Main

Molecular hydrogen (H2) is a central intermediate in gastrointestinal digestive processes. Most bacteria within the gut hydrolyse and ferment dietary carbohydrates to absorbable short-chain fatty acids1,2,3 and large quantities of H2 gas4,5. H2 accumulates to high micromolar levels in the gut, where it is primarily consumed by other microorganisms for energy conservation and carbon fixation6,7, although a large proportion is also expelled as flatus or exhaled8,9,10. Classically, three groups of gut microorganisms are thought to consume H2, namely, acetogenic bacteria, methanogenic archaea and sulfate-reducing bacteria3,11,12,13,14. H2 consumption by gut microorganisms lowers H2 partial pressures, thereby ensuring fermentation remains thermodynamically favourable3,15,16,17,18,19. In turn, many H2-producing and H2-consuming microorganisms form mutualistic relationships by conducting interspecies H2 transfer dependent on physical association15,20. In addition to supporting digestion, gastrointestinal H2 cycling modulates levels of important metabolites in the gut, including butyrate21, hydrogen sulfide22, bile acids2 and host steroids23, with diverse effects on processes such as digestion, inflammation and carcinogenesis. It is also proposed that microbiota-derived or therapeutically supplied H2 may directly benefit human cells as an anti-oxidant24,25. Disruption of the balance between H2-producing and H2-consuming bacteria has been linked to a range of gut and wider disorders19,26; most notably, gas build-up contributes to the symptoms of irritable bowel syndrome and hydrogen breath tests are frequently, if controversially, used to detect disorders such as carbohydrate malabsorption27,28,29. Numerous pathogens also exploit microbiota-derived H2 during invasion, including Helicobacter pylori and Salmonella30,31,32,33,34, or rapidly produce it in the case of pathogenic Clostridia and protists30,35,36.

Despite the central importance of H2 cycling in human health and disease, surprisingly little is known about which microorganisms and enzymes mediate this process. Both the production and consumption of H2 are catalysed by hydrogenases, which fall into three major groups dependent on the metal content of their active sites: the [FeFe]-, [NiFe]-, and [Fe]-hydrogenases, with multiple subgroups37,38. It has been classically thought that most H2 production in the gut is mediated by fermentative bacteria, primarily the class Clostridia, that couple reoxidation of ferredoxin (for example, reduced during acetate fermentation by the pyruvate:ferredoxin oxidoreductase (PFOR) reaction) to the evolution of H2. Some Clostridia use group A1 [FeFe]-hydrogenases, an extensively structurally and mechanistically characterized lineage of enzymes, to rapidly produce H2 (refs. 39,40). Some H2 may also be produced by formate hydrogenlyase complexes (containing a group 4a [NiFe]-hydrogenase) that disproportionate formate during fermentative survival of Enterobacteriaceae41,42. Yet two recent findings suggest that other fermenters are also active in the human gut. Our 2016 survey showed genes encoding a distantly related enzyme called the group B [FeFe]-hydrogenase (28% amino acid identity to the group A1 enzymes) are widespread in diverse gut isolates and abundant in gut metagenomes19,43. Two recent biochemical studies44,45 suggest that these enzymes are active and predominantly mediate H2 production, although their physiological activity and role has yet to be confirmed in any organism. In parallel, electron-confurcating hydrogenase complexes (group A3 [FeFe]-hydrogenases) have been discovered that couple oxidation of NAD(P)H and reduced ferredoxin to the evolution of H2 (refs. 46,47,48,49). We have shown that group A3 [FeFe]-hydrogenases are primarily responsible for H2 production in ruminants17,50,51, although it is unclear if these principles also extend to humans. Similarly, it is unclear whether the paradigms regarding H2 consumption are accurate, given the three classical groups of H2 oxidizers (hydrogenotrophs) are generally in low abundance in the human gut. Indeed, only approximately half of people produce methane gas52,53, and it is becoming increasingly apparent that most hydrogen sulfide is derived from organosulfur compounds rather than sulfate reduction54,55,56. Respiratory hydrogenotrophs that use electron acceptors such as fumarate, nitrate, sulfoxides and inflammation-derived oxygen may also be active but overlooked members of gut microbiota19,31.

Here we have integrated enzymatic and microbial insights to build a detailed picture of H2 cycling in the human gastrointestinal tract. To do so, we holistically profiled the abundance, transcription and distribution of hydrogenases using metagenomes and metatranscriptomes, including original biopsy samples, in both healthy individuals and those with gastrointestinal disorders. We then performed an in-depth analysis of 19 bacterial isolates and 4 heterologously produced hydrogenases to confirm the activity and roles of these enzymes. We reveal that group B [FeFe]-hydrogenase drives most H2 production in the human gut, highlight the overlooked role of Bacteroides as major H2-producing fermenters, and show that hydrogenase genes are differentially abundant between healthy people and those with chronic disease phenotypes, such as Crohn’s disease.

