Presence of diverse nitrate-dependent anaerobic methane oxidizing archaea in sewage sludge.

AIM: The aim of this study was to explore the community diversity and abundance of nitrate-dependent anaerobic methane oxidizing archaea, Candidatus Methanoperedens nitroreducens, in sewage sludge from wastewater treatment plants. METHODS AND RESULTS: Seasonal sampling of the sewage sludge was carried out from two wastewater treatment plants (WWTPs) located in the northern and southern parts of China. Through amplicon sequencing using our newly designed primers, a large number of Candidatus Methanoperedens nitroreducens-like (M. nitroreducens) archaeal sequences (638 743) were generated. These sequences were assigned into 742 operational protein units (OPUs) at 90% cut-off level and classified as Group B member of M. nitroreducens archaea in the phylogenetic tree. More than 80% of the OPUs were not shared between these two WWTPs, showing the M. nitroreducens-like archaeal community in each WWTP was unique. Quantitative PCR assays also confirmed the presence of M. nitroreducens-like archaea and revealed a higher abundance in autumn and winter than other seasons, indicating that the environmental attributes in these seasons might favour the growth of this archaea. Further redundancy analysis revealed that volatile solid and pH were the significant environmental attributes (P < 0·05) in shaping the M. nitroreducens-like archaeal community based on variance inflation factor selection and Monte Carlo permutation test. CONCLUSIONS: The results confirmed the presence of diverse M. nitroreducens-like archaea in sewage sludge using Illumina-based mcrA gene sequencing and quantitative PCR assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study revealed the ecological characteristics of M. nitroreducens-like archaea in sewage sludge that improved our understanding of nitrate-dependent anaerobic methane oxidation process and may be the basis for future application of M. nitroreducens-like archaea for new nitrogen removal in WWTPs.

Microbial community dynamics during the early stages of plant polymer breakdown in paddy soil.

We used paddy soil slurries amended with rice straw to identify the microbial populations involved in the methanogenic breakdown of plant polymers. Rice straw greatly stimulated microbial activity over the 28-day incubation period. On day 7, the transient peak concentration of acetate (24 mM) coincided with the onset of increased methane production. Microbial 16S rRNA transcript numbers increased by one to two orders of magnitude, but not the 16S rRNA gene copy numbers. Using metatranscriptomic rRNA, Clostridiaceae, Lachnospiraceae, Ruminococcaceae, Veillonellaceae and Pseudomonadaceae were identified to be the most abundant and the most dynamic bacterial groups. Changes in methanogen rRNA and mRNA abundances corresponded well with methanogenic activity. Acetate determined the abundance ratio between Methanosarcinaceae and Methanosaetaceae. Methanocellaceae dominated hydrogenotrophic methanogenesis. Transcript levels of mRNA families involved in plant polymer breakdown increased slightly with time. Glycosyl hydrolase (GH) transcripts involved in cellulose and chitin breakdown were predominantly expressed by the Firmicutes, whereas those involved in hemicellulose breakdown exhibited more diverse taxonomic sources, including Acidobacteria, Bacteriodetes and Chloroflexi. Taken together, we observed strong population dynamics and the expression of taxonomically diverse GH families, suggesting that not only Firmicutes, but also less abundant groups play a major functional role in the decomposition of rice straw.

In situ DNA-hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms.

In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non-enzymatic, hybridization chain reaction (HCR)-based signal amplified in situ whole-cell detection technique (in situ DNA-HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA-HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)-FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA-HCR. In summary, in situ DNA-HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.

Metagenome from a Spirulina digesting biogas reactor: analysis via binning of contigs and classification of short reads.

