in enrichments containing acetate or hydrogen/carbon dioxide. Molecular techniques revealed that the archaeal community in enrichment cultures and unamended samples was dominated by members of the Methanosarcinales. The combined geochemical and microbiological investigations identify microbial methanogenesis as a recent source of methane in abandoned coal mines.     

Cultivation of methanogens from shallow marine sediments at Hydrate Ridge, Oregon

Little is known about the methanogenic degradation of acetate, the fate of molecular hydrogen and formate or the ability of methanogens to grow and produce methane in cold, anoxic marine sediments. The microbes that produce methane were examined in permanently cold, anoxic marine sediments at Hydrate Ridge (44 degrees 35' N, 125 degrees 10' W, depth 800 m). Sediment samples (15 to 35 cm deep) were collected from areas of active methane ebullition or areas where methane hydrates occurred. The samples were diluted into enrichment medium with formate, acetate or trimethylamine as catabolic substrate. After 2 years of incubation at 4 degrees C to 15 degrees C, enrichment cultures produced methane. PCR amplification and sequencing of the rRNA genes from the highest dilutions with growth suggested that each enrichment culture contained a single strain of methanogen. The level of sequence similarity (91 to 98%) to previously characterized prokaryotes suggested that these methanogens belonged to novel genera or species within the orders Methanomicrobiales and Methanosarcinales. Analysis of the 16S rRNA gene libraries from DNA extracted directly from the sediment samples revealed phylotypes that were either distantly related to cultivated methanogens or possible anaerobic methane oxidizers related to the ANME-1 and ANME-2 groups of the Archaea. However, no methanogenic sequences were detected, suggesting that methanogens represented only a small proportion of the archaeal community.     

Effects of Temperature on Methanogenesis in a Thermophilic (58 degrees C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO(2) in a thermophilic (58 degrees C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with C-labeled methane precursors (CH(3)COO or CO(2)). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60 degrees C and was completely inhibited at 65 degrees C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58 degrees C and did not grow or produce methane at 65 degrees C. An accidental shift of digestor temperature from 58 to 64 degrees C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from CH(3)COO was optimal at 65 degrees C and completely inhibited at 75 degrees C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70 degrees C. Methanogenesis from CO(2) in the sludge was optimal at 65 degrees C and still proceeded at 75 degrees C. A CO(2)-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75 degrees C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65 degrees C produced more methane than sludge incubated at 60 degrees C, and no acetate accumulated at 65 degrees C. Methanogenesis was severely inhibited in sludge incubated at 70 degrees C, but since neither acetate nor H(2) accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

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.     

Phylogenetic diversity and activity of anaerobic microorganisms of high-temperature horizons of the Dagang Oilfield (China)

The number of microorganisms of major metabolic groups and the rates of sulfate-reducing and methanogenic processes in the formation waters of the high-temperature horizons of Dagang oilfield 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 methanogenic bacteria. 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 radioassays involving NaH14CO3 and 14CH3COONa. Analysis of 16S rDNA of enrichment cultures of methanogens demonstrated that these microorganisms belong to Methanothermobacter sp. (M. thermoautotrophicus), which consumes hydrogen and carbon dioxide as basic substrates. The genes of acetate-utilizing bacteria were not identified. Phylotypes of the representatives of Thermococcus spp. were found among 16S rDNAs of archaea. 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 at 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.     

Use of benzoate as an electron acceptor by Syntrophus aciditrophicus grown in pure culture with crotonate

In methanogenic environments, the main fate of benzoate is its oxidization to acetate, H(2) and CO(2) by syntrophic associations of hydrogen-producing benzoate degraders and hydrogen-using methanogens. Here, we report the use of benzoate as an electron acceptor. Pure cultures of S. aciditrophicus simultaneously degraded crotonate and benzoate when both substrates were present. The growth rate was 0.007 h(-1) with crotonate and benzoate present compared with 0.025 h(-1) with crotonate alone. After 8 days of incubation, 4.12 +/- 0.50 mM of cyclohexane carboxylate and 8.40 +/- 0.61 mM of acetate were formed and 4.0 +/- 0.04 mM of benzoate and 4.8 +/- 0.5 mM of crotonate were consumed. The molar growth yield was 22.7 +/- 2.1 g (dry wt) of cells per mol of crotonate compared with about 14.0 +/- 0.1 g (dry wt) of cells per mol of crotonate when S. aciditrophicus was grown with crotonate alone. Cultures grown with [ring-(13)C]-benzoate and unlabelled crotonate initially formed [ring-(13)C]-labelled cyclohexane carboxylate. No (13)C-labelled acetate was detected. In addition to cyclohexane carboxylate, (13)C-labelled cyclohex-1-ene carboxylate was detected as an intermediate. Once almost all of the benzoate was gone, carbon isotopic analyses showed that cyclohexane carboxylate was formed from both labelled and non-labelled metabolites. Glutarate and pimelate were also detected at this time and carbon isotopic analyses showed that each was made from a mixture labelled and non-labelled metabolites. The increase in molar growth yield with crotonate and benzoate and the formation of [ring-(13)C]-cyclohexane carboxylate from [ring-(13)C]-benzoate in the presence of crotonate are consistent with benzoate serving as an electron acceptor.     

