Selective trace elements significantly enhanced methane production in coal bed methane systems by stimulating microbial activity.

Trace elements are associated with the microbial degradation of organic matter and methanogenesis, as enzymes in metabolic pathways often employ trace elements as essential cofactors. However, only a few studies investigated the effects of trace elements on the metabolic activity of microbial communities associated with biogenic coalbed methane production. We aimed to determine the effects of strategically selected trace elements on structure and function of active bacterial and methanogenic communities to stimulate methane production in subsurface coalbeds. Microcosms were established with produced water and coal from coalbed methane wells located in the Powder River Basin, Wyoming, USA. In initial pilot experiments with eight different trace elements, individual amendments of Co, Cu, and Mo lead to significantly higher methane production. Transcript levels of mcrA, the key marker gene for methanogenesis, positively correlated with increased methane production. Phylogenetic analysis of the mcrA cDNA library demonstrated compositional shifts of the active methanogenic community and increase of their diversity, particularly of hydrogenotrophic methanogens. High-throughput sequencing of cDNA obtained from 16S rRNA demonstrated active and abundant bacterial groups in response to trace element amendments. Active Acetobacterium members increased in response to Co, Cu, and Mo additions. The findings of this study yield new insights into the importance of essential trace elements on the metabolic activity of microbial communities involved in subsurface coalbed methane and provide a better understanding of how microbial community composition is shaped by trace elements.IMPORTANCEMicrobial life in the deep subsurface of coal beds is limited by nutrient replenishment. While coal bed microbial communities are surrounded by carbon sources, we hypothesized that other nutrients such as trace elements needed as cofactors for enzymes are missing. Amendment of selected trace elements resulted in compositional shifts of the active methanogenic and bacterial communities and correlated with higher transcript levels of mcrA. The findings of this study yield new insights to not only identify possible limitations of microbes by replenishment of trace elements within their specific hydrological placement but also into the importance of essential trace elements for the metabolic activity of microbial communities involved in subsurface coalbed methane production and provides a better understanding of how microbial community composition is shaped by trace elements. Furthermore, this finding might help to revive already spent coal bed methane well systems with the ultimate goal to stimulate methane production.

The Complete Genome Sequences of Iris sibirica and Iris virginica (Iridaceae, Asparagales).

We present the complete genome sequences of Iris sibirica and Iris virginica. Illumina sequencing was performed on genetic material from individual cultivated specimens. The reads were assembled using a de novo method followed by a finishing step. The raw and assembled data are publicly available via Genbank.

The Complete Genome Sequence of Lythrypnus dalli, the Bluebanded Goby.

The Bluebanded goby (Lythrypnus dalli) is a small, highly social marine goby. We present the whole genome sequence of this species. A total of 118,266,160 paired end reads consisting of 17.9G bases were obtained by sequencing tissue from a single individual. The reads were assembled by a de novo method followed by alignment to related species. The raw and assembled data is publicly available via Genbank: Sequence Read Archive (SRR5170315) and Assembly (GCA_011763505).

Anaerobic degradation of hexadecane and phenanthrene coupled to sulfate reduction by enriched consortia from northern Gulf of Mexico seafloor sediment.

To advance understanding of the fate of hydrocarbons released from the Deepwater Horizon oil spill and deposited in marine sediments, this study characterized the microbial populations capable of anaerobic hydrocarbon degradation coupled with sulfate reduction in non-seep sediments of the northern Gulf of Mexico. Anaerobic, sediment-free enrichment cultures were obtained with either hexadecane or phenanthrene as sole carbon source and sulfate as a terminal electron acceptor. Phylogenetic analysis revealed that enriched microbial populations differed by hydrocarbon substrate, with abundant SSU rRNA gene amplicon sequences from hexadecane cultures showing high sequence identity (up to 98%) to Desulfatibacillum alkenivorans (family Desulfobacteraceae), while phenanthrene-enriched populations were most closely related to Desulfatiglans spp. (up to 95% sequence identity; family Desulfarculaceae). Assuming complete oxidation to CO2, observed stoichiometric ratios closely resembled the theoretical ratios of 12.25:1 for hexadecane and 8.25:1 for phenanthrene degradation coupled to sulfate reduction. Phenanthrene carboxylic acid was detected in the phenanthrene-degrading enrichment cultures, providing evidence to indicate carboxylation as an activation mechanism for phenanthrene degradation. Metagenome-assembled genomes (MAGs) revealed that phenanthrene degradation is likely mediated by novel genera or families of sulfate-reducing bacteria along with their fermentative syntrophic partners, and candidate genes linked to the degradation of aromatic hydrocarbons were detected for future study.

