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.     

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.     

Methanogenic Pathway and Archaeal Community Structure in the Sediment of Eutrophic Lake Dagow: Effect of Temperature

Methanogenic degradation of organic matter is an important microbial process in lake sediments. Temperature may affect not only the rate but also the pathway of CH₄ production by changing the activity and the abundance of individual microorganisms. Therefore, we studied the function and structure of a methanogenic community in anoxic sediment of Lake Dagow, a eutrophic lake in north-eastern Germany. Incubation of sediment samples (in situ 7.5°C) at increasing temperatures (4, 10, 15, 25, 30°C) resulted in increasing production rates of CH₄ and CO₂ and in increasing steady-state concentrations of H₂. Thermodynamic conditions for H₂/CO₂ -dependent methanogenesis were only exergonic at 25 and 30°C. Inhibition of methanogenesis with chloroform resulted in the accumulation of methanogenic precursors, i.e., acetate, propionate, and isobutyrate. Mass balance calculations indicated that less CH₄ was formed via H₂ at 4°C than at 30°C. Conversion of ¹⁴CO₂ to ¹⁴CH₄ also showed that H₂/CO₂ -dependent methanogenesis contributed less to total CH₄ production at 4°C than at 30°C. [2–¹⁴ C]Acetate turnover rates at 4°C accounted for a higher percentage of total CH₄ production than at 30°C. Collectively, these results showed a higher contribution of H₂-dependent methanogenesis and a lower contribution of acetate-dependent methanogenesis at high versus low temperature. The archaeal community was characterized by cloning, sequencing, and phylogenetic analysis of the 16S rRNA genes retrieved from the sediment. Sequences were affiliated with Methanosaetaceae, Methanomicrobiaceae, and three deeply branching euryarchaeotal clusters, i.e., group III, Rice cluster V, and a novel euryarchaeotal cluster, the LDS cluster. Terminal restriction fragment length polymorphism (T-RFLP) analysis showed that 16S rRNA genes affiliated to Methanosaetaceae (20–30%), Methanomicrobiaceae (35–55%), and group III (10–25%) contributed most to the archaeal community. Incubation of the sediment at different temperatures (4–30°C) did not result in a systematic change of the archaeal community composition, indicating that change of temperature primarily affected the activity rather than the structure of the methanogenic community.     

Methane in maritime Antarctic freshwater lakes

Summary Methane was found to occur in all freshwater lakes, irrespective of trophic status, sampled during this preliminary investigation at Signy Island, South Orkney Islands, Antarctica. Methane accumulated in the water column of these lakes during the winter period when ice cover prevented wind-induced mixing. Maritime Antarctic lakes are usually subject to wind-induced complete mixing during the summer open-water period but two major exceptions to the rule were found during this study. Methanogenesis occurred in both littoral and profundal regions of oligotrophic Sombre Lake. The presence of a substantial algal mat stabilized the Eh status of underlying sediments at the littoral site. Methane production was confined to the sediments in both littoral and profundal sediments during the study period (December–March) but in winter probably migrated to the sediment surface at the profundal site. All Signy Island lakes sampled were sulphate-poor and addition of sulphate markedly inhibited methanogenesis. Radio-isotope studies indicated that the H₂/CO₂ pathway was probably the predominant route for methanogenesis in these sediments through the acetate pathway appeared equally important at the sediment surface. In the absence of sulphate, sulphate reducers probably acted as net hydrogen donors to the methanogens. The process rate was permanently limited by the consistent low temperature (annual range 1–3°C). Rates increased with increasing temperature over the range 4–32°C, but no evidence was found to suggest cold sensitivity or psychrophily. The optimum temperature for methanogenesis was in excess of 30°C, temperatures never experienced at Signy Island. Rates of methanogenesis during the study period (Dec–Mar) ranged from 0.29 to 0.45 mg of carbon m^-2 and on an annual basis methanogenesis was calculated equivalent to 13% of the organic carbon deposition rate.     

