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.     

Methanol as the Primary Methanogenic and Acetogenic Precursor in the Cold Zoige Wetland at Tibetan Plateau

Previous studies suggested that methanol and acetate were the likely methanogenic precursors in the cold Zoige wetland. In this study, the contribution of the two substances to methanogenesis and the conversion in Zoige wetland were analyzed. It was determined that methanol supported the highest CH₄ formation rate in the enrichments of the soil grown with Eleocharis valleculosa, and even higher at 15°C than at 30°C; while hydrogenotrophic methanogenesis was higher at 30°C. Both methanol- and acetate-using methanogens were counted at the highest (10⁷ g⁻¹) in the soil, whereas methanol-using acetogens (10⁸ g⁻¹) were ten times more abundant than either methanol- or acetate-using methanogens. Both methanol and acetate were detected in the methanogenesis-inhibited soil samples, so that both could be the primary methanogenic precursors in E. valleculosa soil. However, the levels of methanol and acetate accumulated in 2-bromoethane-sulfonate (BES)- and CHCl₃-treated soils were in reverse, i.e., higher methanol in CHCl₃- and higher acetate in BES-treated soil, so that methanol-derived methanogenesis could be underestimated due to the consumption by acetogens. Analysis of the soil 16S rRNA genes revealed Acetobacterum bakii and Trichococcus pasteurii to be the dominant methanol-using acetogens in the soil, and a strain of T. pasteurii was isolated, which showed the high conversion of methanol to acetate at 15°C.     

Trophic link between syntrophic acetogens and homoacetogens during the anaerobic acidogenic fermentation of sewage sludge

Acetate production during anaerobic sludge treatment has significant economic and environmental benefits. In this study, trophic links between syntrophic acetogens and homoacetogens in the anaerobic acidogenic fermentation of sewage sludge were investigated using methanogenic inhibitor 2-bromoethanesulfonate (BES) to block the methanogenesis pathway and butyrate to enhance syntrophic acetogenesis. The Gibbs free energies (ΔG) of the butyrate-degrading and homoacetogenic processes were close to the thermodynamic threshold of the reaction activity (−15 kJ/mol). In addition, microbial quantification analysis revealed that the growth of syntrophic acetogenic bacteria and homoacetogens in the treatment incubations was higher than that of the control. The results indicated that hydrogen-producing butyrate degraders are stimulated with homoacetogens when methanogenesis was specifically inhibited.     

2-Bromoethanesulfonate: A selective agent for isolating resistantMethanosarcina mutants

Sodium 2-bromoethanesulfonate (BES), a structural analog of 2-mercaptoethanesulfonate (coenzyme M), inhibited methanogenesis and growth ofMethanosarcina strain 227 in the presence of H₂/CO₂, methanol, or acetate. A single exposure to 24 M BES was sufficient to produce cultures resistant to 240 M BES. Wild-type cultures inhibited by 200 M BES (or less) resumed growth and methane production when coenzyme M (coM) was added to the culture medium. Cultures incubated one week or longer with 200 M BES (or less) spontaneously resumed growth and methanogenesis in the presence of H₂/CO₂, methanol, or acetate without added coM. BES resistance was heritable and not the result of inactivation or decomposition of BES. BES resistance acquired on one methanogenic substrate was retained when cells were grown on a different methanogenic substrate. However, BES resistance did not confer multiple resistance to other halomethane compounds such as chloroform, 2-bromoethanol, 2-bromopropionic acid, and chloramphenicol. BES resistance varied in two other genera of methanogens tested. One strain ofMethanospirillum hungatei was very sensitive to BES, and no resistant mutants were demonstrated. One strain ofMethanobacterium formicicum, however, was resistant to 200 M BES without any known prior exposure to BES.     

