Comparison of enteric methane yield and diversity of ruminal methanogens in cattle and buffaloes fed on the same diet.

An in vivo study was conducted to compare the enteric methane emissions and diversity of ruminal methanogens in cattle and buffaloes kept in the same environment and fed on the same diet. Six cattle and six buffaloes were fed on a similar diet comprising Napier (Pennisetum purpureum) green grass and concentrate in 70:30. After 90 days of feeding, the daily enteric methane emissions were quantified by using the SF6 technique and ruminal fluid samples from animals were collected for the diversity analysis. The daily enteric methane emissions were significantly greater in cattle as compared to buffaloes; however, methane yields were not different between the two species. Methanogens were ranked at different taxonomic levels against the Rumen and Intestinal Methanogen-Database. The archaeal communities in both host species were dominated by the phylum Euryarchaeota; however, Crenarchaeota represented <1% of the total archaea. Methanogens affiliated with Methanobacteriales were most prominent and their proportion did not differ between the two hosts. Methanomicrobiales and Methanomassillicoccales constituted the second largest group of methanogens in cattle and buffaloes, respectively. Methanocellales (Methanocella arvoryza) were exclusively detected in the buffaloes. At the species level, Methanobrevibacter gottschalkii had the highest abundance (55-57%) in both the host species. The relative abundance of Methanobrevibacter wolinii between the two hosts differed significantly. Methanosarcinales, the acetoclastic methanogens were significantly greater in cattle than the buffaloes. It is concluded that the ruminal methane yield in cattle and buffaloes fed on the same diet did not differ. With the diet used in this study, there was a limited influence (<3.5%) of the host on the structure of the ruminal archaea community at the species level. Therefore, the methane mitigation strategies developed in either of the hosts should be effective in the other. Further studies are warranted to reveal the conjunctive effect of diet and geographical locations with the host on ruminal archaea community composition.

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.

The nomenclatural type of the genus Methanocorpusculum Zellner et al. 1988 and the selection of the correct name.

A recent Request for an Opinion has raised the issue of the inter-relationship between Methanocorpusculum parvum Zellner et al. 1988, the type species of the genus Methanocorpusculum Zellner et al. 1988 as defined at the time of valid publication of the genus name and the subsequent recognition of Methanocorpusculum aggregans (Ollivier et al., 1985) Xun et al.1989 as an earlier heterotypic synonym. Examination of the relevant literature indicates that there are a number of misunderstandings that have arisen. In particular misinterpretation of Rule 15 of the International Code of Nomenclature of Prokaryotes continues to be a source of confusion. Additional problems centre on whether the nomenclatural type of a taxon continues to be the nomenclatural type even if that name is not treated as the correct name and would not appear in a list of names in a given classification. It would be appropriate to clarify these issues.

Methanogenic paraffin degradation proceeds via alkane addition to fumarate by 'Smithella' spp. mediated by a syntrophic coupling with hydrogenotrophic methanogens.

Anaerobic microbial biodegradation of recalcitrant, water-insoluble substrates, such as paraffins, presents unique metabolic challenges. To elucidate this process, a methanogenic consortium capable of mineralizing long-chain n-paraffins (C28 -C50 ) was enriched from San Diego Bay sediment. Analysis of 16S rRNA genes indicated the dominance of Syntrophobacterales (43%) and Methanomicrobiales (26%). Metagenomic sequencing allowed draft genome assembly of dominant uncultivated community members belonging to the bacterial genus Smithella and the archaeal genera Methanoculleus and Methanosaeta. Five contigs encoding homologs of the catalytic subunit of alkylsuccinate synthase (assA) were detected. Additionally, mRNA transcripts for these genes, including a homolog binned within the 'Smithella' sp. SDB genome scaffold, were detected via RT-PCR, implying that paraffins are activated via 'fumarate addition'. Metabolic reconstruction and comparison with genome scaffolds of uncultivated n-alkane degrading 'Smithella' spp. are consistent with the hypothesis that syntrophically growing 'Smithella' spp. may achieve reverse electron transfer by coupling the reoxidation of ETFred to a membrane-bound FeS oxidoreductase functioning as an ETF:menaquinone oxidoreductase. Subsequent electron transfer could proceed via a periplasmic formate dehydrogenase and/or hydrogenase, allowing energetic coupling to hydrogenotrophic methanogens such as Methanoculleus. Ultimately, these data provide fundamental insight into the energy conservation mechanisms that dictate interspecies interactions salient to methanogenic alkane mineralization.

