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Dairy cows experience dramatic changes in host physiology from gestation to lactation period and dietary switch from high-forage prepartum diet to high-concentrate postpartum diet over the transition period (parturition +/- three weeks). Understanding the community structure and activity of the rumen microbiota and its associative patterns over the transition period may provide insight for e.g. improving animal health and production. In the present study, rumen samples from ten primiparous Holstein dairy cows were collected over seven weeks spanning the transition period. Total RNA was extracted from the rumen samples and cDNA thereof was subsequently used for characterizing the metabolically active bacterial (16S rRNA transcript amplicon sequencing) and archaeal (qPCR, T-RFLP and mcrA and 16S rRNA transcript amplicon sequencing) communities. The metabolically active bacterial community was dominated by three phyla, showing significant changes in relative abundance range over the transition period: Firmicutes (from prepartum 57% to postpartum 35%), Bacteroidetes (from prepartum 22% to postpartum 18%) and Proteobacteria (from prepartum 7% to postpartum 32%). For the archaea, qPCR analysis of 16S rRNA transcript number, revealed a significant prepartum to postpartum increase in Methanobacteriales, in accordance with an observed increase (from prepartum 80% to postpartum 89%) in relative abundance of 16S rRNA transcript amplicons allocated to this order. On the other hand, a significant prepartum to postpartum decrease (from 15% to 2%) was observed in relative abundance of Methanomassiliicoccales 16S rRNA transcripts. In contrast to qPCR analysis of the 16S rRNA transcripts, quantification of mcrA transcripts revealed no change in total abundance of metabolically active methanogens over the transition period. According to T-RFLP analysis of the mcrA transcripts, two Methanobacteriales genera, Methanobrevibacter and Methanosphaera (represented by the T-RFs 39 and 267 bp), represented more than 70% of the metabolically active methanogens, showing no significant changes over the transition period; minor T-RFs, likely to represent members of the order Methanomassiliicoccales and with a relative abundance below 5% in total, decreased significantly over the transition period. In accordance with the T-RFLP analysis, the mcrA transcript amplicon sequencing revealed Methanobacteriales to cover 99% of the total reads, dominated by the genera Methanobrevibacter (75%) and Methanosphaera (24%), whereas the Methanomassiliicoccales order covered only 0.2% of the total reads. In conclusion, the present study showed that the structure of the metabolically active bacterial and archaeal rumen communities changed over the transition period, likely in response to the dramatic changes in physiology and nutritional factors like dry matter intake and feed composition. It should be noted however that for the methanogens, the observed community changes were influenced by the analyzed gene (mcrA or 16S rRNA).

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.

Abstract Anaerobic digestion (AD) is an attractive wastewater treatment technology, leading to the generation of recoverable biofuel (methane). Most industrial AD applications, carry excessive heating costs, however, as AD reactors are commonly operated at mesophilic temperatures while handling waste streams discharged at ambient or cold temperatures. Consequently, low-temperature AD represents a cost-effective strategy for wastewater treatment. The comparative investigation of key microbial groups underpinning laboratory-scale AD bioreactors operated at 37, 15 and 7°C was carried out. Community structure was monitored using 16S rRNA clone libraries, while abundance of the most prominent methanogens was investigated using qPCR. In addition, metaproteomics was employed to access the microbial functions carried out in situ. While δ-Proteobacteria were prevalent at 37°C, their abundance decreased dramatically at lower temperatures with inverse trends observed for Bacteroidetes and Firmicutes. Methanobacteriales and Methanosaeta were predominant at all temperatures investigated while Methanomicrobiales abundance increased at 15°C compared to 37 and 7°C. Changes in operating temperature resulted in the differential expression of proteins involved in methanogenesis, which was found to occur in all bioreactors, as corroborated by bioreactors’ performance. This study demonstrated the value of employing a polyphasic approach to address microbial community dynamics and highlighted the functional redundancy of AD microbiomes. This study investigated the microbial consortia underpinning anaerobic digestion wastewater treatment and highlighted community shifts associated with functional redundancy as a response to temperature change in these systems. Graphical Abstract Figure. This study investigated the microbial consortia underpinning anaerobic digestion wastewater treatment and highlighted community shifts associated with functional redundancy as a response to temperature change in these systems.

A temporal temperature gradient gel electrophoresis (TTGE) method was developed to determine the diversity of methanogen populations in the rumen. Tests with amplicons from genomic DNA from 12 cultured methanogens showed single bands for all strains, with only two showing apparently comigrating bands. Fingerprints of methanogen populations were analyzed from DNA extracted from rumen contents from two cattle and four sheep grazing pasture. For one sheep, dilution cultures selective for methanogens were grown and the culturable methanogens in each successive dilution examined by TTGE. A total of 66 methanogen sequences were retrieved from bands in fingerprints and analyzed to reveal the presence of methanogens belonging to the Methanobacteriales, the Methanosarcinales, and to an uncultured archaeal lineage. Twenty-four sequences were most similar to Methanobrevibacter ruminantium, five to Methanobrevibacter smithii, four to Methanosphaera stadtmanae, and for three, the nearest match was Methanimicrococcus blatticola. The remaining 30 sequences did not cluster with sequences from cultured archaea, but when combined with published novel sequences from clone libraries formed a monophyletic lineage within the Euryarchaeota, which contained two previously unrecognized clusters. The TTGE bands from this lineage showed that the uncultured methanogens had significant population densities in each of the six rumen samples examined. In cultures of dilutions from one rumen sample, TTGE examination revealed these methanogens at a level of at least 10⁵ g-¹. Band intensities from low-dilution cultures indicated that these methanogens were present at similar densities to Methanobrevibacter ruminantium-like methanogens, the sole culturable methanogens in high dilutions (10⁶-10-¹⁰ g-¹). It is suggested that the uncultured methanogens together with Methanobrevibacter spp. may be the predominant methanogens in the rumen. The TTGE method presented in this article provides a new opportunity for characterizing methanogen populations in the rumen microbial ecosystem.

