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.     

Methanogenic food web in the gut contents of methane-emitting earthworm Eudrilus eugeniae from Brazil

The anoxic saccharide-rich conditions of the earthworm gut provide an ideal transient habitat for ingested microbes capable of anaerobiosis. It was recently discovered that the earthworm Eudrilus eugeniae from Brazil can emit methane (CH₄) and that ingested methanogens might be associated with this emission. The objective of this study was to resolve trophic interactions of bacteria and methanogens in the methanogenic food web in the gut contents of E. eugeniae. RNA-based stable isotope probing of bacterial 16S rRNA as well as mcrA and mrtA (the alpha subunit of methyl-CoM reductase and its isoenzyme, respectively) of methanogens was performed with [¹³C]-glucose as a model saccharide in the gut contents. Concomitant fermentations were augmented by the rapid consumption of glucose, yielding numerous products, including molecular hydrogen (H₂), carbon dioxide (CO₂), formate, acetate, ethanol, lactate, succinate and propionate. Aeromonadaceae-affiliated facultative aerobes, and obligate anaerobes affiliated to Lachnospiraceae, Veillonellaceae and Ruminococcaceae were associated with the diverse fermentations. Methanogenesis was ongoing during incubations, and ¹³C-labeling of CH₄ verified that supplemental [¹³C]-glucose derived carbon was dissimilated to CH₄. Hydrogenotrophic methanogens affiliated with Methanobacteriaceae and Methanoregulaceae were linked to methanogenesis, and acetogens related to Peptostreptoccocaceae were likewise found to be participants in the methanogenic food web. H₂ rather than acetate stimulated methanogenesis in the methanogenic gut content enrichments, and acetogens appeared to dissimilate supplemental H₂ to acetate in methanogenic enrichments. These findings provide insight on the processes and associated taxa potentially linked to methanogenesis and the turnover of organic carbon in the alimentary canal of methane-emitting E. eugeniae.     

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.     

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.     

Effect of cobalt on the anaerobic degradation of methanol

The effect of trace elements on the methanogenesis from methanol and acetate was studied utilizing granular sludge obtained from an anaerobic wastewater treatment plant. The methanogenic activity from methanol was dramatically stimulated by the addition of a cocktail of trace elements in the basal medium. When trace elements were supplied individually, cobalt greatly stimulated methanogenesis which equalled the stimulation observed with the complete trace element mixture. No remarkable influence of any trace element was observed when acetate was used as the substrate. Two Upflow Anaerobic Sludge Blanket (UASB) reactors were operated with and without supplementation of cobalt. Cobalt greatly stimulated both acetogenesis in the initial operational phase and later methanogenesis. The cobalt sufficient column provided almost 3 times the methane productivity compared to the cobalt deprived column. At an organic loading rate of 8 g COD/l·d, 87% of the COD was converted to methane in the cobalt sufficient column. Under low cobalt concentration, methanogens compete better for cobalt than acetogens.     

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.     

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 at low temperatures by microflora of tundra wetland soil

Active methanogenesis from organic matter contained in soil samples from tundra wetland occurred even at 6 °C. Methane was the only end product in balanced microbial community with H₂/CO₂ as a substrate, besides acetate was produced as an intermediate at temperatures below 10°C. The activity of different microbial groups of methanogenic community in the temperature range of 6–28 °C was investigated using 5% of tundra soil as inoculum. Anaerobic microflora of tundra wetland fermented different organic compounds with formation of hydrogen, volatile fatty acids (VFA) and alcohols. Methane was produced at the second step. Homoacetogenic and methanogenic bacteria competed for such substrates as hydrogen, formate, carbon monoxide and methanol. Acetogens out competed methanogens in an excess of substrate and low density of microbial population. Kinetic analysis of the results confirmed the prevalence of hydrogen acetogenesis on methanogenesis. Pure culture of acetogenic bacteria was isolated at 6 °C. Dilution of tundra soil and supply with the excess of substrate disbalanced the methanoigenic microbial community. It resulted in accumulation of acetate and other VFA. In balanced microbial community obviously autotrophic methanogens keep hydrogen concentration below a threshold for syntrophic degradation of VFA. Accumulation of acetate- and H₂/CO₂-utilising methanogens should be very important in methanogenic microbial community operating at low temperatures.     

