Diversity and community of methanogens in the large intestine of finishing pigs.

BACKGROUND: Methane emissions from pigs account for 10% of total methane production from livestock in China. Methane emissions not only contribute to global warming, as it has 25 times the global warming potential (GWP) of CO2, but also represent approximately 0.1~3.3% of digestive energy loss. Methanogens also play an important role in maintaining the balance of the gut microbiome. The large intestines are the main habitat for the microbiome in pigs. Thus, to better understand the mechanism of methane production and mitigation, generic-specific and physio-ecological characteristics (including redox potential (Eh), pH and volatile fatty acids (VFAs)) and methanogens in the large intestine of pig were studied in this paper. Thirty DLY finishing pigs with the same diet and feeding conditions were selected for this experiment. RESULT: A total of 219 clones were examined using the methyl coenzyme reductase subunit A gene (mcrA) and assigned to 43 operational taxonomic units (OTUs) based on a 97% species-level identity criterion. The family Methanobacteriaceae was the dominant methanogen in colonic digesta of finishing pigs, accounting for approximately 70.6% of the identified methanogens, and comprised mainly the genera Methanobrevibacter (57%) and Methanosphaera (14%). The order Methanomassiliicoccales, classified as an uncultured taxonomy, accounted for 15.07%. The methanogenic archaeon WGK1 and unclassified Methanomicrobiales belonging to the order of Methanomicrobiales accounted for 4.57 and 1.37%, respectively. The Eh was negative and within the range - 297.00~423.00 mV and the pH was within the range 5.04~6.97 in the large intestine. The populations of total methanogens and Methanobacteriales were stable in different parts of the large intestine according to real-time PCR. CONCLUSION: The major methanogen in the large intestine of finishing pigs was Methanobrevibacter. The seventh order Methanomassiliicoccales and species Methanosphaera stadtmanae present in the large intestine of pigs might contribute to the transfer of hydrogen and fewer methane emissions. The redox potential (Eh) was higher in the large intestine of finishing pigs, which had a positive correlation with the population of Methanobacteriale.

Comparative Genomic Analysis of Members of the Genera Methanosphaera and Methanobrevibacter Reveals Distinct Clades with Specific Potential Metabolic Functions.

Methanobrevibacter and Methanosphaera species represent some of the most prevalent methanogenic archaea in the gastrointestinal tract of animals and humans and play an important role in this environment. The aim of this study was to identify genomic features that are shared or specific for members of each genus with a special emphasis of the analysis on the assimilation of nitrogen and acetate and the utilization of methanol and ethanol for methanogenesis. Here, draft genome sequences of Methanobrevibacter thaueri strain DSM 11995T, Methanobrevibacter woesei strain DSM 11979T, and Methanosphaera cuniculi strain 4103T are reported and compared to those of 16 other Methanobrevibacter and Methanosphaera genomes, including genomes of the 13 currently available types of strains of the two genera. The comparative genome analyses indicate that among other genes, the absence of molybdopterin cofactor biosynthesis is conserved in Methanosphaera species but reveals also that the three species share a core set of more than 300 genes that distinguishes the genus Methanosphaera from the genus Methanobrevibacter. Multilocus sequence analysis shows that the genus Methanobrevibacter can be subdivided into clades, potentially new genera, which may display characteristic specific metabolic features. These features include not only the potential ability of nitrogen fixation and acetate assimilation in a clade comprised of Methanobrevibacter species from the termite gut and Methanobrevibacter arboriphilus strains but also the potential capability to utilize ethanol and methanol in a clade comprising Methanobrevibacter wolinii strain DSM 11976T, Mbb. sp. AbM4, and Mbb. boviskoreani strain DSM 25824T.

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD: A TOOL FOR REDOX REGULATION IN AN ANAEROBE.