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.     

Methanogen community in Zoige wetland of Tibetan plateau and phenotypic characterization of a dominant uncultured methanogen cluster ZC-I

Zoige wetland of Tibetan plateau is characterized by being located at a low latitude (33°56'N, 102°52'E) region and under the annual temperature around 1°C. Previous studies indicated that Zoige wetland was one of the CH₄ emission centres in Qinghai-Tibetan plateau; in this study, the methanogen community in this low-latitude wetland was analysed based on the homology of 16S rRNA and mcrA genes retrieved from the soil. The results indicated that members of Methanosarcinales and Methanomicrobiales constituted the majority of methanogens, and a novel uncultured methanogen cluster, Zoige cluster I (ZC-I) affiliated to Methanosarcinales , could be dominant. Using quantitative polymerase chain reaction (qPCR) assay, ZC-I methanogens were estimated to be 10⁷ cells per gram of soil, accounting for about 30% of the total Archeae . By combining culturable enrichment with qPCR assay, the quantity of ZC-I methanogens in the methanogenic enrichment with acetate, H2/CO₂, methanol or trimethylamine was determined to increase to 10⁸ cells ml⁻¹, but not with formate, which indicated that ZC-I methanogens could use the four methanogenic substrates. The growth rates at 30°C and 15°C were not pronounced different, implying ZC-I to be the cold-adaptive methanogens. The broad substrate spectrum identified the ZC-I methanogens to be a member of Methanosarcinaceae , and could represent a novel sub-branch specifically inhabited in cold ecosystems. Fluorescence in situ hybridization (FISH) images also visualized ZC-I methanogens the sarcina-like aggregate of the spherical cells. The prevalence and flexibility in substrate utilization and growth temperature suggested ZC-I methanogens to be an important player in the methanogenesis of Zoige wetland.     

Effect of Temperature on Carbon and Electron Flow and on the Archaeal Community in Methanogenic Rice Field Soil

Temperature is an important factor controlling CH₄ production in anoxic rice soils. Soil slurries, prepared from Italian rice field soil, were incubated anaerobically in the dark at six temperatures of between 10 to 37°C or in a temperature gradient block covering the same temperature range at intervals of 1°C. Methane production reached quasi-steady state after 60 to 90 days. Steady-state CH₄ production rates increased with temperature, with an apparent activation energy of 61 kJ mol⁻¹. Steady-state partial pressures of the methanogenic precursor H₂ also increased with increasing temperature from <0.5 to 3.5 Pa, so that the Gibbs free energy change of H₂ plus CO₂-dependent methanogenesis was kept at −20 to −25 kJ mol of CH₄⁻¹ over the whole temperature range. Steady-state concentrations of the methanogenic precursor acetate, on the other hand, increased with decreasing temperature from <5 to 50 μM. Simultaneously, the relative contribution of H₂ as methanogenic precursor decreased, as determined by the conversion of radioactive bicarbonate to ¹⁴CH₄, so that the carbon and electron flow to CH₄ was increasingly dominated by acetate, indicating that psychrotolerant homoacetogenesis was important. The relative composition of the archaeal community was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of the 16S rRNA genes (16S rDNA). T-RFLP analysis differentiated the archaeal Methanobacteriaceae, Methanomicrobiaceae, Methanosaetaceae, Methanosarcinaceae, and Rice clusters I, III, IV, V, and VI, which were all present in the rice field soil incubated at different temperatures. The 16S rRNA genes of Rice cluster I and Methanosaetaceae were the most frequent methanogenic groups. The relative abundance of Rice cluster I decreased with temperature. The substrates used by this microbial cluster, and thus its function in the microbial community, are unknown. The relative abundance of acetoclastic methanogens, on the other hand, was consistent with their physiology and the acetate concentrations observed at the different temperatures, i.e., the high-acetate-requiring Methanosarcinaceae decreased and the more modest Methanosaetaceae increased with increasing temperature. Our results demonstrate that temperature not only affected the activity but also changed the structure and the function (carbon and electron flow) of a complex methanogenic system.     