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.     

Methane production in littoral sediment of Lake Constance

Maximum rates of CH₄ production in the littoral sediment were observed in 2–5 cm depth. The CH₄ production rates increased during the year from about 5 mmol m⁻²d⁻¹ in December to a maximum of about 95 mmol m⁻²d⁻¹ in September. CH₄ production rates showed a temperature optimum at 30°C and an apparent activation energy of 76 kJ mol⁻¹. A large part of the seasonality of CH₄ production could be ascribed to the change of the sediment temperature. Most of the produced CH₄ was lost by ebullition. Gas bubbles contained about 60–70% CH₄ with an average δ¹³C of −56.2% and δD of −354%, and 2% CO₂ with an average δ¹³C of −14.1% indicating that CH₄ was produced from methyl carbon, i.e. mainly using acetate as methanogenic substrate. This result was confirmed by inhibition of methanogenesis with chloroform which resulted in an accumulation rate of acetate equivalent to 81% of the rate of CH₄ production. Most probable numbers of methanogenic bacteria were in the order of 10⁴ bacteria g⁻¹d.w. sediment for acetate-, methanol- or formate-utilizing, and of 10⁵ for H₂-utilizing methanogens. The turnover times of acetate were in the order of 2.3–4.8 h which, with in situ acetate concentrations of about 25–50 μM, resulted in rates of acetate turnover which were comparable to the rates of CH₄ production. The respiratory index (RI) showed that [2−¹⁴C]acetate was mainly used by methanogenesis rather than by respiratory processes, although the zone of CH₄ production in the sediment overlapped with the zone of sulfate reduction.     

低温湿地甲烷古菌及其介导的甲烷产生途径

甲烷是重要的温室气体,低温湿地是大气甲烷的重要来源,因为湿地土壤中生活着大量的微生物包括甲烷古菌,它们将有机物降解转化为甲烷.本文总结了近年来低温湿地甲烷古菌群落组成、甲烷产生途径及其与环境的关系.研究显示,乙酸是低温湿地中主要的产甲烷物质,氢产甲烷过程主要发生在中温地区或酸性泥炭土中,而在盐碱水域中甲醇、甲胺是甲烷的重要底物.位于我国青藏高原的若尔盖湿地具有高海拔但低纬度的地理特征,我们的前期研究却显示甲醇在该湿地的甲烷排放中具有重要贡献.相应地,低温湿地中的甲烷古菌主要是利用甲基类化合物/乙酸的甲烷八叠球菌目和氢营养型的甲烷微球菌目.然而不同类型湿地甲烷排放途径及甲烷古菌的差异主要与环境的土壤类型、pH及植被类型相关,如刚毛荸荠生长的若尔盖湿地土壤中来源于甲醇的甲烷占全部甲烷的l7％;而木里苔草土壤中乙酸是产甲烷的主要前体物质.尽管已知冷适应的甲烷古菌在低温湿地的甲烷排放中发挥重要作用,但目前获得培养的嗜冷甲烷古菌却很少.冷响应的组学研究显示甲烷古菌的冷适应涉及到全局性生物学过程.     

Spatial succession and metabolic properties of functional microbial communities in an anaerobic baffled reactor