Thermophilic sulfate reduction and methanogenesis with methanol in a high rate anaerobic reactor

Sulfate reduction outcompeted methanogenesis at 65 degrees C and pH 7.5 in methanol and sulfate-fed expanded granular sludge bed reactors operated at hydraulic retention times (HRT) of 14 and 3.5 h, both under methanol-limiting and methanol-overloading conditions. After 100 and 50 days for the reactors operated at 14 and 3.5 h, respectively, sulfide production accounted for 80% of the methanol-COD consumed by the sludge. The specific methanogenic activity on methanol of the sludge from a reactor operated at HRTs of down to 3.5 h for a period of 4 months gradually decreased from 0. 83 gCOD. gVSS(-1). day(-1) at the start to a value of less than 0.05 gCOD. gVSS(-1). day(-1), showing that the relative number of methanogens decreased and eventually became very low. By contrast, the increase of the specific sulfidogenic activity of sludge from 0. 22 gCOD. gVSS(-1). day(-1) to a final value of 1.05 gCOD. gVSS(-1). day(-1) showed that sulfate reducing bacteria were enriched. Methanol degradation by a methanogenic culture obtained from a reactor by serial dilution of the sludge was inhibited in the presence of vancomycin, indicating that methanogenesis directly from methanol was not important. H(2)/CO(2) and formate, but not acetate, were degraded to methane in the presence of vancomycin. These results indicated that methanol degradation to methane occurs via the intermediates H(2)/CO(2) and formate. The high and low specific methanogenic activity of sludge on H(2)/CO(2) and formate, respectively, indicated that the former substrate probably acts as the main electron donor for the methanogens during methanol degradation. As sulfate reduction in the sludge was also strongly supported by hydrogen, competition between sulfate reducing bacteria and methanogens in the sludge seemed to be mainly for this substrate. Sulfate elimination rates of up to 15 gSO(4)(2-)/L per day were achieved in the reactors. Biomass retention limited the sulfate elimination rate.     

Source of carbon and hydrogen in methane produced from formate by Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus is able to produce methane either from H2-CO2 or from formate. The route of formate entry into the methanogenic pathway was investigated by using 2H2O or [13C]formate and analysis by mass spectrometry. When cells (H2-CO2 or formate grown) were transferred to formate medium in 95% 2H water, the proportion of 2H in methane was 95%. When cells (H2-CO2 or formate grown) were transferred to media containing [13C]formate in the presence of H2-CO2 or He-CO2, the ratio of 13CH4 to 12CH4 increased over time parallel to the ratio of 13CO2 to 12CO2. The cells catalyzed a significant exchange of label between [13C]formate and 13CO2.     

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.     

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.     

Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice field soil

Turnover of glucose and acetate in the presence of active reduction of nitrate, ferric iron and sulfate was investigated in anoxic rice field soil by using [U-(14)C]glucose and [2-(14)C]acetate. The turnover of glucose was not much affected by addition of ferrihydrite or sulfate, but was partially inhibited (60%) by addition of nitrate. Nitrate addition also strongly reduced acetate production from glucose while ferrihydrite and sulfate addition did not. These results demonstrate that ferric iron and sulfate reducers did not outcompete fermenting bacteria for glucose at endogenous concentrations. Nitrate reducers may have done so, but glucose fermentation may also have been inhibited by accumulation of toxic denitrification intermediates (nitrite, NO, N(2)O). Addition of nitrate resulted in complete inhibition of CH(4) production from [U-(14)C]glucose and [2-(14)C]acetate. However, addition of ferrihydrite or sulfate decreased the production of (14)CH(4) from [U-(14)C]glucose by only 70 and 65%, respectively. None of the electron acceptors significantly increased the production of (14)CO(2) from [U-(14)C]glucose, but all increased the production of (14)CO(2) from [2-(14)C]acetate. Uptake of acetate was faster in the presence of either nitrate, ferrihydrite or sulfate than in the unamended control. Addition of ferrihydrite and sulfate reduced (14)CH(4) production from [2-(14)C]acetate by 83 and 92%, respectively. Chloroform completely inhibited the methanogenic consumption of acetate. It also inhibited the oxidation of acetate, completely in the presence of sulfate, but not in the presence of nitrate or ferrihydrite. Our results show that, besides the possible toxic effect of products of nitrate reduction (NO, NO(2)(-) and N(2)O) on methanogens, nitrate reducers, ferric iron reducers and sulfate reducers were active enough to outcompete methanogens for acetate and channeling the flow of electrons away from CH(4) towards CO(2) production.     