Functional diversity and electron donor dependence of microbial populations capable of U(VI) reduction in radionuclide-contaminated subsurface sediments.

In order to elucidate the potential mechanisms of U(VI) reduction for the optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate. A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas. Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended treatments compared to the results in glucose-amended treatments. Quantification of citrate synthase (gltA) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon-amended microcosms, with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. Our results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.

Retrieval of first genome data for rice cluster I methanogens by a combination of cultivation and molecular techniques.

We report first insights into a representative genome of rice cluster I (RC-I), a major group of as-yet uncultured methanogens. The starting point of our study was the methanogenic consortium MRE50 that had been stably maintained for 3 years by consecutive transfers to fresh medium and anaerobic incubation at 50 degrees C. Process-oriented measurements provided evidence for hydrogenotrophic CO(2)-reducing methanogenesis. Assessment of the diversity of consortium MRE50 suggested members of the families Thermoanaerobacteriaceae and Clostridiaceae to constitute the major bacterial component, while the archaeal population was represented entirely by RC-I. The RC-I population amounted to more than 50% of total cells, as concluded from fluorescence in situ hybridization using specific probes for either Bacteria or Archaea. The high enrichment status of RC-I prompted construction of a large insert fosmid library from consortium MRE50. Comparative sequence analysis of internal transcribed spacer (ITS) regions revealed that three different RC-I rrn operon variants were present in the fosmid library. Three, approximately 40-kb genomic fragments, each representative for one of the three different rrn operon variants, were recovered and sequenced. Computational analysis of the sequence data resulted in two major findings: (i) consortium MRE50 most likely harbours only a single RC-I genotype, which is characterized by multiple rrn operon copies; (ii) seven genes were identified to possess a strong phylogenetic signal (eIF2a, dnaG, priA, pcrA, gatD, gatE, and a gene encoding a putative RNA-binding protein). Trees exemplarily computed for the deduced amino acid sequences of eIF2a, dnaG, and priA corroborated a specific phylogenetic association of RC-I with the Methanosarcinales.

Gene Expression Correlates with Process Rates Quantified for Sulfate- and Fe(III)-Reducing Bacteria in U(VI)-Contaminated Sediments.

Though iron- and sulfate-reducing bacteria are well known for mediating uranium(VI) reduction in contaminated subsurface environments, quantifying the in situ activity of the microbial groups responsible remains a challenge. The objective of this study was to demonstrate the use of quantitative molecular tools that target mRNA transcripts of key genes related to Fe(III) and sulfate reduction pathways in order to monitor these processes during in situ U(VI) remediation in the subsurface. Expression of the Geobacteraceae-specific citrate synthase gene (gltA) and the dissimilatory (bi)sulfite reductase gene (dsrA), were correlated with the activity of iron- or sulfate-reducing microorganisms, respectively, under stimulated bioremediation conditions in microcosms of sediments sampled from the U.S. Department of Energy's Oak Ridge Integrated Field Research Challenge (OR-IFRC) site at Oak Ridge, TN, USA. In addition, Geobacteraceae-specific gltA and dsrA transcript levels were determined in parallel with the predominant electron acceptors present in moderately and highly contaminated subsurface sediments from the OR-IFRC. Phylogenetic analysis of the cDNA generated from dsrA mRNA, sulfate-reducing bacteria-specific 16S rRNA, and gltA mRNA identified activity of specific microbial groups. Active sulfate reducers were members of the Desulfovibrio, Desulfobacterium, and Desulfotomaculum genera. Members of the subsurface Geobacter clade, closely related to uranium-reducing Geobacter uraniireducens and Geobacter daltonii, were the metabolically active iron-reducers in biostimulated microcosms and in situ core samples. Direct correlation of transcripts and process rates demonstrated evidence of competition between the functional guilds in subsurface sediments. We further showed that active populations of Fe(III)-reducing bacteria and sulfate-reducing bacteria are present in OR-IFRC sediments and are good potential targets for in situ bioremediation.

