BinaRena: a dedicated interactive platform for human-guided exploration and binning of metagenomes.

BACKGROUND: Exploring metagenomic contigs and "binning" them into metagenome-assembled genomes (MAGs) are essential for the delineation of functional and evolutionary guilds within microbial communities. Despite the advances in automated binning algorithms, their capabilities in recovering MAGs with accuracy and biological relevance are so far limited. Researchers often find that human involvement is necessary to achieve representative binning results. This manual process however is expertise demanding and labor intensive, and it deserves to be supported by software infrastructure. RESULTS: We present BinaRena, a comprehensive and versatile graphic interface dedicated to aiding human operators to explore metagenome assemblies via customizable visualization and to associate contigs with bins. Contigs are rendered as an interactive scatter plot based on various data types, including sequence metrics, coverage profiles, taxonomic assignments, and functional annotations. Various contig-level operations are permitted, such as selection, masking, highlighting, focusing, and searching. Binning plans can be conveniently edited, inspected, and compared visually or using metrics including silhouette coefficient and adjusted Rand index. Completeness and contamination of user-selected contigs can be calculated in real time. In demonstration of BinaRena's usability, we show that it facilitated biological pattern discovery, hypothesis generation, and bin refinement in a complex tropical peatland metagenome. It enabled isolation of pathogenic genomes within closely related populations from the gut microbiota of diarrheal human subjects. It significantly improved overall binning quality after curating results of automated binners using a simulated marine dataset. CONCLUSIONS: BinaRena is an installation-free, dependency-free, client-end web application that operates directly in any modern web browser, facilitating ease of deployment and accessibility for researchers of all skill levels. The program is hosted at https://github.com/qiyunlab/binarena , together with documentation, tutorials, example data, and a live demo. It effectively supports human researchers in intuitive interpretation and fine tuning of metagenomic data. Video Abstract.

Genes and genome-resolved metagenomics reveal the microbial functional make up of Amazon peatlands under geochemical gradients.

The Pastaza-Marañón Foreland Basin (PMFB) holds the most extensive tropical peatland area in South America. PMFB peatlands store ~7.07 Gt of organic carbon interacting with multiple microbial heterotrophic, methanogenic, and other aerobic/anaerobic respirations. Little is understood about the contribution of distinct microbial community members inhabiting tropical peatlands. Here, we studied the metagenomes of three geochemically distinct peatlands spanning minerotrophic, mixed, and ombrotrophic conditions. Using gene- and genome-centric approaches, we evaluate the functional potential of the underlying microbial communities. Abundance analyses show significant differences in C, N, P, and S acquisition genes. Furthermore, community interactions mediated by toxin-antitoxin and CRISPR-Cas systems were enriched in oligotrophic soils, suggesting that non-metabolic interactions may exert additional controls in low-nutrient environments. Additionally, we reconstructed 519 metagenome-assembled genomes spanning 28 phyla. Our analyses detail key differences across the geochemical gradient in the predicted microbial populations involved in degradation of organic matter, and the cycling of N and S. Notably, we observed differences in the nitric oxide (NO) reduction strategies between sites with high and low N2 O fluxes and found phyla putatively capable of both NO and sulfate reduction. Our findings detail how gene abundances and microbial populations are influenced by geochemical differences in tropical peatlands.

Coupled abiotic-biotic cycling of nitrous oxide in tropical peatlands.

Atmospheric nitrous oxide (N2O) is a potent greenhouse gas thought to be mainly derived from microbial metabolism as part of the denitrification pathway. Here we report that in unexplored peat soils of Central and South America, N2O production can be driven by abiotic reactions (≤98%) highly competitive to their enzymatic counterparts. Extracted soil iron positively correlated with in situ abiotic N2O production determined by isotopic tracers. Moreover, we found that microbial N2O reduction accompanied abiotic production, essentially closing a coupled abiotic-biotic N2O cycle. Anaerobic N2O consumption occurred ubiquitously (pH 6.4-3.7), with proportions of diverse clade II N2O reducers increasing with consumption rates. Our findings show that denitrification in tropical peat soils is not a purely biological process but rather a 'mosaic' of abiotic and biotic reduction reactions. We predict that hydrological and temperature fluctuations differentially affect abiotic and biotic drivers and further contribute to the high N2O flux variation in the region.

Water Vapor Adsorption Provides Daily, Sustainable Water to Soils of the Hyperarid Atacama Desert.

