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In this study, the taxonomic and functional diversity of methanogenic archaea in two parallel 120 l fermenters operated at different temperatures and fed with maize silage was estimated by mcrA metabarcoding analysis using two typical primer pairs (ML and MLA) amplifying part of the functional methyl coenzyme M reductase (mcrA) gene. The alpha diversity indices showed that the ML primer pair detected a higher Operational Taxonomic Unit (OTU) abundance compared to the MLA primer pair and methanogen diversity was significantly lower in the 60 °C fermenters. The beta diversity analysis showed the methanogenic community clustered together at 50 °C and 40° and was statistically different from the 60 °C community. Similar, to alpha diversity, beta diversity was also significantly different between primer pairs. At all temperatures analysed, the primer pairs showed a different abundance of the different methanogenic OTUs, e.g. more OTUs relative to Methanoculleus sp. with the ML primer pair, and more OTUs corresponding to Methanobacterium sp. with the MLA primer pair. Moreover, OTUs corresponding to Methanosphaera sp. and Methanobrevibacter sp. were found only by using ML primer pair, while the MLA primer pair detected sequences corresponding to Methanothrix sp.

Rumen cannulation is a widely employed technique in ruminant nutrition research. However, the gap between skin and rumen cannula can cause leakage of fermentation gases and influx of atmospheric air, which may adversely affect the anaerobic environment in the rumen. The present study was designed to investigate the effects of rumen cannulation on headspace gases, dissolved gases, fermentation end products, and methanogen community in the rumen of dairy cows. Eight Holstein cows were used in the experiment. Four cows were surgically fitted with rumen cannulas, whereas the other 4 intact cows were used as control. Rumen cannulation decreased gaseous hydrogen and methane concentrations, dissolved carbon dioxide concentration, and relative abundances of Methanosphaera, and increased the saturation factor of dissolved hydrogen and dissolved methane, dissolved methane concentration, volatile fatty acid concentration, 16S ribosomal RNA gene copies of methanogens, and Simpson index of methanogen community. In summary, rumen cannulation causes a reduction in headspace gaseous hydrogen and gaseous methane, which may not decrease dissolved gas concentrations due to an increase in saturation factors. Furthermore, rumen cannulation alters methanogen community with increased methanogen population and decreased relative abundances of Methanosphaera.

Environmental microbiome studies rely on fast and accurate bioinformatics tools to characterize the taxonomic composition of samples based on the 16S rRNA gene. MetaGenome Rapid Annotation using Subsystem Technology (MG-RAST) and Quantitative Insights Into Microbial Ecology 2 (QIIME2) are two of the most popular tools available to perform this task. Their underlying algorithms differ in many aspects, and therefore the comparison of the pipelines provides insights into their best use and interpretation of the outcomes. Both of these bioinformatics tools are based on several specialized algorithms pipelined together, but whereas MG-RAST is a user-friendly webserver that clusters rRNA sequences based on their similarity to create Operational Taxonomic Units (OTU), QIIME2 employs DADA2 in the construction of Amplicon Sequence Variants (ASV) by applying an error model that considers the abundance of each sequence and its similarity to other sequences. Taxonomic compositions obtained from the analyses of amplicon sequences of DNA from swine intestinal gut and faecal microbiota samples using MG-RAST and QIIME2 were compared at domain-, phylum-, family- and genus-levels in terms of richness, relative abundance and diversity. We found significant differences between the microbiota profiles obtained from each pipeline. At domain level, bacteria were relatively more abundant using QIIME2 than MG-RAST; at phylum level, seven taxa were identified exclusively by QIIME2; at family level, samples processed in QIIME2 showed higher evenness and richness (assessed by Shannon and Simpson indices). The genus-level compositions obtained from each pipeline were used in partial least squares-discriminant analyses (PLS-DA) to discriminate between sample collection sites (caecum, colon and faeces). The results showed that different genera were found to be significant for the models, based on the Variable Importance in Projection, e.g. when using sequencing data processed by MG-RAST, the three most important genera were Acetitomaculum, Ruminococcus and Methanosphaera, whereas when data was processed using QIIME2, these were Candidatus Methanomethylophilus, Sphaerochaeta and Anaerorhabdus. Furthermore, the application of differential filtering procedures before the PLS-DA revealed higher accuracy when using non-restricted datasets obtained from MG-RAST, whereas datasets obtained from QIIME2 resulted in more accurate discrimination of sample collection sites after removing genera with low relative abundances (

Reducing rumen methane emissions based on the basal diet is one primary approach for livestock production. However, microbiological mechanisms of rumen methane emissions under different diets are very complex and lack comprehensive understanding. This research developed an innovative multilayer network framework to investigate relationships of rumen microbes, transformations of metabolites and functions of rumen microbial genes. The multilayer network model explored the diets-based variation in rumen microbial system differences. The topological structure of the multilayer network reflects volatile fatty acid (VFA) fermentation characteristics between diets. Propionate is the highest degree node of the concentrate-diet (CONC) multilayer network while acetate has the highest degree in the forage-diet (FOR) multilayer network. The microbial communities under FOR diet are more diverse and have more complicated interactions. Methanobrevibacter copresented with Methanosphaera only in the CONC network. There are 33 microbial functional genes in the FOR network that are enriched in the methane synthesize functional module, including the CO2 reduction and the enzymatic reaction of acetyl phosphate to synthesize acetyl-CoA. The microbial genes of the CONC network are found to be associated with the serine biosynthetic and the biochemical process of acetate convert to acetyl-CoA and acetyl phosphate. This study verified the variations of diet-based methane phenotypic are results of systemic changing in the metabolism of the entire rumen microbiome.

The purpose of this study was to investigate the effect of 3-nitrooxypropanol (3-NOP), a potent methane inhibitor, on total and metabolically active methanogens in the rumen of dairy cows over the course of the day and over a 12-wk period. Rumen contents of 8 ruminally cannulated early-lactation dairy cows were sampled at 2, 6, and 10 h after feeding during wk 4, 8, and 12 of a randomized complete block design experiment in which 3-NOP was fed at 60 mg/kg of feed dry matter. Cows (4 fed the control and 4 fed the 3-NOP diet) were blocked based on their previous lactation milk yield or predicted milk yield. Rumen samples were extracted for microbial DNA (total) and microbial RNA (metabolically active), PCR amplified for the 16S rRNA gene of archaea, sequenced on an Illumina platform, and analyzed for archaea diversity. In addition, the 16S copy number and 3 ruminal methanogenic species were quantified using the real-time quantitative PCR assay. We detected a difference between DNA and RNA (cDNA)-based archaea communities, revealing that ruminal methanogens differ in their metabolic activities. Within DNA and cDNA components, methanogenic communities differed by sampling hour, week, and treatment. Overall, Methanobrevibacter was the dominant genus (94.3%) followed by Methanosphaera, with the latter genus having greater abundance in the cDNA component (14.5%) compared with total populations (5.5%). Methanosphaera was higher at 2 h after feeding, whereas Methanobrevibacter increased at 6 and 10 h in both groups, showing diurnal patterns among individual methanogenic lineages. Methanobrevibacter was reduced at wk 4, whereas Methanosphaera was reduced at wk 8 and 12 in cows supplemented with 3-NOP compared with control cows, suggesting differential responses among methanogens to 3-NOP. A reduction in Methanobrevibacter ruminantium in all 3-NOP samples from wk 8 was confirmed using real-time quantitative PCR. The relative abundance of individual methanogens was driven by a combination of dietary composition, dry matter intake, and hydrogen concentrations in the rumen. This study provides novel information on the effects of 3-NOP on individual methanogenic lineages, but further studies are needed to understand temporal dynamics and to validate the effects of 3-NOP on individual lineages of ruminal methanogens.

