Methanobrevibacter Research Articles

Metagenomics reveals differences in microbial composition and metabolic functions in the rumen of dairy cows with different residual feed intake

BackgroundRumen microbial composition and functions have vital roles in feed digestion and fermentation and are linked to feed efficiency in cattle. This study selected Holstein cows, which are high in both milk protein content and milk yield, to analyse the relationship between the rumen microbiota and residual feed intake (RFI). Eighteen multiparous lactating cows were divided into low RFI (LRFI, high efficiency, n = 9) and high RFI (HRFI, low efficiency, n = 9) groups to investigate the differences in microbial composition and functions.ResultsThe relative abundances of butyrate producers, including the Clostridium, Butyrivibrio, Eubacterium and Blautia genera, were higher in HRFI cows than in LRFI cows (P < 0.05). Four carbohydrate metabolic pathways (glycolysis/gluconeogenesis, pentose phosphate pathway, fructose and mannose metabolism, and butanoate metabolism) and one energy metabolism pathway (methane metabolism), were more abundant in HRFI animals (P < 0.05). Quorum sensing and DNA replication pathways were more abundant in HRFI cows. For CAZyme profiles, 14 out of 19 genes encoding carbohydrates-deconstructing enzymes were more abundant in HRFI cows (P < 0.05). Seven Lachnospiraceae species associated with carbohydrate metabolism and quorum sensing may contribute to the difference in feed efficiency. Moreover, the LRFI cows had lower abundances of Methanosphaera (P < 0.01), Methanobrevibacter ruminantium (P = 0.09) and methanogenesis functions (P = 0.04).ConclusionsThe rumen microbiota of low-efficiency cows has stronger abilities to degrade carbohydrates and produce methane, and quorum sensing pathways could also be associated with differences in feed efficiency. This study provides a deeper understanding of the microbial ecology of dairy cows with different feed efficiencies and highlights the possibility of modulating the rumen microbiome or microbial functions to improve the feed efficiency of dairy cows.

Reconstruction and analysis of transcriptome regulatory network of Methanobrevibacter ruminantium M1

Global regulatory network modeling is a great concern of addressing metabolic complexity especially of a non-model organism to prioritize novel drug and vaccine targets. In this study, a knowledge-based bottom-up approach was used to reconstruct a transcriptional regulatory network of Methanobrevibacter ruminantium M1, one of the predominant hydrogenotrophic methanogens, contributing enteric methane emission in ruminants. The network robustness of the resulted model was validated with topological parameters and indicated its stability, consistency, and biological reliability. Each regulon-associated gene was assessed with gene ontology terms and literature support for functional confidence to infer its transcriptional regulatory mechanisms. The inferred genome-wide regulatory network comprises 164 predicted regulons associated with 235 coding genes. The results of this study have identified six DNA-binding motifs in 204 promoter regions interacting with 194 genes. It also described the metabolic gene regulation could be controlled either directly or indirectly under the respective promoter site in the regulatory network. As shown by our analysis, we proposed regulatory mechanisms of 7 important regulons such as PhoB (mru_0135), PrrA (mru_0526), NhaR (mru_0780), SigB (32-mer) (mru_1470), CmbR (mru_1544), MetR (mru_1915) and PpsR (mru_2036) at different environmental niche. Our intensive knowledge about the transcriptional regulatory mechanisms of this organism provides an insight into the understanding of rumen lifestyle and growth physiology in the ruminants.

Characterization of Cyclophilin from Thaumarchaeota Nitrosopumilus maritimus: Implications on the Diversity of Chaperone-like Activity in the Archaeal Domain.

