Functional-Structural Analysis of Nitrogen-Cycle Bacteria in a Hypersaline Mat from the Omani Desert

Anaerobic microbial activity in northern peat soils most often results in more carbon dioxide (CO ₂ ) production than methane (CH₄) production. This study examined why methanogenic conditions (i.e., equal molar amounts of CH₄ production and CO₂ production) prevail so infrequently. We used peat soils from two ombrotrophic bogs and from two rheotrophic fens. The former two represented a relatively dry bog hummock and a wet bog hollow, and the latter two represented a forested fen and a sedge-dominated fen. We quantified gas production rates in soil samples incubated in vitro with and without added metabolic substrates (glucose, ethanol, H₂/CO₂). None of the peat soils exhibited methanogenic conditions when incubated in vitro for a short time (< 5 days) and without added substrates. Incubating some samples > 50 days without added substrates led to methanogenic conditions in only one of four experiments. The anaerobic CO₂:CH₄ production ratio ranged from 5:1 to 40:1 in peat soil without additions and was larger in samples from the dry bog hummock and forested fen than the wet bog hollow and sedge fen. Adding ethanol or glucose separately to peat soils led to methanogenic conditions within 5 days after the addition by stimulating rates of CH₄ production, suggesting CH₄ production from both hydrogenotrophic and acetoclastic methanogenesis. Our results suggest that methanogenic conditions in peat soils rely on a constant supply of easily decomposable metabolic substrates. Sample handling and incubation procedures might obscure methanogenic conditions in peat soil incubated in vitro.     

Methane emissions from fen,bog and swamp peatlands in Quebec

A static chamber technique was used weekly from spring thaw to winter freezing to measure methane emissions from 10 sites representing subarctic fens and temperate swamps and bogs. Rates of < 200 mg CH₄ m^–2 d^–1 were recorded in subarctic fens: within-site emissions were primarily controlled by the evolution of the peat thermal regime, though significant releases during spring thaw were recorded at some sites. Between subarctic fens, topography and water table elevation were important controls on methane emissions, with the general sequence: pool = horizontal fen> string. Emission rates from the 2 swamp sites were lower (< 20 mg CH₄ m^–2 d^–1 ), except during the spring thaw and when the sites were saturated. The low water table ( < 80 cm depth) in abnormally dry years reduced emission rates; rates were also low from a swamp site which had been drained and cleared of vegetation for horticulture. Methane emission rates were also low (< 5 mg CH₄ m^–2 d^–1) from 2 ombrotrophic bog sites. Laboratory measurements of rates of methane production under anaerobic conditions and methane consumption under aerobic conditions revealed that production rates were generally highest in the surface layers (0 to 2.5 cm depth); production was high in the fens and very low in the bogs. The swamp samples were able to produce methane under anaerobic conditions, but were also able to consume methane under aerobic conditions. Annual methane emission rates are estimated to be 1 to 10 g CH₄ m^–2 from the fens, 1 to 4 g CH4 m^–2 from the swamps and <0.2 g CH₄ m^–2 from the bogs and drained swamp.     

Structure,function and resilience to desiccation of methanogenic microbial communities in temporarily inundated soils of the Amazon rainforest (Cunia Reserve,Rondonia)

The floodplain of the Amazon River is a large source for the greenhouse gas methane, but the soil microbial communities and processes involved are little known. We studied the structure and function of the methanogenic microbial communities in soils across different inundation regimes in the Cunia Reserve, encompassing nonflooded forest soil (dry forest), occasionally flooded Igapo soils (dry Igapo), long time flooded Igapo soils (wet Igapo) and sediments from Igarape streams (Igarape). We also investigated a Transect (four sites) from the water shoreline into the dry forest. The potential and resilience of the CH₄ production process were studied in the original soil samples upon anaerobic incubation and again after artificial desiccation and rewetting. Bacterial and archaeal 16S rRNA genes and methanogenic mcrA were always present in the soils, except in dry forest soils where mcrA increased only upon anaerobic incubation. NMDS analysis showed a clear effect of desiccation and rewetting treatments on both bacterial and archaeal communities. However, the effects of the different sites were less pronounced, with the exception of Igarape. After anaerobic incubation, methanogenic taxa became more abundant among the Archaea, while there was only little change among the Bacteria. Contribution of hydrogenotrophic methanogenesis was usually around 40%. After desiccation and rewetting, we found that Firmicutes, Methanocellales and Methanosarcinaceae became the dominant taxa, but rates and pathways of CH₄ production stayed similar. Such change was also observed in soils from the Transects. The results indicate that microbial community structures of Amazonian soils will in general be strongly affected by flooding and drainage events, while differences between specific field sites will be comparatively minor.     