A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavin-containing NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F420 (F420H2), a stronger reductant with a mid-point redox potential (E'0) of -360 mV; E'0 of NAD(P)H is -320 mV. Because F420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F420H2 to thioredoxin via protein-bound flavin; Km values for thioredoxin and F420H2 were 6.3 and 28.6 µm, respectively. The E'0 of DFTR-bound flavin was approximately -389 mV, making electron transfer from NAD(P)H or F420H2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F420/F420H2 pair could be as low as -425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F420-dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F420 systems involved in microbial pathogenesis and antibiotic production.

Influence of periparturient and postpartum diets on rumen methanogen communities in three breeds of primiparous dairy cows.

BACKGROUND: Enteric methane from rumen methanogens is responsible for 25.9 % of total methane emissions in the United States. Rumen methanogens also contribute to decreased animal feed efficiency. For methane mitigation strategies to be successful, it is important to establish which factors influence the rumen methanogen community and rumen volatile fatty acids (VFA). In the present study, we used next-generation sequencing to determine if dairy breed and/or days in milk (DIM) (high-fiber periparturient versus high-starch postpartum diets) affect the rumen environment and methanogen community of primiparous Holstein, Jersey, and Holstein-Jersey crossbreeds. RESULTS: When the 16S rRNA gene sequences were processed and assigned to operational taxonomic units (OTU), a core methanogen community was identified, consisting of Methanobrevibacter (Mbr.) smithii, Mbr. thaueri, Mbr. ruminantium, and Mbr. millerae. The 16S rRNA gene sequence reads clustered at 3 DIM, but not by breed. At 3 DIM, the mean % abundance of Mbr. thaueri was lower in Jerseys (26.9 %) and higher in Holsteins (30.7 %) and Holstein-Jersey crossbreeds (30.3 %) (P < 0.001). The molar concentrations of total VFA were higher at 3 DIM than at 93, 183, and 273 DIM, whereas the molar proportions of propionate were increased at 3 and 93 DIM, relative to 183 and 273 DIM. Rumen methanogen densities, distributions of the Mbr. species, and VFA molar proportions did not differ by breed. CONCLUSIONS: The data from the present study suggest that a core methanogen community is present among dairy breeds, through out a lactation. Furthermore, the methanogen communities were more influenced by DIM and the breed by DIM interactions than breed differences.

Archaeal community in a human-disturbed watershed in southeast China: diversity, distribution, and responses to environmental changes.

The response of freshwater bacterial community to anthropogenic disturbance has been well documented, yet the studies of freshwater archaeal community are rare, especially in lotic environments. Here, we investigated planktonic and benthic archaeal communities in a human-perturbed watershed (Jiulong River Watershed, JRW) of southeast China by using Illumina 16S ribosomal RNA gene amplicon sequencing. The results of taxonomic assignments indicated that SAGMGC-1, Methanobacteriaceae, Methanospirillaceae, and Methanoregulaceae were the four most abundant families in surface waters, accounting for 12.65, 23.21, 18.58 and 10.97 % of planktonic communities, whereas Nitrososphaeraceae and Miscellaneous Crenarchaeotic Group occupied more than 49 % of benthic communities. The compositions of archaeal communities and populations in waters and sediments were significantly different from each other. Remarkably, the detection frequencies of families Methanobacteriaceae and Methanospirillaceae, and genera Methanobrevibacter and Methanosphaera in planktonic communities correlated strongly with bacterial fecal indicator, suggesting some parts of methanogenic Archaea may come from fecal contamination. Because soluble reactive phosphorus (SRP) and the ratio of dissolved inorganic nitrogen to SRP instead of nitrogen nutrients showed significant correlation with several planktonic Nitrosopumilus- and Nitrosotalea-like OTUs, Thaumarchaeota may play an unexplored role in biogeochemical cycling of river phosphorus. Multivariate statistical analyses revealed that the variation of α-diversity of planktonic archaeal community was best explained by water temperature, whereas nutrient concentrations and stoichiometry were the significant drivers of ß-diversity of planktonic and benthic communities. Taken together, these results demonstrate that the structure of archaeal communities in the JRW is sensitive to anthropogenic disturbances caused by riparian human activities.