The Archaea constitute separate domain of life and show resemblance with bacteria in their metabolic pathways while showing similarity with eukaryotes at the level of molecular processes such as cell division, DNA replication, protein synthesis, and proteostasis. However, the molecular machinery of archaea can be considered a simpler version of that found in eukaryotes because of the absence of multiple paralogs for any given molecular factor. Therefore, archaeal systems can possibly be used as a model system for understanding the eukaryotic protein folding machinery and thereby may help to address the molecular mechanism of various protein (mis)foldings and diseases. In the process of protein folding, the cis–trans isomerization of the peptide–prolyl bond is a rate-limiting step for the correct folding of proteins. Different types of peptidyl–prolyl cis–trans isomerases can accelerate this reaction, e.g., cyclophilin, FKBP, and parvulin. Among the five phyla of the archaeal domain, homologs of the cyclophilin protein are found only in two. Here we have characterized a cyclophilin from an archaeal organism, Nitrosopumilus maritimus (NmCyp), belonging to the phylum Thaumarchaeota. Like other known cyclophilins, NmCyp also possesses PPIase activity that can be inhibited by cyclosporine A. Generally, archaeal proteins are expected to possess differential thermal stability due to their adaptation to extreme environmental niche conditions. However, NmCyp exhibits low thermal stability and starts to aggregate beyond 40 °C. The properties of NmCyp are compared to those reported for the cyclophilin from another archaeal organism, Methanobrevibacter ruminantium. The current study sheds light on the differential behavior of cyclophilin proteins from two different phyla of archaea.

The Ability of Hop Extracts to Reduce the Methane Production of Methanobrevibacter ruminantium.

Background Methane emissions from agriculture are responsible for over 40% of the world's greenhouse gas emissions. In the past, antibiotics were used to control methane production by animals, but concerns over the emergence and spread of antibiotic-resistant bacteria to humans have prompted a search for alternative approaches. Hops are the flowers of the hop plant Humulus lupulus. They have been used to feed cattle for many years and are known to contain antibacterial compounds, and their extracts have been shown to kill members of the Mycobacterium spp including Mycobacterium bovis, the causative agent of bovine tuberculosis as well as a number of human pathogens. In this study, hop extracts were studied for their ability to inhibit methane production from Methanobrevibacter ruminantium, a major methane-producing archaeon found in the rumen of cattle. Methods Methanobrevibacter ruminantium M1T (DSM 1093) was grown at 37°C for 30 days, and the amount of methane produced at different time points during this period was measured using gas chromatography. The archaeon was exposed to commercial hop extracts (tetra-hydro-iso-alpha acid and beta acid) and to aqueous hop extracts of a range of hop variants, and their effect on methane production was determined. Results All of the extracts reduced the level of methane production of M. ruminantium over the 30-day period compared to the negative control (sterile distilled water). The commercial hop extracts were the most effective at inhibiting methane production over the course of the experiment in contrast to the aqueous extracts, which showed a gradual reduction of inhibition with time. Conclusions Hops contain compounds which inhibit methane production. Given that hops can be safely fed to cattle, this raises the possibility of rationally designing a feed strategy which could reduce greenhouse gas emissions and protect against bovine tuberculosis. This study recommends that further research be undertaken to further identifying bioactive components from hops and their efficacy against a range of archaea.

Metagenomic Analysis Reveals Microbial Interactions at the Biocathode of a Bioelectrochemical System Capable of Simultaneous Trichloroethylene and Cr(VI) Reduction.

Bioelectrochemical systems (BES) are attractive and versatile options for the bioremediation of organic or inorganic pollutants, including trichloroethylene (TCE) and Cr(VI), often found as co-contaminants in the environment. The elucidation of the microbial players’ role in the bioelectroremediation processes for treating multicontaminated groundwater is still a research need that attracts scientific interest. In this study, 16S rRNA gene amplicon sequencing and whole shotgun metagenomics revealed the leading microbial players and the primary metabolic interactions occurring in the biofilm growing at the biocathode where TCE reductive dechlorination (RD), hydrogenotrophic methanogenesis, and Cr(VI) reduction occurred. The presence of Cr(VI) did not negatively affect the TCE degradation, as evidenced by the RD rates estimated during the reactor operation with TCE (111±2 μeq/Ld) and TCE/Cr(VI) (146±2 μeq/Ld). Accordingly, Dehalococcoides mccartyi, the primary biomarker of the RD process, was found on the biocathode treating both TCE (7.82E+04±2.9E+04 16S rRNA gene copies g−1 graphite) and TCE/Cr(VI) (3.2E+07±2.37E+0716S rRNA gene copies g−1 graphite) contamination. The metagenomic analysis revealed a selected microbial consortium on the TCE/Cr(VI) biocathode. D. mccartyi was the sole dechlorinating microbe with H2 uptake as the only electron supply mechanism, suggesting that electroactivity is not a property of this microorganism. Methanobrevibacter arboriphilus and Methanobacterium formicicum also colonized the biocathode as H2 consumers for the CH4 production and cofactor suppliers for D. mccartyi cobalamin biosynthesis. Interestingly, M. formicicum also harbors gene complexes involved in the Cr(VI) reduction through extracellular and intracellular mechanisms.