Molecular diversity of rumen archaeal populations from bovine rumen fluid incubated with or without condensed tannins was investigated using 16S rRNA gene libraries. The predominant order of rumen archaea in the 16S rRNA gene libraries of the control and condensed tannins treatment was found to belong to a novel group of rumen archaea that is distantly related to the order Thermoplasmatales, with 59.5% (15 phylotypes) and 81.43% (21 phylotypes) of the total clones from the control and treatment clone libraries, respectively. The 16S rRNA gene library of the control was found to have higher proportions of methanogens from the orders Methanomicrobiales (32%) and Methanobacteriales (8.5%) as compared to those found in the condensed tannins treatment clone library in both orders (16.88% and 1.68% respectively). The phylotype distributed in the order Methanosarcinales was only found in the control clone library. The study indicated that condensed tannins could alter the diversity of bovine rumen methanogens.

Hydrogenotrophic methanogens live in a synthrophic relationship with the human gut microbiota as the terminal part of the anaerobic food chain. Methanobrevibacter smithii of the Methanobacteriales is the prevailing archaeal species. Recently, methylotrophic archaea of the novel order Methanomassiliicoccales were isolated from human stools. Few data exist on the prevalence, abundance, persistence and ecology of these methanogens in humans. This study investigated methanogen communities in 26 healthy and obese children (8–14 years) and 18 adults (28–78 years) using quantitative PCR. Samples were obtained from nine females before and after giving birth. Bacterial groups linked to the abundance of methanogens in adult females were identified using a 16S rRNA gene amplicon data set. A total of 89% and 65% of adults and children, respectively, carried Methanobacteriales. Methanomassiliicoccales were recovered from 50% of the adults and one child. Mean relative abundance of Methanomassiliicoccales in adults was lower than that of Methanobacteriales (0.10% versus 0.52%). Both Methanobacteriales and Methanomassiliicoccales formed stable communities in females before and after giving birth. Methanobacteriales co-occurred with bacterial taxonomic groups associated with the trophic chain from carbohydrate degradation to hydrogen and formate formation. Relative abundance was inversely correlated to Blautia. Negative correlation with little characterized groups within the Clostridiales indicated possible interactions of Methanomassiliicoccales with the bacterial community. Methanomassiliicoccales occur frequently and are temporarily persistent in stools of adult humans.

Background The anaerobic digestion is one of the most spread renewable energy technology. The input biomasses included various environmental problematic wastes such as sludge coming from wastewater treatment plant (WWTP) and organic fraction of municipal solid waste (OFMSW). As biomolecular procedures have become important tools for the microbial characterisation of anaerobic samples coming from the reactors, it is crucial sampling and extracting properly DNA in order to employ such types of techniques. The current study is aimed to evaluate how freezing temperature and length of storage at −20 °C influence both the extracted DNA yield and microbial community quantifications from digested sludge samples collected at full-scale plants. Results From WWTP sludge samples, we observed a reduction of DNA concentration comparing fresh and stored samples for 10 days at −20 °C (ANOVA test p  < 0.0001), with an estimated DNA loss of approximately 65 % for such types of samples, however the methanogen communities can be assessed respecting the fresh conditions. From OFMSW sludge samples, we observed a reduction in extracted DNA (−90 %), after 120 frozen days, while microbial communities are determined respecting the fresh conditions within 2 months of frozen storage. Conclusions The remarkable effect of frozen storage on sludge samples suggests as the better procedure to perform the DNA extraction from fresh sample. On the other hand it is not generally possible, so approximately 2 months of storage at −20 °C appears to be suitable time at which DNA concentrations remain sufficient to perform coherent microbial characterization through quantitative qRT-PCR.

Despite the pivotal role of archaea in methane production in landfills, the identity, ecology, and functional diversity of these microorganisms and their link to environmental factors remain largely unknown. We collected 11 landfill leachate samples from six geographically distinct landfills of different ages in China and analyzed the archaeal community by bar-coded 454 pyrosequencing. We retrieved 121,797 sequences from a total of 167,583 sequences (average length of 464 bp). The archaeal community was geographically structured, and nonabundant taxa primarily contributed to the observed dissimilarities. Canonical correlation analysis (CCA) suggested that the total phosphorous (TP), nitrate, and conductivity are important drivers for shaping the archaeal community. The hydrogenotrophic methanogens Methanomicrobiales and Methanobacteriales greatly dominated 9 of 11 samples, ranging from 83.7 to 99.5 %. These methanogens also dominated the remaining two samples, accounting for 70.3 and 58.8 %, respectively. Interestingly, for all of the studied Chinese landfills, 16S rRNA analysis indicated the predominance of hydrogenotrophic methanogens.

The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.

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