Rumen archaea play an important role in scavenging ruminal hydrogen (H2) and thus facilitate rumen fermentation. They require optimum temperature and osmolality for their growth and metabolism; however, a number of external factors may put archaea under heat and osmotic stress. Betaine is an osmolyte, molecular chaperone, and antioxidant; therefore, it bears potential to combat against these stressors. In this in vitro study, three betaine levels, namely, 0 (control), 51 (low), and 286 (high) ppm, were used. Each of these was subjected to two temperatures (39.5 and 42°C) and two osmolality conditions (295 and 420 mOsmol kg-1) with n = 6 per treatment. Sequencing analyses of the solid phase (which use solid materials containing primarily fibrous materials of low-density feed particles) and the liquid phase (rumen fermenter liquid) using 16S rRNA revealed that more than 99.8% of the ruminal archaea in fermenters belong to the phylum Euryarchaeota. At the genus level, Methanobrevibacter was the most prevalent in both phases, and Methanosaeta was only detected in the liquid phase. The genera Methanobrevibacter and Methanobacterium both showed a positive correlation with methane (CH4) formation in the liquid and solid phases, respectively ( P < 0.05 ). Heat stress increased the relative abundance of genus Methanimicrococcus at the expense of candidate archaeal genus Vadin CA11 ( P < 0.05 ). In the solid phase, osmotic stress significantly reduced the Shannon and Simpson indices of diversity, and relative abundance was higher for Methanobrevibacter at the expense of Methanimicrococcus. In the liquid phase, osmotic stress increased not only the abundance-based coverage estimator (ACE) and singles parameters of diversity but also the relative abundances of Methanosphaera and Methanobacterium. The overall decrease in all gas parameters and estimated metabolic hydrogen ([2H]) utilization was observed during osmotic stress conditions ( P < 0.05 ). Betaine enhanced the diversity of solid phase archaea as indicated by the increase in ACE and singles during heat stress, and only a high dose improved all diversity parameters in the liquid phase during osmotic stress ( P < 0.05 ). Thus, betaine alleviates the effects of heat stress and osmotic stress on the archaea community.

Mitigation of greenhouse gas emissions is relevant for reducing the environmental impact of ruminant production. In this study, the rumen microbiome from Holstein cows was characterized through a combination of 16S rRNA gene and shotgun metagenomic sequencing. Methane production (CH4) and dry matter intake (DMI) were individually measured over 4–6 weeks to calculate the CH4 yield (CH4y = CH4/DMI) per cow. We implemented a combination of clustering, multivariate and mixed model analyses to identify a set of operational taxonomic unit (OTU) jointly associated with CH4y and the structure of ruminal microbial communities. Three ruminotype clusters (R1, R2 and R3) were identified, and R2 was associated with higher CH4y. The taxonomic composition on R2 had lower abundance of Succinivibrionaceae and Methanosphaera, and higher abundance of Ruminococcaceae, Christensenellaceae and Lachnospiraceae. Metagenomic data confirmed the lower abundance of Succinivibrionaceae and Methanosphaera in R2 and identified genera (Fibrobacter and unclassified Bacteroidales) not highlighted by metataxonomic analysis. In addition, the functional metagenomic analysis revealed that samples classified in cluster R2 were overrepresented by genes coding for KEGG modules associated with methanogenesis, including a significant relative abundance of the methyl‐coenzyme M reductase enzyme. Based on the cluster assignment, we applied a sparse partial least‐squares discriminant analysis at the taxonomic and functional levels. In addition, we implemented a sPLS regression model using the phenotypic variation of CH4y. By combining these two approaches, we identified 86 discriminant bacterial OTUs, notably including families linked to CH4 emission such as Succinivibrionaceae, Ruminococcaceae, Christensenellaceae, Lachnospiraceae and Rikenellaceae. These selected OTUs explained 24% of the CH4y phenotypic variance, whereas the host genome contribution was ~14%. In summary, we identified rumen microbial biomarkers associated with the methane production of dairy cows; these biomarkers could be used for targeted methane‐reduction selection programmes in the dairy cattle industry provided they are heritable., Y. Ramayo‐Caldas was funded by the European Union H2020 Research and Innovation programme under Marie Skłodowska‐Curie grant (P‐Sphere) agreement no 6655919. L. Zingareti is recipient of a FPI grant to achieve the PhD research from Ministry of Economy and Science (MINECO, Spain), associated with the “Centro de Excelencia Severo Ochoa 2016–2019” award SEV‐2015‐0533 to CRAG. The authors warmly thank all technical staff at the INRA Méjusseaume farm for providing high‐quality measurements. This work was funded by APIS‐GENE and used production data obtained in the Deffilait project cofounded by APIS‐GENE and the Agence Nationale de la Recherche (ANR‐15‐CE20‐0014).

MethanobrevibacterandMethanosphaeraspecies 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 ofMethanobrevibacter thaueristrain DSM 11995T,Methanobrevibacter woeseistrain DSM 11979T, andMethanosphaera cuniculistrain 4103Tare reported and compared to those of 16 otherMethanobrevibacterandMethanosphaeragenomes, 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 inMethanosphaeraspecies but reveals also that the three species share a core set of more than 300 genes that distinguishes the genusMethanosphaerafrom the genusMethanobrevibacter. Multilocus sequence analysis shows that the genusMethanobrevibactercan 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 ofMethanobrevibacterspecies from the termite gut andMethanobrevibacter arboriphilusstrains but also the potential capability to utilize ethanol and methanol in a clade comprisingMethanobrevibacter woliniistrain DSM 11976T,Mbb.sp. AbM4, andMbb. boviskoreanistrain DSM 25824T.

During groundwater recharge with reclaimed water, pentabromodiphenyl ether (BDE-99) might contaminate the underground aquifers. In order to understand the effects of elevated sulfate concentrations on the biodegradation of BDE-99, lab-scale column experiments were performed to study the changes in bacterial and archaeal communities. The results confirmed that the biodegradation rate of BDE-99 improved significantly with the addition of sulfate. Oxygen was replaced by sulfate as the predominant electron acceptor, and the major biodebromination metabolites of BDE-99 were BDE-28 and BDE-47, respectively, The results from 454 pyrosequencing of 16s rRNA genes showed that the microbial community structure shifted toward communities with BDE-99 co-metabolism ability. At the bacterial genus level, Geothrix, Thiobacillus, Sphingomonas, Paenibacillus and Desulforhabdus were the five most abundant genera, wherein, Thiobacillus was able to treat the toxic products generated during polybrominated diphenyl ethers (PBDE) biodegradation. In the case of archaea at the genus level, Methanobrevibacter, Methanomethylovorans, DHVEG-6, Candidatus Nitrososphaera and Methanosphaera were the predominant groups, of which Methanobrevibacter and Methanosphaera showed good ability to metabolize BDE-99 with sulfate amendment.

Heifers emit more enteric methane (CH4) than adult cows and these emissions tend to decrease per unit feed intake as they age. However, common mitigation strategies like expensive high-quality feeds are not economically feasible for these pre-production animals. Given its direct role in CH4 production, altering the rumen microbiota is another potential avenue for reducing CH4 production by ruminants. However, to identify effective microbial targets, a better understanding of the rumen microbiota and its relationship to CH4 production across heifer development is needed. RESULTS Here, we investigate the relationship between rumen bacterial, archaeal, and fungal communities as well as CH4 emissions and a number of production traits in prepubertal (PP), pubertal (PB), and pregnant heifers (PG). Overall, PG heifers emitted the most CH4, followed by PB and PP heifers. The bacterial genus Acetobacter and the archaeal genus Methanobrevibacter were positively associated, while Eubacterium and Methanosphaera were negatively associated with raw CH4 production by heifers. When corrected for dietary intake, both Eubacterium and Methanosphaera remained negatively associated with CH4 production. CONCLUSION We suggest that Eubacterium and Methanosphaera represent likely targets for CH4 mitigation efforts in heifers as they were negatively associated with CH4 production and not significantly associated with production traits. ? 2018 Society of Chemical Industry

Diet impacts the composition of the ruminal microbiota; however, prior to slaughter, cattle are fasted, which may change the ruminal microbial ecosystem structure and lead to dysbiosis. The objective of this study was to determine changes occurring in the rumen after pre-slaughter fasting, which can allow harmful pathogens an opportunity to establish in the rumen. Ruminal samples were collected before and after pre-slaughter fasting from seventeen commercial Angus steers. DNA extraction and 16S rRNA gene sequencing were performed to determine the ruminal microbiota, as well as volatile fatty acid (VFA) concentrations. Microbial richness (Chao 1 index), evenness, and Shannon diversity index all increased after fasting (p ≤ 0.040). During fasting, the two predominant families Prevotellaceae and Ruminococcaceae decreased (p ≤ 0.029), whereas the remaining minor families increased (p < 0.001). Fasting increased Blautia and Methanosphaera (p ≤ 0.003), while Campylobacter and Treponema tended to increase (p ≤ 0.086). Butyrate concentration tended to decrease (p = 0.068) after fasting. The present findings support that fasting causes ruminal nutrient depletion resulting in dysbiosis, allowing opportunistic pathogens to exploit the void in the ruminal ecological niche.