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.     

Effects of Amendment with Ferrihydrite and Gypsum on the Structure and Activity of Methanogenic Populations in Rice Field Soil

Methane emission from paddy fields may be reduced by the addition of electron acceptors to stimulate microbial populations competitive to methanogens. We have studied the effects of ferrihydrite and gypsum (CaSO₄·2H₂O) amendment on methanogenesis and population dynamics of methanogens after flooding of Italian rice field soil slurries. Changes in methanogen community structure were followed by archaeal small subunit (SSU) ribosomal DNA (rDNA)- and rRNA-based terminal restriction fragment length polymorphism analysis and by quantitative SSU rRNA hybridization probing. Under ferrihydrite amendment, acetate was consumed efficiently (<60 μM) and a rapid but incomplete inhibition of methanogenesis occurred after 3 days. In contrast to unamended controls, the dynamics of Methanosarcina populations were largely suppressed as indicated by rDNA and rRNA analysis. However, the low acetate availability was still sufficient for activation of Methanosaeta spp., as indicated by a strong increase of SSU rRNA but not of relative rDNA frequencies. Unexpectedly, rRNA amounts of the novel rice cluster I (RC-I) methanogens increased significantly, while methanogenesis was low, which may be indicative of transient energy conservation coupled to Fe(III) reduction by these methanogens. Under gypsum addition, hydrogen was rapidly consumed to low levels (~0.4 Pa), indicating the presence of a competitive population of hydrogenotrophic sulfate-reducing bacteria (SRB). This was paralleled by a suppressed activity of the hydrogenotrophic RC-I methanogens as indicated by the lowest SSU rRNA quantities detected in all experiments. Full inhibition of methanogenesis only became apparent when acetate was depleted to nonpermissive thresholds (<5 μM) after 10 days. Apparently, a competitive, acetotrophic population of SRB was not present initially, and hence, acetotrophic methanosarcinal populations were less suppressed than under ferrihydrite amendment. In conclusion, although methane production was inhibited effectively under both mitigation regimens, different methanogenic populations were either suppressed or stimulated, which demonstrates that functionally similar disturbances of an ecosystem may result in distinct responses of the populations involved.     

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.     

Methanogenesis in McLean Bog,an Acidic Peat Bog in Upstate New York: Stimulation by H2/CO2 in the Presence of Rifampicin,or by Low Concentrations of Acetate

Acidic peat bog soils produce CH₄ and although molecular biological studies have demonstrated the presence of diverse methano-genic populations in them, few studies have sustained methanogenesis by adding the CH₄ precursors H₂/CO₂ or acetate, and few indigenous methanogens have been cultured. McLean Bog is a small (ca. 70 m across), acidic (pH 3.4–4.3) Sphagnum -dominated bog in upstate New York. Although addition of H₂/CO₂ or 10 mM acetate stimulated methanogenesis in soils from a nearby circumneutral-pH fen, neither of these substrates led to sustained methanogenesis in McLean Bog soil slurries. After a brief period of stimulation by H₂/CO₂, methanogenesis in McLean Bog soil declined, which could be attributed to buildup of large amounts of acetic acid produced from the H₂/CO₂ by acetogens. Addition of the antibiotic rifampicin inhibited acetogenesis (carried out by Bacteria) and allowed methanogenesis (carried out by Archaea) to continue. Using rifampicin, we were able to study effects of temperature, pH, and salts on methanogenesis from H₂/CO₂ in McLean Bog soil samples. The enriched H₂/CO₂-utilizing methanogens showed an optimum for activity near pH 5, and a temperature optimum near 35°C. Methanogenesis was not stimulated by addition of 10 mM acetate, but it was stimulated by 1 mM acetate, and multiple additions were consumed at increasing rates and nearly stoichiometrically converted to CH₄. In conclusion, we have found that both hydrogentrophic and aceticlastic methanogens are present in McLean Bog soils, and that methanogenic activity can be stimulated using H₂/CO₂ in the presence of rifampicin, or using low concentrations of acetate.     