Results

Group B [FeFe]-hydrogenase genes are widespread and transcribed in the human gut

We initially investigated the distribution of hydrogenase genes in the human gut by analysing 300 human stool metagenomes57 (Supplementary Dataset 1). Hydrogenase genes are extremely abundant, occurring on average at 1.44 ± 0.58 copies per genome (cpg) (Fig. 1a). By far the most abundant are the functionally uncharacterized group B [FeFe]-hydrogenases (0.75 ± 0.25 cpg), hypothesized but unproven to mediate fermentative H2 production19 (Fig. 1b). Genes encoding these enzymes are much more abundant than the ferredoxin-dependent group A1 [FeFe]-hydrogenases (0.10 ± 0.09 cpg), which were previously thought to account for most gut H2 production6,58,59, and electron-confurcating group A3 [FeFe]-hydrogenases (0.19 ± 0.11 cpg) that dominate H2 production in ruminants17,50. Other enzymes also potentially play minor roles in H2 production in the human gut, including formate hydrogenlyases (group 4a [NiFe]-hydrogenase genes; 0.02 ± 0.07 cpg) and possibly ferredoxin-dependent energy-converting hydrogenases (group 4e [NiFe]-hydrogenase genes; 0.06 ± 0.04 cpg) (Fig. 1a,b and Supplementary Dataset 1). Consistently, analyses of 78 paired metatranscriptomes confirm that these hydrogenase genes are highly transcribed (RNA:DNA ratios between 1.76 and 6.90, depending on subgroup) (Supplementary Dataset 1 and Fig. 1a). Transcripts for the group B [FeFe]-hydrogenase genes are the most numerous (95 ± 86 reads per kilobase of transcript per million mapped reads (RPKM); RNA:DNA ratio = 2.0) and 3.3-fold, 4.7-fold and 26-fold higher than the well-characterized group A3, A1 and 4a enzymes (Fig. 1a). Given [FeFe]-hydrogenases are usually highly active enzymes39,40, transcription of these levels probably enables rapid H2 production in the gut. There was nevertheless much interindividual variation in expression levels. Genes encoding nitrogenases, which produce H2 during their reaction cycle60, were also widely encoded but minimally transcribed by gut bacteria (Fig. 1a). Altogether, group B [FeFe]-hydrogenases probably drive most H2 production in the gut but operate alongside other H2-producing hydrogenases.

Fig. 1: Abundance, transcription and distribution of hydrogenase genes and H2-related metabolic genes throughout the human gut.

a, Abundance and transcription of the genes encoding the catalytic subunits of the three types of hydrogenases and the terminal reductases known to use H2-derived electrons in faecal metagenomes (left; n = 300), faecal metatranscriptomes (middle; n = 78) and biopsy enrichment metagenomes (right; n = 102). These results summarize homology-based searches against comprehensive reference databases and are shown in average gene copies per organism (normalized to a set of universal single-copy ribosomal genes) for metagenomes and RPKM for metatranscriptomes. b, Proportion of each hydrogenase group present in each sample per dataset. c, Top genera predicted to encode or transcribe hydrogenases for each dataset. The top ten most abundant genera are included for the five most abundant gut hydrogenase lineages, expressed in RPKM. Phyla are represented by the coloured bar above the genera names. Purple represents Actinobacteria; red, Archaea; light green, Bacteroidetes; orange, Firmicutes; dark green, Fusobacteria; yellow, Proteobacteria; pink, Thermotogae; and light blue, Verrucomicrobia.

Source data

To infer which gut microorganisms encode the genes for these enzymes, we mapped the hydrogenase-encoding reads to both our comprehensive hydrogenase database (HydDB)61 and our in-house collection of 812 sequenced gut isolates (Supplementary Dataset 2). Genes encoding the group B [FeFe]-hydrogenases are very widespread among gut bacteria, encoded by 62% of isolates and the dominant gut phyla Firmicutes, Bacteroidetes and Actinobacteria (Supplementary Dataset 2 and Fig. 2). The abundant lineage Bacteroides (Supplementary Dataset 3) accounted for the most group B [FeFe]-hydrogenase reads in the metagenomes and metatranscriptomes, followed by Alistipes and Clostridia lineages such as Faecalibacterium, Agathobacter and Roseburia (Fig. 1c and Supplementary Dataset 1). The group A1 and A3 [FeFe]-hydrogenase genes were also widespread, encoded and transcribed by various Bacteroidia, Clostridia and Fusobacteria, whereas formate hydrogenlyases were restricted to Enterobacteriaceae, Pasteurellaceae and Coriobacteraceae (Figs. 1c and 2 and Supplementary Dataset 1).