Trace elements affect methanogenic activity and diversity in enrichments from subsurface coal bed produced water.

Microbial methane from coal beds accounts for a significant and growing percentage of natural gas worldwide. Our knowledge of physical and geochemical factors regulating methanogenesis is still in its infancy. We hypothesized that in these closed systems, trace elements (as micronutrients) are a limiting factor for methanogenic growth and activity. Trace elements are essential components of enzymes or cofactors of metabolic pathways associated with methanogenesis. This study examined the effects of eight trace elements (iron, nickel, cobalt, molybdenum, zinc, manganese, boron, and copper) on methane production, on mcrA transcript levels, and on methanogenic community structure in enrichment cultures obtained from coal bed methane (CBM) well produced water samples from the Powder River Basin, Wyoming. Methane production was shown to be limited both by a lack of additional trace elements as well as by the addition of an overly concentrated trace element mixture. Addition of trace elements at concentrations optimized for standard media enhanced methane production by 37%. After 7 days of incubation, the levels of mcrA transcripts in enrichment cultures with trace element amendment were much higher than in cultures without amendment. Transcript levels of mcrA correlated positively with elevated rates of methane production in supplemented enrichments (R(2) = 0.95). Metabolically active methanogens, identified by clone sequences of mcrA mRNA retrieved from enrichment cultures, were closely related to Methanobacterium subterraneum and Methanobacterium formicicum. Enrichment cultures were dominated by M. subterraneum and had slightly higher predicted methanogenic richness, but less diversity than enrichment cultures without amendments. These results suggest that varying concentrations of trace elements in produced water from different subsurface coal wells may cause changing levels of CBM production and alter the composition of the active methanogenic community.

Geobacter daltonii sp. nov., an Fe(III)- and uranium(VI)-reducing bacterium isolated from a shallow subsurface exposed to mixed heavy metal and hydrocarbon contamination.

An Fe(III)- and uranium(VI)-reducing bacterium, designated strain FRC-32(T), was isolated from a contaminated subsurface of the USA Department of Energy Oak Ridge Field Research Center (ORFRC) in Oak Ridge, Tennessee, where the sediments are exposed to mixed waste contamination of radionuclides and hydrocarbons. Analyses of both 16S rRNA gene and the Geobacteraceae-specific citrate synthase (gltA) mRNA gene sequences retrieved from ORFRC sediments indicated that this strain was abundant and active in ORFRC subsurface sediments undergoing uranium(VI) bioremediation. The organism belonged to the subsurface clade of the genus Geobacter and shared 92-98 % 16S rRNA gene and 75-81 % rpoB gene sequence similarities with other recognized species of the genus. In comparison to its closest relative, Geobacter uraniireducens Rf4(T), according to 16S rRNA gene sequence similarity, strain FRC-32(T) showed a DNA-DNA relatedness value of 21 %. Cells of strain FRC-32(T) were Gram-negative, non-spore-forming, curved rods, 1.0-1.5 microm long and 0.3-0.5 microm in diameter; the cells formed pink colonies in a semisolid cultivation medium, a characteristic feature of the genus Geobacter. The isolate was an obligate anaerobe, had temperature and pH optima for growth at 30 degrees C and pH 6.7-7.3, respectively, and could tolerate up to 0.7 % NaCl although growth was better in the absence of NaCl. Similar to other members of the Geobacter group, strain FRC-32(T) conserved energy for growth from the respiration of Fe(III)-oxyhydroxide coupled with the oxidation of acetate. Strain FRC-32(T) was metabolically versatile and, unlike its closest relative, G. uraniireducens, was capable of utilizing formate, butyrate and butanol as electron donors and soluble ferric iron (as ferric citrate) and elemental sulfur as electron acceptors. Growth on aromatic compounds including benzoate and toluene was predicted from preliminary genomic analyses and was confirmed through successive transfer with fumarate as the electron acceptor. Thus, based on genotypic, phylogenetic and phenotypic differences, strain FRC-32(T) is considered to represent a novel species of the genus Geobacter, for which the name Geobacter daltonii sp. nov. is proposed. The type strain is FRC-32(T) (=DSM 22248(T)=JCM 15807(T)).

