Clinical evidence of the role of Methanobrevibacter smithii in severe acute malnutrition.

Gut microbial dysbiosis has been shown to be an instrumental factor in severe acute malnutrition (SAM) and particularly, the absence of Methanobrevibacter smithii, a key player in energy harvest. Nevertheless, it remains unknown whether this absence reflects an immaturity or a loss of the microbiota. In order to assess that, we performed a case-control study in Mali using a propensity score weighting approach. The presence of M. smithii was tested using quantitative PCR on faeces collected from SAM children at inclusion and at discharge when possible or at day 15 for controls. M. smithii was highly significantly associated with the absence of SAM, detected in 40.9% controls but only in 4.2% cases (p < 0.0001). The predictive positive value for detection of M. smithii gradually increased with age in controls while decreasing in cases. Among children providing two samples with a negative first sample, no SAM children became positive, while this proportion was 2/4 in controls (p = 0.0015). This data suggests that gut dysbiosis in SAM is not an immaturity but rather features a loss of M. smithii. The addition of M. smithii as a probiotic may thus represent an important addition to therapeutic approaches to restore gut symbiosis.

Co-culture of Methanobrevibacter smithii with enterobacteria during urinary infection.

BACKGROUND: Urinary tract infections are known to be caused by bacteria, but the potential implications of archaea have never been studied in this context. METHODS: In two different university hospital centres we used specific laboratory methods for the detection and culture of archaeal methanogens in 383 urine specimens prospectively collected for diagnosing urinary tract infection (UTI). FINDINGS: Methanobrevibacter smithii was detected by quantitative PCR and sequencing in 34 (9%) of the specimens collected from 34 patients. Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Enterococcus faecium and mixed cultures were detected along with M. smithii in eighteen, six, three, one and six urine samples, respectively. Interestingly, using our specific culture method for methanogens, we also isolated M. smithii in 31 (91%) of the 34 PCR positive urine samples. Genotyping the 31 isolates using multispacer sequence typing revealed three different genotypes which have been previously reported in intestinal microbiota. Antibiotic susceptibility testing found the 31 isolates to be in vitro susceptible to metronidazole (MIC: 1 mg/L) but resistant to fosfomycin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanate and ofloxacin, commonly used to treat bacterial UTI. Finally, 19 (54%) of the 34 patients in whose urine samples M. smithii was detected were diagnosed with UTIs, including cystitis, pyelonephritis and prostatitis. INTERPRETATION: Our results show that M. smithii is part of the urinary microbiota of some individuals and could play a role in community-acquired UTI in association with enteric bacteria. FUND: This study was supported by IHU Méditerranée Infection, Marseille, France.

Isolation and culture of Methanobrevibacter smithii by co-culture with hydrogen-producing bacteria on agar plates.

OBJECTIVES: Methanogenic Archaea are considered as extremely oxygen-sensitive organisms, and their culture is fastidious, requiring specific equipment. We report here conditions allowing the cultivation of Methanobrevibacter smithii in an anaerobic chamber without the addition of hydrogen. METHODS: We first enriched the stool sample in an anaerobic liquid medium. To cultivate M. smithii with Bacteroides thetaiotaomicron and other hydrogen-producing bacteria on solid medium in an anaerobic chamber, we divided the agar plates into two compartments and seeded each strain on each compartment. Methane production was assessed by gas chromatography, and the growing colonies were authenticated by MALDI-TOF MS. RESULTS: We successfully cultured M. smithii from a liquid culture medium inoculated with stool collected from a healthy donor in an anaerobic chamber. The isolation in pure culture permitted successful culture on agar medium by our performing a co-culture with B. thetaiotaomicron. We also successfully tested the co-cultivation of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen in different amounts. Agar colonies of methanogens were obtained by co-culture with these bacteria, and methane production was detected. CONCLUSIONS: We propose a new approach to isolate and cultivate new strains of M. smithii by using a co-culture-based technique that can facilitate and make available the isolation of new methanogenic Archaea strains in clinical microbiology laboratories.

