Enteric methane emissions are energy losses from farmed ruminants and contribute to global warming. Diverting electrons and H2 flow toward beneficial fermentation products would mitigate ruminal methane emissions while improving feed efficiency. Acetogens can direct H2 and electrons to acetate production via the Wood–Ljungdahl pathway, but methanogens have more competitive H2 affinities. Thus, it is unclear how acetogenesis subsists in the rumen. An analysis of 2102 globally derived rumen metagenomes from multiple ruminant species revealed that putative acetogens were phylogenetically diverse and capable of using carbohydrates or H2 as electron donors. The metabolic versatility of these acetogens may enable them to outcompete methanogens with lower versatility. Through animal trials, in vitro experiments, and DNA stable isotope probing, we verified the presence of diverse acetogens in beef cattle rumens and revealed that their niche partitioning is driven by contrasting fiber-rich and starch-rich diets. A fiber-rich diet enriched heterotrophic acetogens, which increased acetate formation while decreasing methane production. Overall, this study highlights the overlooked heterotrophy of acetogens in the rumen and their potential for mitigating enteric methane emissions.

Background: The gastrointestinal tract (GIT) microbiome of ruminants significantly influences their nutrition metabolism and health. Current understanding is extensive for bacterial and archaeal communities, but limited for viral communities within the GIT. Results: We created the Ruminant Gastrointestinal Virome Catalogue (RGVC), which includes 43,981 non-redundant viral Operational Taxonomic Units (vOTUs), with 89.3% newly identified, derived from 370 samples across 10 GIT regions in seven ruminant species. The composition of viral communities is mainly influenced by the GIT regions rather than by the ruminant species. We identified 4,603 putative prokaryotic hosts across 31 bacterial and three archaeal classes for 5,954 viruses, along with significant variations across GIT regions and a strong correlation between hosts and their associated viruses. Lysogeny, constituting 45.6% of survival strategies, was more prevalent than the lytic cycle (4.08%), and the abundances of these viruses varied regionally. The lysogenic viruses encoded 1,805 auxiliary metabolic genes (AMGs) that play key roles in carbohydrates, amino acids, and other metabolisms in their hosts. The variation in AMG abundance across regions highlights the distinct viral communities and the varied virus-host interactions within the GIT. Conclusion: This study offers a comprehensive view of the spatial heterogeneity of viral communities in the ruminant GIT and indicates that this diversity is driven by the interaction of lysogenic viruses with their prokaryotic hosts through AMGs. These findings set the stage for future research into the ecological and nutritional impacts of the ruminant virome, with the potential to uncover novel roles and mechanisms in various GIT regions.

Methane (CH4) from ruminants is a major source of agricultural greenhouse gas and represents a loss of dietary energy. 3-Nitrooxypropanol (3-NOP) is a known methanogenesis inhibitor, but its hydrophilic nature may limit the cellular accessibility to methanogens. Here, we systematically evaluated 1,3-propanediol dinitrate (1,3-PDN), a more hydrophobic derivative of 3-NOP, for its antimethanogenic potential and underlying mode of action using ruminal fermentation, pure-culture assays, multi-omics analyses, and molecular docking. Intracellular accumulation assays indicated greater cellular accumulation of 1,3-PDN than 3-NOP in rumen-derived methanogen Methanobrevibacter olleyae. Ruminal fermentation assays showed that 1,3-PDN reduced CH4 production by ~55%, and altered hydrogen (H2) metabolism, leading to 11-fold increase in H2 accumulation. Metatranscriptomic profiling revealed that 1,3-PDN significantly altered the active archaeal community, with a notable reduction in Methanobrevibacter_A and suppression of hydrogenotrophic methanogenesis. Molecular docking suggested that 1,3-PDN may bind to the conserved active site of methyl-coenzyme M reductase (MCR), potentially contributing to MCR-associated inhibition. Proteomic analysis further indicated that 1,3-PDN supplementation downregulated key MCR subunits and simultaneously affected other redox-sensitive methanogenesis-related processes, including tetrahydromethanopterin S-methyltransferase subunits and proteins involved in the biosynthesis of cofactors F430 and cobalamin. Nitrogen-equivalent assay suggested partial contribution of nitrite to the methanogenesis inhibition and oxidative effects induced by 1,3-PDN. Together, these findings identify 1,3-PDN as an effective inhibitor of ruminal methanogenesis with enhanced cellular enrichment, providing mechanistic insights for the rational development of new CH4 inhibitors.