Methanogen communities in a municipal landfill complex in China.

Landfills are significant global sources of atmospheric methane, but little is known about the ecology and community structure of methanogens in these sites. Here, we investigated the methanogen community based on methyl coenzyme M reductase A gene amplicons in the vertical profiles of three different sites at a municipal landfill complex in China. Links between methanogen communities and refuse properties were explored using multivariate analysis. Clone library results showed that most clones (92%) were related to the hydrogenotrophic methanogens, Methanomicrobiales. Almost all of the Methanomicrobiales clones retrieved in this study are members of the genus Methanoculleus Eight clones were affiliated with the genus Methanofollis The remaining clones were clustered within the genus Methanosarcina Terminal restriction fragment length polymorphism profiles showed that the landfill was predominated by 22 taxa, making up 69%-96% of the community. Of these, a single taxon comprised 36%-65% of the communities across all sites and depths. Principal components analysis separated the methanogen community into three groups, irrespective of site or depth. Redundancy analysis suggested that total phosphorus and pH play roles in structuring methanogen communities in landfills.

Changes in microbial community structures due to varying operational conditions in the anaerobic digestion of oxytetracycline-medicated cow manure.

Management of manure containing veterinary antibiotics is a major concern in anaerobic treatment systems because of their possible adverse effects on microbial communities. Therefore, the aim of study was to investigate how oxytetracycline (OTC) influences bacteria and acetoclastic and hydrogenotrophic methanogens under varying operational conditions in OTC-medicated and non-medicated anaerobic cow manure digesters. Concentrations of OTC and its metabolites throughout the anaerobic digestion were determined using ultraviolet-high-performance liquid chromatography (UV-HPLC) and tandem liquid chromatography-mass spectrometry (LC/MS/MS), respectively. Fluorescent in situ hybridization, denaturing gradient gel electrophoresis, cloning, and sequencing analyses were used to monitor changes in microbial community structures. According to the results of analytical and molecular approaches, operating conditions highly influence active microbial community dynamics and associate with biogas production and elimination of OTC and its metabolites during anaerobic digestion of cow manure in the presence of an average initial concentration of 2.2 mg OTC/L. The impact of operating conditions has a drastic effect on acetoclastic methanogens than hydrogenotrophic methanogens and bacteria.

Abundance and potential metabolic activity of methanogens in well-aerated forest and grassland soils of an alpine region.

Although methanogens were recently discovered to occur in aerated soils, alpine regions have not been extensively studied for their presence so far. Here, the abundance of archaea and the methanogenic guilds Methanosarcinales, Methanococcales, Methanobacteriales, Methanomicrobiales and Methanocella spp. was studied at 16 coniferous forest and 14 grassland sites located at the montane and subalpine belts of the Northern Limestone Alps (calcareous) and the Austrian Central Alps (siliceous) using quantitative real-time PCR. Abundance of archaea, methanogens and the methanogenic potentials were significantly higher in grasslands than in forests. Furthermore, methanogenic potentials of calcareous soils were higher due to pH. Methanococcales, Methanomicrobiales and Methanocella spp. were detected in all collected samples, which indicates that they are autochthonous, while Methanobacteriales were absent from 4 out of 16 forest soils. Methanosarcinales were absent from 10 out of 16 forest soils and 2 out of 14 grassland soils. Nevertheless, together with Methanococcales they represented the majority of the 16S rRNA gene copies quantified from the grassland soils. Contrarily, forest soils were clearly dominated by Methanococcales. Our results indicate a higher diversity of methanogens in well-aerated soils than previously believed and that pH mainly influences their abundances and activities.

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.

Identification and Detection of Prokaryotic Symbionts in the Ciliate Metopus from Anaerobic Granular Sludge.

The aim of the present study was to investigate the prokaryotic community structure of the anaerobic ciliate, Metopus sp. using rRNA sequencing, fluorescence in situ hybridization (FISH), and transmission electron microscopy (TEM). Metopus sp. was physically separated from anaerobic granular sludge in a domestic wastewater treatment plant and anoxically cultivated for 7 d. 16S rRNA gene sequences from the prokaryotes Methanoregula boonei and Clostridium aminobutyricum were abundantly detected in Metopus ciliates. The FISH analysis using the oligonucleotide probes Mg1200b and Cla568 demonstrated that these prokaryotes were localized within Metopus cells. These results identify M. boonei- and C. aminobutyricum-like prokaryotes as novel endosymbionts of Metopus ciliates.