Whole rumen metagenome sequencing allows classifying and predicting feed efficiency and intake levels in cattle.

The current research was carried out to determine the associations between the rumen microbiota and traits related with feed efficiency in a Holstein cattle population (n = 30) using whole metagenome sequencing. Improving feed efficiency (FE) is important for a more sustainable livestock production. The variability for the efficiency of feed utilization in ruminants is partially controlled by the gastrointestinal microbiota. Modulating the microbiota composition can promote a more sustainable and efficient livestock. This study revealed that most efficient cows had larger relative abundance of Bacteroidetes (P = 0.041) and Prevotella (P = 0.003), while lower, but non-significant (P = 0.119), relative abundance of Firmicutes. Methanobacteria (P = 0.004) and Methanobrevibacter (P = 0.003) were also less abundant in the high-efficiency cows. A de novo metagenome assembly was carried out using de Bruijn graphs in MEGAHIT resulting in 496,375 contigs. An agnostic pre-selection of microbial contigs allowed high classification accuracy for FE and intake levels using hierarchical classification. These microbial contigs were also able to predict FE and intake levels with accuracy of 0.19 and 0.39, respectively, in an independent population (n = 31). Nonetheless, a larger potential accuracy up to 0.69 was foreseen in this study for datasets that allowed a larger statistical power. Enrichment analyses showed that genes within these contigs were mainly involved in fatty acids and cellulose degradation pathways. The findings indicated that there are differences between the microbiota compositions of high and low-efficiency animals both at the taxonomical and gene levels. These differences are even more evident in terms of intake levels. Some of these differences remain even between populations under different diets and environments, and can provide information on the feed utilization performance without information on the individual intake level.

Gut Colonization with Methanogenic Archaea Lowers Plasma Trimethylamine N-oxide Concentrations in Apolipoprotein e-/- Mice.

A mechanistic link between trimethylamine N-oxide (TMAO) and atherogenesis has been reported. TMAO is generated enzymatically in the liver by the oxidation of trimethylamine (TMA), which is produced from dietary choline, carnitine and betaine by gut bacteria. It is known that certain members of methanogenic archaea (MA) could use methylated amines such as trimethylamine as growth substrates in culture. Therefore, we investigated the efficacy of gut colonization with MA on lowering plasma TMAO concentrations. Initially, we screened for the colonization potential and TMAO lowering efficacy of five MA species in C57BL/6 mice fed with high choline/TMA supplemented diet, and found out that all five species could colonize and lover plasma TMAO levels, although with different efficacies. The top performing MA, Methanobrevibacter smithii, Methanosarcina mazei, and Methanomicrococcus blatticola, were transplanted into Apoe-/- mice fed with high choline/TMA supplemented diet. Similar to C57BL/6 mice, following initial provision of the MA, there was progressive attrition of MA within fecal microbial communities post-transplantation during the initial 3 weeks of the study. In general, plasma TMAO concentrations decreased significantly in proportion to the level of MA colonization. In a subsequent experiment, use of antibiotics and repeated transplantation of Apoe-/- mice with M. smithii, led to high engraftment levels during the 9 weeks of the study, resulting in a sustained and significantly lower average plasma TMAO concentrations (18.2 ± 19.6 µM) compared to that in mock-transplanted control mice (120.8 ± 13.0 µM, p < 0.001). Compared to control Apoe-/- mice, M. smithii-colonized mice also had a 44% decrease in aortic plaque area (8,570 µm [95% CI 19587-151821] vs. 15,369 µm [95% CI [70058-237321], p = 0.34), and 52% reduction in the fat content in the atherosclerotic plaques (14,283 µm [95% CI 4,957-23,608] vs. 29,870 µm [95% CI 18,074-41,666], p = 0.10), although these differences did not reach significance. Gut colonization with M. smithii leads to a significant reduction in plasma TMAO levels, with a tendency for attenuation of atherosclerosis burden in Apoe-/- mice. The anti-atherogenic potential of MA should be further tested in adequately powered experiments.

Sharpea and Kandleria are lactic acid producing rumen bacteria that do not change their fermentation products when co-cultured with a methanogen.

