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

Hydrogen (H2), produced during serpentinization (hydration and oxidation of ferromanganese minerals), is a crucial link between the geosphere and biosphere, potentially supporting early life. Serpentinization generates hyperalkaline, highly reduced waters with limited dissolved inorganic carbon (DIC), challenging autotrophs like methanogens. While hydrogenotrophic methanogens are considered ancient, their survival in DIC-limited serpentinizing environments is unclear. Previous studies noted isotopically heavy methane (δ13C-CH4) in these systems, suggesting methanogenesis under extreme DIC limitation, with cells potentially prioritizing carbon assimilation over energy production. This study uses metagenomics and single-cell genomics to investigate how autotrophic methanogens in the Samail Ophiolite overcome DIC limitation in hyperalkaline waters.

The literature review extensively cites previous research on serpentinization, hydrogen's role in early life, and the ecology of methanogens in extreme environments. It highlights the paradoxical conditions of abundant reductants (H2, formate) but scarce oxidants (DIC) in serpentinizing systems, which are known to be inhabited by methanogens belonging to the genus *Methanobacterium*. The literature establishes the isotopic signature of methane in these environments as a potential indicator of DIC limitation during methanogenesis, referencing studies that suggest both biotic and abiotic factors may contribute to this signature. Existing work points towards a near-complete consumption of available DIC by methanogens in hyperalkaline serpentinizing systems, emphasizing the need to understand the microbial adaptations to this limitation. This forms the basis for investigating the metabolic strategies of methanogens inhabiting these extreme environments.

Water samples were collected from several wells in the Samail Ophiolite, Oman, representing both circumneutral and hyperalkaline conditions. DNA was extracted, and shotgun metagenomic sequencing was performed. Metagenome assembled genomes (MAGs) were created using MetaBAT. Single-cell genomics (SCG) using fluorescence-activated cell sorting and subsequent sequencing were employed to further refine genomic analysis. Phylogenetic analyses using 103 single-copy phylogenetic marker genes were performed to determine the evolutionary relationships of the identified methanogens. Metabolic reconstructions were conducted using Prokka, KEGG, and BLASTp to identify key genes and pathways. Radiotracer experiments with isotopically labeled substrates (formate and bicarbonate) were performed to assess methane production under different conditions. Average nucleotide identity (ANI) calculations and phylogenetic analysis were also performed to assess genomic diversity and relationships between the identified methanogens.

Two distinct *Methanobacterium* lineages were identified: Type I in circumneutral pH waters and Type II in hyperalkaline waters. Type I possessed typical hydrogenotrophic methanogenesis genes, while Type II lacked key [NiFe]-hydrogenases. Type II instead utilized a novel pathway involving formate oxidation to generate reductant and cytoplasmic CO2. Radiotracer experiments confirmed that Type II produced methane from formate, but not bicarbonate, under conditions resembling its natural environment. Phylogenetic and genomic analyses suggested recent diversification of Type II through gene transfer, loss, and transposition, driven by adaptation to CO2 limitation. The high relative abundance of Type II in the hyperalkaline waters supports the hypothesis that the novel formate oxidation pathway is crucial for survival in these conditions. Analysis of genomic diversity within Type II indicates rapid evolutionary adaptation and diversification in response to selective pressure from the environment.

The findings demonstrate adaptation of methanogens to overcome severe oxidant limitation in hyperalkaline serpentinizing environments. The discovery of the novel formate oxidation pathway in Type II *Methanobacterium* provides a mechanistic explanation for their success in such extreme conditions. This adaptation highlights the evolutionary potential for metabolic innovation in response to environmental challenges. The study's results have implications for understanding the limits of microbial life and the potential for life in similar environments on early Earth or other planets. The rapid diversification within Type II emphasizes the dynamic interplay between evolutionary processes and environmental selection pressures in shaping microbial communities.

This study revealed a novel metabolic strategy employed by methanogens to overcome CO2 limitation in hyperalkaline serpentinizing environments. The adaptation of Type II *Methanobacterium* via a formate-oxidation pathway highlights the adaptability of life in extreme settings. Further research could investigate the prevalence of this pathway in other hyperalkaline environments and explore the potential implications for early life and extraterrestrial life.

The study focused on a single ophiolite site. Extrapolation to other serpentinizing systems requires further investigation. The specific environmental factors beyond CO2 limitation that contribute to the diversification of Type II *Methanobacterium* warrant further study. The resolution of the single-cell genomics data could be limited in certain aspects.