Water is necessary for all life on Earth. Water is so critical that organisms have developed strategies to survive in hyperarid environments. These regions with extremely low water availability are also unique analogs in which to study the physico-chemical conditions of extraterrestrial environments such as Mars. We have identified a daily, sustainable cycle of water vapor adsorption (WVA) and desorption that measurably affects soil water content (SWC) in the hyperarid region of the Atacama Desert in southern Perú. We pair field-based soil temperature and relative humidity soil profiles with laboratory simulations to provide evidence for a daily WVA cycle. Using our WVA model, we estimate that one adsorptive period-one night-increases SWC by 0.2-0.3 mg/g of soil (∼30 µm rainfall). We can plausibly rule out other water inputs during our field campaign that could account for this water input, and we provide evidence that this WVA cycle is driven by solar heating and maintained by atmospheric water vapor. The WVA may also serve to retain water from infrequent rain events in these soils. If the water provided by WVA in these soils is bioavailable, it could have significant implications for the microorganisms that are endemic to hyperarid environments.

Delineating the Drivers and Functionality of Methanogenic Niches within an Arid Landfill.

Microbial communities mediate the transformation of organic matter within landfills into methane (CH4). Yet their ecological role in CH4 production is rarely evaluated. To characterize the microbiome associated with this biotransformation, the overall community and methanogenic Archaea were surveyed in an arid landfill using leachate collected from distinctly aged landfill cells (i.e., younger, intermediate, and older). We hypothesized that distinct methanogenic niches exist within an arid landfill, driven by geochemical gradients that developed under extended and age-dependent waste biodegradation stages. Using 16S rRNA and mcrA gene amplicon sequencing, we identified putative methanogenic niches as follows. The order Methanomicrobiales was the most abundant order in leachate from younger cells, where leachate temperature and propionate concentrations were measured at 41.8°C ± 1.7°C and 57.1 ± 10.7 mg L-1. In intermediate-aged cells, the family Methanocellaceae was identified as a putative specialist family under intermediate-temperature and -total dissolved solid (TDS) conditions, wherein samples had a higher alpha diversity index and near CH4 concentrations. In older-aged cells, accumulating metals and TDS supported Methanocorpusculaceae, "Candidatus Bathyarchaeota," and "Candidatus Verstraetearchaeota" operational taxonomic units (OTUs). Consistent with the mcrA data, we assayed methanogenic activity across the age gradient through stable isotopic measurements of δ13C of CH4 and δ13C of CO2. The majority (80%) of the samples' carbon fractionation was consistent with hydrogenotrophic methanogenesis. Together, we report age-dependent geochemical gradients detected through leachate in an arid landfill seemingly influencing CH4 production, niche partitioning, and methanogenic activity. IMPORTANCE Microbiome analysis is becoming common in select municipal and service ecosystems, including wastewater treatment and anaerobic digestion, but its potential as a microbial-status-informative tool to promote or mitigate CH4 production has not yet been evaluated in landfills. Methanogenesis mediated by Archaea is highly active in solid-waste microbiomes but is commonly neglected in studies employing next-generation sequencing techniques. Identifying methanogenic niches within a landfill offers detail into operations that positively or negatively impact the commercial production of methane known as biomethanation. We provide evidence that the geochemistry of leachate and its microbiome can be a variable accounting for ecosystem-level (coarse) variation of CH4 production, where we demonstrate through independent assessments of leachate and gas collection that the functional variability of an arid landfill is linked to the composition of methanogenic Archaea.

Microbial Communities and Interactions of Nitrogen Oxides With Methanogenesis in Diverse Peatlands of the Amazon Basin.

Tropical peatlands are hotspots of methane (CH4) production but present high variation and emission uncertainties in the Amazon region. This is because the controlling factors of methane production in tropical peats are not yet well documented. Although inhibitory effects of nitrogen oxides (NO x ) on methanogenic activity are known from pure culture studies, the role of NO x in the methane cycling of peatlands remains unexplored. Here, we investigated the CH4 content, soil geochemistry and microbial communities along 1-m-soil profiles and assessed the effects of soil NO x and nitrous oxide (N2O) on methanogenic abundance and activity in three peatlands of the Pastaza-Marañón foreland basin. The peatlands were distinct in pH, DOC, nitrate pore water concentrations, C/N ratios of shallow soils, redox potential, and 13C enrichment in dissolved inorganic carbon and CH4 pools, which are primarily contingent on H2-dependent methanogenesis. Molecular 16S rRNA and mcrA gene data revealed diverse and novel methanogens varying across sites. Importantly, we also observed a strong stratification in relative abundances of microbial groups involved in NO x cycling, along with a concordant stratification of methanogens. The higher relative abundance of ammonia-oxidizing archaea (Thaumarchaeota) in acidic oligotrophic peat than ammonia-oxidizing bacteria (Nitrospira) is noteworthy as putative sources of NO x . Experiments testing the interaction of NO x species and methanogenesis found that the latter showed differential sensitivity to nitrite (up to 85% reduction) and N2O (complete inhibition), which would act as an unaccounted CH4 control in these ecosystems. Overall, we present evidence of diverse peatlands likely differently affected by inhibitory effects of nitrogen species on methanogens as another contributor to variable CH4 fluxes.