Sharpea and Kandleria are associated with rumen samples from low-methane-emitting sheep. Four strains of each genus were studied in culture, and the genomes of nine strains were analysed, to understand the physiology of these bacteria. All eight cultures grew equally well with d-glucose, d-fructose, d-galactose, cellobiose, and sucrose supplementation. d-Lactate was the major end product, with small amounts of the mixed acid fermentation products formate, acetate and ethanol. Genes encoding the enzymes necessary for this fermentation pattern were found in the genomes of four strains of Sharpea and five of Kandleria. Strains of Sharpea produced traces of hydrogen gas in pure culture, but strains of Kandleria did not. This was consistent with finding that Sharpea, but not Kandleria, genomes contained genes coding for hydrogenases. It was speculated that, in co-culture with a methanogen, Sharpea and Kandleria might change their fermentation pattern from a predominately homolactic to a predominately mixed acid fermentation, which would result in a decrease in lactate production and an increase in formation of acetate and perhaps ethanol. However, Sharpea and Kandleria did not change their fermentation products when co-cultured with Methanobrevibacter olleyae, a methanogen that can use both hydrogen and formate, and lactate remained the major end product. The results of this study therefore support a hypothesis that explains the link between lower methane yields and larger populations of Sharpea and Kandleria in the rumens of sheep.

Nitrate decreases ruminal methane production with slight changes to ruminal methanogen composition of nitrate-adapted steers.

BACKGROUND: This study was conducted to examine effects of nitrate on ruminal methane production, methanogen abundance, and composition. Six rumen-fistulated Limousin×Jinnan steers were fed diets supplemented with either 0% (0NR), 1% (1NR), or 2% (2NR) nitrate (dry matter basis) regimens in succession. Rumen fluid was taken after two-week adaptation for evaluation of in vitro methane production, methanogen abundance, and composition measurements. RESULTS: Results showed that nitrate significantly decreased in vitro ruminal methane production at 6 h, 12 h, and 24 h (P < 0.01; P < 0.01; P = 0.01). The 1NR and 2NR regimens numerically reduced the methanogen population by 4.47% and 25.82% respectively. However, there was no significant difference observed between treatments. The alpha and beta diversity of the methanogen community was not significantly changed by nitrate either. However, the relative abundance of the methanogen genera was greatly changed. Methanosphaera (PL = 0.0033) and Methanimicrococcus (PL = 0.0113) abundance increased linearly commensurate with increasing nitration levels, while Methanoplanus abundance was significantly decreased (PL = 0.0013). The population of Methanoculleus, the least frequently identified genus in this study, exhibited quadratic growth from 0% to 2% when nitrate was added (PQ = 0.0140). CONCLUSIONS: Correlation analysis found that methane reduction was significantly related to Methanobrevibacter and Methanoplanus abundance, and negatively correlated with Methanosphaera and Methanimicrococcus abundance.

Mode of action of Saccharomyces cerevisiae in enteric methane mitigation in pigs.

The objectives of this study were to determine the effect and mode of action of Saccharomyces cerevisiae (YST2) on enteric methane (CH4) mitigation in pigs. A total of 12 Duroc×Landrace×Yorkshire male finisher pigs (60±1 kg), housed individually in open-circuit respiration chambers, were randomly assigned to two dietary groups: a basal diet (control); and a basal diet supplemented with 3 g/YST2 (1.8×1010 live cells/g) per kg diet. At the end of 32-day experiment, pigs were sacrificed and redox potential (Eh), pH, volatile fatty acid concentration, densities of methanogens and acetogens, and expression of methyl coenzyme-M reductase subunit A gene were determined in digesta contents from the cecum, colon and rectum. Results showed that S. cerevisiae YST2 decreased (P<0.05) the average daily enteric CH4 production by 25.3%, lowered the pH value from 6.99 to 6.69 in the rectum, and increased the Eh value in cecum and colon by up to -55 mV (P<0.05). Fermentation patterns were also altered by supplementation of YST2 as reflected by the lower acetate, and higher propionate molar proportion in the cecum and colon (P<0.05), resulting in lower acetate : propionate ratio (P<0.05). Moreover, there was a 61% decrease in Methanobrevibacter species in the upper colon (P<0.05) and a 19% increase in the acetogen community in the cecum (P<0.05) of treated pigs. Results of our study concluded that supplementation of S. cerevisiae YST2 at 3 g/kg substantially decreased enteric CH4 production in pigs.