Rumen methanogenic genotypes differ in abundance according to host residual feed intake phenotype and diet type.

Methane is an undesirable end product of rumen fermentative activity because of associated environmental impacts and reduced host feed efficiency. Our study characterized the rumen microbial methanogenic community in beef cattle divergently selected for phenotypic residual feed intake (RFI) while offered a high-forage (HF) diet followed by a low-forage (LF) diet. Rumen fluid was collected from 14 high-RFI (HRFI) and 14 low-RFI (LRFI) animals at the end of both dietary periods. 16S rRNA gene clone libraries were used, and methanogen-specific tag-encoded pyrosequencing was carried out on the samples. We found that Methanobrevibacter spp. are the dominant methanogens in the rumen, with Methanobrevibacter smithii being the most abundant species. Differences in the abundance of Methanobrevibacter smithii and Methanosphaera stadtmanae genotypes were detected in the rumen of animals offered the LF compared to the HF diet while the abundance of Methanobrevibacter smithii genotypes was different between HRFI and LRFI animals irrespective of diet. Our results demonstrate that while a core group of methanogen operational taxonomic units (OTUs) exist across diet and phenotype, significant differences were observed in the distribution of genotypes within those OTUs. These changes in genotype abundance may contribute to the observed differences in methane emissions between efficient and inefficient animals.

Methanothermobacter tenebrarum sp. nov., a hydrogenotrophic, thermophilic methanogen isolated from gas-associated formation water of a natural gas field.

A thermophilic and hydrogenotrophic methanogen, strain RMAS(T), was isolated from gas-associated formation water of a gas-producing well in a natural gas field in Japan. Strain RMAS(T) grew solely on H(2)/CO(2) but required Casamino acids, tryptone, yeast extract or vitamins for growth. Growth of strain RMAS(T) was stimulated by acetate. Cells were non-motile, straight rods (0.5×3.5-10.5 µm) and occurred singly or in pairs. Bundles of fimbriae occurred at both poles of cells and the cell wall was thick (approximately 21 nm, as revealed by ultrathin section electron microscopy). Strain RMAS(T) grew at 45-80 °C (optimum, 70 °C), at pH 5.8-8.7 (optimum, pH 6.9-7.7) and with 0.001-20 g NaCl l(-1) (optimum, 2.5 g NaCl l(-1)). Phylogenetic analysis revealed that Methanothermobacter thermautotrophicus ΔH(T) was most closely related to the isolate (95.7 % 16S rRNA gene sequence similarity). On the basis of morphological, phenotypic and phylogenetic characteristics, it is clear that strain RMAS(T) represents a novel species of the genus Methanothermobacter, for which we propose the name Methanothermobacter tenebrarum sp. nov. The type strain is RMAS(T) ( = DSM 23052(T) = JCM 16532(T) = NBRC 106236(T)).

Cultivation-independent analysis of archaeal and bacterial communities of the formation water in an Indian coal bed to enhance biotransformation of coal into methane.

Biogenic origin of the significant proportion of coal bed methane has indicated the role of microbial communities in methanogenesis. By using cultivation-independent approach, we have analysed the archaeal and bacterial community present in the formation water of an Indian coal bed at 600-700 m depth to understand their role in methanogenesis. Presence of methanogens in the formation water was inferred by epifluorescence microscopy and PCR amplification of mcrA gene. Archaeal 16S rRNA gene clone library from the formation water metagenome was dominated by methanogens showing similarity to Methanobacterium, Methanothermobacter and Methanolinea whereas the clones of bacterial 16S rRNA gene library were closely related to Azonexus, Azospira, Dechloromonas and Thauera. Thus, microbial community of the formation water consisted of predominantly hydrogenotrophic methanogens and the proteobacteria capable of nitrogen fixation, nitrate reduction and polyaromatic compound degradation. Methanogenic potential of the microbial community present in the formation water was elucidated by the production of methane in the enrichment culture, which contained 16S rRNA gene sequences showing close relatedness to the genus Methanobacterium. Microcosm using formation water as medium as well as a source of inoculum and coal as carbon source produced significant amount of methane which increased considerably by the addition of nitrite. The dominance of Diaphorobacter sp. in nitrite amended microcosm indicated their important role in supporting methanogenesis in the coal bed. This is the first study indicating existence of methanogenic and bacterial community in an Indian coal bed that is capable of in situ biotransformation of coal into methane.

