Reductive acetogenesis is a dominant process in the ruminant hindgut

Abstract

Background: The microbes residing in ruminant gastrointestinal tracts play a crucial role in converting plant biomass to volatile fatty acids, which serve as the primary energy source for ruminants. This gastrointestinal tract comprises a foregut (rumen) and hindgut (cecum and colon), which differ in structures and functions, particularly with respect to feed digestion and fermentation. While the rumen microbiome has been extensively studied, the cecal microbiome remains much less investigated and understood, especially concerning the assembling microbial communities and overriding pathways of hydrogen metabolism.

Results: To address this gap, we comparatively investigated the composition, capabilities, and activities of the rumen and the cecum microbiome using goats as an experimental ruminant model. In situ measurements showed significantly higher levels of dissolved hydrogen and acetate in the cecum than in the rumen. Increased dissolved hydrogen indicated distinct processes and reduced coupling between fermentative H₂ production and utilization, whereas higher levels of acetate could be caused by slower VFA absorption through cecal papillae than through the rumen papillae. Microbial profiling indicated that the cecum harbors a greater abundance of mucin-degrading microbes and fermentative hydrogen producers, whereas the rumen contains a higher abundance of fibrolytic fermentative bacteria, hydrogenotrophic respiratory bacteria, and methanogenic archaea. Most strikingly, reductive acetogenic bacteria were 12-fold more abundant in the cecum. Genome-resolved metagenomic analysis unveiled that the cecum acetogens are both phylogenetically and functionally distinct from those found in the rumen. Further supporting these findings, two in vitro experiments demonstrated a marked difference in hydrogen metabolism pathways between the cecum and the rumen, with increased acetate production and reduced methanogenesis in the cecum. Moreover, comparative analysis across multiple ruminant species confirmed a strong enrichment of reductive acetogens in the hindguts, suggesting a conserved functional role.

Conclusions: These findings highlight an enrichment of acetogenesis in a key region of the gastrointestinal tract and reshape our understanding of ruminant hydrogen metabolism and how the H₂ can be managed in accord to livestock methane mitigation efforts. Video Abstract.

Keywords: Acetogens; Fermentation pathway; Hydrogenase; Methanogenesis; Molecular hydrogen.

Fig. 1

Distinct microbiomes, metabolomes, and epithelial functions in the rumen and cecum. A Graphical representation of samples collected from the two distinct digestive tract segments of goats, B Epithelial tissue morphology, C Lumen pH, D lumen dH₂ concentrations, E Total lumen VFA concentrations, F Molar proportions of VFAs, G Protein expression of G-protein coupled receptors 43 (GPR43) as visualized after immunofluorescent staining, and H Top 10 enriched KEGG pathways in the ruminal and the cecal epithelium (other pathways are shown in Additional File S2). The undefined Metabolic pathways category was the result of too many genes being enriched under this category. I Differentially expressed genes (DEGs) involved in VFA absorption and intracellular pH regulation. J 16S rRNA gene copy numbers of total bacteria. K 16S rRNA gene copy numbers of total methanogens. L Relative abundance of major bacterial phyla. M Most differentially abundance bacterial genera identified using LEfSe (only the genera with LDA > 4.2 are shown, with others shown in Additional File S3). Abbreviations: VFA, volatile fatty acid; dH₂, dissolved hydrogen; HMGCS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGCL, 3-hydroxy-3-methylglutaryl-CoA lyase; ATPeV1B, vacuolar H⁺ ATPase subunit B; BDH, β-hydroxybutyrate dehydrogenase. ** p < 0.01, *** p < 0.001, n = 16 per group