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.     

Colonization of rice roots with methanogenic archaea controls photosynthesis‐derived methane emission

The methane emitted from rice fields originates to a large part (up to 60%) from plant photosynthesis and is formed on the rice roots by methanogenic archaea. To investigate to which extent root colonization controls methane (CH₄) emission, we pulse‐labeled rice microcosms with ¹³CO₂ to determine the rates of ¹³CH₄ emission exclusively derived from photosynthates. We also measured emission of total CH₄ (¹²⁺¹³CH₄), which was largely produced in the soil. The total abundances of archaea and methanogens on the roots and in the soil were analysed by quantitative polymerase chain reaction of the archaeal 16S rRNA gene and the mcrA gene coding for a subunit of the methyl coenzyme M reductase respectively. The composition of archaeal and methanogenic communities was determined with terminal restriction fragment length polymorphism (T‐RFLP). During the vegetative growth stages, emission rates of ¹³CH₄ linearly increased with the abundance of methanogenic archaea on the roots and then decreased during the last plant growth stage. Rates of ¹³CH₄ emission and the abundance of methanogenic archaea were lower when the rice was grown in quartz‐vermiculite with only 10% rice soil. Rates of total CH₄ emission were not systematically related to the abundance of methanogenic archaea in soil plus roots. The composition of the archaeal communities was similar under all conditions; however, the analysis of mcrA genes indicated that the methanogens differed between the soil and root. Our results support the hypothesis that rates of photosynthesis‐driven CH₄ emission are limited by the abundance of methanogens on the roots.     

Recovery of Methanogenesis Following Summer Drought in Soils from Two Cool Temperate Peatlands,New York State,USA

Following a summer drought, intact cores of peat soil from two cool temperate peatlands (a rain-fed bog and a groundwater-fed swamp) were exposed experimentally to three different water table levels. The goal was to examine recovery of anaerobic methanogenesis and to evaluate peat soil decomposition to methane (CH₄), carbon dioxide (CO₂), and dissolved organic carbon (DOC) upon rewetting. Methane emission from soils to the atmosphere was greatest (mean = 80 μmol m^?2 s^?1) when the entire peat core was rewetted quickly; emission was negligible at low water level and when peat cores were rewetted gradually. Rates of CO₂ emission (mean = 1.0 μmol m^?2 s^?1) were relatively insensitive to water level. Concentrations of CH₄ in soil air spaces suggest that onset of methanogenesis induces, but later represses, aerobic oxidation of CH₄ above the water table. Concentrations of CO₂ suggest production at the soil surface of swamp peat versus at greater depths in bog peat. Portions of peat soil incubated in vitro without oxygen (O₂) exhibited a lag before the onset of methanogenesis, and the lag time was less in peat from the cores rewetted quickly. The inhibition of methanogenesis by the selective inhibitor 2-bromoethanesulfonic acid (BES) decreased CO₂ production by 20 to 30% but resulted in an increase in concentrations of DOC by 2 to 5 times. The results show that methanogens in peat soils tolerate moderate drought, and recovery varies among different peat types. In peat soils, the inhibition of methanogenesis might enhance DOC availability.     