The spatial successions of bacterial and archaeal communities in anaerobic digestion were investigated in a glucose-degrading five-compartment anaerobic baffled reactor (ABR). The distributions of H₂-producing acetogens, H₂-utilizing acetogens and methanogens in different anaerobic-digestion stages were quantitatively analyzed using functional probes. The results show that the acidogenesis stage and acetogenesis stage were located in the first two compartments, while the methanogenesis were located in the last two compartments. In acidogenesis/acetogenesis stage of anaerobic digestion, H₂-producing acetogens (19.7%) and H₂-utilizing acetogens (8.3%) were the dominant bacterial community. While in methanogenesis stage, methanogens became the dominant (40.2%) with H₂-producing acetogens and H₂-utilizing acetogens only accounting for 6.6% and 4.8%, respectively. With the bacterial population decreasing from 7.2 ± 0.5 × 10¹² cells mL⁻¹ to 0.6 ± 0.3 × 10¹² cells mL⁻¹ along water flowing direction, their diversity increased from 2.79 to 299. The acidogenic bacteria, such as Lactococcus sp., Uncultured Firmicutes bacterium, and Uncultured Clostridium sp., etc., dominated in the acidogenesis/acetogenesis stage, while Uncultured Desulfobacterales bacterium became dominant in the methanogenesis stage. A two-stage anaerobic process may be suitable for easily degradable organic matters removal.     

Investigation of the trophic relations between anaerobic microorganisms from an underground gas repository during methanol utilization

Joint cultivation of the dominant strains of acetogenic, sulfate-reducing and methanogenic microorganisms isolated from water samples of the North Stavropol underground gas storage facility (UGSF) was carried out for revealing their probable trophic relationships. It was shown that acetogenic strains Eubacterium limosum AG12 and Sporomusa sphaeroides AG8-2 growing on methanol could form a considerable pool of hydrogen, which may support development of hydrogenotrophic cultures, the methanogen Methanobacterium formicicum MG134, or the sulfate reducer Desulfovibrio desulfuricans SR12. Growth of this sulfate-reducing strain was not stimulated under joint cultivation with Methanosarcina barkeri MGZ3 on methanol, probably due to its inability to take up low hydrogen concentrations observed during methanosarcina development. The results show that acetogens in the UGSF system are the most important consumers of methanol and hydrogen and after exhaustion of the latter and switching over to methanol utilization they can supply hydrogen to other microorganisms, including methanogens and sulfate reducers. The role of methanosarcina in the UGSF increases as the hydrogen and CO₂ reserves are exhausted, and methanogenesis on methanol becomes the main way of its destruction.     

Synergistic effects of natural and modified zeolites on the methanogenesis of acetate and methanol

The addition of Mg²⁺ modified zeolite at 0.1 g g^–1 of volatile suspended solids (VSS) increased the potential specific methanogenic activity (PSMA) and the apparent kinetic constant, k ₀, showing PSMA and k ₀ values 15 and 2 times higher than those observed for control reactors during the methanogenesis of acetate and methanol, respectively. In addition, the population of hydrolytic, acetogenic and methanogenic bacteria increased 100, 10 and 100 times compared to that observed in the controls. The anaerobic degradation of methanol and acetate occurred in one and two stages, respectively.     

Microbial composition and characterization of prevalent methanogens and acetogens isolated from syntrophic methanogenic granules

The microbial species composition of methanogenic granules developed on an acetate-propionate-butyrate mixture was characterized. The granules contained high numbers of adhesive methanogens (10¹²/g dry weight) and butyrate-, isobutyrate-, and propionate-degrading syntrophic acetogens (10¹¹/g dry weight), but low numbers of hydrolytic-fermentative bacteria (10⁹/g dry weight). Prevalent methanogens in the granules included: Methanobacterium formicicum strain T1N and RF, Methanosarcina mazei strain T18, Methanospirillum hungatei strain BD, and a non-filamentous, bamboo-shaped rod species, Methanothrix/Methanosaeta-like strain M7. Prevalent syntrophic acetogens included: a butyrate-degrading Syntrophospora bryantii-like strain BH, a butyrate-isobutyrate degrading non-spore-forming rod, strain IB, a propionate-degrading sporeforming oval-shaped species, strain PT, and a propionate-degrading none-spore-forming sulfate-reducing rod species, strain PW, which was able to grow syntrophically with an H₂-utilizing methanogen. Sulfate-reducing bacteria did not play a significant role in the metabolism of H₂, formate, acetate and butyrate but they were involved in propionate degradation.Correspondence to: M. K. Jain     

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.     