Syntrophic interactions among anode respiring bacteria (ARB) and Non‐ARB in a biofilm anode: electron balances

We demonstrate that the coulombic efficiency (CE) of a microbial electrolytic cell (MEC) fueled with a fermentable substrate, ethanol, depended on the interactions among anode respiring bacteria (ARB) and other groups of micro‐organisms, particularly fermenters and methanogens. When we allowed methanogenesis, we obtained a CE of 60%, and 26% of the electrons were lost as methane. The only methanogenic genus detected by quantitative real‐time PCR was the hydrogenotrophic genus, Methanobacteriales, which presumably consumed all the hydrogen produced during ethanol fermentation (～30% of total electrons). We did not detect acetoclastic methanogenic genera, indicating that acetate‐oxidizing ARB out‐competed acetoclastic methanogens. Current production and methane formation increased in parallel, suggesting a syntrophic interaction between methanogens and acetate‐consuming ARB. When we inhibited methanogenesis with 50 mM 2‐bromoethane sulfonic acid (BES), the CE increased to 84%, and methane was not produced. With no methanogenesis, the electrons from hydrogen were converted to electrical current, either directly by the ARB or channeled to acetate through homo‐acetogenesis. This illustrates the key role of competition among the various H₂ scavengers and that, when the hydrogen‐consuming methanogens were present, they out‐competed the other groups. These findings also demonstrate the importance of a three‐way syntrophic relationship among fermenters, acetate‐consuming ARB, and a H₂ consumer during the utilization of a fermentable substrate. To obtain high coulombic efficiencies with fermentable substrates in a mixed population, methanogens must be suppressed to promote new interactions at the anode that ultimately channel the electrons from hydrogen to current. Biotechnol. Bioeng. 2009;103: 513–523. © 2009 Wiley Periodicals, Inc.     

Hydrogen metabolism and sulfate-dependent inhibition of methanogenesis in a eutrophic lake sediment (Lake Mendota)

Hydrogen metabolism was studied in anoxic sediments of the stratified Lake Mendota; using a method which allowed the measurement of in situ H₂ concentrations and the headspace-free analysis of turnover of dissolved H₂. Addition of sulfate resulted in partial but immediate inhibition of H₂-dependent methanogenesis. Sulfate addition did not result in an immediate decrease in the steady state concentration of dissolved H₂, nor did it significantly stimulate the rate constant of H₂ turnover. Sulfate-induced decrease in dissolved H₂ was only observed after prolonged incubation or when endogenous H₂ production was stimulated by added glucose. The turnover of the in situ H₂ accounted for only 14% of the H₂-dependent methanogenesis from bicarbonate. While rates of methanogenesis increased during the season, rates of H₂ turnover decreased, accounting for only 2% of the H₂-dependent methanogenesis at the end of summer stratification. These observations indicate that increasing proportions of CH₄ were formed from H₂ being directly transferred in syntrophic methanogenic associations. The rapid inhibition of H₂-dependent methanogenesis by exogenous sulfate may be explained at least partially by assuming methanogenic associations in which syntrophic sulfate reducers change their metabolism from fermentative H₂ production to sulfate reduction.     

Methanogenesis and methanogenic pathways in a peat from subarctic permafrost

Few studies have dealt so far with methanogenic pathways and populations in subarctic and arctic soils. We studied the effects of temperature on rates and pathways of CH4 production and on the relative abundance and structure of the archaeal community in a mildly acidic peat from a permafrost region in Siberia (67 degrees N). We monitored the production of CH4 and CO2 over time and measured the consumption of Fe(II), ethanol and volatile fatty acids. All experiments were performed with and without specific inhibitors [2-bromoethanesulfonate (BES) for methanogenesis and CH3F for acetoclastic methanogenesis]. The optimum temperature for methanogenesis was between 26 degrees C and 28 degrees C [4.3 micromol CH4 (g dry weight)(-1) day(-1)], but the activity was high even at 4 degrees C [0.75 micromol CH4 (g dry weight)(-1) day(-1)], constituting 17% of that at 27 degrees C. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Acetoclastic methanogenesis accounted for about 70% of the total methanogenesis. Most 16S rRNA gene sequences clustered with Methanosarcinales, correlating with the prevalence of acetoclastic methanogenesis. In addition, sequences clustering with Methanobacteriales were recovered. Fe reduction occurred in parallel to methanogenesis. At lower and higher temperatures Fe reduction was not affected by BES. Because butyrate was consumed during methanogenesis and accumulated when methanogenesis was inhibited (BES and CH3F), it is proposed to serve as methanogenic precursor, providing acetate and H2 by syntrophic oxidation. In addition, ethanol and caproate occurred as intermediates. Because of thermodynamic constraints, homoacetogenesis could not compete with hydrogenotrophic methanogenesis.     

Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers

We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate—one where methanogenesis was allowed and another in which it was completely inhibited with 2‐bromoethane sulfonate. We observed a three‐way syntrophy among ethanol fermenters, acetate‐oxidizing anode‐respiring bacteria (ARB), and a H₂ scavenger. When methanogenesis was allowed, H₂‐oxidizing methanogens were the H₂ scavengers, but when methanogenesis was inhibited, homo‐acetogens became a channel for electron flow from H₂ to current through acetate. We established the presence of homo‐acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo‐acetogens. Both methods documented the presence of the homo‐acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol‐fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H₂‐scavenging syntrophic partners that were either H₂‐oxidizing methanogens or homo‐acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron‐flow distribution and H₂‐scavenging mechanism. Biotechnol. Bioeng. 2010;105: 69–78. © 2009 Wiley Periodicals, Inc.     

Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 μmol CH₄ g⁻¹ oil d⁻¹, orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC–MS and FTICR–MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.     

Oxidation of glucose by syntrophic association between <Emphasis Type="Italic">Geobacter</Emphasis> and hydrogenotrophic methanogens in microbial fuel cell

Objective

To investigate a syntrophic interaction between Geobacter sulfurreducens and hydrogenotrophic methanogens in sludge-inoculated microbial fuel cell (MFC) systems running on glucose with an improved electron recovery at the anode.

Results

The presence of archaea in MFC reduces Coulombic efficiency (CE) due to their electron scavenging capability but, here, we demonstrate that a syntrophic interaction can occur between G. sulfurreducens and hydrogenotrophic methanogens via interspecies H₂ transfer with improvement in CE and power density. The addition of the methanogenesis inhibitor, 2-bromoethanesulfonate (BES), resulted in the reduction in power density from 5.29 to 2 W/m³, and then gradually increased to the peak value of 5.5 W/m³ when BES addition was stopped.

Conclusion

Reduction of H₂ partial pressure by archaea is an efficient approach in improving power output in a glucose-fed MFC system using Geobacter sp. as an inoculum.

     

Analysis of alkane-dependent methanogenic community derived from production water of a high-temperature petroleum reservoir

Microbial assemblage in an n-alkanes-dependent thermophilic methanogenic enrichment cultures derived from production waters of a high-temperature petroleum reservoir was investigated in this study. Substantially higher amounts of methane were generated from the enrichment cultures incubated at 55 °C for 528 days with a mixture of long-chain n-alkanes (C₁₅–C₂₀). Stoichiometric estimation showed that alkanes-dependent methanogenesis accounted for about 19.8% of the total amount of methane expected. Hydrogen was occasionally detected together with methane in the gas phase of the cultures. Chemical analysis of the liquid cultures resulted only in low concentrations of acetate and formate. Phylogenetic analysis of the enrichment revealed the presence of several bacterial taxa related to Firmicutes, Thermodesulfobiaceae, Thermotogaceae, Nitrospiraceae, Dictyoglomaceae, Candidate division OP8 and others without close cultured representatives, and Archaea predominantly related to uncultured members in the order Archaeoglobales and CO₂-reducing methanogens. Screening of genomic DNA retrieved from the alkanes-amended enrichment cultures also suggested the presence of new alkylsuccinate synthase alpha-subunit (assA) homologues. These findings suggest the presence of poorly characterized (putative) anaerobic n-alkanes degraders in the thermophilic methanogenic enrichment cultures. Our results indicate that methanogenesis of alkanes under thermophilic condition is likely to proceed via syntrophic acetate and/or formate oxidation linked with hydrogenotrophic methanogenesis.     