Factors influencing Euryarchaeal gut methanogens distribution in dairy cattle in smallholding farms

Guts of ruminants contain symbiotic domains (Eubacteria, Archaea and Eukarya) that aid in the breakdown of consumed carbohydrates from plants to simple molecules that can be absorbed into the ruminant’s bloodstream. Methanogenesis occurs during the gut fermentation and methane gas is released in the final step of biomass degradation from the fermentation chambers. The Archaea that play a major role critical for methane emissions are methanogens and are found freely in the ruminants’ gut. Methane production from ruminants has attracted global attention due to their input on the Green House Gases effect, contribution to global warming and negative effects on farmers’ productivity. The objective of this study was to determine the factors contributing to the methanogens’ gut distribution in dairy cows from smallholder farms using next generation sequencing techniques. A total of 48 samples from smallholding dairy farms were used during this study and were collected from Kenya (Kiambu county) and Tanzania (Lushoto and Rungwe). The collected data samples from the experimental animals were from both the rumen fluid (6) and fecal (42). Samples were analyzed using metagenomic approaches and statistical analysis was undertaken using IBM SPSS statistics software version 28.0.0.0. Results showed that the gut site along the gastrointestinal tract and the feeding regime significantly contributed to the distribution and presence of various methanogenic species (P&lt;0.1). The herd and the genotype had no statistical effect. A total of 12 families were identified. The family Methanobacteriaceae was identified with the leading number (8) of the methanogenic species. A third of the identified families showed presence for at least two methanogenic species with Methanobrevibacter ruminantium being abundant. For proper curbing mechanisms, efforts to reduce methane release should be channeled to the whole gastrointestinal tract and advanced studies carried out on any potential interspecies presence facilitation and/or elimination.

Efficient decolorization of azo dye wastewater with polyaniline/graphene modified anode in microbial electrochemical systems

Azo dye pollution has become a worldwide issue, and the current treatment methods can hardly meet the expected emission standards. Microbial electrochemical systems (MESs) show promising applications for decolorization, but their performance critically depends on the microorganisms. Electrode modification is an interesting method of improving decolorization performance. However, the mechanisms of how the modification can affect microbial communities and the decolorization process remain unclear. Here, a modified anode with polyaniline (PANI) and graphene was fabricated via electro-deposition. Consequently, the highest decolorization efficiency was obtained. The Congo red (CR) decolorization rate of the MESs with the PANI/graphene-modified electrode (PG) reached 90% at 54 h. By contrast, the CR decolorization rates of the MESs with the PANI-modified electrode (P) and those of the MESs with the unmodified electrode (C) only reached 68% and 79%, respectively. Results of the microbial community analysis showed abundant Methanobrevibacter arboriphilus in PG (11%), which was 5.5 times that in C (2%) at 18 h. This phenomenon may be related to the rapid decolorization. The upregulated metabolism pathways, including arginine and proline metabolism, purine metabolism, arginine biosynthesis, and riboflavin metabolism, provided more electron shuttles and redox mediators that facilitated the extracellular electron transfer. Therefore, the PG-modified electrode facilitated the decolorization by altering certain metabolic pathways. This study can help to improve the guideline on the potential application of MESs for wastewater treatment.