BACKGROUND: Anaerobic digestion is a biological process in which a consortium of microorganisms transforms a complex substrate into methane and carbon dioxide. A good understanding of the interactions between the populations that form this consortium can contribute to a successful anaerobic digestion of the substrate. In this study we combine the analysis of the biogas production in a laboratory anaerobic digester fed with the microalgae Spirulina, a protein rich substrate, with the analysis of the metagenome of the consortium responsible for digestion, obtained by high-throughput DNA sequencing. The obtained metagenome was also compared with a metagenome from a full scale biogas plant fed with cellulose rich material. RESULTS: The optimal organic loading rate for the anaerobic digestion of Spirulina was determined to be 4.0 g Spirulina L(-1) day(-1) with a specific biogas production of 350 mL biogas g Spirulina (-1) with a methane content of 68 %. Firmicutes dominated the microbial consortium at 38 % abundance followed by Bacteroidetes, Chloroflexi and Thermotogae. Euryarchaeota represented 3.5 % of the total abundance. The most abundant organism (14.9 %) was related to Tissierella, a bacterium known to use proteinaceous substrates for growth. Methanomicrobiales and Methanosarcinales dominated the archaeal community. Compared to the full scale cellulose-fed digesters, Pfam domains related to protein degradation were more frequently detected and Pfam domains related to cellulose degradation were less frequent in our sample. CONCLUSIONS: The results presented in this study suggest that Spirulina is a suitable substrate for the production of biogas. The proteinaceous substrate appeared to have a selective impact on the bacterial community that performed anaerobic digestion. A direct influence of the substrate on the selection of specific methanogenic populations was not observed.

Metagenome approaches revealed a biological prospect for improvement on mesophilic cellulose degradation.

Improvement on the bioconversion of cellulosic biomass depends much on the expanded knowledge on the underlying microbial structure and the relevant genetic information. In this study, metagenomic analysis was applied to characterize an enriched mesophilic cellulose-converting consortium, to explore its cellulose-hydrolyzing genes, and to discern genes involved in methanogenesis. Cellulose conversion efficiency of the mesophilic consortium enriched in this study was around 70 %. Apart from methane, acetate was the major fermentation product in the liquid phase, while propionate and butyrate were also detected at relatively high concentrations. With the intention to uncover the biological factors that might shape the varying cellulose conversion efficiency at different temperatures, results of this mesophilic consortium were then compared with that of a previously reported thermophilic cellulose-converting consortium. It was found that the mesophilic consortium harbored a larger pool of putative carbohydrate-active genes, with 813 of them in 54 GH modules and 607 genes in 13 CBM modules. Methanobacteriaceae and Methanosaetaceae were the two methanogen families identified, with a preponderance of the hydrogenotrophic Methanobacteriaceae. In contrast to its relatively high diversity and high abundance of carbohydrate-active genes, the abundance of genes involved in the methane metabolism was comparatively lower in the mesophilic consortium. A biological enhancement on the methanogenic process might serve as an effective option for the improvement of the cellulose bioconversion at mesophilic temperature.

Is aceticlastic methanogen composition in full-scale anaerobic processes related to acetate utilization capacity?

In this study, biomass samples were obtained from six municipal and nine industrial full-scale anaerobic processes to investigate whether the aceticlastic methanogen population composition is related to acetate utilization capacity and the nature of the wastewater treated, i.e. municipal sludge or industrial wastewater. Batch serum bottle tests were used to determine the specific acetate utilization rate (AUR), and a quantitative real-time polymerase chain reaction protocol was used to enumerate the acetate-utilizing Methanosaeta and Methanosarcina populations in the biomass samples. Methanosaeta was the dominant aceticlastic methanogen in all samples, except for one industrial wastewater-treating anaerobic process. However, Methanosarcina density in industrial biomass samples was higher than the Methanosarcina density in the municipal samples. The average AUR values of municipal and industrial wastewater treatment plant biomass samples were 10.49 and 10.65 mg CH3COO(-)/log(aceticlastic methanogen gene copy).d, respectively. One-way ANOVA test and principle component analysis showed that the acetate utilization capacities and aceticlastic methanogen community composition did not show statistically significant correlation among the municipal digesters and industrial wastewater-treating processes investigated.

Dominance of Methanosarcinales phylotypes and depth-wise distribution of methanogenic community in fresh water sediments of Sitka stream from Czech Republic.

The variation in the diversity of methanogens in sediment depths from Sitka stream was studied by constructing a 16S rRNA gene library using methanogen-specific primers and a denaturing gradient gel electrophoresis (DGGE)-based approach. A total of nine different phylotypes from the 16S rRNA library were obtained, and all of them were clustered within the order Methanosarcinales. These nine phylotypes likely represent nine new species and at least 5-6 new genera. Similarly, DGGE analysis revealed an increase in the diversity of methanogens with an increase in sediment depth. These results suggest that Methanosarcinales phylotypes might be the dominant methanogens in the sediment from Sitka stream, and the diversity of methanogens increases as the depth increases. Results of the present study will help in making effective strategies to monitor the dominant methanogen phylotypes and methane emissions in the environment.