Genome sequence of the deltaproteobacterial strain NaphS2 and analysis of differential gene expression during anaerobic growth on naphthalene.

BACKGROUND: Anaerobic polycyclic hydrocarbon (PAH) degradation coupled to sulfate reduction may be an important mechanism for in situ remediation of contaminated sediments. Steps involved in the anaerobic degradation of 2-methylnaphthalene have been described in the sulfate reducing strains NaphS3, NaphS6 and N47. Evidence from N47 suggests that naphthalene degradation involves 2-methylnaphthalene as an intermediate, whereas evidence in NaphS2, NaphS3 and NaphS6 suggests a mechanism for naphthalene degradation that does not involve 2-methylnaphthalene. To further characterize pathways involved in naphthalene degradation in NaphS2, the draft genome was sequenced, and gene and protein expression examined. RESULTS: Draft genome sequencing, gene expression analysis, and proteomic analysis revealed that NaphS2 degrades naphthoyl-CoA in a manner analogous to benzoyl-CoA degradation. Genes including the previously characterized NmsA, thought to encode an enzyme necessary for 2-methylnaphthalene metabolism, were not upregulated during growth of NaphS2 on naphthalene, nor were the corresponding protein products. NaphS2 may possess a non-classical dearomatizing enzyme for benzoate degradation, similar to one previously characterized in Geobacter metallireducens. Identification of genes involved in toluene degradation in NaphS2 led us to determine that NaphS2 degrades toluene, a previously unreported capacity. The genome sequence also suggests that NaphS2 may degrade other monoaromatic compounds. CONCLUSION: This study demonstrates that steps leading to the degradation of 2-naphthoyl-CoA are conserved between NaphS2 and N47, however while NaphS2 possesses the capacity to degrade 2-methylnaphthalene, naphthalene degradation likely does not proceed via 2-methylnaphthalene. Instead, carboxylation or another form of activation may serve as the first step in naphthalene degradation. Degradation of toluene and 2-methylnaphthalene, and the presence of at least one bss-like and bbs-like gene cluster in this organism, suggests that NaphS2 degrades both compounds via parallel mechanisms. Elucidation of the key genes necessary for anaerobic naphthalene degradation may provide the ability to track naphthalene degradation through in situ transcript monitoring.

Quantifying expression of a dissimilatory (bi)sulfite reductase gene in petroleum-contaminated marine harbor sediments.

The possibility of quantifying in situ levels of transcripts for dissimilatory (bi)sulfite reductase (dsr) genes to track the activity of sulfate-reducing microorganisms in petroleum-contaminated marine harbor sediments was evaluated. Phylogenetic analysis of the cDNA generated from mRNA for a ca. 1.4 kbp portion of the contiguous dsrA and dsrB genes suggested that Desulfosarcina species, closely related to cultures known to anaerobically oxidize aromatic hydrocarbons, were active sulfate reducers in the sediments. The levels of dsrA transcripts (per mug total mRNA) were quantified in sediments incubated anaerobically at the in situ temperature as well as in sediments incubated at higher temperatures and/or with added acetate to increase the rate of sulfate reduction. Levels of dsrA transcripts were low when there was no sulfate reduction because the sediments were depleted of sulfate or if sulfate reduction was inhibited with added molybdate. There was a direct correlation between dsrA transcript levels and rates of sulfate reduction when sulfate was at ca. 10 mM in the various sediment treatments, but it was also apparent that within a given sediment, dsrA levels increased over time as long as sulfate was available, even when sulfate reduction rates did not increase. These results suggest that phylogenetic analysis of dsr transcript sequences may provide insight into the active sulfate reducers in marine sediments and that quantifying levels of dsrA transcripts can indicate whether sulfate reducers are active in particular sediment. Furthermore, it may only be possible to use dsrA transcript levels to compare the relative rates of sulfate reduction in sediments when sulfate concentrations, and possibly other environmental conditions, are comparable.