Fig. 2: Phylogenomic tree showing distribution of hydrogenase genes among 812 bacterial isolates from the human gut.

Isolates are from the five dominant phyla within the human gut, with branch colours showing their phylum-level taxonomy. Isolates were shown to encode the catalytic subunit genes coding for the major groups of gut hydrogenases and the terminal reductases associated with methanogenesis, acetogenesis, sulfidogenesis, nitrate reduction or succinogenesis (coloured rings). The tree was generated using approximately maximum-likelihood estimation with the Jukes–Cantor model (via FastTree) and standardized ‘bac120’ phylogenetic analysis (via GTDB-Tk) and was midpoint rooted. Results are based on homology-based searches against comprehensive reference databases. Specific isolates were selected for further analysis, including culture-based activity measurements and transcriptome studies (black dots). The tree scale represents the branch length of the tree, as calculated by the number of base substitutions per base position.

Source data

A small but active proportion of the community is predicted to mediate H2 uptake in the human gut. Genes for group 1 [NiFe]-hydrogenases, which support anaerobic respiration using electron acceptors such as fumarate, nitrate, nitrite and sulfite37,38, are encoded by 9% of gut bacteria based on metagenomic short reads (Supplementary Dataset 1 and Fig. 1a,b) and 6% of our isolates (Supplementary Dataset 2 and Fig. 2). As evidenced by the extremely high standard deviations of their metagenome counts (0.09 ± 0.17 cpg) and metatranscriptome reads (51 ± 153 RPKM), the abundance and transcription of these genes greatly vary between individuals (Fig. 1a). They were primarily encoded and transcribed by Enterobacteriaceae ([NiFe] group 1c and 1d), which are known to use gut-derived H2 as a respiratory energy source during colonization30,62,63, and by lineages such as Veillonella (1d), Parabacteroides (1d) and Akkermansia (1f), whose H2 metabolism remains to be investigated (Fig. 1c). Some group A3 [FeFe]-hydrogenase genes were also encoded by hydrogenotrophic acetogens such as Blautia, where these enzymes oxidize H2, rather than produce it, in contrast to fermenters64. Genes encoding the group 3 and 4 [NiFe]-hydrogenases and [Fe]-hydrogenases of methanogenic archaea were also detected in a subset of samples. Consistently, we also detected genes encoding the signature enzymes responsible for fumarate, sulfite, nitrate, and nitrite reduction, acetogenesis, and methanogenesis in the metagenomes and metatranscriptomes (Fig. 1a and Supplementary Dataset 1). Although these genes were in low abundance (except those encoding fumarate reductase), they were often highly transcribed (RNA:DNA ratios of 54 for acetyl-coenzyme A (CoA) synthase, 37 for dissimilatory sulfite reductase and 4 for respiratory nitrate reductase) (Supplementary Dataset 1). Phylogenomic analysis of the gut isolates also revealed frequent co-occurrence of genes encoding group 1 [NiFe]-hydrogenases with respiratory reductases (Fig. 2). However, it should be noted that the respiratory reductases can accept electrons from a range of both organic and inorganic donors other than H2. Also detected were genes encoding putative sensory hydrogenases (group C [FeFe]-hydrogenases, 0.11 ± 0.15 cpg) (Fig. 1a), which are thought to differentially regulate [FeFe]-hydrogenases in response to H2 accumulation in Clostridia and probably other lineages17,37,49.

We tested whether these findings also extend to microbiota sampled within gut tissues, given stool samples provide a biased assessment of gut microbial content65,66,67. To do so, we collected mucosal biopsies from the terminal ileum, caecum and rectum of 42 donors, and then enriched and sequenced their microbiota68 (Supplementary Dataset 1). Concordantly, group B [FeFe]-hydrogenases were by far the most abundant hydrogenase genes detected across these mucosal biopsy samples (0.75 ± 0.25 cpg); they were 3.7-fold more abundant than the next most abundant hydrogenase genes (group A3 [FeFe]-hydrogenases) and primarily encoded by Bacteroides based on read mapping (Fig. 1). The group 1c, 1 d and 4a [NiFe]-hydrogenase genes were also enriched by 6.1-fold, 2.6-fold and 7.0-fold in the biopsy compared with stool metagenomes; this probably reflects the adherence of Enterobacteriaceae to the gut luminal walls, where they potentially use microbiota-derived H2 to support anaerobic and potentially even aerobic respiration (Supplementary Dataset 1 and Fig. 1b,c). Thus, the group B [FeFe]-hydrogenase probably contributes substantially to fermentative H2 production throughout the intestines, much of which is possibly recycled by respiratory hydrogenotrophs. No significant differences in hydrogenase content were found between intestinal regions, which was probably masked by the high degree of interindividual variation.