Biogas production and methanogenic archaeal community in mesophilic and thermophilic anaerobic co-digestion processes.

Over 258 Mt of solid waste are generated annually in Europe, a large fraction of which is biowaste. Sewage sludge is another major waste fraction. In this study, biowaste and sewage sludge were co-digested in an anaerobic digestion reactor (30% and 70% of total wet weight, respectively). The purpose was to investigate the biogas production and methanogenic archaeal community composition in the anaerobic digestion reactor under meso- (35-37 °C) and thermophilic (55-57 °C) processes and an increasing organic loading rate (OLR, 1-10 kg VS m(-3) d(-1)), and also to find a feasible compromise between waste treatment capacity and biogas production without causing process instability. In summary, more biogas was produced with all OLRs by the thermophilic process. Both processes showed a limited diversity of the methanogenic archaeal community which was dominated by Methanobacteriales and Methanosarcinales (e.g. Methanosarcina) in both meso- and thermophilic processes. Methanothermobacter was detected as an additional dominant genus in the thermophilic process. In addition to operating temperatures, the OLRs, the acetate concentration, and the presence of key substrates like propionate also affected the methanogenic archaeal community composition. A bacterial cell count 6.25 times higher than archaeal cell count was observed throughout the thermophilic process, while the cell count ratio varied between 0.2 and 8.5 in the mesophilic process. This suggests that the thermophilic process is more stable, but also that the relative abundance between bacteria and archaea can vary without seriously affecting biogas production.

Community and proteomic analysis of methanogenic consortia degrading terephthalate.

Degradation of terephthalate (TA) through microbial syntrophy under moderately thermophilic (46 to 50°C) methanogenic conditions was characterized by using a metagenomic approach (A. Lykidis et al., ISME J. 5:122-130, 2011). To further study the activities of key microorganisms responsible for the TA degradation, community analysis and shotgun proteomics were used. The results of hierarchical oligonucleotide primer extension analysis of PCR-amplified 16S rRNA genes indicated that Pelotomaculum, Methanosaeta, and Methanolinea were predominant in the TA-degrading biofilms. Metaproteomic analysis identified a total of 482 proteins and revealed a distinctive distribution pattern of microbial functions expressed in situ. The results confirmed that TA was degraded by Pelotomaculum spp. via the proposed decarboxylation and benzoyl-coenzyme A-dependent pathway. The intermediate by-products, including acetate, H(2)/CO(2), and butyrate, were produced to support the growth of methanogens, as well as other microbial populations that could further degrade butyrate. Proteins related to energy production and conservation, and signal transduction mechanisms (that is, chemotaxis, PAS/GGDEF regulators, and stress proteins) were highly expressed, and these mechanisms were important for growth in energy-limited syntrophic ecosystems.

Novel stem-loop probe DNA arrays: detection of specific acetotrophic 16S ribosomal RNA signatures.

In a recent study, we showed how novel stem-loop DNA probes on dendron-modified aldehyde substrates could be used to detect synthetic nucleic acid targets without amplification. In this article, we demonstrate the application of stem-loop DNA probes as arrays for the detection of specific families and genera of methane-producing bacteria from sludge samples harvested from an anaerobic digester using 16S ribosomal RNA (rRNA) signatures. Specific 16S rRNA could be detected in samples that had 0.2ng/µl total sludge RNA without any target amplification.

The effect of managing nutrients in the performance of anaerobic digesters of municipal wastewater treatment plants.

Is it possible to create conditions in the anaerobic digesters to control nutrients without changing the performance of a reactor? This study investigates an answer for this question. To this purpose, anaerobic reactors are operated at high concentrations of Mg(2+) ion to harvest the nutrient ions (NH4 (+) and PO4 (3-)) in the form of struvite, that is, magnesium ammonium phosphate. The effects of this modification on the anaerobic digestion of sewage sludge were investigated in terms of chemical oxygen demand (COD) removal and cumulative CH4 production as well as the changes in the biological diversity. The results showed that approximately 50 % of the nutrients (NH4 (+) and PO4 (3-)) were removed regardless of the method adopted for the addition of Mg(2+) ion, slug or daily dosing. The numbers of Methanosaeta and Methanosarcina in the samples withdrawn prior to and after the addition of Mg(2+) did not show significant difference according to the results obtained from qPCR analyses. The research results showed that the addition of Mg(2+) into the anaerobic digesters in municipal wastewater treatment facilities may help to remove the nutrients from the effluent while recovering in their solid forms.