Methanosalsum natronophilum sp. nov., and Methanocalculus alkaliphilus sp. nov., haloalkaliphilic methanogens from hypersaline soda lakes.

Two groups of haloalkaliphilic methanogenic archaea were dominating in enrichments from hypersaline soda lake sediments at pH 10. At moderate salt concentrations with formate or H2 as electron donor, methanogens belonging to the genus Methanocalculus were enriched, while at high salt concentrations with methylated substrates, a group related to Methanosalsum zhilinae was dominating. For both groups, several pure cultures were obtained including the type strains AMF2T for the Methanocalculus group and AME2T for the Methanosalsum group. The Methanocalculus group is characterized by lithoheterotrophic growth with either formate (preferable substrate) or H2 at moderate salinity up to 1.5-2 M total Na+ and obligate alkaliphilic growth with an optimum at pH 9.5. According to phylogenetic analysis, the group also includes closely related strains isolated previously from the low-salt alkaline Lonar Lake. The novel Methanosalsum group is characterized by high salt tolerance (up to 3.5 M total Na+) and obligate alkaliphilic growth with an optimum at pH 9.5. It has a typical methylotrophic substrate profile, utilizing methanol, methylamines and dimethyl sulfide (at low concentrations) as methanogenic substrates. On the basis of physiological and phylogenetic data, it is proposed that the two groups of soda lake methanogenic isolates are assigned into two novel species, Methanocalculus alkaliphilus sp. nov. (type strain AMF2T = DSM 24457T = UNIQEM U859T) and Methanosalsum natronophilum sp. nov. (type strain AME2T = DSM 24634T = NBRC 110091T).

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.

The correct name of the type species of the genus Methanocorpusculum. Request for an Opinion.

We propose that Methanocorpusculum parvum with type strain XII must remain the nomenclatural type of the genus Methanocorpusculum, in spite of the fact that the description of Methanogenium aggregans with type strain MSt, later transferred to the genus Methanocorpusculum as comb. nov. and then proposed as a heterotypic synonym of Methanocorpusculum parvum, was published before Methanocorpusculum parvum strain XII.

Analysis of methanotroph community structure using a pmoA-based microarray.

The analysis of methanotroph community composition is relevant to studies of methane oxidation in a number of environments where methane is a significant carbon source. The development and application of a microarray targeting the particulate methane monooxygenase gene (pmoA) have allowed a high-throughput, semiquantitative analysis of the major methanotroph groups in a number of different environments. Here we describe the use of a pmoA-based short oligo array for the analysis of methanotroph populations in sediment samples. The method is suitable for analysis of any type of environmental sample from which DNA can be extracted.

Characterization of the methanogen community in a household anaerobic digester fed with swine manure in China.

Household anaerobic digesters have been installed across rural China for biogas production, but information on methanogen community structure in these small biogas units is sparsely available. By creating clone libraries for 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes, we investigated the methanogenic consortia in a household biogas digester treating swine manure. Operational taxonomic units (OTUs) were defined by comparative sequence analysis, seven OTUs were identified in the 16S rRNA gene library, and ten OTUs were identified in the mcrA gene library. Both libraries were dominated by clones highly related to the type strain Methanocorpusculum labreanum Z, 64.0 % for 16S rRNA gene clones and 64.3 % for mcrA gene clones. Additionally, gas chromatography assays showed that formic acid was 84.54 % of the total volatile fatty acids and methane was 57.20 % of the biogas composition. Our results may help further isolation and characterization of methanogenic starter strains for industrial biogas production.

Evidence for microbial carbon and sulfur cycling in deeply buried ridge flank basalt.

Sediment-covered basalt on the flanks of mid-ocean ridges constitutes most of Earth's oceanic crust, but the composition and metabolic function of its microbial ecosystem are largely unknown. By drilling into 3.5-million-year-old subseafloor basalt, we demonstrated the presence of methane- and sulfur-cycling microbes on the eastern flank of the Juan de Fuca Ridge. Depth horizons with functional genes indicative of methane-cycling and sulfate-reducing microorganisms are enriched in solid-phase sulfur and total organic carbon, host δ(13)C- and δ(34)S-isotopic values with a biological imprint, and show clear signs of microbial activity when incubated in the laboratory. Downcore changes in carbon and sulfur cycling show discrete geochemical intervals with chemoautotrophic δ(13)C signatures locally attenuated by heterotrophic metabolism.