This paper studies the abiotic properties and the microbial community of a pilot scale digester with a working volume capacity of 300 L. The system worked at mesophilic operating conditions, with several organic load rates formed by a mixture of different ratios between waste A (fruit and vegetable sludge + tomato waste) and waste B (pig slurry). The core group of microorganisms corresponding to the ratio 70% A:30% B, showed the highest methane production (60%). This core group included bacteria, principally Firmicutes and Bacteroidetes , involved in cellulolytic degradation, proteolysis and acidogenesis, as well as a concomitance between acetotrophic, mainly Methanosaeta methanogens and hydrogenotrophic, principally Methanosphaera and Methanobrevibacter methanogens. An increase of the proportion of waste B (pig slurry) to 50–60% resulted in a decrease of the production of biogas and methane, which unbalanced the methanogenic bacteria, increasing the amount of principally hydrogenotropic methanogens ( Methanosphaera and Methanobrevibacter ).

Rumen in vitro fermentation was used to evaluate the capacity of nitrooxy compounds to mitigate rumen methane production. The following three nitrooxy compounds, each with different molecular structures, were evaluated: 2,2-dimethyl-3-(nitrooxy) propanoic (DNP), N-[2-(Nitrooxy)ethyl]-3-pyridinecarboxamide (NPD), and nitroglycerin (NG). All three compounds substantially decreased the total gas production, methane production, and the acetate:propionate ratio, while increasing hydrogen production. The growth of methanogens was specifically inhibited by all three compounds, without affecting the abundance of bacteria, anaerobic fungi, or protozoa. However, inhibition of methanogenesis required a much higher dose of DNP when compared to NPD or NG. Further investigations were conducted on NG to determine its effects on the methanogenic community. NG reduced the relative abundance of Methanomassiliicoccales, while increasing the relative abundance of Methanobrevibacter and Methanosphaera. Overall, the results suggested that all three of these nitrooxy compounds could specifically inhibit rumen methanogenesis, but NPD and NG were much more efficient than DNP at rumen methane mitigation.

Detailed knowledge of the community structure of methanogens is essential for amelioration of methane emission from livestock species. Several studies have indicated that predominant methanogens of buffalo rumen are different from those in cattle. However, predominant genera of methanogens reported by individual studies varied primarily because of limited scope of sampling, sequencing of limited number of sequences and potential PCR bias in individual studies. In this study, the collective comparative diversity of methanogenic archaea in the rumen of cattle and buffaloes was examined by performing a meta-analysis of all the 16S rRNA (rrn) sequences deposited in GenBank.; Results: Ruminal methanogen sequences of buffalo were clustered into 900 species-level operational taxonomic units (OTUs), and ruminal methanogen sequences of cattle were clustered into 1522 species level OTUs. The number of species-level OTUs shared between cattle and buffaloes was 229 (10.4% of all OTUs), comprising 1746 sequences (27% of the total 6447 sequences). According to taxonomic classification by three different classifiers, Methanobrevibacter was found to be the most predominant genus both in cattle (69-71% of sequences) as well as buffaloes (65.1-68.9% of sequences). Percentage of Methanomicrobium was much higher (P

The greenhouse gases (GHGs) derived from agriculture include carbon dioxide, nitrous oxide, and methane (CH4). Of these GHGs, CH4, in particular, constitutes a major component of the GHG emitted by the agricultural sector. Along with environmental concerns, CH4 emission also leads to losses in gross energy intake with economic implications. While ruminants are considered the main source of CH4 from agriculture, nonruminant animals also contribute substantially, and the CH4 emission intensity of nonruminants remains comparable to that of ruminants. Means of mitigating CH4 emissions from enteric fermentation have therefore been sought. Methane is produced by methanogens-archaeal microorganisms that inhabit the digestive tracts of animals and participate in fermentation processes. As the diversity of methanogen communities is thought to be responsible for the differences in CH4 production among nonruminant animals, it is necessary to investigate the archaeal composition of specific animal species. Methanogens play an important role in energy metabolism and adipose tissue deposition in animals. Higher abundances of methanogens, along with their higher diversity, have been reported to contribute to lean phenotype in pigs. In particular, a greater abundance of Methanosphaera spp. and early dominance of Methanobrevibacter smithii have been reported to correlate with lower body fat formation in pigs. Besides the contribution of methanogens to the metabolic phenotype of their hosts, CH4 release reduces the productivity that could be achieved through other hydrogen (H2) disposal pathways. Enhanced participation of acetogenesis in H2 disposal, leading to acetate formation, could be a more favorable direction for animal production and the environment. Better knowledge and understanding of the archaeal communities of the gastrointestinal tract (GIT), including their metabolism and interactions with other microorganisms, would thus allow the development of new strategies for inhibiting methanogens and shifting toward acetogenesis. There are a variety of approaches to inhibiting methanogens and mitigating methanogenesis in ruminants, which can find an application for nonruminants, such as nutritional changes through supplementation with biologically active compounds and management changes. We summarize the available reports and provide a comprehensive review of methanogens living in the GIT of various nonruminants, such as swine, horses, donkeys, rabbits, and poultry. This review will help in a better understanding of the populations and diversity of methanogens and the implications of their presence in nonruminant animals.

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.

As a major reservoir of antibiotics, animal manure contributes a lot to the augmented environmental pressure of antibiotic resistance genes (ARGs). This might be the first study to explore the effects of different ventilation types on the control of ARGs and to identify the relationships between archaeal communities and ARGs during the composting of dairy manure. Several ARGs were quantified via Real-time qPCR and microbial communities including bacteria and archaea were analyzed by High-throughput sequencing during vacuum-type composting (VTC) and positive-pressure composting (PPC). The total detected ARGs and class I integrase gene (intI1) under VTC were significantly lower than that under PPC during each stage of the composting (p

The risk of overweight or obesity in association with early exposure of antibiotics remains an important public issue for health-care of children. Low-dose antibiotics (LDA) have been widely used to enhance growth rate of pigs, providing a good animal model to study the underlying mechanism. In present study, 28 female piglets, weaned at 21 d, were randomly classified into two groups, receiving either a control diet or a diet supplemented with LDA for 4 weeks. The total bacterial load and intestinal microbiota were determined by qPCR and 16S rRNA amplicon sequencing. UPLC-QTRAP-MS/MS and RNA-seq were further used to determine the colonic SCFAs and transcriptomes. Results showed that LDA significantly increased growth rate and food intake. The F/B index, Methanosphaera species, and the pathway of “carbohydrate metabolism” were improved by LDA exposure, indicating the better carbohydrate degradation and energy utilization. Furthermore, correlation analysis indicated the microbial community contributing to SCFAs production was enriched upon LDA exposure, associating with increased concentrations of short-chain and branched-chain fatty acids (caproate, 2-methyl butyrate and 4-methyl valerate). A multivariate linear fitting model analysis highlighted that caproate was positively correlated with two genera (Faecalibacterium and Allisonella) and four differentially expressed genes (ZNF134, TBX5, NEU4 and SEMA6D), which were all significantly increased upon LDA exposure. Collectively, our study indicates that the growth-promoting effect of LDA exposure in early life is associated with the shifts of colonic microbiota to increase utilization of carbohydrates and energy, enhanced SCFAs production and colonic functions.