Development of Multiwell-Plate Methods Using Pure Cultures of Methanogens To Identify New Inhibitors for Suppressing Ruminant Methane Emissions.

Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H2 and CO2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for medium preparation, assay duration, inhibitor solvents, and culture volume) were optimized to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1,280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and MIC values were confirmed for some of them against M. maripaludis and M. AbM4. The developed method provides a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries to discover novel methanogen-specific inhibitors for the mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions, and new technologies are required to control emissions in the agriculture technology (agritech) sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimized, and validated a high-throughput 96-well microtiter plate assay for growing environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.

Alterations of Intestinal Microbiome by Antibiotic Therapy in Hospitalized Children.

The administration of antimicrobial agents leads to an ecological imbalance of the host-microorganisms relationship, and it causes a rapid and significant reduction in the microbial diversity. The aim of the current study was to evaluate the impact of antibiotic therapy on intestinal microbiota of children between 3 and 12 years of age. The fecal samples were collected from hospitalized children (n = 31) and from healthy untreated children (n = 30). The presence of bacteria and their quantities were assessed by culture-based methods and quantitative polymerase chain reaction (qPCR). By culture method, in the children receiving antibiotics, a low recovery of Bifidobacterium spp. (54.8%), Bacteroides spp./Parabacteroides spp. (54.8%), Clostridium spp. (35.5%), and Escherichia coli (74.2%) was observed compared with the children without antibiotic therapy (100%, 80%, 63.3%, and 86.6%, respectively). By qPCR, the children receiving antibiotics showed a lower copy number for all microorganisms, except to Lactobacillus spp. (p = 0.0092). In comparison to the nontreated children, the antibiotic-treated children showed a significantly lower copy number of Bifidobacterium spp. (p = 0.0002), Clostridium perfringens (p < 0.0001), E. coli (p = 0.0268), Methanobrevibacter smithii (p = 0.0444), and phylum Firmicutes (p = 0.0009). In conclusion, our results obtained through qualitative and quantitative analyses, demonstrate that antibiotic therapy affect the intestinal microbiome of children.

Effect of the Associated Methanogen Methanobrevibacter thaueri on the Dynamic Profile of End and Intermediate Metabolites of Anaerobic Fungus Piromyces sp. F1.

Although the scheme of metabolic pathways involved in the production of the major end products has been described, the dynamic profile of metabolites of anaerobic fungi co-cultured with methanogens is limited, especially for the intermediate metabolites. In the present study, the fermentation of the co-culture of Piromyces sp. F1 and Methanobrevibacter thaueri on glucose was investigated. The presence of methanogens shortened the growth lag time of anaerobic fungi and enhanced the total gas production. The occurrence of the maximum cell dry weight and the disappearance of most of the substrate were observed at 24 h for the co-culture and 48 h for the fungal mono-culture. In the co-culture, hydrogen was detected at a very low level during fermentation, and formate transitorily accumulated at 24 h and disappeared at 48 h, resulting in an increase of pH. Acetate was higher during the fermentation in the co-culture (P < 0.05), while lactate and ethanol were higher only in the initial stage of fermentation (P < 0.05). After 48 h, lactate in the mono-culture became much higher than that in the co-culture (P < 0.05), and ethanol tended to remain the same in both cultures. Moreover, malate tended to be exhausted in the co-culture, while it accumulated in the mono-culture. Citrate was also detected in both co-culture and mono-culture. Collectively, these results suggest that methanogen enhanced the malate pathway and weakened the lactate pathway of anaerobic fungus.

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.

Gut colonization with methanobrevibacter smithii is associated with childhood weight development.