Methane emission from feather moss stands

Data from remote sensing and Eddy towers indicate that forests are not always net sinks for atmospheric CH₄. However, studies describing specific sources within forests and functional analysis of microorganisms on sites with CH₄ turnover are scarce. Feather moss stands were considered to be net sinks for carbon dioxide, but received little attention to their role in CH₄ cycling. Therefore, we investigated methanogenic rates and pathways together with the methanogenic microbial community composition in feather moss stands from temperate and boreal forests. Potential rates of CH₄ emission from intact moss stands (n = 60) under aerobic conditions ranged between 19 and 133 pmol CH₄ h^?1 gdw^?1. Temperature and water content positively influenced CH₄ emission. Methanogenic potentials determined under N₂ atmosphere in darkness ranged between 22 and 157 pmol CH₄ h^?1 gdw^?1. Methane production was strongly inhibited by bromoethane sulfonate or chloroform, showing that CH₄ was of microbial origin. The moss samples tested contained fluorescent microbial cells and between 10⁴ and 10⁵ copies per gram dry weight moss of the mcrA gene coding for a subunit of the methyl CoM reductase. Archaeal 16S rRNA and mcrA gene sequences in the moss stands were characteristic for the archaeal families Methanobacteriaceae and Methanosarcinaceae. The potential methanogenic rates were similar in incubations with and without methyl fluoride, indicating that the CH₄ was produced by the hydrogenotrophic rather than aceticlastic pathway. Consistently, the CH₄ produced was depleted in ¹³C in comparison with the moss biomass carbon and acetate accumulated to rather high concentrations (3–62 mM). The δ¹³C of acetate was similar to that of the moss biomass, indicating acetate production by fermentation. Our study showed that the feather moss stands contained active methanogenic microbial communities producing CH₄ by hydrogenotrophic methanogenesis and causing net emission of CH₄ under ambient conditions, albeit at low rates.     

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.     

Active Methanotrophs in Two Contrasting North American Peatland Ecosystems Revealed Using DNA-SIP

The active methanotroph community was investigated in two contrasting North American peatlands, a nutrient-rich sedge fen and nutrient-poor Sphagnum bog using in vitro incubations and ¹³C-DNA stable-isotope probing (SIP) to measure methane (CH₄) oxidation rates and label active microbes followed by fingerprinting and sequencing of bacterial and archaeal 16S rDNA and methane monooxygenase (pmoA and mmoX) genes. Rates of CH₄ oxidation were slightly, but significantly, faster in the bog and methanotrophs belonged to the class Alphaproteobacteria and were similar to other methanotrophs of the genera Methylocystis, Methylosinus, and Methylocapsa or Methylocella detected in, or isolated from, European bogs. The fen had a greater phylogenetic diversity of organisms that had assimilated ¹³C, including methanotrophs from both the Alpha- and Gammaproteobacteria classes and other potentially non-methanotrophic organisms that were similar to bacteria detected in a UK and Finnish fen. Based on similarities between bacteria in our sites and those in Europe, including Russia, we conclude that site physicochemical characteristics rather than biogeography controlled the phylogenetic diversity of active methanotrophs and that differences in phylogenetic diversity between the bog and fen did not relate to measured CH₄ oxidation rates. A single crenarchaeon in the bog site appeared to be assimilating ¹³C in 16S rDNA; however, its phylogenetic similarity to other CO₂-utilizing archaea probably indicates that this organism is not directly involved in CH₄ oxidation in peat.     

Modeled production,oxidation, and transport processes of wetland methane emissions in temperate,boreal, and Arctic regions

Wetlands are the largest natural source of methane (CH₄) to the atmosphere. The eddy covariance method provides robust measurements of net ecosystem exchange of CH₄, but interpreting its spatiotemporal variations is challenging due to the co-occurrence of CH₄ production, oxidation, and transport dynamics. Here, we estimate these three processes using a data-model fusion approach across 25 wetlands in temperate, boreal, and Arctic regions. Our data-constrained model—iPEACE—reasonably reproduced CH₄ emissions at 19 of the 25 sites with normalized root mean square error of 0.59, correlation coefficient of 0.82, and normalized standard deviation of 0.87. Among the three processes, CH₄ production appeared to be the most important process, followed by oxidation in explaining inter-site variations in CH₄ emissions. Based on a sensitivity analysis, CH₄ emissions were generally more sensitive to decreased water table than to increased gross primary productivity or soil temperature. For periods with leaf area index (LAI) of ≥20% of its annual peak, plant-mediated transport appeared to be the major pathway for CH₄ transport. Contributions from ebullition and diffusion were relatively high during low LAI (<20%) periods. The lag time between CH₄ production and CH₄ emissions tended to be short in fen sites (3 ± 2 days) and long in bog sites (13 ± 10 days). Based on a principal component analysis, we found that parameters for CH₄ production, plant-mediated transport, and diffusion through water explained 77% of the variance in the parameters across the 19 sites, highlighting the importance of these parameters for predicting wetland CH₄ emissions across biomes. These processes and associated parameters for CH₄ emissions among and within the wetlands provide useful insights for interpreting observed net CH₄ fluxes, estimating sensitivities to biophysical variables, and modeling global CH₄ fluxes.     