Methanogens and Methanotrophs Show Nutrient-Dependent Community Assemblage Patterns Across Tropical Peatlands of the Pastaza-Marañón Basin, Peruvian Amazonia.

Tropical peatlands are globally important carbon reservoirs that play a crucial role in fluxes of atmospheric greenhouse gases. Amazon peatlands are expected to be large source of atmospheric methane (CH4) emissions, however little is understood about the rates of CH4 flux or the microorganisms that mediate it in these environments. Here we studied a mineral nutrient gradient across peatlands in the Pastaza-Marañón Basin, the largest tropical peatland in South America, to describe CH4 fluxes and environmental factors that regulate species assemblages of methanogenic and methanotrophic microorganisms. Peatlands were grouped as minerotrophic, mixed and ombrotrophic categories by their general water source leading to different mineral nutrient content (rich, mixed and poor) quantified by trace elements abundance. Microbial communities clustered dependent on nutrient content (ANOSIM p < 0.001). Higher CH4 flux was associated with minerotrophic communities compared to the other categories. The most dominant methanogens and methanotrophs were represented by Methanobacteriaceae, and Methylocystaceae, respectively. Weighted network analysis demonstrated tight clustering of most methanogen families with minerotrophic-associated microbial families. Populations of Methylocystaceae were present across all peatlands. Null model testing for species assemblage patterns and species rank distributions confirmed non-random aggregations of Methylococcacae methanotroph and methanogen families (p < 0.05). We conclude that in studied amazon peatlands increasing mineral nutrient content provides favorable habitats for Methanobacteriaceae, while Methylocystaceae populations seem to broadly distribute independent of nutrient content.

Potential shift from a carbon sink to a source in Amazonian peatlands under a changing climate.

Amazonian peatlands store a large amount of soil organic carbon (SOC), and its fate under a future changing climate is unknown. Here, we use a process-based peatland biogeochemistry model to quantify the carbon accumulation for peatland and nonpeatland ecosystems in the Pastaza-Marañon foreland basin (PMFB) in the Peruvian Amazon from 12,000 y before present to AD 2100. Model simulations indicate that warming accelerates peat SOC loss, while increasing precipitation accelerates peat SOC accumulation at millennial time scales. The uncertain parameters and spatial variation of climate are significant sources of uncertainty to modeled peat carbon accumulation. Under warmer and presumably wetter conditions over the 21st century, SOC accumulation rate in the PMFB slows down to 7.9 (4.3-12.2) g⋅C⋅m-2⋅y-1 from the current rate of 16.1 (9.1-23.7) g⋅C⋅m-2⋅y-1, and the region may turn into a carbon source to the atmosphere at -53.3 (-66.8 to -41.2) g⋅C⋅m-2⋅y-1 (negative indicates source), depending on the level of warming. Peatland ecosystems show a higher vulnerability than nonpeatland ecosystems, as indicated by the ratio of their soil carbon density changes (ranging from 3.9 to 5.8). This is primarily due to larger peatlands carbon stocks and more dramatic responses of their aerobic and anaerobic decompositions in comparison with nonpeatland ecosystems under future climate conditions. Peatland and nonpeatland soils in the PMFB may lose up to 0.4 (0.32-0.52) Pg⋅C by AD 2100 with the largest loss from palm swamp. The carbon-dense Amazonian peatland may switch from a current carbon sink into a source in the 21st century.

Genomic composition and dynamics among Methanomicrobiales predict adaptation to contrasting environments.