Response of Methanogens in Arctic Sediments to Temperature and Methanogenic Substrate Availability

Although cold environments are major contributors to global biogeochemical cycles, comparatively little is known about their microbial community function, structure, and limits of activity. In this study a microcosm based approach was used to investigate the effects of temperature, and methanogenic substrate amendment, (acetate, methanol and H₂/CO₂) on methanogen activity and methanogen community structure in high Arctic wetlands (Solvatnet and Stuphallet, Svalbard). Methane production was not detected in Stuphallet sediment microcosms (over a 150 day period) and occurred within Solvatnet sediments microcosms (within 24 hours) at temperatures from 5 to 40°C, the maximum temperature being at far higher than in situ maximum temperatures (which range from air temperatures of -1.4 to 14.1°C during summer months). Distinct responses were observed in the Solvatnet methanogen community under different short term incubation conditions. Specifically, different communities were selected at higher and lower temperatures. At lower temperatures (5°C) addition of exogenous substrates (acetate, methanol or H₂/CO₂) had no stimulatory effect on the rate of methanogenesis or on methanogen community structure. The community in these incubations was dominated by members of the Methanoregulaceae/WCHA2-08 family-level group, which were most similar to the psychrotolerant hydrogenotrophic methanogen Methanosphaerula palustris strain E1-9c. In contrast, at higher temperatures, substrate amendment enhanced methane production in H₂/CO₂ amended microcosms, and played a clear role in structuring methanogen communities. Specifically, at 30°C members of the Methanoregulaceae/WCHA2-08 predominated following incubation with H₂/CO₂, and Methanosarcinaceaeand Methanosaetaceae were enriched in response to acetate addition. These results may indicate that in transiently cold environments, methanogen communities can rapidly respond to moderate short term increases in temperature, but not necessarily to the seasonal release of previously frozen organic carbon from thawing permafrost soils. However, as temperatures increase such inputs of carbon will likely have a greater influence on methane production and methanogen community structure. Understanding the action and limitations of anaerobic microorganisms within cold environments may provide information which can be used in defining region-specific differences in the microbial processes; which ultimately control methane flux to the atmosphere.     

Activity and Diversity of Methanogens in a Petroleum Hydrocarbon-Contaminated Aquifer

Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H₂ plus CO₂, or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day⁻¹ for methanol, 0.38 mM day⁻¹ for acetate, 0.90 mM day⁻¹ for H₂, and 1.85 mM day⁻¹ for formate. Substrate consumption and CH₄ production during all tests suggested that at least three different physiologic types of methanogens were present: H₂ plus CO₂ or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H₂ and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4′,6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO₂-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.     

Methanosarcinaceae and Acetate-Oxidizing Pathways Dominate in High-Rate Thermophilic Anaerobic Digestion of Waste-Activated Sludge

This study investigated the process of high-rate, high-temperature methanogenesis to enable very-high-volume loading during anaerobic digestion of waste-activated sludge. Reducing the hydraulic retention time (HRT) from 15 to 20 days in mesophilic digestion down to 3 days was achievable at a thermophilic temperature (55°C) with stable digester performance and methanogenic activity. A volatile solids (VS) destruction efficiency of 33 to 35% was achieved on waste-activated sludge, comparable to that obtained via mesophilic processes with low organic acid levels (<200 mg/liter chemical oxygen demand [COD]). Methane yield (VS basis) was 150 to 180 liters of CH₄/kg of VS_added. According to 16S rRNA pyrotag sequencing and fluorescence in situ hybridization (FISH), the methanogenic community was dominated by members of the Methanosarcinaceae, which have a high level of metabolic capability, including acetoclastic and hydrogenotrophic methanogenesis. Loss of function at an HRT of 2 days was accompanied by a loss of the methanogens, according to pyrotag sequencing. The two acetate conversion pathways, namely, acetoclastic methanogenesis and syntrophic acetate oxidation, were quantified by stable carbon isotope ratio mass spectrometry. The results showed that the majority of methane was generated by nonacetoclastic pathways, both in the reactors and in off-line batch tests, confirming that syntrophic acetate oxidation is a key pathway at elevated temperatures. The proportion of methane due to acetate cleavage increased later in the batch, and it is likely that stable oxidation in the continuous reactor was maintained by application of the consistently low retention time.     