Climate change effects on carbon and nitrogen mineralization in peatlands through changes in soil quality

Climate change will directly affect carbon and nitrogen mineralization through changes in temperature and soil moisture, but it may also indirectly affect mineralization rates through changes in soil quality. We used an experimental mesocosm system to examine the effects of 6‐year manipulations of infrared loading (warming) and water‐table level on the potential anaerobic nitrogen and carbon (as carbon dioxide (CO₂) and methane (CH₄) production) mineralization potentials of bog and fen peat over 11 weeks under uniform anaerobic conditions. To investigate the response of the dominant methanogenic pathways, we also analyzed the stable isotope composition of CH₄ produced in the samples. Bog peat from the highest water‐table treatment produced more CO₂ than bog peat from drier mesocosms. Fen peat from the highest water‐table treatment produced the most CH₄. Cumulative nitrogen mineralization was lowest in bog peat from the warmest treatment and lowest in the fen peat from the highest water‐table treatment. As all samples were incubated under constant conditions, observed differences in mineralization patterns reflect changes in soil quality in response to climate treatments. The largest treatment effects on carbon mineralization as CO₂ occurred early in the incubations and were ameliorated over time, suggesting that the climate treatments changed the size and/or quality of a small labile carbon pool. CH₄ from the fen peat appeared to be predominately from the acetoclastic pathway, while in the bog peat a strong CH₄ oxidation signal was present despite the anaerobic conditions of our incubations. There was no evidence that changes in soil quality have lead to differences in the dominant methanogenic pathways in these systems. Overall, our results suggest that even relatively short‐term changes in climate can alter the quality of peat in bogs and fens, which could alter the response of peatland carbon and nitrogen mineralization to future climate change.     

Pathways for Methanogenesis and Diversity of Methanogenic Archaea in Three Boreal Peatland Ecosystems

The main objectives of this study were to uncover the pathways used for methanogenesis in three different boreal peatland ecosystems and to describe the methanogenic populations involved. The mesotrophic fen had the lowest proportion of CH₄ produced from H₂-CO₂. The oligotrophic fen was the most hydrogenotrophic, followed by the ombrotrophic bog. Each site was characterized by a specific group of methanogenic sequences belonging to Methanosaeta spp. (mesotrophic fen), rice cluster-I (oligotrophic fen), and fen cluster (ombrotrophic bog).     

Methane sources in arctic thermokarst lake sediments on the North Slope of Alaska

The permafrost on the North Slope of Alaska is densely populated by shallow lakes that result from thermokarst erosion. These lakes release methane (CH₄) derived from a combination of ancient thermogenic pools and contemporary biogenic production. Despite the potential importance of CH₄ as a greenhouse gas, the contribution of biogenic CH₄ production in arctic thermokarst lakes in Alaska is not currently well understood. To further advance our knowledge of CH₄ dynamics in these lakes, we focused our study on (i) the potential for microbial CH₄ production in lake sediments, (ii) the role of sediment geochemistry in controlling biogenic CH₄ production, and (iii) the temperature dependence of this process. Sediment cores were collected from one site in Siqlukaq Lake and two sites in Sukok Lake in late October to early November. Analyses of pore water geochemistry, sedimentary organic matter and lipid biomarkers, stable carbon isotopes, results from CH₄ production experiments, and copy number of a methanogenic pathway‐specific gene (mcrA) indicated the existence of different sources of CH₄ in each of the lakes chosen for the study. Analysis of this integrated data set revealed that there is biological CH₄ production in Siqlukaq at moderate levels, while the very low levels of CH₄ detected in Sukok had a mixed origin, with little to no biological CH₄ production. Furthermore, methanogenic archaea exhibited temperature‐dependent use of in situ substrates for methanogenesis, and the amount of CH₄ produced was directly related to the amount of labile organic matter in the sediments. This study constitutes an important first step in better understanding the actual contribution of biogenic CH₄ from thermokarst lakes on the coastal plain of Alaska to the current CH₄ budgets.     

