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Anthelmintic treatment of adult ewes is widely practiced to remove parasite burdens in the expectation of increased ruminant productivity. However, the broad activity spectra of many anthelmintic compounds raises the possibility of impacts on the rumen microbiota. To investigate this, 300 grazing ewes were allocated to treatment groups that included a 100-day controlled release capsule (CRC) containing albendazole and abamectin, a long-acting moxidectin injection (LAI), and a non-treated control group (CON). Rumen bacterial, archaeal and protozoal communities at day 0 were analysed to identify 36 sheep per treatment with similar starting compositions. Microbiota profiles, including those for the rumen fungi, were then generated for the selected sheep at days 0, 35 and 77. The CRC treatment significantly impacted the archaeal community, and was associated with increased relative abundances of Methanobrevibacter ruminantium, Methanosphaera sp. ISO3-F5, and Methanomassiliicoccaceae Group 12 sp. ISO4-H5 compared to the control group. In contrast, the LAI treatment increased the relative abundances of members of the Veillonellaceae and resulted in minor changes to the bacterial and fungal communities by day 77. Overall, the anthelmintic treatments resulted in few, but highly significant, changes to the rumen microbiota composition.

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.

The present study aimed to determine whether dietary supplementation with methanogen inhibitors during early life may lead to an imprint on the rumen microbial community and change the rumen function and performance of calves to 49-weeks of rearing. Twenty-four 4-day-old Friesian x Jersey cross calves were randomly assigned into a control and a treatment group. Treated calves were fed a combination of chloroform (CF) and 9,10-anthraquinone (AQ) in the solid diets during the first 12 weeks of rearing. Afterward, calves were grouped by treatments until week 14, and then managed as a single group on pasture. Solid diets and water were offered ad libitum. Methane measurements, and sample collections for rumen metabolite and microbial community composition were carried out at the end of weeks 2, 4, 6, 8, 10, 14, 24 and 49. Animal growth and dry matter intake (DMI) were regularly monitored over the duration of the experiment. Methane emissions decreased up to 90% whilst hydrogen emissions increased in treated compared to control calves, but only for up to 2 weeks after treatment cessation. The near complete methane inhibition did not affect calves’ DMI and growth. The acetate:propionate ratio decreased in treated compared to control calves during the first 14 weeks but was similar at weeks 24 and 49. The proportions of Methanobrevibacter and Methanosphaera decreased in treated compared to control calves during the first 14 weeks; however, at week 24 and 49 the archaea community was similar between groups. Bacterial proportions at the phylum level and the abundant bacterial genera were similar between treatment groups. In summary, methane inhibition increased hydrogen emissions, altered the methanogen community and changed the rumen metabolite profile without major effects on the bacterial community composition. This indicated that the main response of the bacterial community was not a change in composition but rather a change in metabolic pathways. Furthermore, once methane inhibition ceased the methanogen community, rumen metabolites and hydrogen emissions became similar between treatment groups, indicating that perhaps using the treatments tested in this study, it is not possible to imprint a low methane microbiota into the rumen in the solid feed of pre-weaned calves.

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 (

Fat rich microorganisms, such as microalgae Schizochytrium spp., are potential biotechnological tools in the modulation of rumen microbiome towards ecofriendly and high nutritional value end-products. However, limited in vivo trials have been reported on the topic. The aim of this study was to contribute to the knowledge on the effect of fat rich microalgae on the methanogenic and feed degrading particle-associated microbes in goats&rsquo, rumen content. For the trial, twenty-four goats were divided into four homogenous clusters (six goats/treatment) according to their fat corrected (4%) milk yield, body weight and age and individually were fed with alfalfa hay and concentrate feeds (F/C = 50/50). The concentrate of the control group (CON) contained no microalgae, while those of the treated groups were supplemented daily with 20 (ALG20), 40 (ALG40), and 60 (ALG60) g of Schizochytrium spp./goat. The relative abundances of total Archaea, methanogens, Methanomassiliicoccales, Methanobrevibacter spp., Methanosphaera stadmanae and Methanobacterium formicicum were significantly (p &lt, 0.05) decreased in microalgae-fed goats compared to the CON ones. Moreover, a significant decline in the relative abundances of Firmicutes, Ruminococcus flavefaciens, Butyrivibrio fibrosolvents, and Neocallimastigales in the rumen particle-associated microbiota of microalgae supplemented goats were observed. In conclusion, goats&rsquo, diets supplementation with Schizochytrium spp., could be considered a sustainable nutritional strategy for methanogens inhibition in their rumen particle-associated microbiota.