The effect of enzymatic pre-hydrolysis of dairy wastewater on the granular and immobilized microbial community in anaerobic bioreactors.

The effect of a lipase-rich enzyme preparation produced by the fungus Penicillium sp. on solid-state fermentation was evaluated in two anaerobic bioreactors (up-flow anaerobic sludge blanket (UASB) and horizontal-flow anaerobic immobilized biomass (HAIB)) treating dairy wastewater with 1200 mg oil and grease/L. The oil and grease hydrolysis step was carried out with 0.1% (w/v) of the solid enzymatic preparation at 30 degrees C for 24 h. This resulted in a final concentration of free acids eight times higher than the initial value. The bioreactors operated at 30 degrees C with hydraulic retention times of 12 h (HAIB) and 20 h (UASB) for a period of 430 days, and had high chemical oxygen demand (COD) removal efficiencies (around 90%) when fed with pre-hydrolyzed wastewater. There was, however, an increase in the effluent oil and grease concentration (from values as low as 17 mg/L to values above 150 mg/L in the UASB bioreactor, and from 38-242 mg/L in the HAIB bioreactor), and oil and grease accumulation in the biomass throughout the operational period (the oil and grease content reached 1.7 times that found in the inoculum of the UASB bioreactor). The HAIB bioreactor gave better results because the support for biomass immobilization acted as a filter, retaining oil and grease at the entry of the bioreactor. The molecular analysis of the Bacteria and Archaea domains revealed significant differences in the microbial profiles in experiments conducted with and without the pre-hydrolysis step. The differences observed in the overall parameters could be related to the microbial diversity of the anaerobic sludge.

Performance and dynamic characteristics of microbial communities in an internal circulation reactor for treating brewery wastewater.

A laboratory-scale internal circulation (IC) anaerobic reactor fed with brewery wastewater was operated at 35 degrees C + 1 degrees C. The influent was pumped into the bottom of the IC reactor by a pulse pump, whereas the effluent was drawn from the upper outlet and allowed to flow into the effluent tank. The biogas volume was recorded using a gas container connected to a biogas metre. The results indicated that the maximum organic loading rate (OLR) of the IC reactor was 19.5 kg chemical oxygen demand (COD)/m3/day; at which point, the dominant archaeal populations found in the sludge using the polymerase chain reaction with denaturing gradient gel electrophoresis were Methanosaeta species. The COD removal efficiencies of the reactor exceeded 85%, with a maximum specific methane production rate of 210 mL CH4/g volatile suspended solids (VSS)/day and a coenzyme F420 content of 0.16 micromol/g VSS, respectively. The main archaeal species in the sludge samples at different OLRs varied greatly, as compared with the organisms in the inoculated sludge. The dominant archaeal species in the treated sludge at low OLRs were Methanosarcina species, whereas those at high OLRs were Methanosaeta species.

Aceticlastic and NaCl-requiring methanogen "Methanosaeta pelagica" sp. nov., isolated from marine tidal flat sediment.

Among methanogens, only 2 genera, Methanosaeta and Methanosarcina, are known to contribute to methanogenesis from acetate, and Methanosaeta is a specialist that uses acetate specifically. However, Methanosaeta strains so far have mainly been isolated from anaerobic digesters, despite the fact that it is widespread, not only in anaerobic methanogenic reactors and freshwater environments, but also in marine environments, based upon extensive 16S rRNA gene-cloning analyses. In this study, we isolated an aceticlastic methanogen, designated strain 03d30q(T), from a tidal flat sediment. Phylogenetic analyses based on 16S rRNA and mcrA genes revealed that the isolate belongs to the genus Methanosaeta. Unlike the other known Methanosaeta species, this isolate grows at Na(+) concentrations of 0.20 to 0.80 M, with an optimum concentration of 0.28 M. Quantitative estimation using real-time PCR detected the 16S rRNA gene of the genus Methanosaeta in the marine sediment, and relative abundance ranged from 3.9% to 11.8% of the total archaeal 16S rRNA genes. In addition, the number of Methanosaeta organisms increased with increasing depth and was much higher than that of Methanosarcina organisms, suggesting that aceticlastic methanogens contribute to acetate metabolism to a greater extent than previously thought in marine environments, where sulfate-reducing acetate oxidation prevails. This is the first report on marine Methanosaeta species, and based on phylogenetic and characteristic studies, the name "Methanosaeta pelagica" sp. nov. is proposed for this novel species, with type strain 03d30q.