Effects of cofD gene knock-out on the methanogenesis of Methanobrevibacter ruminantium

This study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P < 0.05) than those of wild-type, and the maximum biomass of mutant M. ruminantium was approximately half of the wild-type; meanwhile, the proliferation was reduced. The synthesis amount of coenzyme F420 of M. ruminantium was significantly decreased (P < 0.05) after the cofD gene knock-out. Moreover, the maximum amount of H2 consumed and CH4 produced by mutant were 14 and 2% of wild-type M. ruminantium respectively. In conclusion, cofD gene knock-out induced the decreased growth rate and reproductive ability of M. ruminantium. Subsequently, the synthesis of coenzyme F420 was decreased. Ultimately, the production capacity of CH4 in M. ruminantium was reduced. Our research provides evidence that cofD gene plays an indispensable role in the regulation of coenzyme F420 synthesis and CH4 production in M. ruminantium.

Temporal changes in total and metabolically active ruminal methanogens in dairy cows supplemented with 3-nitrooxypropanol

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.

Hydrogen sulfide affects the performance of a methanogenic bioelectrochemical system used for biogas upgrading

Methanogenic bioelectrochemical systems (BESs) can convert carbon dioxide (CO2) to methane (CH4) and may be used for anaerobic digester biogas upgrading. However, the effect of hydrogen sulfide (H2S), a common biogas component, on BES performance is unknown. Thus, the objective of this study was to assess the effect of H2S addition to the cathode headspace on BES performance at a range of initial gas-phase H2S concentrations (0–6% v/v), as well as its effect on the anode and cathode microbial communities. As the initial cathode headspace H2S increased from 0 to 2% (v/v), biocathodic CH4 production increased by two-fold to 3.56 ± 0.36 mmol/L-d, due to dissolved H2S transport from the cathode to the anode where H2S was oxidized. Elemental sulfur and sulfate were H2S oxidation products detected in the anode. Above 3% initial cathode headspace H2S, biocathodic CH4 production declined due to inhibition. A phylotype most closely related to Methanobrevibacter arboriphilus dominated the cathode archaeal communities. In the sulfide-amended BES, a phylotype similar to the exoelectrogen Ochrobactrum anthropi was enriched in both the anode and cathode, whereas phylotypes related to sulfate-reducing and sulfur oxidizing Bacteria were detected in the bioanode. Thus, sulfide transport and oxidation in the anode play an important role in methanogenic BESs treating sulfide-bearing biogas.

Effects of Starch Level and a Mixture of Sunflower and Fish Oils on Nutrient Intake and Digestibility, Rumen Fermentation, and Ruminal Methane Emissions in Dairy Cows.

Simple SummaryMethane produced by ruminants contributes to increased greenhouse gas effect. There are various nutritional strategies to reduce methane emission, such as supplementing fat or changing starch levels in the diet. Understanding the interactions of these strategies on methane emission, as well as performance, digestibility, and rumen fermentation is important. The present study aimed to assess the effects of starch level with or without a mixture of sunflower and fish oils on nutrient intake and digestibility, milk yield and composition, rumen fermentation, ruminal CH4 emissions and microbial ecology in dairy cows. Oil mixture rich in polyunsaturated fatty acids supplemented to low- or high-starch diets reduced dry matter intake and increased energy digestibility of lactating cows. High starch level improved nutrient digestibility and tended to reduce ruminal acetate:propionate ratio but did not affect rumen pH, molar propionate ratio, or ruminal CH4 emissions. Oil decreased absolute ruminal CH4 emission or tended to decrease CH4 per energy corrected milk.Four multiparous dairy cows were used in a 4 × 4 Latin square to examine how starch level and oil mixture impact dry matter (DM) intake and digestibility, milk yield and composition, rumen fermentation, ruminal methane (CH4) emissions, and microbial diversity. Experimental treatments comprised high (HS) or low (LS) levels of starch containing 0 or 30 g of a mixture of sunflower and fish oils (2:1 w/w) per kg diet DM (LSO and HSO, respectively). Intake of DM did not differ between cows fed LS and HS diets while oil supplementation reduced DM intake. Dietary treatments did not affect milk and energy corrected milk yields. There was a tendency to have a lower milk fat concentration due to HSO compared with other treatments. Both high starch level and oil supplementation increased digestibility of gross energy. Cows receiving HS diets had higher levels of total rumen VFA while acetate was lower than LS without any differences in rumen pH, or ruminal CH4 emissions. Although dietary oil supplementation had no impact on rumen fermentation, decreased CH4 emissions (g/day and g/kg milk) were observed with a concomitant increase in Anoplodinium-Diplodinium sp. and Epidinium sp. but a decrease in Christensenellaceae, Ruminococcus sp., Methanobrevibacter ruminantium and Mbb. gottschalkii clades.