Isolation and characterization of Methanothermobacter crinale sp. nov., a novel hydrogenotrophic methanogen from the Shengli oil field.

Syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis is an alternative methanogenic pathway in certain thermophilic anaerobic environments such as high-temperature oil reservoirs and thermophilic biogas reactors. In these environments, the dominant thermophilic methanogens were generally related to uncultured organisms of the genus Methanothermobacter. Here we isolated two representative strains, Tm2(T) and HMD, from the oil sands and oil production water in the Shengli oil field in the People's Republic of China. The type strain, Tm2(T), was nonmotile and stained Gram positive. The cells were straight to slightly curved rods (0.3 µm in width and 2.2 to 5.9 µm in length), but some of them possessed a coccal shape connecting with the rods at the ends. Strain Tm2(T) grew with H(2)-CO(2), but acetate is required. Optimum growth of strain Tm2(T) occurred in the presence of 0.025 g/liter NaCl at pH 6.9 and a temperature of 65°C. The G+C content of the genomic DNA was 40.1 mol% ± 1.3 mol% (by the thermal denaturation method) or 41.1 mol% (by high-performance liquid chromatography). Analysis of the 16S rRNA gene sequence indicated that Tm2(T) was most closely related to Methanothermobacter thermautotrophicus ΔH(T) and Methanothermobacter wolfeii VKM B-1829(T) (both with a sequence similarity of 96.4%). Based on these phenotypic and phylogenic characteristics, a novel species was proposed and named Methanothermobacter crinale sp. nov. The type strain is Tm2(T) (ACCC 00699(T) = JCM 17393(T)).

Archaea as emerging organisms in complex human microbiomes.

In this work, we review the state of knowledge of Archaea associated with the human microbiome. These prokaryotes, initially discovered in extreme environments, were named Archaea because these environments were thought to be the most primitive on Earth. Further research revealed that this terminology is misleading because these organisms were later found in various non-extreme environments, including the human host. Further examination of the human microbiome has enabled the isolation of three archaeal species, Methanobrevibacter smithii, Methanosphaera stadtmanae and Methanobrevibacter oralis, which are associated with oral, intestinal and vaginal mucosae in humans. Moreover, molecular studies including metagenomic analyses detected DNA sequences indicative of the presence of additional methanogenic and non-methanogenic Archaea in the human intestinal tract. All three culturable Archaea are strict anaerobes, although their potential role in human diseases remains to be established. Future research aims to detect and culture additional human mucosa-associated Archaea and to look for their presence in additional human tissues, to establish their role in human infections involving complex flora.

Isolation and characterization of a new strain of Methanothermobacter marburgensis DX01 from hot springs in China.

Strain DX01, a thermophilic methanogen, was isolated from a hot spring in China. Strain DX01 grew only on H2/CO2. The DNA G+C content is 52 mol% and optimal growth temperature is 65 degrees C. The cell pellet is brick red. By analyzing 16S rRNA sequence, methyl-coenzyme M reductase I, gamma subunit protein sequences, we determined the DX01 strain to be closely related to the species of Methanothermobacter marburgensis. In addition, Methanothermobacter thermautotrophicus delta H(T) and strain DX01 had clear differences in their biochemical composition and protein expression profiles. Based on the above analysis, we propose that strain DX01 is a novel strain within thermoautotrophicus the species of M. marburgensis, namely M. marburgensis DX01. The isolation and characterization of the new M. marburgensis DX01 strain expands the known range of the Methanothermobacter genus.