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 performance of ruminant livestock has been shown to benefit from the enhanced nutritive value and herbage yield associated with clover incorporation in the grazing sward. However, little research to date has been conducted investigating the effects of mixed swards containing white clover on the composition of the rumen microbiome. In this study, the rumen microbial composition of late lactation dairy cows grazing perennial ryegrass only (PRG; n = 20) or perennial ryegrass and white clover (WCPRG; n = 19) swards, was characterised using 16S rRNA amplicon sequencing. PERMANOVA analysis indicated diet significantly altered the composition of the rumen microbiome (P = 0.024). Subtle shifts in the relative abundance of 14 bacterial genera were apparent between diets, including an increased relative abundance of Lachnospira (0.04 vs. 0.23%) and Pseudobutyrivibrio (1.38 vs. 0.81%) in the WCPRG and PRG groups, respectively. The composition of the archaeal community was altered between dietary groups, with a minor increase in the relative abundance of Methanosphaera in the WCPRG observed. Results from this study highlight the potential for sward type to influence the composition of the rumen microbial community.

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.

Methanogenesis is the final step in the anaerobic degradation of organic matter. The most important substrates of methanogens are hydrogen plus carbon dioxide and acetate, but also the use of methanol, methylated amines, and aromatic methoxy groups appears to be more widespread than originally thought. Except for most members of the family Methanosarcinaceae, all methylotrophic methanogens require external hydrogen as reductant and therefore compete with hydrogenotrophic methanogens for this common substrate. Since methanogenesis from carbon dioxide consumes four molecules of hydrogen per molecule of methane, whereas methanogenesis from methanol requires only one, methyl-reducing methanogens should have an energetic advantage over hydrogenotrophic methanogens at low hydrogen partial pressures. However, experimental data on their hydrogen threshold is scarce and suffers from relatively high detection limits. Here, we show that the methyl-reducing methanogens Methanosphaera stadtmanae (Methanobacteriales), Methanimicrococcus blatticola (Methanosarcinales), and Methanomassiliicoccus luminyensis (Methanomassiliicoccales) consume hydrogen to partial pressures, Based on their extremely low hydrogen threshold, methylotrophic methanogens (MM) should always outcompete hydrogenotrophic methanogens (HM) for hydrogen, provided that methyl groups are available in sufficient amounts.

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.

This study was conducted to investigate the phylogenetic diversity of archaea in the rumen of adult and elderly yaks. Six domesticated female yaks, 3 adult yaks ((5.3±0.6) years old), and 3 elderly yaks ((10.7±0.6) years old), were used for the rumen contents collection. Illumina MiSeq high-throughput sequencing technology was applied to examine the archaeal composition of rumen contents. A total of 92 901 high-quality archaeal sequences were analyzed, and these were assigned to 2 033 operational taxonomic units (OTUs). Among these, 974 OTUs were unique to adult yaks while 846 OTUs were unique to elderly yaks; 213 OTUs were shared by both groups. At the phylum level, more than 99% of the obtained OTUs belonged to the Euryarchaeota phylum. At the genus level, the archaea could be divided into 7 archaeal genera. The 7 genera (i.e., Methanobrevibacter, Methanobacterium, Methanosphaera, Thermogymnomonas, Methanomicrobiu, Methanimicrococcus and the unclassified genus) were shared by all yaks, and their total abundance accounted for 99% of the rumen archaea. The most abundant archaea in elderly and adult yaks were Methanobrevibacter and Thermogymnomonas, respectively. The abundance of Methanobacteria (class), Methanobacteriales (order), Methanobacteriaceae (family), and Methanobrevibacter (genus) in elderly yaks was significantly higher than in adult yaks. In contrast, the abundance of Thermogymnomonas in elderly yaks was 34% lower than in adult yaks, though the difference was not statistically significant. The difference in abundance of other archaea was not significant between the two groups. These results suggested that the structure of archaea in the rumen of yaks changed with age. This is the first study to compare the phylogenetic differences of rumen archaeal structure and composition using the yak model.

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.