Holstein and Jersey Steers Differ in Rumen Microbiota and Enteric Methane Emissions Even Fed the Same Total Mixed Ration.

Previous studies have focused on the rumen microbiome and enteric methane (CH4) emissions in dairy cows, yet little is known about steers, especially steers of dairy breeds. In the present study, we comparatively examined the rumen microbiota, fermentation characteristics, and CH4 emissions from six non-cannulated Holstein (710.33 ± 43.02 kg) and six Jersey (559.67 ± 32.72 kg) steers. The steers were fed the same total mixed ration (TMR) for 30 days. After 25 days of adaptation to the diet, CH4 emissions were measured using GreenFeed for three consecutive days, and rumen fluid samples were collected on last day using stomach tubing before feeding (0 h) and 6 h after feeding. CH4 production (g/d/animal), CH4 yield (g/kg DMI), and CH4 intensity (g/kg BW0.75) were higher in the Jersey steers than in the Holstein steers. The lowest pH value was recorded at 6 h after feeding. The Jersey steers had lower rumen pH and a higher concentration of ammonia-nitrogen (NH3-N). The Jersey steers had a numerically higher molar proportion of acetate than the Holstein steers, but the opposite was true for that of propionate. Metataxonomic analysis of the rumen microbiota showed that the two breeds had similar species richness, Shannon, and inverse Simpson diversity indexes. Principal coordinates analysis showed that the overall rumen microbiota was different between the two breeds. Both breeds were dominated by Prevotella ruminicola, and its highest relative abundance was observed 6 h after feeding. The genera Ethanoligenens, Succinivibrio, and the species Ethanoligenens harbinense, Succinivibrio dextrinosolvens, Prevotella micans, Prevotella copri, Prevotella oris, Prevotella baroniae, and Treponema succinifaciens were more abundant in Holstein steers while the genera Capnocytophaga, Lachnoclostridium, Barnesiella, Oscillibacter, Galbibacter, and the species Capnocytophaga cynodegmi, Galbibacter mesophilus, Barnesiella intestinihominis, Prevotella shahii, and Oscillibacter ruminantium in the Jersey steers. The Jersey steers were dominated by Methanobrevibacter millerae while the Holstein steers by Methanobrevibacter olleyae. The overall results suggest that sampling hour has little influence on the rumen microbiota; however, breeds of steers can affect the assemblage of the rumen microbiota and different mitigation strategies may be needed to effectively manipulate the rumen microbiota and mitigate enteric CH4 emissions from these steers.

Effects of long-acting, broad spectra anthelmintic treatments on the rumen microbial community compositions of grazing sheep

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.

Inoculation with rumen fluid in early life accelerates the rumen microbial development and favours the weaning process in goats

BackgroundNewborn ruminants possess an underdeveloped rumen which is colonized by microorganisms acquired from adult animals and the surrounding environment. This microbial transfer can be limited in dairy systems in which newborns are separated from their dams at birth. This study explores whether the direct inoculation of fresh or autoclaved rumen fluid from adult goats to newborn kids has a beneficial effect on rumen microbial development and function.ResultsRepetitive inoculation of young kids with fresh rumen fluid from adult goats adapted to forage (RFF) or concentrate diets (RFC) accelerated microbial colonization of the rumen during the pre-weaning period leading to high protozoal numbers, a greater diversity of bacterial (+ 234 OTUs), methanogens (+ 6 OTUs) and protozoal communities (+ 25 OTUs) than observed in control kids (CTL) without inoculation. This inoculation also increased the size of the core bacterial and methanogens community and the abundance of key rumen bacteria (Ruminococcaceae, Fibrobacteres, Veillonellaceae, Rikenellaceae, Tenericutes), methanogens (Methanobrevibacter ruminantium, Methanomicrobium mobile and Group 9), anaerobic fungi (Piromyces and Orpinomyces) and protozoal taxa (Enoploplastron, Diplodinium, Polyplastron, Ophryoscolex, Isotricha and Dasytricha) before weaning whereas CTL kids remained protozoa-free through the study. Most of these taxa were positively correlated with indicators of the rumen microbiological and physiological development (higher forage and concentrate intakes and animal growth during the post-weaning period) favoring the weaning process in RFF and RFC kids in comparison to CTL kids. Some of these microbiological differences tended to decrease during the post-weaning period, although RFF and RFC kids retained a more complex and matured rumen microbial ecosystem than CTL kids. Inoculation with autoclaved rumen fluid promoted lower development of the bacterial and protozoal communities during the pre-weaning period than using fresh inocula, but it favored a more rapid microbial development during the post-weaning than observed for CTL kids.ConclusionsThis study demonstrated that inoculation of young ruminants with fresh rumen fluid from adult animals accelerated the rumen microbial colonization which was associated with an earlier rumen functional development. This strategy facilitated a smoother transition from milk to solid feed favoring the animal performance during post-weaning and minimizing stress.

