Diversification of methanogens into hyperalkaline serpentinizing environments through adaptations to minimize oxidant limitation

Serpentinization of ultramafic rocks generates abundant molecular hydrogen (H₂) that can abiotically reduce dissolved inorganic carbon (DIC) to reduced carbon species (CO, formate, CH₄), creating energy-rich but highly reduced, hyperalkaline waters. While such environments can support autotrophic metabolisms like hydrogenotrophic methanogenesis, they are typically DIC- and oxidant-limited, posing a challenge to organisms that use CO₂ as both carbon source and terminal electron acceptor. Methanobacterium spp. are consistently detected in serpentinizing systems, yet how they persist under extreme DIC limitation remains unclear. The study investigates whether and how hydrogenotrophic methanogens adapt to oxidant (CO₂) limitation in hyperalkaline, H₂-rich waters of the Samail Ophiolite (Oman) by combining metagenomics, single-cell genomics, phylogenomics, and radiotracer microcosm assays. The central hypothesis is that lineages inhabiting hyperalkaline waters evolved metabolic strategies to minimize oxidant limitation, potentially by leveraging formate to generate intracellular CO₂ and reductant for methanogenesis. Understanding these adaptations informs models of subsurface life in serpentinizing crust today and offers insights into potential metabolisms on early Earth.

Study site and sampling: Subsurface fracture waters were sampled from previously drilled wells in the Samail Ophiolite (Oman). In 2017, waters were collected from seven wells, including WAB188 (circumneutral; sampled at 78 m) and NSHQ14 (hyperalkaline; sampled at 85 m [NSHQ14C] and another depth [NSHQ14B]). In 2020, waters for methanogenic activity assays were collected from WAB188 (50 m) and from NSHQ14 using a double packer to isolate 9–30 m waters (insufficient flow prevented isolation of deeper waters). Approximately 100 L were purged prior to sampling. Biomass for DNA work was collected on 0.2 μm filters, immediately flash-frozen with cryoprotectants (5% glycerol, 1× TE), and stored at −80 °C. Field measurements included temperature and pH; geochemical data (e.g., H₂, DIC, formate, CH₄) were compiled from 2015–2017 campaigns where available. Geochemistry (selected): WAB188: pH 7.5–7.6; DIC ~3.0 mM (2017); H₂ 0–2.1 μM; CH₄ 1.69 μM (2017). NSHQ14: pH 11.1–11.3; DIC 0.05–0.13 mM; H₂ up to 164 μM (2017); CH₄ 12.6–34.6 μM. DNA extraction and sequencing: DNA was extracted with the Zymo Xpedition Soil/Fecal kit. Shotgun metagenomic libraries (Nextera XT) were sequenced on Illumina HiSeq 2500 (2 × 250 bp). Assembly and binning: Reads were quality-filtered/trimmed and assembled as previously described. Contigs were binned into MAGs with MetaBAT (v0.26.3) using tetranucleotide frequencies and coverage. Contigs with ≥98% nucleotide identity to a WAB188 Methanobacterium MAG were classified as Type I; those with ≥98% identity to an NSHQ14C Methanobacterium MAG were classified as Type II. Read recruitment to Type I/II MAGs was used to estimate relative abundances. Single-cell genomics (SAGs): Cells were sorted by FACS, processed, and sequenced at Bigelow Laboratory Single Cell Genomics Center. MAGs/SAGs are under BioProject PRJNA631088. Phylogenomics: Genomes of Methanobacteria (study MAGs and references) were analyzed using a concatenation of 103 single-copy phylogenetic marker genes (identified via Amphora2), aligned with Clustal Omega, and reconstructed with IQ-TREE (LG+F+R10; model selected by ModelFinder). Metabolic reconstruction: Gene prediction/annotation used Prokka and KEGG (KAAS), with targeted BLASTp searches (E < 1e−6; >30% aa identity; >60% query length) to confirm presence/absence of key enzymes (e.g., hydrogenases, dehydrogenases, transporters). Missing functions were queried in full metagenome assemblies and single-enrichment datasets; contigs were assigned to MAGs based on coverage/GC content. Hydrogenase classification: Putative [NiFe]-hydrogenase catalytic subunits were identified by BLASTp, aligned, evaluated for conserved CxxC motifs, placed phylogenetically, and functionally inferred using L1/L2 motif conservation and gene-neighborhood analyses (e.g., F420-binding partners). Comparative genomics (MAGs vs SAGs): Average nucleotide identity (ANI) among genomes was computed (MUSCLE script). Protein ortholog identities were assessed relative to the NSHQ14C Type II MAG. Transposase orthologs were classified (ACLAME), aligned (Clustal Omega), and analyzed phylogenetically (PhyML). Synteny around transposases was examined with progressiveMauve and Gene Graphics. Mantel tests evaluated correlations between divergence in orthologs and whole-genome ANI (vegan in R). Microcosm radiotracer assays: N₂-purged, sterile serum vials were filled directly at the manifold for methanogenic activity assays (2020). Radiotracer amendments tested biological CH₄ production from formate vs bicarbonate, with incubation and detection to assess substrate utilization linked to methanogenesis in waters where Type II was most abundant.