The diversity of the gastrointestinal microbiome is closely associated with human health. In the present study, the gastrointestinal microbiome and tumor-infiltrating lymphocytes (TILs) were compared in patients with breast cancer (BC). A total of 80 patients with BC were divided into three groups based on the expression of TILs, as follows: High expression of TILs (TIL-H), medium expression of TILs (TIL-M) and low expression of TILs (TIL-L). DNA of the gastrointestinal microbiome was determined by Illumina sequencing and taxonomy of 16S ribosomal RNA genes. A χ2 test and UniFrac analysis of β-diversity were applied to assess the association between clinical characteristics and diversity of the gastrointestinal microbiome. The β-diversity distribution was statistically significant (weighted UniFrac, P

The bovine gastrointestinal (GI) tract microbiota has important influences on animal health and production. Presently, a large number of studies have used high-throughput sequencing of the archaeal and bacteria 16S rRNA gene to characterize these microbiota under various experimental parameters. By aggregating publically available archaeal and bacterial 16S rRNA gene datasets from 52 studies we were able to determine taxa that are common to nearly all microbiota samples from the bovine GI tract as well as taxa that are strongly linked to either the rumen or feces. The methanogenic genera Methanobrevibacter and Methanosphaera were identified in nearly all fecal and rumen samples (> 99.1%), as were the bacterial genera Prevotella and Ruminococcus (≥ 92.9%). Bacterial genera such as Alistipes, Bacteroides, Clostridium, Faecalibacterium and Escherichia/Shigella were associated with feces and Fibrobacter, Prevotella, Ruminococcus and Succiniclasticum with the rumen. As expected, individual study strongly affected the bacterial community structure, however, fecal and rumen samples did appear separated from each other. This meta-analysis provides the first comparison of high-throughput sequencing 16S rRNA gene datasets generated from the bovine GI tract by multiple studies and may serve as a foundation for improving future microbial community research with cattle.

This work investigated the changes of the rumen microbiome of goats switched from a forage to a concentrate diet with special attention to anaerobic fungi (AF). Female goats were fed an alfalfa hay (AH) diet (0% grain, n = 4) for 20 days and were then abruptly shifted to a high-grain (HG) diet (40% corn grain, 60% AH, n = 4) and treated for another 10 days. Rumen content samples were collected from the cannulated animals at the end of each diet period (day 20 and 30). The microbiome structure was studied using high-throughput sequencing for bacteria, archaea (16S rRNA gene) and fungi (ITS2), accompanied by qPCR for each group. To further elucidate unclassified AF, clone library analyses were performed on the ITS1 spacer region. Rumen pH was significantly lower in HG diet fed goats, but did not induce subacute ruminal acidosis. HG diet altered prokaryotic communities, with a significant increase of Bacteroidetes and a decrease of Firmicutes. On the genus level Prevotella 1 was significantly boosted. Methanobrevibacter and Methanosphaera were the most abundant archaea regardless of the diet and HG induced a significant augmentation of unclassified Thermoplasmatales. For anaerobic fungi, HG triggered a considerable rise in Feramyces observed with both ITS markers, while a decline of Tahromyces was detected by ITS2 and decrease of Joblinomyces by ITS1 only. The uncultured BlackRhino group revealed by ITS1 and further elucidated in one sample by LSU analysis, formed a considerable part of the AF community of goats fed both diets. Results strongly indicate that the rumen ecosystem still acts as a source for novel microorganisms and unexplored microbial interactions and that initial rumen microbiota of the host animal considerably influences the reaction pattern upon diet change.

• Methane emissions from ruminant livestock are proposed to contribute ~10 to 20% of annual global greenhouse gas emissions. • Recent efforts have shown that "low methane emitting" animals can be identified both within and across species, including nondomesticated herbivores. • Methanogens are also present within the human gut and some other body sites, and variations in their diversity and/or abundance has been linked to some functional gastrointestinal disorders and non-communicable chronic diseases. • Recent advances in "-omics" technologies have provided new insights of the roles that methanogens play in both feed digestion and host-microbe interactions. • Although much research and understanding has been derived from the study of the numerically dominant Methanobrevibacter genus, the recent studies outlined above have shown that other, "heterotrophic" methanogens warrant greater attention. • Here, we provide a brief overview of our recent research of the Methanosphaera genus and outline the possible consequences associated with its presence in the gut of livestock, native Australian herbivores, and humans.

peer-reviewed 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.

The aim of this study was to investigate the effects of 3-nitrooxypropanol (3-NOP) and chloroform on methane (CH4) and H2 production, ruminal metabolites and microbial community structure in cattle fed a tropical forage diet. Eight rumen-fistulated steers were fed a roughage hay diet (Rhodes grass; Chloris gayana) for 31 days (control period). Four animals received the antimethanogenic compound chloroform (1.6 g chloroform–cyclodextrin/100 kg live weight (LW)) while the other four received 3-NOP (2.5 g 3-NOP/animal/day) for 21 days. Methane decrease compared with control period was similar for both treatments (30–38%) with no differences for expelled H2 between controls and treatments. Daily weight gain (DWG) was significantly increased when animals were treated with 3-NOP compared with chloroform and control. Regarding the ruminal fermentation parameters increases in ammonia, acetate and branched chain fatty acids were observed with both compounds compared with the controls. Also, methylamines, alcohols and dimethyl sulfone (DMSO2) concentrations were significantly increased with the treatments compared with control, being greater with 3-NOP. The rumen microbial analyses revealed a similar profile for both treatments, with a shift in operational taxonomic units (OTUs) assigned to the Prevotellaceae and Campylobacteraceae family. Moreover, major archaeal OTUs associated with Methanobrevibacter and Methanosphaera were significantly affected to varying extents based on the inhibitory treatments compared to the control. The abundance of the Methanobrevibacter spp. was decreased by 3-NOP and chloroform, while the Methanomassiliicoccaceae family was inhibited only by 3-NOP. The results suggest that despite the specific mode of action of 3-NOP on methanogens, inhibition of methanogenesis by both compounds resulted in similar responses in metabolism and microbial community structure in the rumen. We hypothesized that these changes were driven by the redirection of metabolic hydrogen ([H]) by both treatments. Therefore results from previous publications using chloroform as an inhibitor of methanogenesis may be useful in predicting ruminal microbiota and fermentation responses to 3-NOP.

Limit-feeding of a high concentrate diet has been proposed as an effective method for improving feed efficiency and reducing total manure output of dairy heifers; meanwhile the effects of this method on hindgut microbiota are still unclear. This study investigated the effects of a wide range of dietary forage:concentrate ratios (F:C) on the fecal composition of bacteria and archaea in heifers using next-generation sequencing. Four diets with different F:C (80:20, 60:40, 40:60, and 20:80) were limit-fed to 24 Holstein heifers, and the fecal fermentation parameters and bacterial and archaeal communities were investigated. With increasing dietary concentrate levels, the fecal dry matter output, neutral detergent fiber (NDF) content, and proportion of acetate decreased linearly (P < 0.01), while the fecal starch content and proportions of propionate, butyrate, and total branched-chain volatile fatty acids (TBCVFAs) were increased (P ≤ 0.05). An increased concentrate level linearly increased (P = 0.02) the relative abundance of Proteobacteria, and linearly decreased (P = 0.02) the relative abundance of Bacteroidetes in feces. At the genus level, the relative abundance of unclassified Ruminococcaceae and Paludibacter which may have the potential to degrade forage decreased linearly (q ≤ 0.02) with increasing dietary concentrate levels, while the relative abundance of Roseburia and Succinivibrio which may be non-fibrous carbohydrate degrading bacteria increased linearly (q ≤ 0.05). Some core microbiota operational taxonomic units (OTUs) also showed significant association with fecal VFAs, NDF, and/or acid detergent fiber (ADF) content. Meanwhile, the relative abundance of most detected taxa in archaea were similar across different F:C, and only Methanosphaera showed a linear decrease (P = 0.01) in high concentrate diets. Our study provides a better understanding of fecal fermentation parameters and microbiota under a wide range of dietary F:C. These findings support the potential for microbial manipulation by diet, which could enhance feed digestibility and relieve environmental problems associated with heifer rearing.