Members of the order Methanomicrobiales are abundant, and sometimes dominant, hydrogenotrophic (H2-CO2 utilizing) methanoarchaea in a broad range of anoxic habitats. Despite their key roles in greenhouse gas emissions and waste conversion to methane, little is known about the physiological and genomic bases for their widespread distribution and abundance. In this study, we compared the genomes of nine diverse Methanomicrobiales strains, examined their pangenomes, reconstructed gene flow and identified genes putatively mediating their success across different habitats. Most strains slowly increased gene content whereas one, Methanocorpusculum labreanum, evidenced genome downsizing. Peat-dwelling Methanomicrobiales showed adaptations centered on improved transport of scarce inorganic nutrients and likely use H+ rather than Na+ transmembrane chemiosmotic gradients during energy conservation. In contrast, other Methanomicrobiales show the potential to concurrently use Na+ and H+ chemiosmotic gradients. Analyses also revealed that the Methanomicrobiales lack a canonical electron bifurcation system (MvhABGD) known to produce low potential electrons in other orders of hydrogenotrophic methanogens. Additional putative differences in anabolic metabolism suggest that the dynamics of interspecies electron transfer from Methanomicrobiales syntrophic partners can also differ considerably. Altogether, these findings suggest profound differences in electron trafficking in the Methanomicrobiales compared with other hydrogenotrophs, and warrant further functional evaluations.

Complete Genome Sequence of Methanosphaerula palustris E1-9CT, a Hydrogenotrophic Methanogen Isolated from a Minerotrophic Fen Peatland.

Here, we report the complete genome sequence (2.92 Mb) of Methanosphaerula palustris E1-9C(T), a methanogen isolated from a minerotrophic fen. This is the first genome report of the Methanosphaerula genus, within the Methanoregulaceae family, in the Methanomicrobiales order. E1-9C(T) relatives are found in a wide range of ecological and geographical settings.

Exometabolite niche partitioning among sympatric soil bacteria.

Soils are arguably the most microbially diverse ecosystems. Physicochemical properties have been associated with the maintenance of this diversity. Yet, the role of microbial substrate specialization is largely unexplored since substrate utilization studies have focused on simple substrates, not the complex mixtures representative of the soil environment. Here we examine the exometabolite composition of desert biological soil crusts (biocrusts) and the substrate preferences of seven biocrust isolates. The biocrust's main primary producer releases a diverse array of metabolites, and isolates of physically associated taxa use unique subsets of the complex metabolite pool. Individual isolates use only 13-26% of available metabolites, with only 2 out of 470 used by all and 40% not used by any. An extension of this approach to a mesophilic soil environment also reveals high levels of microbial substrate specialization. These results suggest that exometabolite niche partitioning may be an important factor in the maintenance of microbial diversity.

Genome of Methanoregula boonei 6A8 reveals adaptations to oligotrophic peatland environments.

Analysis of the genome sequence of Methanoregula boonei strain 6A8, an acidophilic methanogen isolated from an ombrotrophic (rain-fed) peat bog, has revealed unique features that likely allow it to survive in acidic, nutrient-poor conditions. First, M. boonei is predicted to generate ATP using protons that are abundant in peat, rather than sodium ions that are scarce, and the sequence of a membrane-bound methyltransferase, believed to pump Na+ in all methanogens, shows differences in key amino acid residues. Further, perhaps reflecting the hypokalemic status of many peat bogs, M. boonei demonstrates redundancy in the predicted potassium uptake genes trk, kdp and kup, some of which may have been horizontally transferred to methanogens from bacteria, possibly Geobacter spp. Overall, the putative functions of the potassium uptake, ATPase and methyltransferase genes may, at least in part, explain the cosmopolitan success of group E1/E2 and related methanogenic archaea in acidic peat bogs.

Isolation of a significant fraction of non-phototroph diversity from a desert Biological Soil Crust.

Biological Soil Crusts (BSCs) are organosedimentary assemblages comprised of microbes and minerals in topsoil of terrestrial environments. BSCs strongly impact soil quality in dryland ecosystems (e.g., soil structure and nutrient yields) due to pioneer species such as Microcoleus vaginatus; phototrophs that produce filaments that bind the soil together, and support an array of heterotrophic microorganisms. These microorganisms in turn contribute to soil stability and biogeochemistry of BSCs. Non-cyanobacterial populations of BSCs are less well known than cyanobacterial populations. Therefore, we attempted to isolate a broad range of numerically significant and phylogenetically representative BSC aerobic heterotrophs. Combining simple pre-treatments (hydration of BSCs under dark and light) and isolation strategies (media with varying nutrient availability and protection from oxidative stress) we recovered 402 bacterial and one fungal isolate in axenic culture, which comprised 116 phylotypes (at 97% 16S rRNA gene sequence homology), 115 bacterial and one fungal. Each medium enriched a mostly distinct subset of phylotypes, and cultivated phylotypes varied due to the BSC pre-treatment. The fraction of the total phylotype diversity isolated, weighted by relative abundance in the community, was determined by the overlap between isolate sequences and OTUs reconstructed from metagenome or metatranscriptome reads. Together, more than 8% of relative abundance of OTUs in the metagenome was represented by our isolates, a cultivation efficiency much larger than typically expected from most soils. We conclude that simple cultivation procedures combined with specific pre-treatment of samples afford a significant reduction in the culturability gap, enabling physiological and metabolic assays that rely on ecologically relevant axenic cultures.