Effects of amendment with ferrihydrite and gypsum on the structure and activity of methanogenic populations in rice field soil

Methane emission from paddy fields may be reduced by the addition of electron acceptors to stimulate microbial populations competitive to methanogens. We have studied the effects of ferrihydrite and gypsum (CaSO(4). 2H(2)O) amendment on methanogenesis and population dynamics of methanogens after flooding of Italian rice field soil slurries. Changes in methanogen community structure were followed by archaeal small subunit (SSU) ribosomal DNA (rDNA)- and rRNA-based terminal restriction fragment length polymorphism analysis and by quantitative SSU rRNA hybridization probing. Under ferrihydrite amendment, acetate was consumed efficiently (<60 microM) and a rapid but incomplete inhibition of methanogenesis occurred after 3 days. In contrast to unamended controls, the dynamics of Methanosarcina populations were largely suppressed as indicated by rDNA and rRNA analysis. However, the low acetate availability was still sufficient for activation of Methanosaeta spp., as indicated by a strong increase of SSU rRNA but not of relative rDNA frequencies. Unexpectedly, rRNA amounts of the novel rice cluster I (RC-I) methanogens increased significantly, while methanogenesis was low, which may be indicative of transient energy conservation coupled to Fe(III) reduction by these methanogens. Under gypsum addition, hydrogen was rapidly consumed to low levels ( approximately 0.4 Pa), indicating the presence of a competitive population of hydrogenotrophic sulfate-reducing bacteria (SRB). This was paralleled by a suppressed activity of the hydrogenotrophic RC-I methanogens as indicated by the lowest SSU rRNA quantities detected in all experiments. Full inhibition of methanogenesis only became apparent when acetate was depleted to nonpermissive thresholds (<5 microM) after 10 days. Apparently, a competitive, acetotrophic population of SRB was not present initially, and hence, acetotrophic methanosarcinal populations were less suppressed than under ferrihydrite amendment. In conclusion, although methane production was inhibited effectively under both mitigation regimens, different methanogenic populations were either suppressed or stimulated, which demonstrates that functionally similar disturbances of an ecosystem may result in distinct responses of the populations involved.     

Biogenic methane production in formation waters from a large gas field in the North Sea

Methanogenesis was investigated in formation waters from a North Sea oil rimmed gas accumulation containing biodegraded oil, which has not been subject to seawater injection. Activity and growth of hydrogenotrophic methanogens was measured but acetoclastic methanogenesis was not detected. Hydrogenotrophic methanogens showed activity between 40 and 80°C with a temperature optimum (ca. 70°C) consistent with in situ reservoir temperatures. They were also active over a broad salinity range, up to and consistent with the high salinity of the waters (90 g l⁻¹). These findings suggest the methanogens are indigenous to the reservoir. The conversion of H₂ and CO₂ to CH₄ in methanogenic enrichments was enhanced by the addition of inorganic nutrients and was correlated with cell growth. Addition of yeast extract also stimulated methanogenesis. Archaeal 16S rRNA gene sequences recovered from enrichment cultures were closely related to Methanothermobacter spp. which have been identified in other high-temperature petroleum reservoirs. It has recently been suggested that methanogenic oil degradation may be a major factor in the development of the world’s heavy oils and represent a significant and ongoing process in conventional deposits. Although an oil-degrading methanogenic consortium was not enriched from these samples the presence and activity of communities of fermentative bacteria and methanogenic archaea was demonstrated. Stimulation of methanogenesis by addition of nutrients suggests that in situ methanogenic biodegradation of oil could be harnessed to enhance recovery of stranded energy assets from such petroleum systems.     