Propionate formation by Opitutus terrae in pure culture and in mixed culture with a hydrogenotrophic methanogen and implications for carbon fluxes in anoxic rice paddy soil.

Propionate-forming bacteria seem to be abundant in anoxic rice paddy soil, but biogeochemical investigations show that propionate is not a correspondingly important intermediate in carbon flux in this system. Mixed cultures of Opitutus terrae strain PB90-1, a representative propionate-producing bacterium from rice paddy soil, and the hydrogenotrophic Methanospirillum hungatei strain SK maintained hydrogen partial pressures similar to those in the soil. The associated shift away from propionate formation observed in these cultures helps to reconcile the disparity between microbiological and biogeochemical studies.

Community analysis of methanogenic archaea within a riparian flooding gradient.

Anoxic soils in river floodplains (or riparian soils) are a source of methane emission. However, little is known about the ecology and community structure of archaeal methanogenic microbes, which are a crucial component of methane flux in those habitats. We studied the archaeal community in the vertical profile of four different sites along the River Waal in the Netherlands. These sites differ in their annual flooding regime ranging from never or seldom to permanently flooded. The archaeal community structure has been characterized by terminal restriction fragment length polymorphism (T-RFLP) and comparative sequence analysis of the archaeal SSU rRNA gene and the mcrA gene. The latter gene codes for the alpha-subunit of methyl-coenzyme M reductase. Additionally, the potential methanogenic activity was determined by incubation of soil slurries under anoxic conditions. The community composition differed only slightly with the depth of the soil (0-20 cm). However, the diversity of archaeal SSU rRNA genes increased with the frequency of flooding. Terminal restriction fragment length polymorphism analysis of mcrA gene amplicons confirmed the results concerning methanogenic archaea. In the never and rarely flooded soils, crenarchaeotal sequences were the dominant group. In the frequently and permanently flooded soils, Methanomicrobiaceae, Methanobacteriaceae, Methanosarcinaceae and the uncultured Rice Clusters IV and VI (Crenarchaeota) were detectable independently from duration of anoxic conditions. Methanosaetaceae, on the other hand, were only found in the permanently and frequently flooded soils under conditions where concentrations of acetate were < 30 microM. The results indicate that methanogens as well as other archaea occupy characteristic niches according to the flooding conditions in the field. Methanosaetaceae, in particular, seem to be adapted (or proliferate at) to low acetate concentrations.

Effect of temperature stress on structure and function of the methanogenic archaeal community in a rice field soil.

The effect of a brief (24 h) temperature shift to 4 degrees C (low-temperature stress) or 50 degrees C (high-temperature stress) was studied in methanogenic slurries of Italian rice field soil incubated at either constant 30 degrees C or 15 degrees C. Low-temperature (low-T) stress showed no effect in either the 30 degrees C or the 15 degrees C incubations. High-temperature (high-T) stress, on the other hand, generally resulted in an increase in the partial pressure of H(2), and the concentrations of acetate and propionate, which accumulated to about 100-200 Pa, 4-6 mM and 0.7-0.9 mM, respectively. The increase in H(2) was transient for 8-15 days. The increases in acetate and propionate were transient for about 20 days only in the 30 degrees C incubations, but persisted in the 15 degrees C incubations until the end of the experiment. The high-T stress did not result in inhibition of CH(4) production in the 30 degrees C incubations, but transiently inhibited the 15 degrees C incubations for about 25-30 days. The archaeal community in the soils was analyzed by terminal restriction fragment length polymorphism of the gene of the SSU rRNA. In the 15 degrees C incubations, the relative gene frequency of members of the Methanosarcinaceae decreased over an incubation period of 54 days, while those of Methanosaetaceae and of methanogenic rice cluster I increased. Temperature stress, high-T stress in particular, tended to reverse this trend. In the 30 degrees C incubations, on the other hand, the relative gene frequency of archaeal members showed the opposite temporal trend or remained constant unlike the 15 degrees C incubations. Again, high-T stress tended to reverse these trends, but the observed effects were much smaller in the 30 degrees C incubations than in the 15 degrees C incubations. In conclusion, a brief high-T stress affected structure and function of the methanogenic archaeal community of rice field soil.

