Hydrogen and acetate levels are elevated in the cecum compared to the rumen
We collected epithelium and digesta samples from the rumen and cecum of 16 growing healthy goats before the morning feeding (Fig. 1A). The cecum had a distinct epithelial structure and papillary morphology, including rougher and rounder papillae, in comparison with the rumen (Fig. 1B). Although the cecum had a slightly higher pH (p < 0.001, Fig. 1C), it contained a higher concentration of total volatile fatty acids (VFAs) (p < 0.01) and acetate concentration and molar proportion (p < 0.001), but a lower molar proportion of propionate (p < 0.01) and butyrate (p < 0.001) (Fig. 1E, F; Additional Fig S1). These differences may reflect increased VFA production within the cecal microbiome and or reduced absorption through the cecal epithelium. Measurement of dissolved H2 (dH2), a central electron carrier produced during fermentation and mainly consumed by methanogens in the rumen [2], revealed a four-fold higher dH2 concentration in the cecum compared to the rumen (p < 0.001, Fig. 1D). Such greatly increased dH2 concentrations indicates a distinct processes and reduced coupling between fermentative H2 production and utilization in the cecum.
To understand the different VFA concentrations between the rumen and the cecum, we compared the expression of genes involved in VFA absorption. Immunofluorescent staining revealed that cecum papillae exhibited a lower level of GPR43 (Fig. 1G), a VFA receptor widely distributed throughout the gastrointestinal tract involved in controlling host energy accumulation [29]. Proteomic analysis identified 2,276 differentially expressed proteins (fold-change > 2, FDR < 0.05) in the epithelia of the rumen and cecum (Additional File S1). Principal component analysis (PCA) further showed that these proteins were distinct between the rumen and cecum (Additional Fig S2) and were primarily associated with metabolic pathway (Fig. 1H, Additional File S2). For example, proteins involved in VFA absorption and intracellular pH regulation, including 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS), 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL), vacuolar H+ ATPase subunit B (ATPeV1B), and β-hydroxybutyrate dehydrogenase (BDH) [30–32], were expressed at a higher level in the rumen than in the cecum (p < 0.001, Fig. 1I). These results indicate that VFA absorption through cecal papillae is probably slower than that through the rumen papillae, explaining the relatively higher VFA concentration in the cecum, at least in part.
We analyzed the rumen and cecum microbiomes through 16S rRNA gene amplicon sequencing and qPCR to understand their contribution to the observed VFA disparity. Compared to the rumen, the cecum contained a significantly lower population of total bacteria (6.7-fold lower, p < 0.01, Fig. 1J) and archaeal methanogens (2.8-fold lower, p < 0.01, Fig. 1K), but a higher ratio of methanogens to bacteria (3.1% vs 1.3%). Principal coordinates analysis (PCoA) based on Bray-Curtis dissimilarity revealed distinct bacterial communities (p < 0.001) between the rumen and cecum (Additional Fig S3). At the phylum level, Bacteroidota and Spirochaetota were enriched in rumen microbiota, whereas Firmicutes,Verrucomicrobiota, Proteobacteria and Actinobacteriota were relatively more abundant in cecum microbiome (p < 0.001, Fig. 1L, Additional File S3). Linear discriminant analysis (LDA) identified 15 key bacterial genera with relative abundance differentiated in both gastrointestinal regions [33] (p < 0.001, Fig. 1M, Additional File S4). Among these genera, Prevotella and Lachnospiraceae XPB1014, two genera known functions in facilitating forage fiber degradation [34, 35], In contrast, Ruminococcaceae UCG-005 and Christensenellaceae R7 were the most abundant and representative cecal genera (Additional Fig S4), previously shown to mediate sugar degradation and maintain gut health [36, 37]. Acetate levels positively correlated with these two genera and several other genera, most strongly for Akkermansia, Bacteroidales UCG_010_ge, Ruminococcaceae UCG_002, and Lachnospiraceae unclassified (Additional Fig S5). dH2 concentration also exhibited a positive correlation with several genera of microbes, most strongly for Christensenellaceae R7, Monoglobus, and Ruminococaceae unclassified (Additional Fig S5). Thus, these differentially enriched taxa may contribute to the differential electron flows and H2 metabolism observed between the rumen and the cecum.
