Links between the rumen microbiota, methane emissions and feed efficiency of finishing steers offered dietary lipid and nitrate supplementation.

Ruminant methane production is a significant energy loss to the animal and major contributor to global greenhouse gas emissions. However, it also seems necessary for effective rumen function, so studies of anti-methanogenic treatments must also consider implications for feed efficiency. Between-animal variation in feed efficiency represents an alternative approach to reducing overall methane emissions intensity. Here we assess the effects of dietary additives designed to reduce methane emissions on the rumen microbiota, and explore relationships with feed efficiency within dietary treatment groups. Seventy-nine finishing steers were offered one of four diets (a forage/concentrate mixture supplemented with nitrate (NIT), lipid (MDDG) or a combination (COMB) compared to the control (CTL)). Rumen fluid samples were collected at the end of a 56 d feed efficiency measurement period. DNA was extracted, multiplexed 16s rRNA libraries sequenced (Illumina MiSeq) and taxonomic profiles were generated. The effect of dietary treatments and feed efficiency (within treatment groups) was conducted both overall (using non-metric multidimensional scaling (NMDS) and diversity indexes) and for individual taxa. Diet affected overall microbial populations but no overall difference in beta-diversity was observed. The relative abundance of Methanobacteriales (Methanobrevibacter and Methanosphaera) increased in MDDG relative to CTL, whilst VadinCA11 (Methanomassiliicoccales) was decreased. Trimethylamine precursors from rapeseed meal (only present in CTL) probably explain the differences in relative abundance of Methanomassiliicoccales. There were no differences in Shannon indexes between nominal low or high feed efficiency groups (expressed as feed conversion ratio or residual feed intake) within treatment groups. Relationships between the relative abundance of individual taxa and feed efficiency measures were observed, but were not consistent across dietary treatments.

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.

Process performance and microbial community structure in thermophilic trickling biofilter reactors for biogas upgrading.

This study evaluated the process performance and determined the microbial community structure of two lab-scale thermophilic trickling biofilter reactors used for biological methanation of hydrogen and carbon-dioxide for a total period of 94 days. Stable and robust operation was achieved by means of a single-pass gas flow. The quality of the output gas (>97%) was comparable to the methane purity achieved by commercial biogas upgrading systems fulfilling the specifications to be used as substitute to natural gas. The reactors' methane productivity reached >1.7 LCH4/(LR·d) at hydrogen loading rate of 7.2 LH2/(LR·d). The spatial distribution of the microbial consortia localized in the liquid media and biofilm enabled us to gain a deeper understanding on how the microbiome is structured inside the trickling biofilter. Sequencing results revealed a significant predominance of Methanothermobacter sp. in the biofilm. Unknown members of the class Clostridia were highly abundant in biofilm and liquid media, while acetate utilising bacteria predominated in liquid samples.

Effects of different levels of methionine on sow health and plasma metabolomics during late gestation.

Fetal growth, survival, and development are benchmarks for the production performance of sows, and methionine has been shown to impact fetal protein mass and the transport of nutrients through the uteroplacental vasculature. This study evaluated the effects of dietary methionine, administered during the late gestation period, on the production performance of sows. Specifically, it measured the effect of methionine on biochemical indicators in the plasma, plasma metabolites, and fecal bacterial communities. Thirty Landrace × Large White sows at day 90 of gestation were randomly assigned to three groups and fed the following diets: (1) a basal diet containing 0.36% methionine; (2) a basal diet + 0.12% methionine (0.48% methionine); and (3) a basal diet + 0.24% methionine (0.60% methionine). The results showed that the 0.48% methionine diet significantly (P < 0.05) increased piglets' birth weight, and the 0.60% methionine diet significantly (P < 0.05) improved the survival ratio. Dietary methionine lowered the triglyceride (TG) levels (P < 0.05), total bilirubin (BILT3) (P < 0.001) concentration, and gamma-glutamyl transferase (GGT) (P < 0.05) enzyme activity in the plasma at farrowing. In the plasma metabolomics, dietary methionine increased plasma pyroglutamic acid and decreased 2-pyrrolidinone, hypotaurine, and anyl-histidine in both the 0.48% methionine and 0.60% methionine groups. In addition, the bacteria richness (Chao1 and ACE) and diversity (Shannon) were reduced in the 0.48% methionine group. For the microbiota composition, at the family level, the 0.48% methionine group had a significant increase (P < 0.05) in the relative abundance of Methanobacteriaceae compared to the other two groups, but a decrease in the relative abundance of Enterobacteriaceae, Ruminococcaceae and Erysipelotrichaceae compared to the 0.60% methionine group. In conclusion, a diet consisting of 0.48% methionine administered during the late gestation period can improve the production performance of sows and maintain their health.