Direct correlation between rates of anaerobic respiration and levels of mRNA for key respiratory genes in Geobacter sulfurreducens.

The predominance of Geobacter species in environments in which Fe(III) reduction is important has suggested that Fe(III) reduction rates might be estimated in Geobacter-dominated environments by assessing in situ activity with molecular techniques. To determine whether mRNA levels of key respiratory genes might be correlated with respiration rates in Geobacter sulfurreducens, studies were conducted with fumarate as the electron acceptor and acetate as the limiting electron donor in anaerobic continuous cultures. Levels of mRNA for a fumarate reductase gene, frdA, quantified by real-time reverse transcription-PCR were directly correlated with fumarate reduction rates. In similar studies with Fe(III) as the electron acceptor, mRNA levels for omcB, a gene for an outer membrane c-type cytochrome involved in Fe(III) reduction, were positively correlated with Fe(III) reduction rates. Levels of mRNA for frdA and omcB were also positively correlated with fumarate and Fe(III) reduction rates, respectively, when growth was limited by the availability of fumarate or Fe(III), but mRNA levels were higher than in acetate-limited cultures. Levels of mRNA for omcC, which encodes a c-type cytochrome highly similar to OmcB but not necessary for Fe(III) reduction, followed patterns different than those of omcB. This agrees with the previous finding that OmcC is not involved in Fe(III) reduction and suggests that changes in mRNA levels of omcB are related to its role in Fe(III) reduction. These results demonstrate that mRNA levels for respiratory genes might be used to estimate in situ Fe(III) reduction rates in Geobacter-dominated environments but suggest that information on environmental conditions and/or the metabolic state of Geobacter species is also required for accurate rate estimates.

Acetoclastic and hydrogenotrophic methane production and methanogenic populations in an acidic West-Siberian peat bog.

Sites in the West Siberian peat bog 'Bakchar' were acidic (pH 4.2-4.8), low in nutrients, and emitted CH4 at rates of 0.2-1.5 mmol m(-2) h(-1). The vertical profile of delta13CH4 and delta13CO2 dissolved in the porewater indicated increasing isotope fractionation and thus increasing contribution of H2/CO2-dependent methanogenesis with depth. The anaerobic microbial community at 30-50 cm below the water table produced CH4 with optimum activity at 20-25 degrees C and pH 5.0-5.5 respectively. Inhibition of methanogenesis with 2-bromo-ethane sulphonate showed that acetate, phenyl acetate, phenyl propionate and caproate were important intermediates in the degradation pathway of organic matter to CH4. Further degradation of these intermediates indicated that 62-72% of the CH4 was ultimately derived from acetate, the remainder from H2/CO2. Turnover times of [2-14C]acetate were on the order of 2 days (15, 25 degrees C) and accounted for 60-65% of total CH4 production. Conversion of 14CO2 to 14CH4 accounted for 35-43% of total CH4 production. These results showed that acetoclastic and hydrogenotrophic methanogenesis operated closely at a ratio of approximately 2 : 1 irrespective of the incubation temperature (4, 15 and 25 degrees C). The composition of the archaeal community was determined in the peat samples by terminal restriction fragment length polymorphism (T-RFLP) analysis and sequencing of amplified SSU rRNA gene fragments, and showed that members of Methanomicrobiaceae, Methanosarcinaceae and Rice cluster II (RC-II) were present. Other, presumably non-methanogenic archaeal clusters (group III, RC-IV, RC-V, RC-VI) were also detected. Fluorescent in situ hybridization (FISH) showed that the number of Bacteria decreased (from 24 x 10(7) to 4 x 10(7) cells per gram peat) with depth (from 5 to 55 cm below the water table), whereas the numbers of Archaea slightly increased (from 1 x 10(7) to 2 x 10(7) cells per gram peat). Methanosarcina spp. accounted for about half of the archaeal cells. Our results show that both hydrogenotrophic and acetoclastic methanogenesis are an integral part of the CH4-producing pathway in acidic peat and were represented by appropriate methanogenic populations.