An Aggregation-defective Mutant of Methanothermobacter sp. CaT2 Reveals Unique Protein-dependent Aggregation.

The thermophilic hydrogenotrophic methanogen, Methanothermobacter sp. CaT2, which possesses an extracellular sugar layer, commonly aggregates by itself or with other microorganisms. To elucidate the molecular mechanisms responsible for this aggregation, the aggregation-defective mutant, CLA160, was isolated. Optical and electron microscopy observations revealed that the mutant exhibited a significant reduction in aggregation. Genomic sequencing showed that CLA160 has a single point mutation, causing a nonsense mutation in MTCT_1020, which encodes a hypothetical protein. Motif and domain analyses indicated that the hypothetical protein bears two membrane-spanning segments at the N- and C-terminal regions and a large middle repeat-containing region. The results of a bioinformatic analysis suggested that the first middle region (RII) of the protein or the whole structure is responsible for the function of the product of MTCT_1020 in the aggregation of CaT2. A treatment with proteinase K suppressed sedimentation in CaT2, indicating a reduction in aggregation, with almost no effect on sedimentation in CLA160. The addition of Ca2+ or Mg2+ ions enhanced sedimentation in CaT2, whereas a DNase treatment had no effect on sedimentation in either strain. These results suggest that the hypothetical protein encoded by MTCT_1020 plays a key role as a membrane-bound adhesion protein in the aggregation of CaT2, which is enhanced by the addition of Ca2+ or Mg2+ ions.

Evaluations of different hypervariable regions of archaeal 16S rRNA genes in profiling of methanogens by Archaea-specific PCR and denaturing gradient gel electrophoresis.

Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.

Evaluating the potential of indigenous methanogenic consortium for enhanced oil and gas recovery from high temperature depleted oil reservoir.

In past years, lots of research has been focused on the indigenous bacteria and their mechanisms, which help in enhanced oil recovery. Most of the oil wells in Indian subcontinent have temperature higher than 60 °C. Also, the role of methanogenic consortia from high temperature petroleum reservoir for enhanced oil recovery (EOR) has not been explored much. Hence, in the present study methanogens isolated from thermophilic oil wells (70 °C) were evaluated for enhanced oil recovery. Methane gas is produced by methanogens, which helps in oil recovery from depleted oil wells through reservoir re-pressurization and also can be recovered from reservoir along with crude oil as alternative energy source. Therefore, in this study indigenous methanogenic consortium (TERIL146) was enriched from high temperature oil reservoir showing (12 mmol/l) gas production along with other metabolites. Sequencing analysis revealed the presence of Methanothermobacter sp., Thermoanaerobacter sp., Gelria sp. and Thermotoga sp. in the consortium. Furthermore, the developed indigenous consortium TERIL146 showed 8.3% incremental oil recovery in sandpack assay. The present study demonstrates successful recovery of both oil and energy (gas) by the developed indigenous methanogenic consortium TERIL146 for potential application in thermophilic depleted oil wells of Indian subcontinent.

Isolation and characterization of an amiloride-resistant mutant of Methanothermobacter thermautotrophicus possessing a defective Na+/H+ antiport.

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to the Na+/H+ antiporter inhibitor amiloride was isolated. The Na+/H+ exchanger activity in the mutant cells was remarkably decreased in comparison with wild-type cells. Methanogenesis rates in the mutant strain were higher than wild-type cells and resistant to the inhibitory effect of 2 mM amiloride. In contrast, methanogenesis in wild-type cells was completely inhibited by the same amiloride concentration. ATP synthesis driven by methanogenic electron transport or by an electrogenic potassium efflux in the presence of sodium ions was significantly enhanced in the mutant cells. ATP synthesis driven by potassium diffusion potential was profoundly inhibited in wild-type cells by the presence of uncoupler 3,3',4',5- tetrachlorosalicylanilide and sodium ions, whereas c. 50% inhibition was observed in the mutant cells under the same conditions.