Influence of temperature and high acetate concentrations on methanogenesis in lake sediment slurries

Methanogenesis from main methane precursors H(2)/CO(2) and acetate was investigated in a temperature range of 2-70 degrees C using sediments from Lake Baldegg, Switzerland. Psychrophilic, psychrotrophic, mesophilic, and thermophilic methanogenic microbial communities were enriched by incubations for 1-3 months of nonamended sediment slurries at 5, 15, 30, and 50 degrees C. Isotope experiments with slurries amended with (14)C-labeled bicarbonate and (14)C-2-acetate showed that in the psychrophilic community (enriched at 5 degrees C), about 95% of methane originated from acetate, in contrast to the thermophilic community (50 degrees C) where up to 98% of methane was formed from bicarbonate. In the mesophilic community (30 degrees C), acetate was the precursor of about 80% of the methane produced. When the hydrogen-carbon dioxide mixture (H(2)/CO(2)) was used as a substrate, it was directly converted to methane under thermophilic conditions (70 and 50 degrees C). Under mesophilic conditions (30 degrees C), both pathways, hydrogenotrophic and acetoclastic, were observed. At low temperatures (5 and 15 degrees C), H(2)/CO(2) was converted into methane by a two-step process; first acetate was formed, followed by methane production from acetate. When slurries were incubated at high partial pressures of H(2)/CO(2), the high concentrations of acetate produced of more than 20 mM inhibited acetoclastic methanogenesis at a temperature below 15 degrees C. However, slow adaptation of the psychrophilic microbial community to high acetate concentrations was observed.     

CO(2) Incorporation and 4-Hydroxy-2-Methylbenzoic Acid Formation during Anaerobic Metabolism of m-Cresol by a Methanogenic Consortium

The metabolism of m-cresol by methanogenic cultures enriched from domestic sewage sludge was investigated. In the initial studies, bromoethanesulfonic acid was used to inhibit methane production. This led to the accumulation of 4.0 +/- 0.8 mol of acetate per mol of m-cresol metabolized. These results suggested that CO(2) incorporation occurred because each molecule of m-cresol contained seven carbon atoms, whereas four molecules of acetate product contained a total of eight carbon atoms. To verify this, [C]bicarbonate was added to bromoethanesulfonic acid-inhibited cultures, and those cultures yielded [C]acetate. Of the label recovered as acetate, 89% was found in the carboxyl position. Similar cultures fed [methyl-C]m-cresol yielded methyl-labeled acetate. A C-labeled transient intermediate was detected in cultures given either m-cresol and [C]bicarbonate or bicarbonate and [methyl-C]m-cresol. The intermediate was identified as 4-hydroxy-2-methylbenzoic acid. In addition, another metabolite was detected and identified as 2-methylbenzoic acid. This compound appeared to be produced only sporadically, and it accumulated in the medium, suggesting that the dehydroxylation of 4-hydroxy-2-methylbenzoic acid led to an apparent dead-end product.     

Stable-isotope probing of microorganisms thriving at thermodynamic limits: syntrophic propionate oxidation in flooded soil

Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H(2)-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [(13)C]propionate (<10 mM) and the oxidation of approximately 30 micromol of (13)C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were (13)C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in "heavy" (13)C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured "rice cluster I" lineage had incorporated substantial amounts of (13)C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [(13)C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.     

Isolation of Methanobrevibacter smithii from human feces.

Fecal specimens from nine adults were examined for the presence of methanogenic bacteria. Enrichment cultures of five specimens produced methane in 5 days. Of these five specimens, three were tested and produced methane during a short-term incubation. Four specimens did not produce methane in either short-term incubation or in enrichment culture. Each methanogenic culture contained methanogens similar in morphology to organisms of the genus Methanobrevibacter and showed factor-420 fluorescence by fluorescence microscopy. Pure cultures were obtained from four of the five methanogenic enrichment cultures. Each isolate grew and formed methane from either H2-CO2 or formate, but growth obtained with formate was poor. None of the isolates used acetate, methanol, or trimethylamine. All isolates grew in the presence of bile salts. In immunological studies, each isolate was closely related to the type strain of Methanobrevibacter smithii, a finding consistent with the physiological and morphological similarities between the isolates and the type strain.