Distinct processes of carbohydrate, VFA, and hydrogen metabolism in the cecum
We screened carbohydrate-active enzymes (CAZymes), the microbial enzymes responsible for degrading plant biomass to VFAs [38–40], in the assembled metagenomic contigs from cecum and rumen microbiota. CAZymes were distinct in composition between both gastrointestinal regions (principal coordinate analysis) and were more abundant in the rumen microbiome (p < 0.05, Additional Fig S6). The most abundant family of CAZymes in both regions were glycoside hydrolases (GH) (Additional File S5), which hydrolyze glycosidic linkages within a wide range of carbohydrate substrates in a substrate-specific manner [41]. The rumen selected for more fiber-degrading enzymes, such as β-xylosidase GH43 (p < 0.001, 2.2-fold higher, Fig. 2A; Additional Fig S7, Additional File S6), whereas the cecum selected for greater abundance of mucin-degrading enzymes, including α-N-acetylgalactosaminidase GH109 (p < 0.001, 2.4-fold higher, Fig. 2A; Additional Fig S7, Additional File S6). While mucin degradation reduces the protective layer of complex mucus layer, there is increasing evidence that this process may stimulate mucin growth, renewal, and thickening, as a key mechanism that the host uses to select favorable bacteria [19, 42]. There were also strong differences in the types of hemicellulose-degrading enzymes encoded in the two regions, with the rumen enriched in 15 GH (e.g., GH10, GH11) and the cecum enriched in 10 GH (e.g., GH2, GH42). Such distinct enrichments of CAZymes family were further upheld by the subsequent pathway of fermentative VFA production, which had differed the abundance of genes related to the production of VFA from carbohydrates between the gastrointestinal regions (Additional Fig S8). Cecum microbiome had more the abundance of four key genes involved in fermentative acetate production (i.e. porA, porD, porG, and ackA, p < 0.05) (Fig. 2B, Additional Fig S9, Additional File S7). Rumen microbiome showed greater the abundance of six key genes involved in butyrate production (i.e. crt, fabV, atoA, atoD, ptb, and buk genes, p < 0.001) and three key genes involved in propionate production via succinate (i.e. fumA, sucC, and MCEE genes, p < 0.001) (Fig. 2B, Additional Fig S9, Additional File S7). These results suggest that the cecum selected distinct microorganisms that favor fermentative acetate production, which may be driven by the enrichment of host-glycan in the hindgut [24].
We subsequently examined the pathways of H2 production and consumption by identifying the hydrogenase genes. A total of 16,883 contigs encoded hydrogenase-encoding genes, which were distributed among 306 genera across 23 phyla (Additional File S8). The trimeric group A3 [FeFe]-hydrogenases (identified based on the presence of genes encoding the hydrogenase subunit hydA and diaphorase subunit hydB) were the most abundant hydrogenases in both gastrointestinal regions, and were highly enriched in the cecum (Fig. 2C). Primarily encoded by the classes Clostridia and Bacteroidia (Fig. 2C, Additional File S8), these hydrogenases are thought to predominate H2 production in rumen [12]. The group B [FeFe]-hydrogenases was similarly abundant and highly enriched (8-fold) in the cecum; this functionally unresolved enzyme has previously been implicated in fermentative H2 production in the human colon, where it is the most abundant hydrogenase [43]. Additionally, several other hydrogenases were also more enriched in the cecum (sensory group C1 and C3 [FeFe]-, energy-converting group 4g [NiFe]-, and hypothetical group A2 [FeFe]-hydrogenases (p < 0.001, Fig. 2C, Additional File S8). In contrast, the rumen selected for greater abundance of sensory group C2 [FeFe]-hydrogenases (4.1-fold higher) and energy-converting 4e [NiFe]-hydrogenases (1.7-fold higher) (p < 0.001, Fig. 2C, Additional File S8). Altogether, these findings suggest elevated H2 production and distinct H2 cycling processes in the cecum, which aligns with observed higher dH2 concentrations detected in the cecal samples (Fig. 1D).
The gastrointestinal regions also contained distinct hydrogenotrophic microorganisms, based on profiling of their hydrogenases and terminal reductases. The capacity for methanogenesis appears to be similar between the rumen and the cecum, given the similar abundance of genes encoding for methyl-CoM reductase and methanogen-specific hydrogenases ([Fe]-hydrogenases, groups 3a, 3c, 4h, and 4i [NiFe]-hydrogenases) (Fig. 2C, Additional File S8, Additional Fig S10, Additional File S8). Several hydrogenotrophic bacteria compete with methanogens for the supply of H2 in the gastrointestinal region of ruminants, including those that mediate reductive acetogenesis, fumarate reduction, nitrate ammonification and sulfate reduction [12, 44]. The signature gene for reductive acetogenesis (acetyl-CoA synthase, acsB, p < 0.001) was 12-fold more abundant in the cecum than the rumen (Fig. 2C, Additional File S8). This enrichment of reductive acetogenesis is also reflected by increased levels of nine genes (i.e. fhs, acsB, acsC, acsD, acsE, cooS, metF, fchA, folD) involved in the Wood-Ljungdahl pathway (Additional Fig S9, Additional File S7). In contrast, the rumen microbiome encoded a greater proportion of genes for fumarate reduction (fumarate reductase, frdA, p < 0.001, 2-folder higher), nitrate ammonification (ammonia-forming nitrite reductase, nrfA and dissimilatory nitrate reductase, narG, p < 0.001, 51-folder and 10-folder higher), and sulfate reduction (adenylylsulfate reductase, aprA, p < 0.001, 7.4-folder higher) (Fig. 2C, Additional File S8). Altogether, this suggests that the cecum favors reductive acetogenesis over hydrogenotrophic respiration, which is consistent with the higher concentration of dH2 in the cecum (Fig. 1D) required for reductive acetogenesis to be thermodynamically viable [45]. A relative predominance of reductive acetogenesis over hydrogenotrophic respiration in the cecum compared to the rumen is also consistent with the higher molar proportion of acetate and acetate to propionate ratio (Fig. 1F).