- Two distinct Methanobacterium lineages (Type I and Type II) were recovered from Oman subsurface waters; Type I dominated circumneutral waters (e.g., WAB188, pH ~7.5–7.6; DIC ~3.0 mM), while Type II dominated H₂-rich, hyperalkaline waters (e.g., NSHQ14, pH ~11.1–11.3; H₂ up to 164 μM; DIC 0.05–0.13 mM). - Type I genomes encoded canonical hydrogenotrophic methanogenesis coupling H₂ oxidation to CO₂ reduction, including key oxidative [NiFe]-hydrogenases. - Type II, branching from Type I, lacked homologs of two key oxidative [NiFe]-hydrogenases, indicating a divergent strategy in high-pH, oxidant-limited waters. - Genomic reconstructions indicate Type II repurposed/former dehydrogenases to oxidize formate, generating intracellular reductant and cytoplasmic CO₂, a unique pathway among characterized Methanobacteria that mitigates CO₂ (oxidant) limitation. - Microcosm radiotracer assays showed significant biological methane production from formate, but not from bicarbonate, in waters where Type II was most abundant, empirically supporting the predicted metabolic adaptation. - Phylogenomic and comparative analyses revealed signatures of recent and ongoing diversification of Type II via gene transfer, gene loss, and transposition, with variability in gene content (notably transposases) across SAGs. - Geochemical context supports selective pressures: hyperalkaline waters were highly reduced (elevated H₂) and DIC-limited, conditions that would favor strategies generating cytoplasmic CO₂ from formate over reliance on scarce extracellular DIC.

The study addressed how hydrogenotrophic methanogens persist in serpentinization-driven hyperalkaline waters where DIC (CO₂) is scarce and serves as both carbon source and electron acceptor. The discovery that the Type II Methanobacterium lineage lacks key oxidative [NiFe]-hydrogenases yet retains robust methanogenic capacity via formate oxidation indicates an adaptive response to oxidant limitation: by oxidizing formate, cells produce intracellular reductant and cytoplasmic CO₂, sidestepping constraints of low external DIC and high pH. Radiotracer evidence (methane generation from formate but not bicarbonate) directly supports this metabolic shift in situ. Phylogenomic signals of recent diversification mediated by horizontal transfer, gene loss, and transposition suggest rapid evolutionary responses to the redox and pH extremes imposed by serpentinization. These findings reconcile repeated observations of Methanobacterium persistence in DIC-poor, H₂-rich fluids and imply that oxidant availability (CO₂) is a primary selective driver shaping methanogen metabolic repertoires in such environments. The work has implications for early Earth habitability, where similar geochemical regimes may have favored methanogens capable of generating cytoplasmic CO₂ from reduced carbon intermediates like formate.

This work identifies two Methanobacterium lineages partitioned by geochemical niche in the Samail Ophiolite and demonstrates that the hyperalkaline-associated Type II lineage has diversified from a Type I ancestor by losing key oxidative [NiFe]-hydrogenases and adapting to oxidant (CO₂) limitation through a unique, formate-oxidizing pathway that supplies intracellular CO₂ and reductant for methanogenesis. Radiotracer microcosms corroborate this adaptation, and comparative genomics implicates gene transfer, loss, and transposition in ongoing diversification. These findings advance understanding of how methanogens exploit serpentinization-derived reductants under DIC limitation and inform models of subsurface and early Earth ecosystems. Future research should: (i) isolate Type II representatives to experimentally validate predicted pathways and enzyme directionality; (ii) quantify in situ fluxes of formate, CO₂, and CH₄ across pH gradients; (iii) resolve regulatory controls and transport systems for formate/CO₂ handling; and (iv) assess the prevalence of similar adaptations across global serpentinizing systems.

- Affiliations between specific authors and institutions are not fully enumerated in the provided excerpt. Methodologically, several constraints may affect interpretation: (i) some 2020 geochemical measurements were unavailable, limiting direct contemporaneous geochemistry–activity linkages; (ii) attempts to isolate deep waters at NSHQ14 in 2020 were unsuccessful, so activity assays represent a 9–30 m interval rather than deeper horizons; (iii) metabolic inferences rely on MAGs/SAGs that may be incomplete, potentially obscuring rare genes; (iv) microcosm assays, while supportive, may not fully replicate in situ conditions and kinetics.