OBJECTIVE: To prospectively investigate the presence and counts of archaea in feces of 472 children in association with weight development from 6 to 10 years of age. METHODS: Within the KOALA Birth Cohort Study, a single fecal sample from each child was analyzed by quantitative polymerase chain reaction to quantify archaea (Methanobrevibacter smithii, Methanosphera stadtmanae). Anthropometric outcomes (overweight [body mass index {BMI} ≥ 85th percentile], age- and sex-standardized BMI, weight, and height z-scores) were repeatedly measured at ages (mean ± SD) of 6.2 ± 0.5, 6.8 ± 0.5, 7.8 ± 0.5, and 8.8 ± 0.5 years. Generalized estimating equation was used for statistical analysis while controlling for confounders. RESULTS: Methanobrevibacter smithii colonization was associated with an increased risk of overweight (adjusted odds ratio [OR] = 2.69; 95% confidence interval [CI] 0.96-7.54) from 6 to 10 years of age. Children with high levels (>7 log10 copies/g feces) of this archaeon were at highest risk for overweight (OR = 3.27; 95% CI 1.09-9.83). Moreover, M. smithii colonization was associated with higher weight z-scores (adj. ß 0.18; 95% CI 0.00-0.36), but not with height. For BMI z-scores, the interaction (P = 0.008) between M. smithii and age was statistically significant, implying children colonized with M. smithii had increasing BMI z-scores with age. CONCLUSIONS: Presence and higher counts of M. smithii in the gut of children are associated with higher weight z-scores, higher BMI z-scores, and overweight.

Archaeal microbiota population in piglet feces shifts in response to weaning: Methanobrevibacter smithii is replaced with Methanobrevibacter boviskoreani.

Methanogens commonly inhabit swine intestine. We analyzed the gut archaeal population by extracting DNA from the feces of nine piglets. We performed PCR to target the V6-V8 region of the 16S rRNA gene. Subsequent denaturing gradient gel electrophoresis (DGGE) revealed the presence of Methanobrevibacter boviskoreani, which has not previously been identified in pigs. We confirmed these data with a PCR-DGGE analysis of the mcrA gene, and subsequent sequencing. At 63 days old, the only band in fecal samples corresponded to M. boviskoreani. The DGGE analysis also showed that Methanobrevibacter smithii, which was abundant at 28 days, was dramatically reduced at 42 days, and it completely disappeared at 63 days. To confirm these data, we quantified M. smithii and the total archaeal population by quantitative PCR (qPCR); moreover, we designed a new set of species-specific primers based on the 16S rRNA gene of M. boviskoreani. The qPCR results confirmed the reduction in M. smithii over time and a simultaneous increase in M. boviskoreani. At 63 days, the total numbers of archaea and M. boviskoreani genomes were comparable, which suggested that M. boviskoreani represented the dominant archaea. This work showed that the archaeal population shifted during weaning, and M. boviskoreani replaced M. smithii.

A high-throughput screening assay for identification of inhibitors of the A1AO-ATP synthase of the rumen methanogen Methanobrevibacter ruminantium M1.

We report the development of a high-throughput screening platform to identify inhibitors of the membrane-bound A1Ao-ATP synthase from the rumen methanogen Methanobrevibacter ruminantium M1. Inhibitors identified in the screen were tested against growing cultures of M. ruminantium, validating the approach to identify new inhibitors of methanogens.

Impact of the hydrogen partial pressure on lactate degradation in a coculture of Desulfovibrio sp. G11 and Methanobrevibacter arboriphilus DH1.

