Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail...

Serpentinization can generate highly reduced fluids replete with hydrogen (H₂) and methane (CH₄), potent reductants capable of driving microbial methanogenesis and methanotrophy, respectively. However, CH₄ in serpentinized waters is thought to be primarily abiogenic, raising key questions about the relative importance of methanogens and methanotrophs in the production and consumption of CH₄ in these systems. Herein, we apply molecular approaches to examine the functional capability and activity of microbial CH₄ cycling in serpentinization-impacted subsurface waters intersecting multiple rock and water types within the Samail Ophiolite of Oman. Abundant 16S rRNA genes and transcripts affiliated with the methanogenic genus Methanobacterium were recovered from the most alkaline (pH, >10), H₂- and CH₄-rich subsurface waters. Additionally, 16S rRNA genes and transcripts associated with the aerobic methanotrophic genus Methylococcus were detected in wells that spanned varied fluid geochemistry. Metagenomic sequencing yielded genes encoding homologs of proteins involved in the hydrogenotrophic pathway of microbial CH₄ production and in microbial CH₄ oxidation. Transcripts of several key genes encoding methanogenesis/methanotrophy enzymes were identified, predominantly in communities from the most hyperalkaline waters. These results indicate active methanogenic and methanotrophic populations in waters with hyperalkaline pH in the Samail Ophiolite, thereby supporting a role for biological CH₄ cycling in aquifers that undergo low-temperature serpentinization.IMPORTANCE Serpentinization of ultramafic rock can generate conditions favorable for microbial methane (CH₄) cycling, including the abiotic production of hydrogen (H₂) and possibly CH₄ Systems of low-temperature serpentinization are geobiological targets due to their potential to harbor microbial life and ubiquity throughout Earth's history. Biomass in fracture waters collected from the Samail Ophiolite of Oman, a system undergoing modern serpentinization, yielded DNA and RNA signatures indicative of active microbial methanogenesis and methanotrophy. Intriguingly, transcripts for proteins involved in methanogenesis were most abundant in the most highly reacted waters that have hyperalkaline pH and elevated concentrations of H₂ and CH₄ These findings suggest active biological methane cycling in serpentinite-hosted aquifers, even under extreme conditions of high pH and carbon limitation. These observations underscore the potential for microbial activity to influence the isotopic composition of CH₄ in these systems, which is information that could help in identifying biosignatures of microbial activity on other planets.