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

The cecum plays an important role in the feed fermentation of ruminants. However, information is very limited regarding the cecal microbiota and their methane production. In the present study, the cecal content from twelve local Chinese goats, fed with either a hay diet (0% grain) or a high-grain diet (71.5% grain), were used to investigate the bacterial and archaeal community and their methanogenic potential. Microbial community analysis was determined using high-throughput sequencing of 16S rRNA genes and real-time PCR, and the methanogenesis potential was assessed by in vitro fermentation with ground corn or hay as substrates. Compared with the hay group, the high-grain diet significantly increased the length and weight of the cecum, the proportions of starch and crude protein, the concentrations of volatile fatty acids and ammonia nitrogen, but decreased the pH values (P

Murrah and Nili-Ravi are water buffalo breeds widely used as dairy animals in Asian countries. In this study, we investigated the diversity of ruminal microbes in six Murrah and six Nili-Ravi water buffaloes consuming diets typical of those used in Southern China which had forage to concentrate ratios of 3.2 and 1.6. After feeding the diets for 25 days, ruminal fluid was sampled by stomach tube before the morning feeding. Bacterial and archaeal 16S rRNA genes and the ciliate protozoal 18S rRNA genes were PCR-amplified from DNA extracted from rumen samples, sequenced using 454 Titanium pyrosequencing, and analyzed using the QIIME software package. Our results showed that, at the phylum level, Bacteroidetes was the predominant bacterial group, accounting for 42–72% of total bacteria, followed by Firmicutes, Fibrobacteres, Proteobacteria and Spirochaetes. At genus level, Prevotella dominated, accounting for 22–58% of total bacteria, followed by Fibrobacter, Paludibacter, and Ruminococcus. While there were differences between the bacterial community compositions of different animals, there was no obvious correlation of bacterial community composition at the phylum or genus level with the diets or with buffalo breeds. Methanobrevibacter-related organisms were the dominant archaeal group, accounting for around 80% of the total, followed by Methanomassiliicoccales (Rumen Cluster C (RCC), 15%) and Methanosphaera (3%). Similar to the bacterial community, there was no obvious correlation of archaeal community profiles with diet or buffalo breed. The ciliate protozoal communities differed between the samples analyzed, and Entodinium was the most abundant group of ciliates in every sample, accounting for more than 40% of total protozoa. The second largest ciliate group varied in different samples, with Isotricha, Polyplastron or Dasytricha being the most dominant genera after Entodinium.

Wheat straw (WS) was pretreated with four concentrations of H2O2 (1%, 2%, 3%, and 4%) and was anaerobically codigested with dairy cattle manure (CM) at various ratios from 100:0 to 0:100. Wet-state H2O2 pretreatment effectively enhanced the biodegradability and methane yield of the WS. The optimal concentration of H2O2 for treating WS was 3%. The methane yield was higher with the codigestion of CM and H2O2-treated WS than with untreated WS and higher than with H2O2-treated WS alone or CM alone. A 40:60 ratio of H2O2-treated WS mixed with CM produced the highest yield of methane (320.8 mL g volatile solid (VS)−1). Results of high-throughput sequencing indicated that the methanogenic community shifted during the codigestion from the acetoclastic methanogens, Methanosarcina, to the hydrogenotrophic methanogens, Methanosphaera and Methanoculleus.

The methanogenic archaea are responsible for maintaining an efficient scheme of fermentation in many environments and habitats, including the gastrointestinal tracts of animals and humans. The principal members of gut methanogenic communities are members of the Methanobrevibacter genus, with lesser numbers of Methanosphaera and Methanomassiliicoccus spp. Much of our understanding of gut methanogens has been produced using axenic cultures and genomic data for ~30 Methanobrevibacter spp. but the functional relevance of these other archaeal lineages to digestive function remains poorly understood. With this background, the goals of my PhD research are: i) to increase the biotic representation of the Methanosphaera genus through the recovery of axenic isolates from different environments; ii) characterise the metabolic properties of these isolates in terms of their methanogenic pathways and; iii) expand our functional understanding of this genus via reconstruction of “population genomes” from existing metagenomic datasets and comparative genome analyses. During the latter stages of my PhD, I chose to make a transition in my research activities to include some biomedical focus and take advantage of my relocation to the University of Queensland Diamantina Institute. Here, I have examined variations in methanogenic archaeal populations in some clinical studies, as well as evaluated the immunostimulatory properties of some gut archaea. Chapter Two describes the enrichment, isolation, and pathways of methane formation by a strain of Methanosphaera sp. (WGK6) recovered from the Western Grey kangaroo (Macropus fulginosus). In contrast with the human isolate, strain WGK6 was found to utilize ethanol to support growth, but principally as a source of reducing power. Both the WGK6 and Msp. stadtmanae DSMZ 3091T genomes are very similar in terms of their size (~1.7 Mbp), synteny, and G:C content. However, the WGK6 genome was found to encode contiguous genes encoding putative alcohol- and aldehyde- dehydrogenases, which are absent from the Msp. stadtmanae genome. These two genes provide a plausible explanation for the ability of WGK6 to utilize ethanol for methanol reduction to methane. Furthermore, my in vitro studies suggest that ethanol supports a greater cell yield per mol of methane formed compared to hydrogen-dependent growth. Taken together, this expansion in metabolic versatility can explain the persistence of these archaea in the kangaroo foregut, and their abundance in these “low methane emitting” herbivores. Chapter Three describes the isolation of a hydrogen-dependent methylotrophic archaeon assigned to the Methanosphaera genus from bovine animals in northern Australia (strain BMS). The BMS genome was sequenced to closure and surprisingly, found to be substantially larger (2.9 Mbp) than the WGK6 and Msp. stadtmanae genomes (~1.8 Mbp). I then interrogated metagenomic datasets produced from human stool and ruminant digesta samples, to recover 7 “population genomes” assigned to the Methanosphaera genus, with 5 of these genomes also predicted to be large (> 2.1 Mbp). The Methanosphaera pan-genome consists of 5321 genes and 305 of these were assigned to the core-genome, which principally consists of the genes coordinating methanogenesis, anaerobic metabolism, and related functions. The whole genome phylogeny analysis of all genomes suggests a monophyletic origin for the genus Methanosphaera, with those isolates possessing smaller genomes being the most recently evolved lineages. Chapter Four investigates whether and how the composition of the methanogenic archaea in patients with chronic kidney disease is altered in response to synbiotic administration, as an intervention designed to mitigate uremic toxin production. The synbiotic intervention was found to have no measurable impact on the methanogen profiles of the patients, with Methanobrevibacter smithii the predominant archaeon and an infrequent occurrence of Methanomassiliicoccus, but no detectable populations of Methanosphaera spp. I also have assessed the immunogenic properties of Mbb. smithii DSMZ 861T, Msp. stadtmanae DSMZ 3091T, Mbb. ruminantium DSMZ 1093T, and my new Methanosphaera isolates, using human peripheral blood mononuclear cells (PBMC) and a mouse macrophage cell line. I first showed that Msp. stadtmanae DSMZ 3091T cell preparations produce a stronger TNF-α response from PBMC than Mbb. smithii DSMZ 861T, as well as the other Methanosphaera isolates WGK6 and BMS, but Mbb. ruminantium DSMZ 1093T cells produced the strongest TNF-α response. In subsequent studies using methanogen cell preparations and different sources of PBMC (also from healthy subjects) I measured the release of a broader range of cytokines, using a multi-bead array and flow cytometry techniques. In these studies, Mbb. smithii DSMZ 861T was more immunogenic compared to Msp. stadtmanae DSMZ 3091T, but both types of cells induce a similar cytokine profile from the PBMC preparations. Last, I used a murine macrophage cell line bearing a reporter gene activated by the NF-κB transcription system, which further confirmed that cell preparations of all these archaea induce reporter gene activation via this important pathway of stress response. Collectively, these results suggest all these methanogens possess immunostimulatory properties that remain cell associated, and the findings are discussed with respect to gastrointestinal inflammation and also the development of anti-methanogen vaccines. Chapter 5 provides an integrative overview of the findings arising from my research, which provide a deeper understanding into the genus Methanosphaera, in terms of its evolutionary development, nutritional ecology, and host adaptation. I believe my findings are novel and provide new insights into the role of methanogenic archaea in gut function; and this knowledge will be useful to address key challenges facing agriculture (e.g. methane abatement strategies) and human health (e.g. as triggers of inflammation and immune response in gut and respiratory disease).