Group B [FeFe]-hydrogenases are expressed and active in diverse gut isolates

To confirm whether the group B [FeFe]-hydrogenase is active, we used gas chromatography to test H2 production of 19 phylogenetically and physiologically diverse bacterial gut isolates each grown on standard yeast casitone fatty acids (YCFA) medium under fermentative conditions (Supplementary Table 1 and Supplementary Fig. 1). Of these isolates, 13 encoded group B [FeFe]-hydrogenase genes, either individually or together with other hydrogenases, all but 1 of which produced high levels of H2 (Fig. 3a and Supplementary Fig. 2). This collection included 7 Bacteroides isolates that each rapidly produced headspace H2 to average maximum levels of 3.0 ± 0.6% during fermentative growth, and 4 genera from the class Clostridia (Fig. 3b, Supplementary Fig. 1 and Supplementary Table 1). We compared these activities with those of six positive and negative control isolates (Fig. 3a and Supplementary Fig. 1): no H2 was detected in the three isolates lacking hydrogenases; high levels of H2 were produced during fermentative growth of a Fusobacterium isolate with a prototypical group A1 [FeFe]-hydrogenase gene; and H2 was produced during stationary phase in bacteria encoding formate hydrogenlyases, in line with their confirmed roles42,69. Altogether, these analyses show that H2 production is a widespread trait among gut bacteria that encode group B [FeFe]-hydrogenase genes.

Fig. 3: Hydrogenase transcription and activity across 18 bacterial gut isolates.

a, A heat map showing the average transcription levels (expressed as log10 TPM) of the catalytic subunit genes for hydrogenases and the terminal reductases associated with sulfidogenesis, succinogenesis, nitrate reduction and aerobic respiration. b, A heat map showing the average maximum H2 production for each isolate (expressed as log10 (ppm + 1)). In both heat maps, results show means from biologically independent triplicates. Bifidobacterium longum and Catenibacterium mitsuokai do not encode hydrogenase genes and so are used as negative controls. c, Bacterial growth measured by OD600 (green lines) and H2 production (% of headspace; pink lines) of representative isolates from chosen phyla over 24–72 hour periods (n = 3), where the lower detection threshold of the gas chromatograph is 1,000 ppm (dashed red line). A. hadrus, Anaerostipes hadrus; B. dorei, Bacteroides dorei; C. aerofaciens, Collinsella aerofaciens; C. baratii, Clostridium baratii; D. longicatena, Dorea longicatena; F. varium, Fusobacterium varium; G. formicilis, Gemmiger formicilis; N. rosorum, Necropsobacter rosorum; O. umbonata, Olsenella umbonata.

Source data

We performed transcriptome sequencing to confirm whether the group B [FeFe]-hydrogenase genes are expressed and likely responsible for the observed activities (Supplementary Dataset 4 and Supplementary Note 1). After 6 hours of fermentative growth, the 7 hydrogenase-encoding Bacteroides species had each produced an average of 1.51 ± 0.6% H2 in their headspace (Supplementary Figs. 1 and 2). The group B [FeFe]-hydrogenase gene was transcribed at high levels during growth of each strain, averaging 180 transcripts per million (TPM) (ranging from 71 ± 18 TPM for Bacteroides fragilis to 345 ± 17 TPM for Bacteroides plebeius) (Fig. 3a and Supplementary Dataset 4). Four of these strains encoded genes for the group B [FeFe]-hydrogenase as their sole H2-metabolizing enzyme. Three other strains (Bacteroides caccae, Bacteroides thetaiotaomicron and Bacteroides faecis) also encoded genes for the electron-confurcating group A3 [FeFe]-hydrogenase complex, although the transcription of this enzyme was minimal during these growth conditions (average of 3.5 TPM) (Fig. 3a and Supplementary Dataset 4). Importantly, we observed no H2 production by Bacteroides stercoris, the only Bacteroides species in our isolate collection that consistently lacked detection of any hydrogenase genes (Supplementary Fig. 2). Altogether, these results show that the group B [FeFe]-hydrogenases account for H2 production of Bacteroides during fermentative growth and are highly conserved, expressed and active in this genus. Although some Bacteroides species have previously been shown to produce H2 (refs. 70,71,72,73), our culture-dependent studies indicated that fermentative H2 production is a key feature of their physiology. As elaborated in Supplementary Note 1, patterns of hydrogenase expression and activity varied between species within the class Clostridia; whereas some species appear to primarily dispose of H2 using group B [FeFe]-hydrogenases, fast-growing species (for example, Clostridium perfringens) instead appear to be reliant on canonical rapid-acting group A1 [FeFe]-hydrogenases.