Diversity of Methanogens in Animals' Gut.

Methanogens are members of anaerobe microbiota of the digestive tract of mammals, including humans. However, the sources, modes of acquisition, and dynamics of digestive tract methanogens remain poorly investigated. In this study, we aimed to expand the spectrum of animals that could be sources of methanogens for humans by exploring methanogen carriage in animals. We used real-time PCR, PCR-sequencing, and multispacer sequence typing to investigate the presence of methanogens in 407 fecal specimens collected from nine different mammalian species investigated here. While all the negative controls remained negative, we obtained by PCR-sequencing seven different species of methanogens, of which three (Methanobrevibacter smithii, Methanobrevibacter millerae and Methanomassiliicoccus luminyensis) are known to be part of the methanogens present in the human digestive tract. M. smithii was found in 24 cases, including 12/24 (50%) in pigs, 6/24 (25%) in dogs, 4/24 (16.66%) in cats, and 1/24 (4.16%) in both sheep and horses. Genotyping these 24 M. smithii revealed five different genotypes, all known in humans. Our results are fairly representative of the methanogen community present in the digestive tract of certain animals domesticated by humans, and other future studies must be done to try to cultivate methanogens here detected by molecular biology to better understand the dynamics of methanogens in animals and also the likely acquisition of methanogens in humans through direct contact with these animals or through consumption of the meat and/or milk of certain animals, in particular cows.

Functional Prediction and Assignment of Methanobrevibacter ruminantium M1 Operome Using a Combined Bioinformatics Approach.

Methanobrevibacter ruminantium M1 (MRU) is a rod-shaped rumen methanogen with the ability to use H2 and CO2, and formate as substrates for methane formation in the ruminants. Enteric methane emitted from this organism can also be influential to the loss of dietary energy in ruminants and humans. To date, there is no successful technology to reduce methane due to a lack of knowledge on its molecular machinery and 73% conserved hypothetical proteins (HPs; operome) whose functions are still not ascertained perceptively. To address this issue, we have predicted and assigned a precise function to HPs and categorize them as metabolic enzymes, binding proteins, and transport proteins using a combined bioinformatics approach. The results of our study show that 257 (34%) HPs have well-defined functions and contributed essential roles in its growth physiology and host adaptation. The genome-neighborhood analysis identified 6 operon-like clusters such as hsp, TRAM, dsr, cbs and cas, which are responsible for protein folding, sudden heat-shock, host defense, and protection against the toxicities in the rumen. The functions predicted from MRU operome comprised of 96 metabolic enzymes with 17 metabolic subsystems, 31 transcriptional regulators, 23 transport, and 11 binding proteins. Functional annotation of its operome is thus more imperative to unravel the molecular and cellular machinery at the systems-level. The functional assignment of its operome would advance strategies to develop new anti-methanogenic targets to mitigate methane production. Hence, our approach provides new insight into the understanding of its growth physiology and lifestyle in the ruminants and also to reduce anthropogenic greenhouse gas emissions worldwide.

Feed Intake, Methane Emissions, Milk Production and Rumen Methanogen Populations of Grazing Dairy Cows Supplemented with Various C 18 Fatty Acid Sources.