Suppression of methanogenesis by dissimilatory Fe(III)-reducing bacteria in tropical rain forest soils: implications for ecosystem methane flux

Tropical forests are an important source of atmospheric methane (CH₄), and recent work suggests that CH₄ fluxes from humid tropical environments are driven by variations in CH₄ production, rather than by bacterial CH₄ oxidation. Competition for acetate between methanogenic archaea and Fe(III)‐reducing bacteria is one of the principal controls on CH₄ flux in many Fe‐rich anoxic environments. Upland humid tropical forests are also abundant in Fe and are characterized by high organic matter inputs, steep soil oxygen (O₂) gradients, and fluctuating redox conditions, yielding concomitant methanogenesis and bacterial Fe(III) reduction. However, whether Fe(III)‐reducing bacteria coexist with methanogens or competitively suppress methanogenic acetate use in wet tropical soils is uncertain. To address this question, we conducted a process‐based laboratory experiment to determine if competition for acetate between methanogens and Fe(III)‐reducing bacteria influenced CH₄ production and C isotope composition in humid tropical forest soils. We collected soils from a poor to moderately drained upland rain forest and incubated them with combinations of ¹³C‐bicarbonate, ¹³C‐methyl labeled acetate (¹³CH₃COO^?), poorly crystalline Fe(III), or fluoroacetate. CH₄ production showed a greater proportional increase than Fe²⁺ production after competition for acetate was alleviated, suggesting that Fe(III)‐reducing bacteria were suppressing methanogenesis. Methanogenesis increased by approximately 67 times while Fe²⁺ production only doubled after the addition of ¹³CH₃COO^?. Large increases in both CH₄ and Fe²⁺ production also indicate that the two process were acetate limited, suggesting that acetate may be a key substrate for anoxic carbon (C) metabolism in humid tropical forest soils. C isotope analysis suggests that competition for acetate was not the only factor driving CH₄ production, as ¹³C partitioning did not vary significantly between ¹³CH₃COO^? and ¹³CH₃COO^?+Fe(III) treatments. This suggests that dissimilatory Fe(III)‐reduction suppressed both hydrogenotrophic and aceticlastic methanogenesis. These findings have implications for understanding the CH₄ biogeochemistry of highly weathered wet tropical soils, where CH₄ efflux is driven largely by CH₄ production.     

Quantitative Determination of H2-Utilizing Acetogenic and Sulfate-Reducing Bacteria and Methanogenic Archaea from Digestive Tract of Different Mammals

Total number of bacteria, cellulolytic bacteria, and H₂-utilizing microbial populations (methanogenic archaea, acetogenic and sulfate-reducing bacteria) were enumerated in fresh rumen samples from sheep, cattle, buffaloes, deer, llamas, and caecal samples from horses. Methanogens and sulfate reducers were found in all samples, whereas acetogens were not detected in some samples of each animal. Archaea methanogens were the largest H₂-utilizing populations in all animals, and a correlation was observed between the numbers of methanogens and those of cellulolytic microorganisms. Higher counts of acetogens were found in horses and llamas (1 × 10⁴ and 4 × 10⁴ cells ml⁻¹ respectively).     

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

The emission of methane (1.3 mmol of CH₄ m⁻² day⁻¹), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH₄ (0.4 to 1.7 μmol g [dry wt] of soil⁻¹ day⁻¹) under anoxic conditions. At in situ pH, supplemental H₂-CO₂, ethanol, and 1-propanol all increased CH₄ production rates while formate, acetate, propionate, and butyrate inhibited the production of CH₄; methanol had no effect. H₂-dependent acetogenesis occurred in H₂-CO₂-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H₂ that were subsequently consumed. The accumulation of H₂ was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H₂; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H₂-utilizing methanogens. A total of 10⁹ anaerobes and 10⁷ hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H₂-CO₂-supplemented, fatty acid-enriched defined medium. CH₄ production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH₄-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H₂ that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H₂ is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

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.