Assessing the impact of rumen microbial communities on methane emissions and production traits in Holstein cows in a tropical climate.

The evaluation of how the gut microbiota affects both methane emissions and animal production is necessary in order to achieve methane mitigation without production losses. Toward this goal, the aim of this study was to correlate the rumen microbial communities (bacteria, archaea, and fungi) of high (HP), medium (MP), and low milk producing (LP), as well as dry (DC), Holstein dairy cows in an actual tropical production system with methane emissions and animal production traits. Overall, DC cows emitted more methane, followed by MP, HP and LP cows, although HP and LP cow emissions were similar. Using next-generation sequencing, it was found that bacteria affiliated with Christensenellaceae, Mogibacteriaceae, S24-7, Butyrivibrio, Schwartzia, and Treponema were negatively correlated with methane emissions and showed positive correlations with digestible dry matter intake (dDMI) and digestible organic matter intake (dOMI). Similar findings were observed for archaea in the genus Methanosphaera. The bacterial groups Coriobacteriaceae, RFP12, and Clostridium were negatively correlated with methane, but did not correlate with dDMI and dOMI. For anaerobic fungal communities, no significant correlations with methane or animal production traits were found. Based on these findings, it is suggested that manipulation of the abundances of these microbial taxa may be useful for modulating methane emissions without negatively affecting animal production.

PCR-based DGGE fingerprinting and identification of methanogens detected in three different types of UASB granules.

Methane is produced by various methanogenic bacteria present in upflow anaerobic sludge blanket (UASB) bioreactors. Methane can be used to predict and improve UASB bioreactor efficiency. The methanogen population in the granules can be influenced by the composition of the substrate. The aim of this study was to fingerprint and identify the methanogens present in three different types of UASB granules that had been used to treat winery, brewery and peach-lye canning effluents. This was done using polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis. The DGGE fingerprints obtained from the methanogen reference cultures of Methanosaeta concilii, Methanosaeta thermophila, Methanosarcina barkeri, Methanosarcina mazeii and Methanobacterium formicicum were compared to the DGGE profiles of the Archaea in the different granules. The positions of the DGGE bands that did not correspond well to the bands of the known species were sequenced and compared to sequences available on GenBank using the Blastn search option. The aligned DNA sequences were used to construct a phylogenetic tree. Based on the data obtained, a DGGE marker was constructed which was used to provide a quick method to identify the Archaeal members of the microbial consortium in UASB granules.

[Reclassification of thermophylic methane-oxidizing bacteria with the use of sequence-analysis of 16S rRNA genes].

The authors have performed sequence-analysis of 16S rRNA genes of thermophylic methane-oxidizing bacteria UCM B-3026, UCM B-3032, UCM B-3109, UCM B-3014 which were isolated from sludge pond of different regions in Ukraine and deposited at Ukrainian Collection of Microorganisms (UCM) as Methylococcus thermophilus and "M. gracilis". A comparative analysis of 16S rRNA gene sequences of the studied bacteria with those sequences of various strains of bacteria in the GenBank databases has shown that the level of similarity of the strains UCM B-3026, UCM B-3032, UCM B-3109 with Methylocaldum szegediense was 98.0-98.7%, strain UCM B-3014 with Methylocaldum gracile--99.1%, that allows to relate them to these species. As to other species of the genus Methylocaldum, these strains had a lower level of similarity (94.8-97.2 %) and with Methylococcus thermophilus ACM 3585T (= IMV-B-3037T = UCM B-3037T)--less than 90%. Thus, the strains UCM B-3026, UCM B-3032, UCM-3109 were reclassified as Methylocaldum szegediense, strain UCM B-3014--as Methylocaldum gracile. When constructing phylogenetic tree based on the comparison of 16S rRNA genes with the use of various algorithms realized in the packages of programs Tree View (version 1.5.2) and ClastalX (version 1.81) certain clusters have formed genera Methylocaldum, Methylococcus and Methylomonas. Genera Methylobacter and Methylomicrobium have formed three clusters, one of the clusters contained two species of Methylobacter and two species of Methylomicrobium. Apparently, the taxonomy of genera Methylobacter and Methylomicrobium is to be specified.