Genotypic distribution of a specialist model microorganism, Methanosaeta, along an estuarine gradient: does metabolic restriction limit niche differentiation potential?

A reductionist ecological approach of using a model genus was adopted in order to understand how microbial community structure is driven by metabolic properties. The distribution along an estuarine gradient of the highly specialised genus Methanosaeta was investigated and compared to the previously determined distribution of the more metabolically flexible Desulfobulbus. Methanosaeta genotypic distribution along the Colne estuary (Essex, UK) was determined by DNA- and RNA-based denaturing gradient gel electrophoresis and 16S rRNA gene sequence analyses. Methanosaeta distribution was monotonic, with a consistently diverse community and no apparent niche partitioning either in DNA or RNA analyses. This distribution pattern contrasts markedly with the previously described niche partitioning and sympatric differentiation of the model generalist, Desulfobulbus. To explain this difference, it is hypothesised that Methanosaeta's strict metabolic needs limit its adaptation potential, thus populations do not partition into spatially distinct groups and so do not appear to be constrained by gross environmental factors such as salinity. Thus, at least for these two model genera, it appears that metabolic flexibility may be an important factor in spatial distribution and this may be applicable to other microbes.

Analyses of n-alkanes degrading community dynamics of a high-temperature methanogenic consortium enriched from production water of a petroleum reservoir by a combination of molecular techniques.

Despite the knowledge on anaerobic degradation of hydrocarbons and signature metabolites in the oil reservoirs, little is known about the functioning microbes and the related biochemical pathways involved, especially about the methanogenic communities. In the present study, a methanogenic consortium enriched from high-temperature oil reservoir production water and incubated at 55 °C with a mixture of long chain n-alkanes (C(15)-C(20)) as the sole carbon and energy sources was characterized. Biodegradation of n-alkanes was observed as methane production in the alkanes-amended methanogenic enrichment reached 141.47 µmol above the controls after 749 days of incubation, corresponding to 17 % of the theoretical total. GC-MS analysis confirmed the presence of putative downstream metabolites probably from the anaerobic biodegradation of n-alkanes and indicating an incomplete conversion of the n-alkanes to methane. Enrichment cultures taken at different incubation times were subjected to microbial community analysis. Both 16S rRNA gene clone libraries and DGGE profiles showed that alkanes-degrading community was dynamic during incubation. The dominant bacterial species in the enrichment cultures were affiliated with Firmicutes members clustering with thermophilic syntrophic bacteria of the genera Moorella sp. and Gelria sp. Other represented within the bacterial community were members of the Leptospiraceae, Thermodesulfobiaceae, Thermotogaceae, Chloroflexi, Bacteroidetes and Candidate Division OP1. The archaeal community was predominantly represented by members of the phyla Crenarchaeota and Euryarchaeota. Corresponding sequences within the Euryarchaeota were associated with methanogens clustering with orders Methanomicrobiales, Methanosarcinales and Methanobacteriales. On the other hand, PCR amplification for detection of functional genes encoding the alkylsuccinate synthase α-subunit (assA) was positive in the enrichment cultures. Moreover, the appearance of a new assA gene sequence identified in day 749 supported the establishment of a functioning microbial species in the enrichment. Our results indicate that n-alkanes are converted to methane slowly by a microbial community enriched from oilfield production water and fumarate addition is most likely the initial activation step of n-alkanes degradation under thermophilic methanogenic conditions.

Acetogens and acetoclastic methanosarcinales govern methane formation in abandoned coal mines.

In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [(13)C]acetate and H(2)-(13)CO(2), were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [(13)C]methane was linked to acetoclastic methanogenesis in both the [(13)C]acetate- and the H(2)-(13)CO(2)-amended cultures of coal and timber. H(2)-(13)CO(2) was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H(2) conditions found in coal mines.