Genome binning confirms novel acetogens are enriched in the cecum
To gain insights into the aforementioned differences at the bacterial species level, we performed metagenomic binning, yielding 2,358 metagenome-assembled genomes (MAGs, completeness ≥ 50% and contamination ≤ 10%) after quality control and dereplication (Additional File S9). The 2,324 bacterial MAGs were distributed through 19 phyla, including Firmicutes (1,463), Bacteroidota (637) and Spirochaetota (70) (Fig. 3A, Additional File S9). Also detected were 34 methanogenic MAGs from two archaeal phyla, Methanobacteriota (primarily Methanobrevibacter spp.) and Halobacteriota (primarily Methanocorpusculum spp.) (Additional Fig S11, Additional Fig S12, Additional File S9); although these MAGs were distributed in both regions, all Halobacteriota MAGs were enriched in the cecum, in line with their reported roles in low-methane produced during hindgut fermentation in monogastric animals such as horses [46].
The MAGs were functionally annotated to comparatively examine their potential capacity in carbohydrate degradation, fermentation, and H2 cycling between the gastrointestinal regions. Most assembled MAGs encoded for GH, including those responsible for the degradation of cellulose (86% MAGs), hemicellulose (83%), and host glycans (96%). In the cecum, there was an enrichment of MAGs classified to Faecousia, Akkermansia, and Alistipes that encoded GH109, a GH enzyme hydrolyzing host glycans (p < 0.05, Fig. 3B, Additional File S10). The rumen-enriched MAGs were assigned to Prevotella, Cryptobacteroides, Ruminococcus, and Butyrivibrio (p < 0.01), including MAGs encoding cellulases (e.g. GH3) and hemicellulases (e.g. GH43) (Fig. 3B, Additional File S10). The MAG-level analysis also confirmed that fermentative acetate-producing bacterial were enriched in the cecum (e.g. MAG587, MAG985).
Two thirds of the MAGs also carried hydrogenases genes encompassing a range of subgroups, and MAGs encoding H2-producing [FeFe]-hydrogenases (1488 MAGs to be exact, 63% of the total) were particularly prevalent in both gastrointestinal regions (Fig. 3A, Additional File S10 and S11). The cecum was enriched with MAGs predicted to produce H2 using the electron-confurcating trimeric group A3 [FeFe]-hydrogenases (e.g. MAG264) and fermentative group B [FeFe]-hydrogenases (e.g. MAG270). A total of 300 MAGs (13%) encoded both H2-consuming hydrogenases and terminal reductases, including those involved in reductive acetogenesis (52 MAGs), fumarate reduction (176 MAGs), nitrate ammonification (34 MAGs), sulfate reduction (65 MAGs) (Additional File S11). In the rumen microbiome, the most enriched were Desulfovibrio and Succiniclasticum MAGs capable of hydrogenotrophic sulfate and fumarate reduction, respectively (p < 0.001, Fig. 3B, Additional File S11). In contrast, putative reductive acetogens from the class Clostridia were enriched in the cecum microbiome, each encoding both [FeFe]-hydrogenases and the acetyl-CoA synthase (p < 0.001, Fig. 3B, Additional File S11). These results point to distinct microbes selected in the rumen and the cecum that utilize different pathways of H2 metabolism, with acetogenesis likely being a major H2 sink within the cecal microbiome.