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.

Biphasic Study to Characterize Agricultural Biogas Plants by High-Throughput 16S rRNA Gene Amplicon Sequencing and Microscopic Analysis.

Process surveillance within agricultural biogas plants (BGPs) was concurrently studied by high-throughput 16S rRNA gene amplicon sequencing and an optimized quantitative microscopic fingerprinting (QMF) technique. In contrast to 16S rRNA gene amplicons, digitalized microscopy is a rapid and cost-effective method that facilitates enumeration and morphological differentiation of the most significant groups of methanogens regarding their shape and characteristic autofluorescent factor 420. Moreover, the fluorescence signal mirrors cell vitality. In this study, four different BGPs were investigated. The results indicated stable process performance in the mesophilic BGPs and in the thermophilic reactor. Bacterial subcommunity characterization revealed significant differences between the four BGPs. Most remarkably, the genera Defluviitoga and Halocella dominated the thermophilic bacterial subcommunity, whereas members of another taxon, Syntrophaceticus, were found to be abundant in the mesophilic BGP. The domain Archaea was dominated by the genus Methanoculleus in all four BGPs, followed by Methanosaeta in BGP1 and BGP3. In contrast, Methanothermobacter members were highly abundant in the thermophilic BGP4. Furthermore, a high consistency between the sequencing approach and the QMF method was shown, especially for the thermophilic BGP. The differences elucidated that using this biphasic approach for mesophilic BGPs provided novel insights regarding disaggregated single cells of Methanosarcina and Methanosaeta species. Both dominated the archaeal subcommunity and replaced coccoid Methanoculleus members belonging to the same group of Methanomicrobiales that have been frequently observed in similar BGPs. This work demonstrates that combining QMF and 16S rRNA gene amplicon sequencing is a complementary strategy to describe archaeal community structures within biogas processes.

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.

Evidence for the involvement of two heterodisulfide reductases in the energy-conserving system of Methanomassiliicoccus luminyensis.

Methanomassiliicoccus luminyensis was isolated from the human gut, and requires H2 and methanol or methylamines to produce methane. The organism lacks cytochromes, indicating that it cannot couple membrane-bound electron transfer reactions with extrusion of H(+) or Na(+) ions using known methanogenic pathways. Furthermore, M. luminyensis contains a soluble hydrogenase/heterodisulfide reductase complex (MvhAGD/HdrABC) as found in obligate hydrogenotrophic methanogens, but the energy-conserving methyltransferase (MtrA-H) is absent. Thus, the question arises as to how this species synthesizes ATP. We present evidence that M. luminyensis uses two types of heterodisulfide reductases (HdrABC and HdrD) in a novel process for energy conservation. Quantitative RT-PCR studies revealed that genes encoding these heterodisulfide reductases showed high expression levels. Other genes with high transcript abundance were fpoA (part of the operon encoding the 'headless' F420 H2 dehydrogenase) and atpB (part of the operon encoding the A1 Ao ATP synthase). High activities of the soluble heterodisulfide reductase HdrABC and the hydrogenase MvhADG were found in the cytoplasm of M. luminyensis. Also, heterologously produced HdrD was able to reduce CoM-S-S-CoB using reduced methylviologen as an electron donor. We propose that membrane-bound electron transfer is based on conversion of two molecules of methanol and concurrent formation of two molecules of the heterodisulfide CoM-S-S-CoB. First the HdrABC/MvhADG complex catalyzes the H2 -dependent reduction of CoM-S-S-CoB and formation of reduced ferredoxin. In a second cycle, reduced ferredoxin is oxidized by the 'headless' F420 H2 dehydrogenase, thereby translocating up to 4 H(+) across the membrane, and electrons are channeled to HdrD for reduction of the second heterodisulfide.

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.

Substrate sources regulate spatial variation of metabolically active methanogens from two contrasting freshwater wetlands.

There is ample evidence that methane (CH4) emissions from natural wetlands exhibit large spatial variations at a field scale. However, little is known about the metabolically active methanogens mediating these differences. We explored the spatial patterns in active methanogens of summer inundated Calamagrostis angustifolia marsh with low CH4 emissions and permanently inundated Carex lasiocarpa marsh with high CH4 emissions in Sanjiang Plain, China. In C. angustifolia marsh, the addition of (13)C-acetate significantly increased the CH4 production rate, and Methanosarcinaceae methanogens were found to participate in the consumption of acetate. In C. lasiocarpa marsh, there was no apparent increase in the CH4 production rate and no methanogen species were labeled with (13)C. When (13)CO2-H2 was added, however, CH4 production was found to be due to Fen Cluster (Methanomicrobiales) in C. angustifolia marsh and Methanobacterium Cluster B (Methanobacteriaceae) together with Fen Cluster in C. lasiocarpa marsh. These results suggested that CH4 was produced primarily by hydrogenotrophic methanogens using substrates mainly derived from plant litter in C. lasiocarpa marsh and by both hydrogenotrophic and acetoclastic methanogens using substrates mainly derived from root exudate in C. angustifolia marsh. The significantly lower CH4 emissions measured in situ in C. angustifolia marsh was primarily due to a deficiency of substrates compared to C. lasiocarpa marsh. Therefore, we speculate that the substrate source regulates both the type of active methanogens and the CH4 production pathway and consequently contributes to the spatial variations in CH4 productions observed in these freshwater marshes.