This work was aimed to investigate the effects of the different particle size of ground alfalfa hay on caecal microbial and archeal communities of rabbits. One hundred-twenty New Zealand rabbits (950.3 ± 8.82 g) were allocated into four treatments, with five replicates in each treatment and six rabbits in each replicate. The particle sizes of the alfalfa meal in the four treatment diets were 2,500, 1,000, 100 and 10 µm respectively, while the other ingredients were ground through a 2.5 mm sieve. High-throughput sequencing technology was applied to examine the differences in bacteria and methanogenic archaea diversity in the caecum of the four treatment groups of rabbits. A total of 745,946 bacterial sequences (a mean of 31,081 ± 13,901 sequences per sample) and 539,227 archaeal sequences (a mean of 22,468 ± 2,443 sequences per sample) were recovered from twenty-four caecal samples, and were clustered into 9,953 and 2,246 OTUs respectively. A total of 26 bacterial phyla with 465 genera and three archaeal phyla with 10 genera were identified after taxonomic summarization. Bioinformatic analyses illustrated that Firmicutes (58.69% ∼ 68.50%) and Bacteroidetes (23.96% ∼ 36.05%) were the two most predominant bacterial phyla and Euryarchaeota (over 99.9%) was the most predominant archaeal phyla in the caecum of all rabbits. At genus level, as the particle size of alfalfa decreased from 2,500 to 10 µm, the relative abundances of Ruminococcaceae UCG-014 (P Lactobacillus (P = 0.043) were increased and Ruminococcaceae UCG-005 (P = 0.012) was increased first and then decreased when the alfalfa particle size decreased, while Lachnospiraceae NK4A136 group (P = 0.016), Ruminococcaceae NK4A214 (P = 0.044), Christensenellaceae R-7 group (P = 0.019), Lachnospiraceae other (Family) (P = 0.011) and Ruminococcaceae UCG-013 (P = 0.021) were decreased. The relative abundance of Methanobrevibacter was increased from 62.48% to 90.40% (P Methanosphaera was reduced from 35.47% to 8.62% (P

Development of alternative forage resources is of great importance to provide necessary nutrients and minimize greenhouse gas emissions in ruminant production. The aim of this study was to examine the effects of dietary supplementation of Moringa oleifera on the production performance and fecal methanogenic community in dairy cows using methyl-coenzyme M reductase &alpha, subunit gene. Sixty-four cows were allocated to one of four treatments: basal diet without M. oleifera (control) or low (3% w/w, M3), medium (6%, M6), or high (9%, M9) supplementation with M. oleifera. This study demonstrated that different supplementation levels of Moringa oleifera in the diet achieved similar feed intake and milk production, but adding 6% of Moringa oleifera improved milk fat content. Two families, two phyla, three genera, and three species in total were identified among the four treatments. The fecal archaeal community in the control treatment was predominated by Methanobrevibacter (39.1% of the total sequence reads) followed by Methanosphaera and Methanocorpusculum at the genus level. The increased abundance of the Methanosphaera genus and Methanosphaera sp. ISO3-F5 species was induced by secondary metabolites of Moringa oleifera in the diet. Results indicated that Moringa oleifera supplementation not only improved dairy product quality but could also potentially reduce methane emissions.

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.

Background Dexamethasone (Dex), an artificially synthetic cortisol substitute, is commonly used as an anti-inflammatory drug, and is also employed to mimic the stress state experimentally. It is well known that chronic stress disturbs the gut microbiota community and digestive functions. However, no relevant studies have been conducted in ruminants. Results In this study, a low dosage of Dex (0.2 mg/kg body weight, Dex group, n = 5) was consecutively injected intramuscularly for 21 days to simulate chronic stress in growing goats. Goats were injected with saline (0.2 mg/kg body weight) as the control group (Con, n = 5). Dex-treated goats showed a higher number of white blood cells and blood glucose levels (p 0.05); however, ruminal VFA concentrations decreased dramatically 2, 4, 6, and 8 h after the morning feeding on day 21 of the Dex injections. In this study, chronic Dex exposure did not alter the community structure of microbes or methanogenes in the rumen, caecum, or colonic digesta. Only Prevotella increased on days 7 and 14 of Dex treatment, but decreased on day 21, and Methanosphaera was the only genus of methanogene that decreased. Conclusions Our results suggest that chronic Dex exposure retards growth by decreasing DMI, which may be mediated by higher levels of blood glucose and lower ruminal VFA production. Microbiota in the digestive tract was highly resistant to chronic Dex exposure. Electronic supplementary material The online version of this article (10.1186/s12866-018-1253-1) contains supplementary material, which is available to authorized users.