We used structural modelling, biochemical measurements and spectroscopy to confirm that group B [FeFe]-hydrogenases are active H2-producing enzymes (Fig. 4). AlphaFold2 modelling (Supplementary Figs. 3 and 4) predicts group B [FeFe]-hydrogenases are structurally conserved among Bacteroides species (Supplementary Fig. 5) and distinct from canonical group A1 [FeFe]-hydrogenases (Fig. 4a). As elaborated in Supplementary Note 2, they contain two distinct globular domains: an H-cluster domain (containing the typical catalytic H2-binding H-cluster of [FeFe]-hydrogenases and two [4Fe–4S] clusters) and an unusual smaller ferredoxin-like domain (containing two off-pathway [4Fe–4S] clusters) connected through a short flexible linker (Fig. 4b). We validated that the Bacteroides [FeFe]-hydrogenases could bind the catalytic H-cluster and produce H2 by heterologously expressing and semi-synthetically maturing them as previously described (Supplementary Note 3, Supplementary Table 2 and Fig. 7). The group B [FeFe]-hydrogenases expressed from three different species all produced H2 (Fig. 4c), whereas the catalytic subunit of the group A3 enzyme from B. thetaiotaomicron showed low activity in this set-up (Fig. 4c and Supplementary Table 2). Despite extensive effort, we were unable to purify stable or active group B [FeFe]-hydrogenases due to the low solubility of these enzymes. This has so far prevented detailed comparisons of their kinetics, electrochemistry or experimental structures compared with group A1 [FeFe]-hydrogenases. Nevertheless, we were able to show through whole-cell X-band electron paramagnetic resonance (EPR) spectroscopy that the B. fragilis group B [FeFe]-hydrogenase produced spectroscopic signatures consistent with a typical H-cluster74,75 (Supplementary Fig. 8, Supplementary Table 3 and Supplementary Note 3). In combination, the structural predictions and recombinant analysis suggest that group B [FeFe]-hydrogenases are true hydrogenases that bind the H-cluster and produce H2, although they differ from other hydrogenases in their redox centres and electron flow pathways.

Fig. 4: Metabolic integration, predicted structure and biochemical activity of the group B [FeFe]-hydrogenases from Bacteroides.

a, Structural superposition of group A [FeFe]-hydrogenase (Clostridium pasteurianum CpI; X-ray crystallography; PDB:6GM2) and group B [FeFe]-hydrogenase (B. fragilis; AlphaFold2). Structurally similar regions (root mean square deviation (RMSD) < 2 Å) are outlined in black, whereas divergent regions (RMSD > 2 Å) are semi-transparent. b, Top-ranked AlphaFold2 model of the B. fragilis group B [FeFe]-hydrogenase with modelled putative cofactors. The predicted H-cluster site with coordinating conserved residues is highlighted (green). Four Fe–S clusters (A1–A4) are predicted to coordinate with conserved cysteines throughout the protein. c, Average H2 production (gas chromatography) from lysates (n = 3) activated with [2Fe]adt. Results are shown for the group B [FeFe]-hydrogenases of B. fragilis, B. vulgatus and B. thetaiotaomicron (pink), and the group A3 [FeFe]-hydrogenase of B. thetaiotaomicron (blue). Activities were normalized for number of cells used (nmol H2 min−1 OD600−1) and error bars reflect s.d. from biological triplicates. All enzymes were expressed in E. coli BL21(DE3) cells. ‘Blank’ represents the same strain but contains an empty vector that was also added with [2Fe]adt (grey). d, Average transcription (TPM) of fermentation-associated genes, including group B and A3 [FeFe]-hydrogenases, across seven enteric Bacteroides isolates, in triplicate. e, Average B. fragilis H2 production (ppm) relative to growth (OD600) with (pink) and without (blue) hemin (n = 3). f, H2 production (log10 ppm) of a B. fragilis cell extract (CE)-only control (light blue), upon stimulation of the PFOR with CoA, and p