Complete Genome Sequence of Methanoregula formicica SMSPT, a Mesophilic Hydrogenotrophic Methanogen Isolated from a Methanogenic Upflow Anaerobic Sludge Blanket Reactor.

Methanoregula formicica SMSP(T) is a mesophilic H2/formate-utilizing methanogenic archaeon and a representative of the family Methanoregulaceae, a recently proposed novel family within the order Methanomicrobiales. Here, we report a 2.8-Mb complete genome sequence of this methanogenic archaeon.

Complete Genome Sequence of Methanolinea tarda NOBI-1T, a Hydrogenotrophic Methanogen Isolated from Methanogenic Digester Sludge.

Here, we report a 2.0-Mb complete genome sequence of Methanolinea tarda NOBI-1(T), a methanogenic archaeon isolated from an anaerobic digested sludge. This is the first genome report of the genus Methanolinea isolate belonging to the family Methanoregulaceae, a recently proposed novel family within the order Methanomicrobiales.

Methanobacterium paludis sp. nov. and a novel strain of Methanobacterium lacus isolated from northern peatlands.

Two mesophilic, hydrogenotrophic methanogens, designated strains SWAN1T and AL-21, were isolated from two contrasting peatlands: a near circumneutral temperate minerotrophic fen in New York State, USA, and an acidic boreal poor fen site in Alaska, USA, respectively. Cells of the two strains were rod-shaped, non-motile, stained Gram-negative and resisted lysis with 0.1% SDS. Cell size was 0.6×1.5-2.8 µm for strain SWAN1T and 0.45-0.85×1.5-35 µm for strain AL-21. The strains used H2/CO2 but not formate or other substrates for methanogenesis, grew optimally around 32-37 °C, and their growth spanned through a slightly low to neutral pH range (4.7-7.1). Strain AL-21 grew optimally closer to neutrality at pH 6.2, whereas strain SWAN1T showed a lower optimal pH at 5.4-5.7. The two strains were sensitive to NaCl with a maximal tolerance at 160 mM for strain SWAN1T and 50 mM for strain AL-21. Na2S was toxic at very low concentrations (0.01-0.8 mM), resulting in growth inhibition above these values. The DNA G+C content of the genomes was 35.7 mol% for strain SWAN1T and 35.8 mol% for strain AL-21. Phylogenetic analysis of the 16S rRNA gene sequences showed that the strains are members of the genus Methanobacterium. Strain SWAN1T shared 94-97% similarity with the type strains of recognized species of the genus Methanobacterium, whereas strain AL-21 shared 99% similarity with Methanobacterium lacus 17A1T. On the basis of phenotypic, genomic and phylogenetic characteristics, strain SWAN1T (=DSM 25820T=JCM 18151T) is proposed as the type strain of a novel species, Methanobacterium paludis sp. nov., while strain AL-21 is proposed as a second strain of Methanobacterium lacus.

Recombination shapes genome architecture in an organism from the archaeal domain.

Variation in recombination rates across chromosomes has been shown to be a primary force shaping the architecture of genome divergence. In archaea, little is known about variation in recombination across the chromosome or how it shapes genome evolution. We identified significant variations in polymorphism occurring across the chromosomes of ten closely related sympatric strains of the thermoacidophilic archaeon Sulfolobus islandicus. Statistical analyses show that recombination varies across the genome and interacts with selection to define large genomic regions with reduced polymorphism, particularly in the regions surrounding the three origins of replication. Our findings demonstrate how recombination defines the mosaic of variation in this asexually reproducing microorganism and provide insight into the evolutionary origins of genome architecture in this organism from the Archaeal domain.

Methane emissions from wetlands: biogeochemical, microbial, and modeling perspectives from local to global scales.