Simple SummaryReducing methane emissions from dairy cows is environmentally important. In this experiment, pasture fed dairy cows offered concentrates containing linseed oil emitted 18% less methane per kg of milk solids produced than those offered concentrates containing either stearic acid or soy oil. Additionally, cows fed linseed oil or soy oil produced more milk than those fed stearic acid. These results may contribute to the development of strategies to reduce methane emissions from pasture-based livestock whilst maintaining or improving animal productivity.Emissions of methane (CH4) from dairy production systems are environmentally detrimental and represent an energy cost to the cow. This study evaluated the effect of varying C18 fatty acid sources on CH4 emissions, milk production and rumen methanogen populations in grazing lactating dairy cows. Forty-five Holstein Friesian cows were randomly allocated to one of three treatments (n = 15). Cows were offered 15 kg dry matter (DM)/d of grazed pasture plus supplementary concentrates (4 kg DM/d) containing either stearic acid (SA), linseed oil (LO), or soy oil (SO). Cows offered LO and SO had lower pasture DM intake (DMI) than those offered SA (11.3, 11.5 vs. 12.6 kg/d). Cows offered LO and SO had higher milk yield (21.0, 21.3 vs. 19.7 kg/d) and milk protein yield (0.74, 0.73 vs. 0.67 kg/d) than those offered SA. Emissions of CH4 (245 vs. 293, 289 g/d, 12.4 vs. 15.7, 14.8 g/kg of milk and 165 vs. 207, 195 g/kg of milk solids) were lower for cows offered LO than those offered SA or SO. Methanobrevibacter ruminantium abundance was reduced in cows offered LO compared to SA. Offering supplementary concentrates containing LO can reduce enteric CH4 emissions from pasture fed dairy cows.

192 Abundance of rumen methanogens did not differ in cattle that differed in residual feed intake

Abstract Methane represents an energetic loss, and it has been proposed that its reduction may be associated with improved feed efficiency. Rumen methanogens produce most of the methane. Forty-nine crossbred heifers were individually fed a ration that consisted of 67.75% rolled corn, 20% wet distillers grain with solubles, 8% chopped alfalfa and 4.25% mineral/vitamin/rumensin mix for 84 d. Residual feed intake was the residual of the observed – predicted FI for the multivariate regression of FI on ADG and mid-metabolic BW for the population. Six heifers with the greatest RFI (1.75 ± 0.31 kg DM/d) and 6 with least RFI (-1.48 ± 0.31 kg DM/d) were selected. Rumen fluid was sampled from a tube passed through the mouth into the rumen. Rumen fluid was flash frozen in liquid N2 and stored at -80°C. After the first collection, heifers with greater RFI were pair fed to the less RFI heifers and rumen fluid was sampled 5 wk later. DNA was isolated from the rumen fluid and primer sets targeting total methanogens, Methanomicrobiales, Methanobacteriales, Methanosarcina, Methanobacterium, Methanobrevibacter ruminantium + Mbb. Cuticularis, and Methanobrevibacter smithii + Mbb. wolinii + Mbb. thaueri + Mbb. gottschalkii + Mbb. Woesei were amplified with real-time quantitative PCR (qPCR). Standard curves were generated to quantify copy number. There were no differences in copy number for the interaction of RFI classifications and sample period for all primer sets (P &amp;gt; 0.18). There were no differences in (P &amp;gt; 0.15) in RFI classification for all qPCR primer sets. There were fewer copies for the Methanobrevibacter ruminantium + Mbb. Cuticularis qPCR for samples collected in the first week compared to the 5 wk sample (P = 0.03); however, there were no difference for collection week for the other qPCR assays (P &amp;gt; 0.16). USDA is an equal opportunity provider and employer.