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.     

Methanogen diversity and community composition in peatlands of the central to northern Appalachian Mountain region, North America

Methanogenic archaea are ubiquitous in peat soils; however, their diversity and distributions within and among peatland ecosystems are not well known. We used comprehensive clone libraries of 16S rRNA gene sequences to investigate spatial patterns in diversity (richness, evenness of taxa) and composition (taxonomic, phylogenetic) of the methanogenic community in six peatlands arrayed 775?km from eastern Ontario, Canada to West Virginia, USA. Five sites were Sphagnum (moss) and shrub dominated; one site was sedge dominated; and, potential rates of methane (CH₄) production ranged from 15 to 450?nmol/g?day. The gradient allowed us to examine influences of site conditions, site history, and climate on community composition. The region had representatives of methanogens from four taxonomic orders. We observed 29 operationally defined units (OTUs) based on >97% sequence identity. One OTU accounted for 43% of all clones, whereas 15 OTUs were rare with <1% of the total number of clones. The number of OTUs per site ranged from 4 to 21, and statistical analysis suggested diversity of 4–43 per site. Eighteen of the OTUs were endemic to one site; albeit, most endemics occurred in the sedge dominated site. One OTU was cosmopolitan, occurring in all six sites. We found a positive relationship between methanogen diversity and rates of CH₄ production per site (Pearson r?=?0.93). Turnover in community composition between sites was weakly related to geographic distance between sites, whereas variation in soil pH and annual temperature played larger roles. About 50% of the variation in community composition was unexplained by distance, pH, mean climate, and site age. We conclude that methanogen diversity in peatlands of the central Appalachian region is shaped by present-day environmental conditions, suggesting an influence of impending climatic and environmental changes.     

Oxygen effects on methane production and oxidation in humid tropical forest soils

We investigated the effects of oxygen (O₂) concentration on methane (CH₄) production and oxidation in two humid tropical forests that differ in long‐term, time‐averaged soil O₂ concentrations. We identified sources and sinks of CH₄ through the analysis of soil gas concentrations, surface emissions, and carbon isotope measurements. Isotope mass balance models were used to calculate the fraction of CH₄ oxidized in situ. Complementary laboratory experiments were conducted to determine the effects of O₂ concentration on gross and net rates of methanogenesis. Field and laboratory experiments indicated that high levels of CH₄ production occurred in soils that contained between 9±1.1% and 19±0.2% O₂. For example, we observed CH₄ concentrations in excess of 3% in soils with 9±1.1% O₂. CH₄ emissions from the lower O₂ sites were high (22–101 nmol CH₄ m^?2 s^?1), and were equal in magnitude to CH₄ emissions from natural wetlands. During peak periods of CH₄ efflux, carbon dioxide (CO₂) emissions became enriched in ¹³C because of high methanogenic activity. Gross CH₄ production was probably greater than flux measurements indicated, as isotope mass balance calculations suggested that 48–78% of the CH₄ produced was oxidized prior to atmospheric egress. O₂ availability influenced CH₄ oxidation more strongly than methanogenesis. Gross CH₄ production was relatively insensitive to O₂ concentrations in laboratory experiments. In contrast, methanotrophic bacteria oxidized a greater fraction of total CH₄ production with increasing O₂ concentration, shifting the δ¹³C composition of CH₄ to values that were more positive. Isotopic measurements suggested that CO₂ was an important source of carbon for methanogenesis in humid forests. The δ¹³C value of methanogenesis was between ?84‰ and ?98‰, which is well within the range of CH₄ produced from CO₂ reduction, and considerably more depleted in ¹³C than CH₄ formed from acetate.     

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.