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

The genus Methanosphaera is a well-recognized but poorly characterized member of the mammalian gut microbiome, and distinctive from Methanobrevibacter smithii for its ability to induce a pro-inflammatory response in humans. Here we have used a combination of culture- and metagenomics-based approaches to expand the representation and information for the genus, which has supported the examination of their phylogeny and physiological capacity. Novel isolates of the genus Methanosphaera were recovered from bovine rumen digesta and human stool, with the bovine isolate remarkable for its large genome size relative to other Methanosphaera isolates from monogastric hosts. To substantiate this observation, we then recovered seven high-quality Methanosphaera-affiliated population genomes from ruminant and human gut metagenomic datasets. Our analyses confirm a monophyletic origin of Methanosphaera spp. and that the colonization of monogastric and ruminant hosts favors representatives of the genus with different genome sizes, reflecting differences in the genome content needed to persist in these different habitats. Refereed/Peer-reviewed

Ruminant animals, such as cows, live in a tight symbiotic association with microorganisms, allowing them to feed on otherwise indigestible plant biomass as food sources. Methane is produced as an end product of the anaerobic feed degradation in ruminants and is emitted to the atmosphere, making ruminant animals among the major anthropogenic sources of the potent greenhouse gas methane. Using newly developed quantitative metatranscriptomics for holistic microbiome analysis, we here identified bacterial, archaeal, and eukaryotic key players and the short-term dynamics of the rumen microbiome during anaerobic plant biomass degradation and subsequent methane emissions. These novel insights might pave the way for novel ecologically and economically sustainable methane mitigation strategies, much needed in times of global climate change., Ruminant livestock is a major source of the potent greenhouse gas methane. The complex rumen microbiome, consisting of bacteria, archaea, and microbial eukaryotes, facilitates anaerobic plant biomass degradation in the cow rumen, leading to methane emissions. Using an integrated approach combining multidomain quantitative metatranscriptomics with gas and volatile fatty acid (VFA) profiling, we aimed at obtaining the most comprehensive picture of the active rumen microbiome during feed degradation to date. Bacterial, archaeal, and eukaryotic biomass, but also methane emissions and VFA concentrations, increased drastically within an hour after feed intake. mRNA profiling revealed a dynamic response of carbohydrate-active enzyme transcripts, transcripts involved in VFA production and methanogenesis. While the relative abundances of functional transcripts did not mirror observed processes, such as methane emissions, transformation to mRNA abundance per gram of rumen fluid echoed ruminant processes. The microbiome composition was highly individual, with, e.g., ciliate, Neocallimastigaceae, Prevotellaceae, Succinivibrionaceae, and Fibrobacteraceae abundances differing between cows. Microbiome individuality was accompanied by inter- and intradomain multifunctional redundancy among microbiome members during feed degradation. This likely enabled the robust performance of the anaerobic degradation process in each rumen. Neocallimastigaceae and ciliates contributed an unexpectedly large share of transcripts for cellulose- and hemicellulose-degrading enzymes, respectively. Methyl-reducing but not CO2-reducing methanogens were positively correlated with methane emissions. While Methanomassiliicoccales switched from methanol to methylamines as electron acceptors, Methanosphaera became the dominating methanol-reducing methanogen. This study for the first time linked rumen meta-omics with processes and enabled holistic insights into the contribution of all microbiome members to feed degradation. IMPORTANCE Ruminant animals, such as cows, live in a tight symbiotic association with microorganisms, allowing them to feed on otherwise indigestible plant biomass as food sources. Methane is produced as an end product of the anaerobic feed degradation in ruminants and is emitted to the atmosphere, making ruminant animals among the major anthropogenic sources of the potent greenhouse gas methane. Using newly developed quantitative metatranscriptomics for holistic microbiome analysis, we here identified bacterial, archaeal, and eukaryotic key players and the short-term dynamics of the rumen microbiome during anaerobic plant biomass degradation and subsequent methane emissions. These novel insights might pave the way for novel ecologically and economically sustainable methane mitigation strategies, much needed in times of global climate change.