Fig. 2

Distinct functions in the rumen and cecum microbiomes. A Abundance of GH families and their enrichment in various prokaryotic phyla. B Relative abundance of genes involved in acetate, propionate, butyrate production, and the Wood-Ljungdahl pathway, and their enrichment in various bacterial phyla. C Relative abundance and phylum-level distribution of hydrogenases and terminal reductases expressed as transcripts per million (TPM). Classifications: Bifurcating hydrogenases (group A3 and A4 [FeFe]-hydrogenases), fermentative hydrogenases (groups B, A1 and A2 [FeFe]-hydrogenases), energy-converting hydrogenases (groups 4a, 4c, 4e, 4f, 4 g [NiFe]-hydrogenases), sensory hydrogenases (group C [FeFe]-hydrogenases), methanogenic hydrogenases (group 3a, 3c, 4h, 4i [NiFe]-hydrogenases, [Fe]-hydrogenases). HydB, hydrogenase-associated diaphorase. H₂ uptake pathways can be coupled to methanogenesis (McrA, methyl-CoM reductase), reductive acetogenesis (AcsB, acetyl-CoA synthase), fumarate reduction (FrdA, fumarate reductase), nitrate ammonification (NrfA, ammonia-forming nitrite reductase; NarG, dissimilatory nitrate reductase; NapA, periplasmic nitrate reductase), sulfate and sulfite reduction (AprA, adenylylsulfate reductase; AsrA, alternative sulfite reductase; DsrA, dissimilatory sulfite reductase), dimethyl sulfoxide and trimethylamine N-oxide reduction (DmsA, DMSO and TMAO reductase). Only genes with an average abundance > 5 TPM were shown, while others are shown in Additional File S8. Abbreviations: AA, acetyl-CoA to acetate; AB, acetyl-CoA to butyrate; PLP, propionate formation via the lactate pathway; PSP, propionate formation via the succinate pathway; WLP, Wood-Ljungdahl pathway. Data with error bars are expressed as mean ± standard error. Significance was tested using independent two-group Wilcoxon rank-sum tests. * p < 0.05, *** p < 0.001, n = 16 per group

Fig. 3

Phylogenetically and functionally distinct microbial MAGs are enriched in the rumen and the cecum microbiomes. A A phylogenetic tree of 2324 MAGs encoding CAZymes or hydrogenases. B A heatmap depicting the microbial phyla and their enzymes identified from the 2324 MAGs. Z-scores between alignments of rumen/cecum samples are used for correcting abundance, and major phyla are expressed as log₁₀ (TPM + 1). Significance was tested using independent two-group Wilcoxon rank-sum tests. * p < 0.05, ** p < 0.01, *** p < 0.001, n = 16 per group

Fig. 4

Maximum-likelihood phylogenetic trees of acetogenic MAGs, the AcsB gene sequences, and the Wood-Ljungdahl pathway reconstructed from the identified genes. A A maximum-likelihood phylogenetic tree of nine acetogenic MAGs obtained from this study and 15 reference genomes of acetogens. The heatmap indicates the relative abundance of MAG based on short reads in the rumen and the cecum samples. B A maximum-likelihood phylogenetic tree based on 36 AcsB sequences encoded by the 24 MAGs. C The Wood-Ljungdahl pathway reconstructed from nine novel acetogenic MAGs at the genus level. For both trees, the triangles show the reference genomes of acetogens, and the pentagrams show acetogenic MAGs assembled in this study. MAGs are colored based on their phylogenetic affiliation at the family level. Bootstrap values > 70% are indicated as black circles at the nodes, and scale bars indicate the mean number of substitutions per site. Detailed data are presented in Additional File S12. Abbreviations: HDCR, hydrogen-dependent carbon dioxide reductase; Fhs, formate-tetrahydrofolate (THF) ligase; Fch, methenyl-THF cyclohydrolase; Fol, methylene-THF dehydrogenase; Met, methylene-THF reductase; MT, methyltransferase; CODH/ACS, carbon monoxide dehydrogenase/acetyl-CoA synthetase

Fig. 5

In vitro metabolic activities of the rumen and the cecum microbiomes. A Feed degradation, methane (CH₄) production, gaseous hydrogen (gH₂) accumulation, and VFA profiles after 72-h in vitro incubation with corn stover as a substrate. B Utilization of headspace H₂ and CH₄ production during 48-h incubation in experiment 2 without adding any substrate. * p < 0.05, ** p < 0.01, *** p < 0.001, n = 6 per group

Fig. 6

Distinct hydrogen metabolism in the rumen and cecum of different ruminants. A Relative abundance of bacterial phyla whose genomes carry acsB, the marker gene of the reductive acetogenesis pathway. The data were obtained from Xie et al. [19]. B Summary of key pathways, enzymes, microorganisms, and metabolites involved in hydrogen metabolism in the rumen (left-hand part) and the cecum (right-hand part) microbiomes. The data are presented in detail in Additional File S14