Assessment of Reductive Acetogenesis with Indigenous Ruminal Bacterium Populations and Acetitomaculum ruminis

The objective of this study was to evaluate the role of reductive acetogenesis as an alternative H₂ disposal mechanism in the rumen. H₂/CO₂-supported acetogenic ruminal bacteria were enumerated by using a selective inhibitor of methanogenesis, 2-bromoethanesulfonic acid (BES). Acetogenic bacteria ranged in density from 2.5 × 10⁵ cells/ml in beef cows fed a high-forage diet to 75 cells/ml in finishing steers fed a high-grain diet. Negligible endogenous acetogenic activity was demonstrated in incubations containing ruminal contents, NaH¹³CO₃, and 100% H₂ gas phase since [U-¹³C]acetate, as measured by mass spectroscopy, did not accumulate. Enhancement of acetogenesis was observed in these incubations when methanogenesis was inhibited by BES and/or by the addition of an axenic culture of the rumen acetogen Acetitomaculum ruminis 190A4 (10⁷ CFU/ml). To assess the relative importance of population density and/or H₂ concentration for reductive acetogenesis in ruminal contents, incubations as described above were performed under a 100% N₂ gas phase. Both selective inhibition of methanogenesis and A. ruminis 190A4 fortification (>10⁵ CFU/ml) were necessary for the detection of reductive acetogenesis under H₂-limiting conditions. Under these conditions, H₂ accumulated to 4,800 ppm. In contrast, H₂ accumulated to 400 ppm in incubations with active methanogenesis (without BES). These H₂ concentrations correlated well with the pure culture H₂ threshold concentrations determined for A. ruminis 190A4 (3,830 ppm) and the ruminal methanogen 10-16B (126 ppm). The data demonstrate that ruminal methanogenic bacteria limited reductive acetogenesis by lowering the H₂ partial pressure below the level necessary for H₂ utilization by A. ruminis 190A4.     

Predominance of Methanolobus spp. and Methanoculleus spp. in the Archaeal Communities of Saline Gas Field Formation Fluids

The microbial communities in sulfate-rich, saline formation fluids of a natural gas reservoir in Lower Saxony, Germany were investigated to enhance the knowledge about microbial communities in potential carbon dioxide sequestration sites. This investigation of the initial state of the deep subsurface microbiota is necessary to predict their influence on the long-term stability and storage capacity of such sites. While the bacterial 16S rDNA gene library was comprised of sequences affiliating with the Firmicutes, the Alphaproteobacteria, the Gammaproteobacteria and the Thermotogales, the archaeal 16S rDNA libraries were simply dominated by two phylotypes related to the genera Methanolobus and Methanoculleus. The monitoring of the archaeal communities in different formation fluid samples by T-RFLP and Real-Time-PCR indicated that these two methanogenic genera dominated at all, whereas the proportion of the two groups varied. Thus, methylotrophic and autotrophic methanogenesis seems to be of importance in the reservoir fluids, dependent on the provided reduction equivalents and substrates and it also may influence the fate of CO₂ in the subsurface.     

Functional and structural response of the methanogenic microbial community in rice field soil to temperature change

The microbial community in anoxic rice field soil produces CH₄ over a wide temperature range up to 55°C. However, at temperatures higher than about 40°C, the methanogenic path changes from CH₄ production by hydrogenotrophic plus acetoclastic methanogenesis to exclusively hydrogenotrophic methanogenesis and simultaneously, the methanogenic community consisting of Methanosarcinaceae, Methanoseataceae, Methanomicrobiales, Methanobacteriales and Rice Cluster I (RC‐1) changes to almost complete dominance of RC‐1. We studied changes in structure and function of the methanogenic community with temperature to see whether microbial members of the community were lost or their function impaired by exposure to high temperature. We characterized the function of the community by the path of CH₄ production measuring δ¹³C in CH₄ and CO₂ and calculating the apparent fractionation factor (α_app) and the structure of the community by analysis of the terminal restriction fragment length polymorphism (T‐RFLP) of the microbial 16S rRNA genes. Shift of the temperature from 45°C to 35°C resulted in a corresponding shift of function and structure, especially when some 35°C soil was added to the 45°C soil. The bacterial community (T‐RFLP patterns), which was much more diverse than the archaeal community, changed in a similar manner upon temperature shift. Incubation of a mixture of 35°C and 50°C pre‐incubated methanogenic rice field soil at different temperatures resulted in functionally and structurally well‐defined communities. Although function changed from a mixture of acetoclastic and hydrogenotrophic methanogenesis to exclusively hydrogenotrophic methanogenesis over a rather narrow temperature range of 42–46°C, each of these temperatures also resulted in only one characteristic function and structure. Our study showed that temperature conditions defined structure and function of the methanogenic microbial community.     