We comprehensively compared the nine acetogenic MAGs obtained from this study with 15 reference acetogen genomes of acetogens [47, 48]. Genome-based phylogenetic analysis showed that these MAGs were not closely related to cultured acetogens, and they instead fell into five novel genera, namely SIG483 and RGIG7114 in the family Oscillospiraceae and HGM12587, SIG280, and RGIG5612 in the family Lachnospiraceae (Fig. 4A, Additional File S12). Nevertheless, a tree based on the acsB gene showed that they each encoded either one or two copies of the canonical acetyl-CoA synthase gene, suggesting they are capable of acetogenesis (Fig. 4B). Further annotation revealed that the MAGs from each of the five novel genera encoded all the enzymes of the Wood-Ljungdahl pathway, together with the group A3 [FeFe]-hydrogenase, Nfn transhydrogenase, and energy-converting Rnf or Ech enzymes, arguing or their capability of both assimilating carbon and conserving energy through reductive acetogenesis (Fig. 4C). The CO2-reducing formate dehydrogenase was absent in all but one MAG, implying potential assimilation of formate rather than CO2. This premise concurs with the adaptation of gastrointestinal acetogens to their formate-rich habitats [48]. The HGM12587 MAGs also lacked the genes involved in the further conversion of acetyl-CoA to acetate. Finally, we compared the composition of the acetogenic communities between rumen and cecum microbiota based on both the novel and reference MAGs [47, 48]. The Bray-Curtis analysis confirmed that the two communities were distinctly structured (p < 0.001, Additional Fig S13), with 15 MAGs (including HGM12587 and SIG483) enriched in the cecum and 9 others (primarily SIG280 and RGIG7114) enriched in the rumen (Fig. 4A).
In vitro experiments show reductive acetogens are more active in the cecum
To confirm the enrichment of acetogens in the cecal microbiota, we conducted two in vitro experiments comparing the biochemical activities of the cecum and rumen microbiomes, specifically focusing on their activity of reductive acetogenesis. In the first experiment, using corn stover as the nutrient, the rumen inocula achieved a higher dry matter degradability (DMD), VFA concentration, and methane production, whereas the cecum inocula resulted in a higher H2 accumulation (p < 0.05, Fig. 5A). These results indicate that the rumen microbiome possesses a greater capacity for fiber breakdown and hydrogenotrophic methanogenesis, leading to less H2 accumulated. Analysis of the VFA profiles confirmed that the cecal microbiome produced more acetate than the rumen microbiome, whereas the latter produced more propionate (p < 0.05, Fig. 5A). The heightened acetate production and reduced methane production by the cecal microbiome suggest that the elevated acetate production in the in vitro cecal culture might stem from reductive acetogenesis rather than fermentative processes. This aligns with the enrichment MAGs classified as acetogens in the cecal microbiome.
In the second in vitro experiment, we compared the rumen and the cecum microbiome for their capability to utilize H2. Both microbiomes significantly decreased H2 compared to the uninoculated control and increased headspace methane concentration towards an asymptote (p < 0.05, Fig. 5B). After 48 hours, the rumen microbiota consumed more gaseous H2 and produced more methane than the cecum microbiome (p < 0.05, Fig. 5B), suggesting a greater capacity of hydrogenotrophic methanogenesis. Adding H2 to the headspace also increased total VFA production, possibly due to increased reductive acetogenesis or fumarate reduction, as proposed previously [17]. The rumen inocula led to a higher molar proportion of propionate and butyrate. In comparison, the cecum inocula resulted in a higher molar proportion of acetate with concurrently increased acetate to propionate ratio (Fig. 5C). These results further confirm that the rumen microbiome selects for hydrogenotrophic respiratory bacteria and methanogenic archaea. In contrast, the cecal microbiome selects for reductive acetogens.
Acetogens are enriched in the hindguts of diverse ruminants
Leveraging on the metagenomic data reported in our previous study [18], we tested whether our findings could be extended to other ruminants, including dairy cattle, goat, roe deer, sheep, buffalo, water deer, and yak. We found that the abundance of the signature gene of the reductive acetogenesis pathway, acsB, was enriched in the cecal microbiome of all the ruminants relative to their rumen microbiome (p < 0.001): enriched by 22-fold higher in dairy cows, 58-fold higher in goats, 111-fold higher in roe deer, 14-fold higher in sheep, 10-fold higher in water buffalo, 133-fold higher in water deer, and 20-fold higher yak (Fig. 6A). Reductive acetogens were particularly abundant in cow and buffalo samples, whereas their abundance was relatively low in water deer. Nearly all the acetogens were taxonomically assigned to the class Clostridia. Such enrichment of reductive acetogenesis in the cecal microbiome was further upheld by a high abundance of genes involved in the Wood-Ljungdahl pathway (Additional File S13), which included six genes (i.e., fhs, acsA, acsB, acsC, acsD, acsE) encoding Fhs, methyltransferase, and CODH/ACS. These results suggest a generalized phenomenon of more significant enrichment of reductive acetogenesis in ruminants’ cecum.