In the present study, we hypothesized that the rumen bacterial and archaeal communities would change significantly over the transition period of dairy cows, mainly as an adaptation to the classical use of low-grain prepartum and high-grain postpartum diets. Bacterial 16S rRNA gene amplicon sequencing of rumen samples from 10 primiparous Holstein dairy cows revealed no changes over the transition period in relative abundance of genera such as Ruminococcus, Butyrivibrio, Clostridium, Coprococcus, and Pseudobutyrivibrio. However, other dominant genus-level taxa, such as Prevotella, unclassified Ruminococcaceae, and unclassified Succinivibrionaceae, showed distinct changes in relative abundance from the prepartum to the postpartum period. Overall, we observed individual fluctuation patterns over the transition period for a range of bacterial taxa that, in some cases, were correlated with observed changes in the rumen short-chain fatty acids profile. Combined results from clone library and terminal-restriction fragment length polymorphism (T-RFLP) analyses, targeting the methyl-coenzyme M reductase α-subunit (mcrA) gene, revealed a methanogenic archaeal community dominated by the Methanobacteriales and Methanomassiliicoccales orders, particularly the genera Methanobrevibacter, Methanosphaera, and Methanomassiliicoccus. As observed for the bacterial community, the T-RFLP patterns showed significant shifts in methanogenic community composition over the transition period. Together, the composition of the rumen bacterial and archaeal communities exhibited changes in response to particularly the dietary changes of dairy cows over the transition period.

The ecological processes underlying the observed patterns in community composition of archaea in swine manure slurry are poorly understood. We studied the archaeal communities from six swine manure slurry storage tanks using paired-end Illumina sequencing of the v3 hypervariable region of 16S rRNA gene. Across all samples, the archaeal community was dominated by methanogens related toThermoplasmata, Methanomicrobia, andMethanobacteriaclasses. At the genus level, the archaeal community was dominated by a single uncultured lineage of archaea,vadinCA11, followed by methanogenic generaMethanobrevibacter,Methanosarcina,Methanosphaera,Methanogenium,Methanocorpusculum,Methanoculleus, andMethanomicrococcus. Significant phylogenetic signals were detected across relatively short phylogenetic distances, indicating that closely related archaeal operational taxonomic units (OTUs) tend to have similar niches. The standardized effect sizes of mean nearest taxon distance (SES.MNTD) showed that archaeal community was phylogenetically clustered, suggesting that environmental filtering deterministically influence the within-community composition of archaea. However, between-community analysis based onβ-nearest taxon index (βNTI) revealed that both deterministic selection and stochastic processes operate simultaneously to govern the assembly of archaeal communities. These findings significantly enhance our understanding of archaeal community assembly and underlying ecological processes is swine manure slurry.

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.

Diet has been shown to have a significant impact on microbial community composition in the rumen and could potentially be used to manipulate rumen microbiome structure to achieve specific outcomes. There is some evidence that a window may exist in early life, while the microbiome is being established, where manipulation through diet could lead to long-lasting results. The aim of this study was to test the hypothesis that dietary supplementation in early life will have an effect on rumen microbial composition that will persist even once supplementation is ceased. Twenty-seven new-born lambs were allocated to one of three dietary treatments; a control group receiving standard lamb meal, a group receiving lamb meal supplemented with 40 g kg-1 DM of linseed oil and a group receiving the supplement pre-weaning and standard lamb meal post weaning. The supplement had no effect on average daily feed intake or average daily weight gain of lambs. Bacterial and archaeal community composition was significantly (p = 0.033 and p = 0.005, respectively) different in lambs fed linseed oil throughout the study compared to lambs on the control diet. Succinivibrionaceae, succinate producers, and Veillonellaceae, propionate producers, were in a higher relative abundance in the lambs fed linseed oil while Ruminococcaceae, a family linked with high CH4 emitters, were in a higher relative abundance in the control group. The relative abundance of Methanibrevibacter was reduced in the lambs receiving linseed compared to those that didn’t. In contrast, the relative abundance of Methanosphaera was significantly higher in the animals receiving the supplement compared to animals receiving no supplement (40.82% and 26.67% respectively). Furthermore, lambs fed linseed oil only in the pre-weaning period had a bacterial community composition significantly (p = 0.015) different to that of the control group, though archaeal diversity and community structure did not differ. Again, Succinivibrionaceae and Veillonellaceae were in a higher relative abundance in the group fed linseed oil pre-weaning while Ruminococcaceae were in a higher relative abundance in the control group. This study shows that lambs fed the dietary supplement short-term had a rumen microbiome that remained altered even after supplementation had ceased.