Noteworthy Facts about a Methane-Producing Microbial Community Processing Acidic Effluent from Sugar Beet Molasses Fermentation

Anaerobic digestion is a complex process involving hydrolysis, acidogenesis, acetogenesis and methanogenesis. The separation of the hydrogen-yielding (dark fermentation) and methane-yielding steps under controlled conditions permits the production of hydrogen and methane from biomass. The characterization of microbial communities developed in bioreactors is crucial for the understanding and optimization of fermentation processes. Previously we developed an effective system for hydrogen production based on long-term continuous microbial cultures grown on sugar beet molasses. Here, the acidic effluent from molasses fermentation was used as the substrate for methanogenesis in an upflow anaerobic sludge blanket bioreactor. This study focused on the molecular analysis of the methane-yielding community processing the non-gaseous products of molasses fermentation. The substrate for methanogenesis produces conditions that favor the hydrogenotrophic pathway of methane synthesis. Methane production results from syntrophic metabolism whose key process is hydrogen transfer between bacteria and methanogenic Archaea. High-throughput 454 pyrosequencing of total DNA isolated from the methanogenic microbial community and bioinformatic sequence analysis revealed that the domain Bacteria was dominated by Firmicutes (mainly Clostridia), Bacteroidetes, δ- and γ-Proteobacteria, Cloacimonetes and Spirochaetes. In the domain Archaea, the order Methanomicrobiales was predominant, with Methanoculleus as the most abundant genus. The second and third most abundant members of the Archaeal community were representatives of the Methanomassiliicoccales and the Methanosarcinales. Analysis of the methanogenic sludge by scanning electron microscopy with Energy Dispersive X-ray Spectroscopy and X-ray diffraction showed that it was composed of small highly heterogeneous mineral-rich granules. Mineral components of methanogenic granules probably modulate syntrophic metabolism and methanogenic pathways. A rough functional analysis from shotgun data of the metagenome demonstrated that our knowledge of methanogenesis is poor and/or the enzymes responsible for methane production are highly effective, since despite reasonably good sequencing coverage, the details of the functional potential of the microbial community appeared to be incomplete.     

Characteristics and metabolic patterns of soil methanogenic archaea communities in the high‐latitude natural forested wetlands of China

Soil methanogenic microorganisms are one of the primary methane‐producing microbes in wetlands. However, we still poorly understand the community characteristic and metabolic patterns of these microorganisms according to vegetation type and seasonal changes. Therefore, to better elucidate the effects of the vegetation type and seasonal factors on the methanogenic community structure and metabolic patterns, we detected the characteristics of the soil methanogenic mcrA gene from three types of natural wetlands in different seasons in the Xiaoxing''an Mountain region, China. The results indicated that the distribution of Methanobacteriaceae (hydrogenotrophic methanogens) was higher in winter, while Methanosarcinaceae and Methanosaetaceae accounted for a higher proportion in summer. Hydrogenotrophic methanogenesis was the dominant trophic pattern in each wetland. The results of principal coordinate analysis and cluster analysis showed that the vegetation type considerably influenced the methanogenic community composition. The methanogenic community structure in the Betula platyphylla–Larix gmelinii wetland was relatively different from the structure of the other two wetland types. Indicator species analysis further demonstrated that the corresponding species of indicator operational taxonomic units from the Alnus sibirica wetland and the Betula ovalifolia wetland were similar. Network analysis showed that cooperative and competitive relationships exist both within and between the same or different trophic methanogens. The core methanogens with higher abundance in each wetland were conducive to the adaptation to environmental disturbances. This information is crucial for the assessment of metabolic patterns of soil methanogenic archaea and future fluxes in the wetlands of the Xiaoxing''an Mountain region given their vulnerability.