Understanding the dynamics of methane (CH4 ) emissions is of paramount importance because CH4 has 25 times the global warming potential of carbon dioxide (CO2 ) and is currently the second most important anthropogenic greenhouse gas. Wetlands are the single largest natural CH4 source with median emissions from published studies of 164 Tg yr(-1) , which is about a third of total global emissions. We provide a perspective on important new frontiers in obtaining a better understanding of CH4 dynamics in natural systems, with a focus on wetlands. One of the most exciting recent developments in this field is the attempt to integrate the different methodologies and spatial scales of biogeochemistry, molecular microbiology, and modeling, and thus this is a major focus of this review. Our specific objectives are to provide an up-to-date synthesis of estimates of global CH4 emissions from wetlands and other freshwater aquatic ecosystems, briefly summarize major biogeophysical controls over CH4 emissions from wetlands, suggest new frontiers in CH4 biogeochemistry, examine relationships between methanogen community structure and CH4 dynamics in situ, and to review the current generation of CH4 models. We highlight throughout some of the most pressing issues concerning global change and feedbacks on CH4 emissions from natural ecosystems. Major uncertainties in estimating current and future CH4 emissions from natural ecosystems include the following: (i) A number of important controls over CH4 production, consumption, and transport have not been, or are inadequately, incorporated into existing CH4 biogeochemistry models. (ii) Significant errors in regional and global emission estimates are derived from large spatial-scale extrapolations from highly heterogeneous and often poorly mapped wetland complexes. (iii) The limited number of observations of CH4 fluxes and their associated environmental variables loosely constrains the parameterization of process-based biogeochemistry models.

Contribution of transcriptomics to systems-level understanding of methanogenic Archaea.

Methane-producing Archaea are of interest due to their contribution to atmospheric change and for their roles in technological applications including waste treatment and biofuel production. Although restricted to anaerobic environments, methanogens are found in a wide variety of habitats, where they commonly live in syntrophic relationships with bacterial partners. Owing to tight thermodynamic constraints of methanogenesis alone or in syntrophic metabolism, methanogens must carefully regulate their catabolic pathways including the regulation of RNA transcripts. The transcriptome is a dynamic and important control point in microbial systems. This paper assesses the impact of mRNA (transcriptome) studies on the understanding of methanogenesis with special consideration given to how methanogenesis is regulated to cope with nutrient limitation, environmental variability, and interactions with syntrophic partners. In comparison with traditional microarray-based transcriptome analyses, next-generation high-throughput RNA sequencing is greatly advantageous in assessing transcription start sites, the extent of 5' untranslated regions, operonic structure, and the presence of small RNAs. We are still in the early stages of understanding RNA regulation but it is already clear that determinants beyond transcript abundance are highly relevant to the lifestyles of methanogens, requiring further study.

Seasonal changes in methanogenesis and methanogenic community in three peatlands, new york state.

Fluctuating environmental conditions can promote diversity and control dominance in community composition. In addition to seasonal temperature and moisture changes, seasonal supply of metabolic substrates selects populations temporally. Here we demonstrate cascading effects in the supply of metabolic substrates on methanogenesis and community composition of anaerobic methanogenic archaea in three contrasting peatlands in upstate New York. Fresh samples of peat soils, collected about every 3 months for 20 months and incubated at 22 ± 2°C regardless of the in situ temperature, exhibited potential rates of methane (CH(4)) production of 0.02-0.2 mmol L(-1) day(-1) [380-3800 nmol g(-1) (dry) day(-1)). The addition of acetate stimulated rates of CH(4) production in a fen peatland soil, whereas addition of hydrogen (H(2)), and simultaneous inhibition of H(2)-consuming acetogenic bacteria with rifampicin, stimulated CH(4) production in two acidic bog soils, especially, in autumn and winter. The methanogenic community structure was characterized using T-RFLP analyses of SSU rRNA genes. The E2 group of methanogens (Methanoregulaceae) dominated in the two acidic bog peatlands with relatively greater abundance in winter. In the fen peatland, the E1 group (Methanoregulaceae) and members of the Methanosaetaceae were co-dominant, with E1 having a high relative abundance in spring. Change in relative abundance profiles among methanogenic groups in response to added metabolic substrates was as predicted. The acetate-amendment increased abundance of Methanosarcinaceae, and H(2)-amendment enhanced abundance of E2 group in all peat soils studied, respectively. Additionally, addition of acetate increased abundance of Methanosaetaceae only in the bog soils. Variation in the supply of metabolic substrates helps explain the moderate diversity of methanogens in peatlands.