Diversity and quantitative analysis of Archaea in aggressive periodontitis and periodontally healthy subjects.

AIM: To investigate the diversity, levels and proportions of Archaea in the subgingival biofilm of generalized aggressive periodontitis (GAgP; n=30) and periodontally healthy (PH; n=30) subjects. MATERIALS AND METHODS: Diversity was determined by sequencing archaeal 16S rRNA gene libraries from 20 samples (10/group). The levels and proportions of Archaea were analysed by quantitative PCR (qPCR) in four and two samples/subject in GAgP and PH groups, respectively. RESULTS: Archaea were detected in 27/28 subjects and 68% of the sites of the GAgP group, and in 26/30 subjects and 58.3% sites of the PH group. Methanobrevibacter oralis was found in all 20 samples studied, Methanobacterium curvum/congolense in three GAgP and six PH samples, and Methanosarcina mazeii in four samples from each group. The levels and proportions of Archaea were higher in GAgP than in PH, whereas no differences were observed between the two probing depth category sites from the GAgP group. CONCLUSION: Archaea were frequently found in subjects with periodontal health and GAgP, especially M. oralis. However, the higher levels and proportions (Archaea/total prokaryotes) of this domain observed in GAgP in comparison with PH subjects indicate a possible role of some of these microorganisms as an environmental modifier in GAgP.

Phylogenetic comparison of the methanogenic communities from an acidic, oligotrophic fen and an anaerobic digester treating municipal wastewater sludge.

Methanogens play a critical role in the decomposition of organics under anaerobic conditions. The methanogenic consortia in saturated wetland soils are often subjected to large temperature fluctuations and acidic conditions, imposing a selective pressure for psychro- and acidotolerant community members; however, methanogenic communities in engineered digesters are frequently maintained within a narrow range of mesophilic and circumneutral conditions to retain system stability. To investigate the hypothesis that these two disparate environments have distinct methanogenic communities, the methanogens in an oligotrophic acidic fen and a mesophilic anaerobic digester treating municipal wastewater sludge were characterized by creating clone libraries for the 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes. A quantitative framework was developed to assess the differences between these two communities by calculating the average sequence similarity for 16S rRNA genes and mcrA within a genus and family using sequences of isolated and characterized methanogens within the approved methanogen taxonomy. The average sequence similarities for 16S rRNA genes within a genus and family were 96.0 and 93.5%, respectively, and the average sequence similarities for mcrA within a genus and family were 88.9 and 79%, respectively. The clone libraries of the bog and digester environments showed no overlap at the species level and almost no overlap at the family level. Both libraries were dominated by clones related to uncultured methanogen groups within the Methanomicrobiales, although members of the Methanosarcinales and Methanobacteriales were also found in both libraries. Diversity indices for the 16S rRNA gene library of the bog and both mcrA libraries were similar, but these indices indicated much lower diversity in the 16S digester library than in the other three libraries.

Dynamics of the methanogenic archaeal community during plant residue decomposition in an anoxic rice field soil.

Incorporation of plant residues strongly enhances the methane production and emission from flooded rice fields. Temperature and residue type are important factors that regulate residue decomposition and CH(4) production. However, the response of the methanogenic archaeal community to these factors in rice field soil is not well understood. In the present experiment, the structure of the archaeal community was determined during the decomposition of rice root and straw residues in anoxic rice field soil incubated at three temperatures (15 degrees C, 30 degrees C, and 45 degrees C). More CH(4) was produced in the straw treatment than root treatment. Increasing the temperature from 15 degrees C to 45 degrees C enhanced CH(4) production. Terminal restriction fragment length polymorphism analyses in combination with cloning and sequencing of 16S rRNA genes showed that Methanosarcinaceae developed early in the incubations, whereas Methanosaetaceae became more abundant in the later stages. Methanosarcinaceae and Methanosaetaceae seemed to be better adapted at 15 degrees C and 30 degrees C, respectively, while the thermophilic Methanobacteriales and rice cluster I methanogens were significantly enhanced at 45 degrees C. Straw residues promoted the growth of Methanosarcinaceae, whereas the root residues favored Methanosaetaceae. In conclusion, our study revealed a highly dynamic structure of the methanogenic archaeal community during plant residue decomposition. The in situ concentration of acetate (and possibly of H(2)) seems to be the key factor that regulates the shift of methanogenic community.