Physiological and genetic basis for self-aggregation of a thermophilic hydrogenotrophic methanogen, Methanothermobacter strain CaT2.

Several thermophilic hydrogenotrophic methanogens naturally aggregate in their habitats in association with hydrogen-producing bacteria for efficient transfer of the methane fermentation intermediates to produce methane. However, physiology of aggregation and the identity of aggregation-specific genes remain to be elucidated. Here, we isolated and characterized a hydrogen and formate-utilizing Methanothermobacter sp. CaT2 that is capable of self-aggregation and utilizing formate. CaT2 produced methane from propionate oxidation in association with a syntrophic propionate-oxidizing bacterium faster than other methanogens, including Methanothermobacter thermautotrophicus ΔH and Methanothermobacter thermautotrophicus Z-245. CaT2 also aggregated throughout the culture period and was coated with polysaccharides, which was not found on the ΔH and Z-245 cells. Sugar content (particularly of rhamnose and mannose) was also higher in the CaT2 cells than the ΔH and Z-245 cells. Comparative genomic analysis of CaT2 indicated that four candidate genes, all of which encode glycosyltransferase, were involved in aggregation of CaT2. Transcriptional analysis showed that one glycosyltransferase gene was expressed at relatively high levels under normal growth conditions. The polysaccharide layer on the CaT2 cell surface, which is probably assembled by these glycosyltransferases, may be involved in cell aggregation.

Increased prevalence of Methanosphaera stadtmanae in inflammatory bowel diseases.

BACKGROUND: The gut microbiota is associated with the modulation of mucosal immunity and the etiology of inflammatory bowel diseases (IBD). Previous studies focused on the impact of bacterial species on IBD but seldom suspected archaea, which can be a major constituent of intestinal microbiota, to be implicated in the diseases. Recent evidence supports that two main archaeal species found in the digestive system of humans, Methanobrevibacter smithii (MBS) and Methanosphaera stadtmanae (MSS) can have differential immunogenic properties in lungs of mice; with MSS but not MBS being a strong inducer of the inflammatory response. We thus aimed at documenting the immunogenic potential of MBS and MSS in humans and to explore their association with IBD. METHODS: To validate the immunogenicity of MBS and MSS in humans, peripheral blood mononuclear cells from healthy subjects were stimulated with these two microorganisms and the production of inflammatory cytokine TNF was measured by ELISA. To verify MBS and MSS prevalence in IBD, stool samples from 29 healthy control subjects and 29 patients suffering from IBD were collected for DNA extraction. Plasma was also collected from these subjects to measure antigen-specific IgGs by ELISA. Quantitative PCR was used for bacteria, methanogens, MBS and MSS quantification. RESULTS: Mononuclear cells stimulated with MSS produced higher concentrations of TNF (39.5 ng/ml) compared to MBS stimulation (9.1 ng/ml). Bacterial concentrations and frequency of MBS-containing stools were similar in both groups. However, the number of stool samples positive for the inflammatory archaea MSS was higher in patients than in controls (47% vs 20%). Importantly, only IBD patients developed a significant anti-MSS IgG response. CONCLUSION: The prevalence of MSS is increased in IBD patients and is associated with an antigen-specific IgG response.

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.

Technical note: Comparison of biomarker and molecular biological methods for estimating methanogen abundance.

Quantitative real-time PCR (qPCR) has become a popular method for estimation of methanogen abundance in the ruminant digestive tract. However, there is no established method in terms of primer choice and quantification, which means that results are variable and not directly comparable between studies. Archaeol has been proposed as an alternative marker for methanogen abundance, as it is ubiquitous in methanogenic Archaea, and can be quantified by gas chromatography-mass spectrometry (GC-MS). The aim of this experiment was to compare total methanogen populations estimated using the new archaeol approach with estimates based on qPCR. Specific primer sets and probes were used to detect dominant ruminal methanogen species Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanosphaera stadtmanae, and total methanogen populations. There was variation in the relationships among total methanogen abundance estimates based on archaeol and qPCR. In addition, the universal methanogen primers appeared to preferentially amplify genes from M. smithii. Archaeol had the strongest relationship with the dominant rumen methanogen M. ruminantium, whereas the total methanogen primers had a comparatively weak relationship with archaeol. Archaeol analysis was a useful adjunct to molecular biology methods, but it seems that a valid specific primer for M. ruminantium would be more useful than a biased primer for total methanogens.