Although the rumen microbiome of domesticated ruminants has been evaluated, few studies have explored the rumen microbiome of wild ruminants, and no studies have identified the rumen microbiome in the impala (Aepyceros melampus melampus). In the present study, next-generation sequencing and real-time polymerase chain reaction were used to investigate the diversity and density of the bacteria and methanogenic archaea residing in the rumen of five adult male impalas, culled during the winter dry season in Pongola, South Africa. A total of 15,323 bacterial 16S rRNA gene sequences (from five impala), representing 3,892 different phylotypes, were assigned to 1,902 operational taxonomic units (OTUs). A total of 20,124 methanogen 16S rRNA gene sequence reads (from four impala), of which 5,028 were unique, were assigned to 344 OTUs. From the total sequence reads, Bacteroidetes, Proteobacteria, and Firmicutes were the most abundant bacterial phyla. While the majority of the bacterial genera found were unclassified, Prevotella and Cupriavidus were the most abundant classified genera. For methanogens, the genera Methanobrevibacter and Methanosphaera represented 94.3% and 4.0% of the classified sequences, respectively. Most notable was the identification of Methanobrevibacter thaueri-like 16S rRNA gene sequence reads in all four impala samples, representing greater than 30% of each individual's total sequences. Both data sets are accessible through NCBI's Sequence Read Archive (SRA), under study accession number SRP [048619]. The densities of bacteria (1.26 × 10(10)-3.82 × 10(10) cells/ml whole rumen contents) and methanogens (4.48 × 10(8)-7.2 × 10(9) cells/ml of whole rumen contents) from five individual impala were similar to those typically observed in domesticated ruminants.

Ruminant livestock represent the single largest anthropogenic source of the potent greenhouse gas methane, which is generated by methanogenic archaea residing in ruminant digestive tracts. While differences between individual animals of the same breed in the amount of methane produced have been observed, the basis for this variation remains to be elucidated. To explore the mechanistic basis of this methane production, we measured methane yields from 22 sheep, which revealed that methane yields are a reproducible, quantitative trait. Deep metagenomic and metatranscriptomic sequencing demonstrated a similar abundance of methanogens and methanogenesis pathway genes in high and low methane emitters. However, transcription of methanogenesis pathway genes was substantially increased in sheep with high methane yields. These results identify a discrete set of rumen methanogens whose methanogenesis pathway transcription profiles correlate with methane yields and provide new targets for CH4 mitigation at the levels of microbiota composition and transcriptional regulation.

Erhualian and Landrace breeds are typical genetically obese and lean pigs, respectively. To compare the fecal methanogenic Archaeal community between these two pig breeds, fecal samples from different growth phase pigs were collected and used for PCR-denaturing gradient gel electrophoresis (DGGE) with two primer pairs (344fGC/519r and 519f/915rGC) and real-time PCR analysis. Results showed that a better separation and higher quality of bands pattern were obtained in DGGE profiles using primers 344fGC/519r as compared with primers 519f/915rGC. Sequencing of DGGE bands showed that the predominant methanogens in the feces of Erhualian and Landrace pigs belonged to Methanobrevibacter spp. and Methanosphaera spp. Real-time PCR analysis revealed that there was no significant difference in the numbers of fecal total methanogens between Erhualian and Landrace pigs; however, pig growth phase affected the numbers of 16S rRNA genes of total methanogens and Methanobrevibacter smithii. Dissociation curves of methyl coenzyme-M reductase subunit A (mcrA) gene fragments amplified with real-time PCR showed all samples possessed a single peak at 82°C, which might be associated with M. smithii. Samples from the same growth phase of each breed showed good replicative dissociation curves. The results suggest that the growth phase (including diet factor) other than genotype of pig may affect the fecal methanogenic Archaeal community of pigs.

Anaerobic digestion (AD) with thermal hydrolysis pretreatment is widely used as an efficient sludge treatment nowadays. However, the evolution of microbial community (especially for the archaea community), the fate of antibiotic resistance genes (ARGs), and their associations during such process in full-scale sludge treatment plants are rarely reported. Therefore, these scientific questions were explored at two full-scale sludge treatment plants through high-throughput sequencing and quantitative PCR. Results showed that Methanobacterium and Methanosphaera were the dominant archaea in thermal hydrolyzed sludge. The predominant bacteria in the sludge first shifted from nutrients removal functional bacteria to spore-forming bacteria after thermal hydrolysis, and then shifted to fermentative bacteria after AD. The full-scale plants could select ermB, ermF, mefA/E, qnrS and tetM. Though the bacteria and archaea biomass and community largely influenced the fate of ARGs, multiple linear regression analysis showed that the total ARGs were mainly affected by mobile genetic elements (MGEs).

Quantitative real-time PCR (qPCR) has become a popular method for estimation of meth- anogen 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 metha- nogen populations estimated using the new archaeol approach with estimates based on qPCR. Specific prim- er sets and probes were used to detect dominant ruminal methanogen species Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanosphaera stadt- manae, 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 metha- nogen 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 use- ful than a biased primer for total methanogens.

This study was performed to investigate the initial colonization of metabolically active methanogens and subsequent changes in four fractions: the rumen solid-phase (RS), liquid-phase (RL), protozoa-associated (RP), and epithelium-associated (RE) from 1 to 60 d after birth, and manipulate methanogen community by early weaning on 40 d and supplementing rhubarb from 40 to 60 d in black goats. The RNA-based real-time quantitative PCR and 16S rRNA amplicon sequencing were employed to indicate the metabolically active methanogens. Results showed that active methanogens colonized in RL and RE on 1 d after birth. RP and RE contained the highest and lowest density of methanogens, respectively. Methanobrevibacter, Candidatus Methanomethylophilus, and Methanosphaera were the top three genera. The methanogen communities before weaning differed from those post weaning and the structure of the methanogen community in RE was distinct from those in the other three fractions. The discrepancies in the distribution of methanogens across four fractions, and various fluctuations in abundances among four fractions according to age were observed. The addition of rhubarb significantly (P Methanimicrococcus spp. in four fractions on 50 d, but did not change the methanogen community composition on 60 d.

Summary In rabbits, many studies have investigated the effect of diet, including the fibre intake, on caecal microbiota; however, there are no direct measurements of the influence of diets with different dietary fibre-to-starch ratios and the archaeal community composition in the caecum. We used 16S rDNA sequencing to investigate the impact of different dietary neutral detergent fibre (NDF)-to-starch ratios (1.0–2.3) on the caecal archaeal community in rabbits. The results revealed that the archaeal community from all experimental rabbits was relatively less complex than intestinal bacterial community. High-throughput 16S rDNA sequencing of the gut archaea indicated that the rabbit caecum was inhabited by Methanobrevibacter and Methanosphaera species, and therefore is possibly dominated by two species of archaea. Here, we compared the effect of different diets on the archaeal community in the rabbit caecum and found no significant differences in the diversity and abundance of caecal archaeal community of rabbits that were fed diets with different dietary fibre-to-starch ratios. Therefore, we suggest that the contribution of host-derived substrates to caecal archaea constitution is insignificant.

Aims To investigate, using culture-independent techniques, the presence and diversity of methanogenic archaea in the foregut of kangaroos. Methods and Results DNA was extracted from forestomach contents of 42 kangaroos (three species), three sheep and three cattle. Four qualitative and quantitative PCR assays targeting the archaeal domain (16S rRNA gene) or the functional methanogenesis gene, mcrA, were used to determine the presence and population density of archaea in kangaroos and whether they were likely to be methanogens. All ruminal samples were positive for archaea, produced PCR product of expected size, contained high numbers of archaea and high numbers of cells with mcrA genes. Kangaroos were much more diverse and contradictory. Fourteen kangaroos had detectable archaea with numbers 10- to 1000-fold fewer than sheep and cattle. Many kangaroos that did not possess archaea were positive for the mcrA gene and had detectable numbers of cells with this gene and vice versa. DNA sequence analysis of kangaroos' archaeal 16S rRNA gene clones show that many methanogens were related to Methanosphaera stadmanae. Other sequences were related to non-methanogenic archaea (Thermoplasma sp.), and a number of kangaroos had mcrA gene sequences related to methane oxidising archaea (ANME). Conclusions Discrepancies between qualitative and quantitative PCR assays for archaea and the mcrA gene suggest that the archaeal communities are very diverse and it is possible that novel species exist. Significance and Impact of the Study Archaea (in general) were below detectable limits in many kangaroos, especially Red kangaroos; when present they are in lower numbers than in ruminants, and the archaea are not necessarily methanogenic. The determination of why this is the case in the kangaroo foregut could assist in reducing emissions from other ecosystems in the future.