Potential functional gene diversity involved in methanogenesis and methanogenic community structure in Indian buffalo (Bubalus bubalis) rumen.

Understanding the methanogen community structure and methanogenesis from Bubalus bubalis in India may be beneficial to methane mitigation. Our current understanding of the microbial processes leading to methane production is incomplete, and further advancement in the knowledge of methanogenesis pathways would provide means to manipulate its emission in the future. In the present study, we evaluated the methanogenic community structure in the rumen as well as their potential genes involved in methanogenesis. The taxonomic and metabolic profiles of methanogens were assessed by shotgun sequencing of rumen metagenome by Ion Torrent semiconductor sequencing. The buffalo rumen contained representative genera of all the families of methanogens. Members of Methanobacteriaceae were found to be dominant, followed by Methanosarcinaceae, Methanococcaceae, Methanocorpusculaceae, and Thermococcaceae. A total of 60 methanogenic genera were detected in buffalo rumen. Methanogens related to the genera Methanobrevibacter, Methanosarcina, Methanococcus, Methanocorpusculum, Methanothermobacter, and Methanosphaera were predominant, representing >70 % of total archaeal sequences. The metagenomic dataset indicated the presence of genes involved in the methanogenesis and acetogenesis pathways, and the main functional genes were those of key enzymes in the methanogenesis. Sequences related to CoB--CoM heterodisulfide reductase, methyl coenzyme M reductase, f420-dependent methylenetetrahydromethanopterin reductase, and formylmethanofuran dehydrogenase were predominant in rumen. In addition, methenyltetrahydrofolate cyclohydrolase, methylenetetrahydrofolate dehydrogenase, 5,10-methylenetetrahydrofolate reductase, and acetyl-coenzyme A synthetase were also recovered.

Ammonia effect on hydrogenotrophic methanogens and syntrophic acetate-oxidizing bacteria.

Ammonia-rich substrates can cause inhibition on anaerobic digestion process. Syntrophic acetate-oxidizing bacteria (SAOB) and hydrogenotrophic methanogens are important for the ammonia inhibitory mechanism on anaerobic digestion. The roles and interactions of SAOB and hydrogenotrophic methanogens to ammonia inhibition effect are still unclear. The aim of the current study was to determine the ammonia toxicity levels of various pure strains of SAOB and hydrogenotrophic methanogens. Moreover, ammonia toxicity on the syntrophic-cultivated strains of SAOB and hydrogenotrophic methanogens was tested. Thus, four hydrogenotrophic methanogens (i.e. Methanoculleus bourgensis, Methanobacterium congolense, Methanoculleu thermophilus and Methanothermobacter thermautotrophicus), two SAOB (i.e. Tepidanaerobacter acetatoxydans and Thermacetogenium phaeum) and their syntrophic cultivation were assessed under 0.26, 3, 5 and 7 g NH4 (+)-N L(-1). The results showed that some hydrogenotrophic methanogens were equally, or in some cases, more tolerant to high ammonia levels compared to SAOB. Furthermore, a mesophilic hydrogenotrophic methanogen was more sensitive to ammonia toxicity compared to thermophilic methanogens tested in the study, which is contradicting to the general belief that thermophilic methanogens are more vulnerable to high ammonia loads compared to mesophilic. This unexpected finding underlines the fact that the complete knowledge of ammonia inhibition effect on hydrogenotrophic methanogens is still absent.