Motivation: Gene abundance in metagenome datasets is commonly represented in terms of Counts or Copies Per Million. However, above term lack the consideration of the size of the microbial communities. To reflect the gene abundance in the microbial communities (GAM), GCompip, a comprehensive pipeline for estimating GAM, was developed based on specialized universal single copy genes (USCG) database, stringent alignment parameters, and rigorous filtering criteria. Results: GCompip showed high specificity without compromising computational efficiency, and improved the precision of downstream GAM estimations across diverse six ecological environments (i.e. human gut, rumen, freshwater, marine, hydrothermal sediment, and glacier). In contrast, the comparative annotation tools (i.e. KofamScan, eggNOG-mapper and HUMAnN3) showed larger error intervals, higher susceptibility to false positives, or overestimation of USCG abundance, primarily due to more relaxed thresholds, multifamily matches, or less stringent alignment settings. To facilitating the applicability of GCompip, we provided both Linux command line and R package versions. Overall, this GCompip presented an accurate, robust, user-friendly, and efficient computational pipeline designed to calculate GAM using metagenomic sequencing data. The developed pipeline makes it accessible to researchers seeking to evaluate the metabolic capabilities of microbial communities, and improve the capacity of interpreting metagenomic data related to microbial communities. Availability and implementation: GCompip package source code and documentation are freely available for download at https://github.com/XiangZhouCAS/GCompip. A separate Linux command line version is available at https://github.com/XiangZhouCAS/GCompip_onlinux.

Mixed silage can disrupt the girder structure of rape straw, and thus facilitate ruminal degradation. Further investigation is warranted to validate this observation in vivo. The objective of this study was to investigate the degradation kinetics and bacterial colonization of mixed silage during digestion using an in situ ruminal incubation technique. The experiment comprised two treatments: a mixture of rape straw and corn silage (control), and a mixed silage treatment of rape straw and whole crop corn (mixed silage). Three ruminally cannulated Holstein bulls were employed. Substrates were incubated for varying durations (4, 12, 24, 48, 72, 96, 120 and 216 h) to assess substrate degradation kinetics. Bacterial colonization were analyzed after 4- and 48-h incubation time. Mixed ensiling disrupted the fiber structure of rape straw, and thus had lower fiber content compared to the control, as NDF and ADF content ‌decreased by 55 g/kg (678 vs. 623 g/kg) and 27 g/kg (440 vs. 413 g/kg), respectively. Compared to the control group, ruminal DM disappearance of mixed silage significantly (p ≤ 0.05) increased from 315 to 366 g/kg (+16.2%) at an incubation time of 4 h, 552 to 638 g/kg (+15.6%) at 120 h, and 563 to 651 g/kg (+15.6%) at 216 h. Similarly, compared to the control group, NDF disappearance of mixed silage significantly (p ≤ 0.05) rose from 112 to 201 g/kg (+79.5%) at 4 h, 405 to 517 g/kg (+27.7%) at 120 h, and 429 to 532 g/kg (+24.0%) at 216 h. Compared to the control group, soluble and washout nutrient fractions (a) of DM or NDF fraction in mixed silage significantly (p ≤ 0.05) rose from 289 to 340 g/kg (+17.6%), potentially degradable fractions (b) of NDF increased from 310 to 370 g/kg (+19.4%), and the undegraded fraction of NDF (μNDF) decreased from 582 to 471 g/kg (−19.1%). Incubation time, apart from in the mixed ensiling treatment, altered the bacterial community. The study highlights that higher total potentially degradable fractions account for enhanced ruminal substrate degradation of mixed silage.

Ruminants are important for global food security but are major sources of the greenhouse gas methane. Methane yield is controlled by the cycling of molecular hydrogen (H₂), which is produced during carbohydrate fermentation and consumed by methanogenic, acetogenic, and respiratory microorganisms. However, we lack a holistic understanding of the mediators and pathways of H₂ metabolism, and how this varies between ruminants with different methane-emitting phenotypes. Here we used metagenomic, metatranscriptomic, metabolomics, and biochemical approaches to compare H₂ cycling and reductant disposal pathways between low-methane-yield Holstein and high-methane-yield Jersey dairy cattle. The microbiota of Holstein cattle disposed of reductant via propionate and amino acid production and expressed uptake [NiFe]-hydrogenases to use H₂ to support sulfate and nitrate respiration. In contrast, Jersey microbiota primarily disposed of H2 through methanogenesis via methanogenic [NiFe]-hydrogenases and acetogenesis via [FeFe]-hydrogenases, resulting in enhanced methane and acetate production. These findings were supported by two in vitro measurements of microbiota activities and metabolites, as well as public global rumen microbiome data from sheep and beef cattle. Overall, this study highlights the importance of promoting alternative H2 consumption and reductant disposal pathways for the synthesis of host-beneficial metabolites and the reduction of methane production in ruminants.