Condensed tannins (CT) and corn distillers grains from ethanol production have the potential to reduce enteric CH4 production from ruminants because of their anti-microbial properties and high lipid contents, respectively. However, their effects on the community of rumen methanogens in cattle rumens have not been investigated. Our objective was to evaluate effects of dry corn distillers grain with solubles (DDG) and CT from Acacia mearnsii added to the diet of cattle on rumen methanogens using PCR denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time (qRT)-PCR. Eight ruminally cannulated beef heifers were used in a replicated 4 × 4 Latin square design with four 35 d periods. Diets were (g DDG dry matter [DM]/kg DM): control (Ctrl; 0 DDG), 200 DDG, 400 DDG and 400 DDG plus 25 g CT (400 DDG + CT). Rumen digesta samples were collected before feeding on d 25 and 28 of each period from 4 sites in the rumen. Total DNA extracted from rumen digesta samples (i.e., 0.5 ml liquid + 0.5 g solid pooled by heifer for each period) was used to generate amplicons of partial 16S rRNA genes. The PCR products obtained were subjected to PCR-DGGE. Detectable methanogenic PCR-DGGE profiles from periods 1 and 2 clustered while periods 3 and 4 formed a second cluster. A total of 25 distinct PCR-DGGE bands were identified, of which 16 bands were successfully cloned and sequenced. The sequences of 11 bands had 97–100% similarity with Methanobrevibacter sp., and this was supported by clustering of 10 of these bands with this species upon phylogenetic analysis of the sequences. The remaining bands clustered with Methanosphaera stadmanae. Although no effect of diet on total methanogenic PCR-DGGE profiles occurred, the proportion of bands 4, 5, 6, 7, 9 and 11 increased and bands 16 and 18 decreased with increased DDG in the diet. Bands 13 and 19 were in all diets except 400 DDG + CT. Quantitative RT-PCR analysis revealed that the copy numbers of total methanogenic 16S rRNA gene did not differ among diets. Findings from PCR-DGGE and qRT-PCR analysis suggest that inclusion of DDG or a mixture of DDG and CT altered the diversity of rumen methanogens without affecting total methanogen associated 16S rRNA copy numbers thereby indicating that copy numbers of particular species of methanogens may be required to explain the altered diversity of rumen methanogens. This article is part of the special issue entitled: Greenhouse Gases in Animal Agriculture – Finding a Balance between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors; K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.

Methane emissions from ruminant livestock is generated by the action of methanogenic archaea, mainly in the rumen. A variety of methanogen genera are responsible for CH4 production, including a large group that lacks cultivated representatives. To be generally effective, technologies for reducing ruminant CH4 emissions must target all rumen methanogens to prevent any unaffected methanogen from expanding to occupy the vacated niche. Interventions must also be specific for methanogens so that other rumen microbes can continue normal digestive functions. Thus a detailed knowledge of the diversity and physiology of rumen methanogens is required to define conserved features that can be targeted for methanogen inhibition. Genome sequencing projects are underway in New Zealand and Australia on several ruminal methanogen groups, including representatives of the genera Methanobrevibacter, Methanobacterium, Methanosphaera, Methanosarcina, and the uncultured group, Rumen Cluster C. The completed Methanobrevibacter ruminantium genome and draft sequences from other rumen methanogen species are beginning to allow identification of underlying cellular processes that define these organisms, and is leading to a better understanding of their lifestyles within the rumen. Although the research is mainly at the explorative stage, two types of opportunities for inhibiting methanogens are emerging, being inactivation of conserved methanogen enzymes by screening for, or designing, small inhibitors via a chemogenomics approach, and identifying surface proteins shared among rumen methanogens that can be used as antigens in an anti-methanogen vaccine. Many of the conserved enzyme targets are involved in energy generation via the methanogenesis pathway, while the majority of the conserved surface protein targets are of unknown function. An understanding of the expression and accessibility of these targets within methanogen cells and/or microbial biofilms under ruminal conditions will be required for their development as CH4 production mitigations.This paper is part of the special issue entitled: Greenhouse Gases in Animal Agriculture - Finding a Balance Between Food and Emissions, Guest Edited by T.A. McAllister, Section Guest Editors: K.A. Beauchemin, X. Hao, S. McGinn and Editor for Animal Feed Science and Technology, P.H. Robinson.

Methyl coenzyme-M reductase A (mcrA) clone libraries were generated from microbial DNA extracted from the rumen of cattle fed a roughage diet with and without supplementation of the antimethanogenic compound bromochloromethane. Bromochloromethane reduced total methane emissions by c. 30%, with a resultant increase in propionate and branched chain fatty acids. The mcrA clone libraries revealed that Methanobrevibacter spp. were the dominant species identified. A decrease in the incidence of Methanobrevibacter spp. from the clone library generated from bromochloromethane treatment was observed. In addition, a more diverse methanogenic population with representatives from Methanococcales, Methanomicrobiales and Methanosacinales orders was observed for the bromochloromethane library. Sequence data generated from these libraries aided in the design of an mcrA-targeted quantitative PCR (qPCR) assay. The reduction in methane production by bromochloromethane was associated with an average decrease of 34% in the number of methanogenic Archaea when monitored with this qPCR assay. Dissociation curve analysis of mcrA amplicons showed a clear difference in melting temperatures for Methanobrevibacter spp. (80-82 °C) and all other methanongens (84-86 °C). A decrease in the intensity of the Methanobrevibacter spp. specific peak and an increase for the other peak in the bromochloromethane-treated animals corresponded with the changes within the clone libraries.

The effects of microwave pretreatment (MW) on co-digestion of food waste (FW) and sewage sludge (SS) have never been investigated. In this study, a series of mesophilic biochemical methane potential (BMP) tests were conducted to determine the optimized ratio of FW and SS based on MW, and the evolution of bacterial and archaeal community was investigated through high-throughput sequencing method. Results showed that the optimized ratio was 3:2 for co-digestion of FW and SS based on MW, and the methane production was 316.24 and 338.44mLCH4/gVSadded for MW-FW and MW-SS, respectively. The MW-SS was superior for methane production compared to MW-FW, in which accumulation of propionic acid led to the inhibition of methanogenesis. Proteiniborus and Parabacteroides were responsible for proteins and polysaccharides degradation for all, respectively, while Bacteroides only dominated in co-digestion. Methanosphaera dominated in MW-FW at the active methane production phase, while it was Methanosarcina in MW-SS and mono-SS.

The Australian macropodids (kangaroos and wallabies) possess a distinctive foregut microbiota that contributes to their reduced methane emissions. However, methanogenic archaea are present within the macropodid foregut, although there is scant understanding of these microbes. Here, an isolate taxonomically assigned to the Methanosphaera genus (Methanosphaera sp. WGK6) was recovered from the anterior sacciform forestomach contents of a Western grey kangaroo (Macropus fuliginosus). Like the human gut isolate Methanosphaera stadtmanae DSMZ 3091(T), strain WGK6 is a methylotroph with no capacity for autotrophic growth. In contrast, though with the human isolate, strain WGK6 was found to utilize ethanol to support growth, but principally as a source of reducing power. Both the WGK6 and DSMZ 3091(T) genomes are very similar in terms of their size, synteny and G:C content. However, the WGK6 genome was found to encode contiguous genes encoding putative alcohol and aldehyde dehydrogenases, which are absent from the DSMZ 3091(T) genome. Interestingly, homologs of these genes are present in the genomes for several other members of the Methanobacteriales. In WGK6, these genes are cotranscribed under both growth conditions, and we propose the two genes provide a plausible explanation for the ability of WGK6 to utilize ethanol for methanol reduction to methane. Furthermore, our in vitro studies suggest that ethanol supports a greater cell yield per mol of methane formed compared to hydrogen-dependent growth. Taken together, this expansion in metabolic versatility can explain the persistence of these archaea in the kangaroo foregut, and their abundance in these 'low-methane-emitting' herbivores.