The pterin lumazine inhibits growth of methanogens and methane formation

The pterin compound lumazine [2, 4-(1H, 3H)-pteridinedione] inhibited the growth of several methanogenic archaea completely at a concentration of ≤ 0.6 mM and was bacteriocidal for Methanobacterium thermoautotrophicum strain Marburg. In contrast, growth of two non-methanogenic archaea, several eubacteria, and one eukaryote was not strongly affected at much higher concentrations. In washed-cell suspensions, methanogenesis from H₂ and CO₂ by Mb. thermoautotrophicum or from H₂ and methanol by Methanosarcina barkeri was inhibited by addition of lumazine. In cell-free extracts of Mb. thermoautotrophicum, H₂-driven methane production from CO₂ or CH₃-S-CoM was completely inhibited by 0.6 mM lumazine. The results suggest that the compound may be useful in probing the methanogenesis pathway or in selecting against methanogens. Received: 30 January 1996 / Accepted 15 May 1996     

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.     

Determination of the G + C content of twoSyntrophus buswellii strains by ultracentrifugation techniques

An anaerobic syntrophic bacterial culture degrading benzoate was isolated from a river sediment. The syntrophic organism was grown in coculture in the presence of a hydrogenotrophic strain,Desulfovibrio fructosovorans orMethanospirillum hungatei. The G+C content of the syntrophic benzoate degrader determined by density gradient ultracentrifugation was similar to that ofSyntrophus buswellii (54.3%). A method ensuring the G+C% determination of syntrophic bacteria is presented.     

Effect of Temperature on Anaerobic Ethanol Oxidation and Methanogenesis in Acidic Peat from a Northern Wetland

The effects of temperature on rates and pathways of CH₄ production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68°N). We monitored the production of CH₄ and CO₂ over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH₃F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25°C (2.3 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹), but the activity was relatively high even at 4°C (0.25 μmol CH₄ · g [dry weight]⁻¹ · day⁻¹). The theoretical lower limit for methanogenesis was calculated to be at −5°C. The optimum temperature for growth as revealed by real-time PCR was 25°C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order Methanobacteriales, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H₂ sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.     

Archaeal Community Structure and Pathway of Methane Formation on Rice Roots

The community structure of methanogenic Archaea on anoxically incubated rice roots was investigated by amplification, sequencing, and phylogenetic analysis of 16S rRNA and methyl-coenzyme M reductase (mcrA) genes. Both genes demonstrated the presence of Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae, Methanosaetaceae, and Rice cluster I, an uncultured methanogenic lineage. The pathway of CH₄ formation was determined from the ¹³C-isotopic signatures of the produced CH₄, CO₂ and acetate. Conditions and duration of incubation clearly affected the methanogenic community structure and the pathway of CH₄ formation. Methane was initially produced from reduction of CO₂ exclusively, resulting in accumulation of millimolar concentrations of acetate. Simultaneously, the relative abundance of the acetoclastic methanogens (Methanosarcinaceae, Methanosaetaceae), as determined by T-RFLP analysis of 16S rRNA genes, was low during the initial phase of CH₄ production. Later on, however, acetate was converted to CH₄ so that about 40% of the produced CH₄ originated from acetate. Most striking was the observed relative increase of a population of Methanosarcina spp. (but not of Methanosaeta spp.) briefly before acetate concentrations started to decrease. Both acetoclastic methanogenesis and Methanosarcina populations were suppressed by high phosphate concentrations, as observed under application of different buffer systems. Our results demonstrate the parallel change of microbial community structure and function in a complex environment, i.e., the increase of acetoclastic Methanosarcina spp. when high acetate concentrations become available.