Novel bacterial groups dominate in a thermophilic methanogenic hexadecane-degrading consortium.

A methanogenic hexadecane-degrading consortium designated SK originating from the Shengli oil field was cultured at 55 °C. The structure and dynamics of the microbial community during successive transfers were examined using terminal restriction fragment length polymorphism (T-RFLP) fingerprinting and sequencing of 16S rRNA gene fragments. The archaeal community was mainly composed of hydrogenotrophic methanogens affiliated with Methanothermobacter crinale and acetoclastic methanogens related to Methanosaeta thermophila. Over four-fifths of the bacterial clones in the hexadecane-degrading subcultures exhibited < 90% similarity to sequences of known type strains, and clones were mainly grouped into unclassified bacteria (66.3-66.7%), Firmicutes (9.6-10.6%), Thermotogae (7.0-7.7%), and Nitrospira (5.3-5.8%). The dominant operating taxonomic unit (OTU) (41.3-43.0% of all clones) representing terminal restriction fragment (T-RF) 125 bp exhibited only 82.6% sequence similarity to Thermotoga maritime and clustered in a monophyletic, deep-branching lineage (designated Shengli cluster). Two other OTUs (T-RFs 66 and 67 bp) were assigned to uncultured members of the candidate phylum OP8 and Firmicutes, respectively. These novel bacterial assemblages are likely to be involved in the process of hexadecane degradation because of their high abundance in the enrichments. These result substantially expand the knowledge of the extent of bacterial diversity associated with the anaerobic degradation of alkanes under thermophilic conditions.

Biodiversity and composition of methanogenic populations in the rumen of cows fed alfalfa hay or triticale straw.

It is clear that methanogens are responsible for ruminal methane emissions, but quantitative information about the composition of the methanogenic community in the bovine rumen is still limited. The diversity and composition of rumen methanogens in cows fed either alfalfa hay or triticale straw were examined using a full-cycle rRNA approach. Quantitative fluorescence in situ hybridization undertaken applying oligonucleotide probes designed here identified five major methanogenic populations or groups in these animals: the Methanobrevibacter TMS group (consisting of Methanobrevibacter thaueri, Methanobrevibacter millerae and Methanobrevibacter smithii), Methanbrevibacter ruminantium-, Methanosphaera stadtmanae-, Methanomicrobium mobile-, and Methanimicrococcus-related methanogens. The TMS- and M. ruminantium-related methanogens accounted for on average 46% and 41% of the total methanogenic cells in liquid (Liq) and solid (Sol) phases of the rumen contents, respectively. Other prominent methanogens in the Liq and Sol phases included members of M. stadtmanae (15% and 33%), M. mobile (17% and 12%), and Methanimicrococcus (23% and 9%). The relative abundances of these methanogens in the community varied among individual animals and across diets. No clear differences in community composition could be observed with dietary change using cloning techniques. This study extends the known biodiversity levels of the methanogenic communities in the rumen of cows.

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)).

Age-related prevalence of Methanomassiliicoccus luminyensis in the human gut microbiome.

A 16S rDNA sequence-based investigation of methanogenic Archaea in the human stools found Methanobrevibacter smithii in 99.2% and Methanosphaera stadtmanae in 32.6%. The recently described Methanomassiliicoccus luminyensis found by others to be representative of a new order of methanogenic Archaea was found in 4% of stool specimens. The prevalence of M. luminyensis significantly increased with age, contrary to M. smithii and M. stadtmanae.

Laboratory tools for detection of archaea in humans.

This work represents an update of knowledge regarding the detection methods for human microbiome-associated archaea. Despite the fact that, during the last three decades, only four methanoarchaeal species have been isolated from the human mucosa, including faeces, subgingival plaque, and vaginal mucosa (Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanobrevibacter oralis and, most recently, 'Methanomassiliicoccus luminyensis'), molecular studies, including PCR and metagenomic analyses, have detected DNA sequences indicative of the presence of additional methanoarchaea, as well as non-methanogenic archaea, in the human intestinal tract. Opinion is divided on the roles (if any) of these organisms in human disease, and certainly the data are still unclear. Future research and recently reported data highlighting the antimicrobial susceptibility of the human methanoarchaea could help in the design of selective media to discover additional human mucosa-associated archaea and ascertain their role in human infections involving complex flora.