Study of the efficacy of methanogenesis inhibitors in the rumen has given inconsistent results, mainly due to poorly understood effects on the key microbial groups involved in pathways for methane (CH4) synthesis. The experiment described in this report was designed to assess the effect of propyl propane thiosulfinate (PTS), diallyl disulfide (DDS) and bromochloromethane (BCM) on rumen fermentation, methane production and microbial populations in continuous culture fermenters. No effects on total volatile fatty acids (VFA) were observed with PTS or DDS, but VFA were decreased with BCM. Amylase activity increased with BCM as compared with the other treatments. A decrease in methane production was observed with PTS (48%) and BCM (94%) as compared with control values. The concentration of methanogenic archaea decreased with BCM from day 4 onward and with PTS on days 4 and 8. Pyrosequencing analysis revealed that PTS and BCM decreased the relative abundance of Methanomicrobiales and increased that of Methanobrevibacter and Methanosphaera. The total concentration of bacteria was not modified by any treatment, although treatment with BCM increased the relative abundance of Prevotella and decreased that of Ruminococcus. These results suggest that the inhibition of methane production in the rumen by PTS and BCM is associated with a shift in archaeal biodiversity and changes in the bacterial community with BCM, This research was supported by the Science and Innovation Spanish Ministry (Project AGL2008-04707-C02-01). We gratefully acknowledge T. García, E. Jiménez and P. Rufino for their technical assistance and the DMC Research Center S.L. for providing PTS. Gonzalo Martínez-Fernandez gratefully acknowledges an FPI grant from the Spanish Science Ministry

Sequencing and analyses of 16S rRNA gene amplicons were performed to estimate the composition of the rumen methanogen community in 252 samples from eight cohorts of sheep and cattle, separated into 16 different sample groups by diet, and to determine which methanogens are most prominent in the rumens of farmed New Zealand ruminants. Methanobacteriales (relative abundance ± standard deviation, 89.6% ± 9.8%) and Methanomassiliicoccales (10.4% ± 9.8%) were the two major orders and contributed 99.98% (±0.1%) to the rumen methanogen communities in the samples. Sequences from Methanobacteriales were almost entirely from only four different species (or clades of very closely related species). Each was detectable in at least 89% of the samples. These four species or clades were the Methanobrevibacter gottschalkii clade and Methanobrevibacter ruminantium clade with a mean abundance of 42.4% (±19.5% standard deviation) and 32.9% (±18.8%), respectively, and Methanosphaera sp. ISO3-F5 (8.2% ± 6.7%) and Methanosphaera sp. group5 (5.6% ± 5.7%). These four species or clades appeared to be primarily represented by only one or, in one case, two dominant sequence types per species or clade when the sequences were grouped into operational taxonomic units (OTUs) at 99% sequence identity. The mean relative abundance of Methanomassiliicoccales in the samples was relatively low but exceeded 40% in some of the treatment groups. Animal feed affected the apparent methanogen community structure of both orders, as evident from differences in relative abundances of the major OTUs in animals under different feeding regimens.

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.

Aims To develop and test a fluorescence in situ hybridization (FISH) based technique and to identify and quantify simultaneously those methanogenic populations colonizing Entodinium spp. in the rumen of cows fed different forages. Methods and Results New FISH probes targeting protozoal Entodinium spp. were designed and used together with FISH probes for methanogens in the cow rumen. The composition and relative abundance of methanogenic populations colonizing Entodinium simplex-, E. caudaum- and Entodinium furca-related populations were similar. Methanogens including Methanobrevibacter thaueri, Methanobrevibacter millerae and Methanobrevibacter smithii, and members of Methanomicrobium and Methanosphaera were generally the predominant colonizers of protozoa, regardless of the forage fed to cattle. Individual animals appeared to differ in which ruminal methanogenic populations colonized each of the individual Entodinium spp. Conclusions Simultaneous FISH probing is shown here to be a reliable and effective approach to investigate the dynamics of symbiotic relationships between ruminal protozoa and methanogens at a single cell level. Phylogenetically closely related Entodinium spp. were colonized by similar methanogenic populations regardless of the forage fed. Significance and Impact of the Study This is the first report of the methanogenic archaeal populations that specifically colonize Entodinium spp. as identified using simultaneous FISH probing.

Most studies present in the literature about the rumen microbiome have focused on cattle and sheep. This is the first report of the characterization of the bacterial and archaeal communities present in the liquid and solid-associated fractions of the rumen from free ranging Moxoto breed goats using 16S rRNA gene libraries. PCR was used to amplify the 16S rRNA gene with bacterial and archaeal universal primers and sequences from each library constructed were obtained. Sequences of Bacteria from the phyla Bacteroidetes and Firmicutes were predominant. The overall dominant classes in the rumen were Clostridia and Bacteroidia, which are known to play a role in plant fiber degradation in other ruminants. Unclassified Bacteria accounted for 4.7% of the liquid fraction sequences and 16.4% of the solid fraction sequences. From the archaeal libraries only sequences from the phylum Euryarcheota were identified and were assigned to the class Methanobacteria of the genera Methanobrevibacter and Methanosphaera. A group of Archaea not previously known to be associated with the rumen was identified: uncultured methanogens belonging to the "uncultured marine bacteria" groups II and III. The local water contained high salt concentrations and this may explain the presence of these groups in the Moxoto goat rumen.

The aim of this study was to design a microarray targeting methanogens found in anaerobic digesters, and to apply this chip together with a cloning approach to investigate the methanogenic community present in an anaerobic digester. Oligonucleotide probes were designed based on sequence differences in the 16S rRNA genes in order to target microorganisms in situ. For microarray hybridisations, DNA was subjected to PCR amplification of the 16S rRNA gene and Cy5-labeled. The microarray was tested with pure cultures, and of the 1854 individual probe-target hybridisation reactions performed, there were only 28 false positive (1.5%) and 16 false negative signals (0.86%). The sensitivity of the array was also tested, and it was found that when 0.4 pg of DNA from a pure culture was subjected to PCR amplification, signals above the detection limit were obtained. Also, the application of 25 ng of PCR product from a pure culture to an array resulted in detectable signals. The ANAEROCHIP was hybridised with DNA from an anaerobic sludge. Strong hybridisation signals were obtained for Methanoculleus, and weaker signals, in decreasing order, were obtained for Methanosarcina, Methanobacterium, Methanobrevibacter, and Methanosphaera. In order to check the results obtained with the microarray, the archaeal community structure of the same digester was analysed by 16S rRNA gene cloning and sequencing. Community structure was determined by restriction digestion of almost 200 clones and by sequencing of the 15 different resulting patterns. Methanoculleus was the dominant (84.1%) microorganism in the anaerobic sludge, and Methanobrevibacter (5.8%), Methanobacterium (3.7%), Methanosarcina (2.1%), Methanosphaera (1.6%), an uncultured archaeon (1.6%) and Methanothermobacter (1%) were also detected. These results showed the microarray to be a suitable tool for studying methanogenic communities in sludge.

This study evaluated the effects of selected essential oils on archaeal communities using the ovine rumen model. Forty weaned Canadian Arcott ewes, fed with barley-based diet, were allotted to one of three essential oil supplementation treatments or a control (10 ewes per treatment) for 13 weeks. The treatments were cinnamaldehyde, garlic oil, juniper berry oil, and a control with no additive. Rumen content was sampled after slaughter and grouped by treatment by combining subsamples from each animal. DNA was extracted from the pooled samples and analyzed for methanogenic archaea using quantitative polymerase chain reaction, denaturing gradient gel electrophoresis, cloning, and sequencing. Our results suggest that the total copy number of archaeal 16S rRNA was not significantly affected by the treatments. The phylogenetic analysis indicated a trend toward an increased diversity of methanogenic archaea related to Methanosphaera stadtmanae, Methanobrevibacter smithii, and some uncultured groups with cinnamaldehyde, garlic, and juniper berry oil supplementation. The trends in the diversity of methanogenic archaea observed with the essential oil supplementation may have resulted from changes in associated protozoal species. Supplementation of ruminant diets with essential oils may alter the diversity of rumen methanogens without affecting the methanogenic capacity of the rumen.