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.

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.

Feasibility tests for treating shampoo and hair colorant wastewaters using anaerobic processes.

Wastes from the personal care product (PCP) industry are often high in biodegradable carbon, which makes them amenable to aerobic biological treatment, although process costs are usually high due to aeration inefficiencies, high electricity demand and production of large amounts of sludge. As such, anaerobic treatment technologies are being considered to lower net energy costs by reducing air use and increasing methane production. To assess the amenability of PCP wastes to anaerobic treatment, methane yields and rates were quantified in different anaerobic reactors treating typical PCP wastes, including wastes from shampoo and hair colorant products. Overall, shampoo wastes were more amenable to methanogenesis with almost double the methane yields compared with colour wastes. To assess relevant microbial guilds, qPCR was performed on reactor biomass samples. Methanosaetaceae abundances were always significantly higher than Methanosarcinaceae and Methanomicrobiales abundances (P < 0.05), and did not differ significantly between waste types. Although colour wastes were less amenable to anaerobic treatment than shampoo wastes, differences cannot be explained by relative microbial abundances and probably result from the presence of inhibiting compounds in hair colorants (e.g., oxidants) at higher levels. Results showed that anaerobic technologies have great potential for treating PCP wastes, but additional work is needed to establish the basis of elevated methane yields and inhibition, especially when colorant wastes are present.

Molecular diversity of methanogens and identification of Methanolobus sp. as active methylotrophic Archaea in Lonar crater lake sediments.

Soda lakes constitute extreme aquatic ecosystems with remarkably high primary productivity rates, but information on the diversity and activity of methanogens in such environments is sparse. Using 16S rRNA and functional genes, we investigated the diversity of methanogens in the sediments of Lonar Lake, a unique saline and alkaline ecosystem formed by meteorite impact in the Deccan basalts. Although domain and phylum level 16S rRNA gene libraries were dominated by phylotypes related to Halobacteriales, sequences related to potentially novel Archaea within the orders Methanosarcinales and Methanomicrobiales were obtained together with a significant fraction of sequences representing uncultivated Euryarchaeota [Correction added after online publication 16 April 2012: orders 'Methanosarcina and Methanomicrobiaceae' changed to 'Methanosarcinales and Methanomicrobiales']. To identify the active methylotrophic Archaea involved in methanogenesis, mRNA transcripts of mcrA were retrieved from methanol consuming and methane emitting sediment microcosms at two different time points. Reverse-transcription PCR, qPCR, DGGE fingerprint, and clone library analysis showed that the active Archaea were closely related to Methanolobus oregonensis. To our knowledge, this is the first study identifying active methylotrophic methanogens in such an environment.

Field and laboratory studies on the bioconversion of coal to methane in the San Juan Basin.

The bioconversion of coal to methane in the San Juan Basin, New Mexico, was investigated. Production waters were analyzed via enrichment studies, metabolite-profiling, and culture-independent methods. Analysis of 16S rRNA gene sequences indicated the presence of methanogens potentially capable of acetoclastic, hydrogenotrophic, and methylotrophic metabolisms, predominantly belonging to the Methanosarcinales and Methanomicrobiales. Incubations of produced water and coal readily produced methane, but there was no correlation between the thermal maturity and methanogenesis. Coal methanogenesis was greater when samples with a greater richness of Firmicutes were utilized. A greater archaeal diversity was observed in the presence of several aromatic and short-chain fatty acid metabolites. Incubations amended with lactate, hydrogen, formate, and short-chain alcohols produced methane above un-amended controls. Methanogenesis from acetate was not observed. Metabolite profiling showed the widespread occurrence of putative aromatic ring intermediates including benzoate, toluic acids, phthalic acids, and cresols. The detection of saturated and unsaturated alkylsuccinic acids indicated n-alkane and cyclic alkane/alkene metabolism. Microarray analysis complemented observations based on hybridization to functional genes related to the anaerobic metabolism of aromatic and aliphatic substrates. These data suggest that coal methanogenesis is unlikely to be limited by methanogen biomass, but rather the activation and degradation of coal constituents.