PSXII-6 Supplementation of Angus crossbred steers with avian-derived polyclonal antibody preparations against the ruminal methanogen Methanobrevibacter ruminantium M1 does not alter in vivo methane emissions

Abstract An experiment was conducted to evaluate the effect of supplementing twenty Angus crossbred steers with avian-derived polyclonal antibody preparations (PAP) against the ruminal methanogen Methanobrevibacter ruminantium M1 on in vivo methane production, using the sulfur hexafluoride (SF6) tracer technique (Johnson et al., 1994). Steers were fed chopped bermudagrass hay (BGH) ad libitum and 0.7 kg d-1 of corn gluten feed. The experiment followed a change-over design. Treatments were 1) supplementation of 3 mL d-1 of PAP against M. ruminantium M1 (PAP-M1), and 2) supplementation of 3 mL d-1 of a non-immunized egg product (CON). Individual BGH intake was recorded using an electronic radio-frequency monitoring system (GrowSafe System Ltd., Airdrie, Alberta, Canada). There was a 14-d adaptation period to the feeding regime, with no PAP supplementation, followed by an 18-d treatment period. Steers were dosed with brass permeation tubes with a known release rate of SF6 on d 7 of treatment period. Enteric methane emissions were sampled from d 13 to 18 of the treatment period, into N-rinsed pre-evacuated U-shaped polyvinyl chloride canisters (2 L) through a capillary tube. Methane emissions were averaged per animal within period. Data were analyzed as a change-over design using a model with fixed effects of order, period, and treatment and random effect of steer within order. Dry matter intake (DMI) was not different (P = 0.44) between treatments. Methane emissions, expressed as grams per day (P = 0.86), as grams per kilogram of DMI (P = 0.78), or in terms of methane emission factors (Ym, P = 0.78) were not different between PAP-M1 and CON treatments. Supplementation of steers with PAP against M. ruminantium M1 did not decrease enteric methane emissions. Based on preliminary ex situ trials, evaluation of different doses and combinations of PAP against other methanogenic species warrant further investigation.

Rumen Microbiome Composition Is Altered in Sheep Divergent in Feed Efficiency.

Rumen microbiome composition and functionality is linked to animal feed efficiency, particularly for bovine ruminants. To investigate this in sheep, we compared rumen bacterial and archaeal populations (and predicted metabolic processes) of sheep divergent for the feed efficiency trait feed conversion ratio (FCR). In our study 50 Texel cross Scottish Blackface (TXSB) ram lambs were selected from an original cohort of 200 lambs. From these, 26 were further selected for experimentation based on their extreme FCR (High Feed Efficiency, HFE = 13; Low Feed Efficiency, LFE = 13). Animals were fed a 95% concentrate diet ad libitum over 36 days. 16S rRNA amplicon sequencing was used to investigate the rumen bacterial and archaeal communities in the liquid and solid rumen fractions of sheep divergent for FCR. Weighted UniFrac distances separated HFE and LFE archaea communities from the liquid rumen fraction (Permanova, P < 0.05), with greater variation observed for the LFE cohort (Permdisp, P < 0.05). LFE animals exhibited greater Shannon and Simpson diversity indices, which was significant for the liquid rumen fraction (P < 0.05). Methanobrevibacter olleyae (in liquid and solid fractions) and Methanobrevibacter millerae (liquid fraction) were differentially abundant, and increased in the LFE cohort (P.adj < 0.05), while Methanobrevibacter wolinii (liquid fraction) was increased in the HFE cohort (P.adj < 0.05). This suggests that methanogenic archaea may be responsible for a potential loss of energy for the LFE cohort. Bacterial community composition (Permanova, P > 0.1) and diversity (P > 0.1) was not affected by the FCR phenotype. Only the genus Prevotella 1 was differentially abundant between HFE and LFE cohorts. Although no major compositional shifts of bacterial populations were identified amongst the feed efficient cohorts (FDR > 0.05), correlation analysis identified putative drivers of feed efficiency with Ruminococcaceae UCG-014 (liquid, rho = −0.53; solid, rho = −0.56) and Olsenella (solid, rho = −0.40) exhibiting significant negative association with FCR (P < 0.05). Bifidobacterium and Megasphaera showed significant positive correlations with ADG. Major cellulolytic bacteria Fibrobacter (liquid, rho = 0.43) and Ruminococcus 1 (liquid, rho = 0.41; solid, rho = 41) correlated positively with FCR (P < 0.05). Our study provides evidence that feed efficiency in sheep is likely influenced by compositional changes to the archaeal community, and abundance changes of specific bacteria, rather than major overall shifts within the rumen microbiome.