At birth, calves display an underdeveloped rumen that eventually matures into a fully functional rumen as a result of solid food intake and microbial activity. However, little is known regarding the gradual impact of pre-weaning diet on the establishment of the rumen microbiota. Here, we employed next-generation sequencing to investigate the effects of the inclusion of starter concentrate (M: milk-fed vs. MC: milk plus starter concentrate fed) on archaeal, bacterial and anaerobic fungal communities in the rumens of 45 crossbred dairy calves across pre-weaning development (7, 28, 49, and 63 days). Our results show that archaeal, bacterial, and fungal taxa commonly found in the mature rumen were already established in the rumens of calves at 7 days old, regardless of diet. This confirms that microbiota colonization occurs in the absence of solid substrate. However, diet did significantly impact some microbial taxa. In the bacterial community, feeding starter concentrate promoted greater diversity of bacterial taxa known to degrade readily fermentable carbohydrates in the rumen (e.g., Megasphaera, Sharpea, and Succinivribrio). Shifts in the ruminal bacterial community also correlated to changes in fermentation patterns that favored the colonization of Methanosphaera sp. A4 in the rumen of MC calves. In contrast, M calves displayed a bacterial community dominated by taxa able to utilize milk nutrients (e.g., Lactobacillus, Bacteroides, and Parabacteroides). In both diet groups, the dominance of these milk-associated taxa decreased with age, suggesting that diet and age simultaneously drive changes in the structure and abundance of bacterial communities in the developing rumen. Changes in the composition and abundance of archaeal communities were attributed exclusively to diet, with more highly abundant Methanosphaera and less abundant Methanobrevibacter in MC calves. Finally, the fungal community was dominated by members of the genus SK3 and Caecomyces. Relative anaerobic fungal abundances did not change significantly in response to diet or age, likely due to high inter-animal variation and the low fiber content of starter concentrate. This study provides new insights into the colonization of archaea, bacteria, and anaerobic fungi communities in pre-ruminant calves that may be useful in designing strategies to promote colonization of target communities to improve functional development.

Gastrointestinal microbiota may play an important role in regulating host mucosal innate immune function. This study was conducted to test the hypothesis that age (non-rumination, transition and rumination) and feeding type [Supplemental feeding (S) vs. Grazing (G)] could alter ruminal microbial diversity and maturation of host mucosal innate immune system in goat kids. MiSeq sequencing was applied to investigate ruminal microbial composition and diversity, and RT-PCR was used to test expression of immune-related genes in ruminal mucosa. Results showed that higher (P < 0.05) relative abundances of Prevotella, Butyrivibrio, Pseudobutyrivibrio, Methanobrevibacter.gottschalkii, Neocallimastix, Anoplodinium–Diplodinium, and Polyplastron, and lower relative abundance of Methanosphaera (P = 0.042) were detected in the rumen of S kids when compared to those in G kids. The expression of genes encoding TLRs, IL1α, IL1β and TICAM2 was down-regulated (P < 0.01), while expression of genes encoding tight junction proteins was up-regulated (P < 0.05) in the ruminal mucosa of S kids when compared to that in G kids. Moreover, irrespective of feeding type, relative abundances of ruminal Prevotella, Fibrobacter, Ruminococcus, Butyrivibrio, Methanobrevibacter, Neocallimastix, and Entodinium increased with age. The expression of most genes encoding TLRs and cytokines increased (P < 0.05) from day 0 to 7, while expression of genes encoding tight junction proteins declined with age (P < 0.05). This study revealed that the composition of each microbial domain changed as animals grew, and these changes might be associated with variations in host mucosal innate immune function. Moreover, supplementing goat kids with concentrate could modulate ruminal microbial composition, enhance barrier function and decrease local inflammation. The findings provide useful information in interpreting microbiota and host interactions, and developing nutritional strategies to improve the productivity and health of rumen during early life.

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).