In this study, the impact of the hydrogen partial pressure on lactate degradation was investigated in a coculture of Desulfovibrio sp. G11 and Methanobrevibacter arboriphilus DH1. To impose a change of the hydrogen partial pressure, formate was added to the reactor. Hydrogen results from the bioconversion of formate besides lactate in the liquid phase. In the presence of a hydrogen-consuming methanogen, this approach allows for a better estimation of low dissolved hydrogen concentrations than under conditions where hydrogen is supplied externally from the gas phase, resulting in a more accurate determination of kinetic parameters. A change of the hydrogen partial pressure from 1,200 to 250 ppm resulted in a threefold increase of the biomass-specific lactate consumption rate. The 50 % inhibition constant of hydrogen on lactate degradation was determined as 0.692 ± 0.064 µM dissolved hydrogen (831 ± 77 ppm hydrogen in the gas phase). Moreover, for the first time, the maximum biomass-specific lactate consumption rate of Desulfovibrio sp. G11 (0.083 ± 0.006 mol-Lac/mol-XG11/h) and the affinity constant for hydrogen uptake of Methanobrevibacter arboriphilus DH1 (0.601 ± 0.022 µM dissolved hydrogen) were determined. Contrary to the widely established view that the biomass-specific growth rate of a methanogenic coculture is determined by the hydrogen-utilizing partner; here, it was found that the hydrogen-producing bacterium determined the biomass-specific growth rate of the coculture grown on lactate and formate.

Diversity and community composition of methanogenic archaea in the rumen of Scottish upland sheep assessed by different methods.

Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥ 91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6-V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to 16S rRNA gene references produced taxonomic identification to Order level including 2-3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19-35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10-18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6-V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.

Windrow composting mitigated CH4 emissions: characterization of methanogenic and methanotrophic communities in manure management.

With increasing livestock breeding, methane (CH4 ) emissions from manure management will increasingly contribute more to atmospheric CH4 concentration. The dynamics of methanogens and methanotrophs have not yet been studied in the manure environment. The current study combines surface CH4 emissions with methanogenic and methanotrophic community analyses from two management practices, windrow composting (WCOM) and solid storage (SSTO). Our results showed that there was an c. 50% reduction of CH4 emissions with WCOM compared with SSTO over a 50-day period. A sharp decrease in the quantities of both methanogens and methanotrophs in WCOM suggested that CH4 mitigation was mainly due to decreased CH4 production rather than increased CH4 oxidation. Pyrosequencing analysis demonstrated that aeration caused a clear shift of dominant methanogens in the manure, with specifically a significant decrease in Methanosarcina and increase in Methanobrevibacter. The composition of methanogenic community was influenced by manure management and regulated CH4 production. A sharp increase in the quantity of methanotrophs in SSTO suggested that microbial CH4 oxidation is an important sink for the CH4 produced. The increased abundance of Methylococcaceae in SSTO suggested that Type I methanotrophs have an advantage in CH4 oxidation in occupying niches under low CH4 and high O2 conditions.

Intestinal Methanobrevibacter smithii but not total bacteria is related to diet-induced weight gain in rats.

UNLABELLED: It is increasingly understood that gastrointestinal (GI) methanogens, including Methanobrevibacter smithii, influence host metabolism. OBJECTIVE: Therefore, we compared M. smithii colonization and weight gain in a rat model under different dietary conditions. DESIGN AND METHODS: Sprague-Dawley rats were inoculated with M. smithii or vehicle (N = 10/group), fed normal chow until day 112 postinoculation, high-fat chow until day 182, then normal chow until day 253. Thereafter, five rats from each group were fed high-fat and normal chow until euthanasia. RESULTS: Both groups exhibited M. smithii colonization, which increased following inoculation only for the first 9 days. Change to high-fat chow correlated with significant increases in weight (P < 0.00001) and stool M. smithii (P < 0.01) in all rats, with stool M. smithi decreasing on return to normal chow. Rats switched back to high-fat on day 253 further increased weight (P < 0.001) and stool M. smithii (P = 0.039). Euthanasia revealed all animals had higher M. smithii, but not total bacteria, in the small intestine than in the colon. Rats switched back to high-fat chow had higher M. smithii levels in the duodenum, ileum, and cecum than those fed normal chow; total bacteria did not differ in any bowel segment. Rats which gained more weight had more bowel segments colonized, and the lowest weight recorded was in a rat on high-fat chow which had minimal M. smithii colonization. CONCLUSIONS: We conclude that M. smithii colonization occurs in the small bowel as well as in the colon, and that the level and extent of M. smithii colonization is predictive of degree of weight gain in this animal model.