Microbial abundance and diversity in deep subsurface environments is dependent upon the availability of energy and carbon. However, supplies of oxidants and reductants capable of sustaining life within mafic and ultramafic continental aquifers undergoing low-temperature water-rock reaction are relatively unknown. We conducted an extensive analysis of the geochemistry and microbial communities recovered from fluids sampled from boreholes hosted in peridotite and gabbro in the Tayin block of the Samail Ophiolite in the Sultanate of Oman. The geochemical compositions of subsurface fluids in the ophiolite are highly variable, reflecting differences in host rock composition and the extent of fluid-rock interaction. Principal component analysis of fluid geochemistry and geologic context indicate the presence of at least four fluid types in the Samail Ophiolite (“gabbro,” “alkaline peridotite,” “hyperalkaline peridotite,” and “gabbro/peridotite contact”) that vary strongly in pH and the concentrations of H2, CH4, Ca2+, Mg2+, NO3-, SO42-, trace metals, and DIC. Geochemistry of fluids is strongly correlated with microbial community composition; similar microbial assemblages group according to fluid type. Hyperalkaline fluids exhibit low diversity and are dominated by taxa related to the Deinococcus-Thermus genus Meiothermus, candidate phyla OP1, and the family Thermodesulfovibrionaceae. Gabbro- and alkaline peridotite- aquifers harbor more diverse communities and contain abundant microbial taxa affiliated with Nitrospira, Nitrosospharaceae, OP3, Parvarcheota, and OP1 order Acetothermales. Wells that sit at the contact between gabbro and peridotite host microbial communities distinct from all other fluid types, with an enrichment in betaproteobacterial taxa. Together the taxonomic information and geochemical data suggest that several metabolisms may be operative in subsurface fluids, including methanogenesis, acetogenesis, and fermentation, as well as the oxidation of methane, hydrogen and small molecular weight organic acids utilizing nitrate and sulfate as electron acceptors. Dynamic nitrogen cycling may be especially prevalent in gabbro and alkaline peridotite fluids. These data suggest water-rock reaction, as controlled by lithology and hydrogeology, constrains the distribution of life in terrestrial ophiolites.

Hydration of ultramafic rock during the geologic process of serpentinization can generate reduced substrates that microorganisms may use to fuel their carbon and energy metabolisms. However, serpentinizing environments also place multiple constraints on microbial life by generating highly reduced hyperalkaline waters that are limited in dissolved inorganic carbon. To better understand how microbial life persists under these conditions, we performed geochemical measurements on waters from a serpentinizing environment and subjected planktonic microbial cells to metagenomic and physiological analyses. Metabolic potential inferred from metagenomes correlated with fluid type, and genes involved in anaerobic metabolisms were enriched in hyperalkaline waters. The abundance of planktonic cells and their rates of utilization of select single-carbon compounds were lower in hyperalkaline waters than alkaline waters. However, the ratios of substrate assimilation to dissimilation were higher in hyperalkaline waters than alkaline waters, which may represent adaptation to minimize energetic and physiologic stress imposed by highly reducing, carbon-limited conditions. Consistent with this hypothesis, estimated genome sizes and average oxidation states of carbon in inferred proteomes were lower in hyperalkaline waters than in alkaline waters. These data suggest that microorganisms inhabiting serpentinized waters exhibit a unique suite of physiological adaptations that allow for their persistence under these polyextremophilic conditions.

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 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, information that could help in identifying biosignatures of microbial activity on other planets.

In hyperalkaline (normalpH>0.25em10) fluids that have participated in low‐temperature (<1500.17em°normalC) serpentinization reactions, the dominant form of C is often methane (CH4), but the origin of this CH4 is uncertain. To assess CH4 origin in serpentinite aquifers within the Samail Ophiolite, Oman, we determined fluid chemical compositions, analyzed taxonomic profiles of fluid‐hosted microbial communities, and measured isotopic compositions of hydrocarbon gases. We found that 16S rRNA gene sequences affiliated with methanogens were widespread in the aquifer. We measured clumped isotopologue (CH313D and CH212D2) relative abundances less than equilibrium, consistent with substantial microbial CH4 production. Furthermore, we observed an inverse relationship between dissolved inorganic C concentrations and δ13CnormalCH4 across fluids bearing microbiological evidence of methanogenic activity, suggesting that the apparent C isotope effect of microbial methanogenesis is modulated by C availability. An additional source of CH4 is evidenced by the presence of CH4‐bearing fluid inclusions in the Samail Ophiolite and our measurement of high δ13normalC values of ethane and propane, which are similar to those reported in studies of CH4‐rich inclusions in rocks from the oceanic lithosphere. In addition, we observed 16S rRNA gene sequences affiliated with aerobic methanotrophs and, in lower abundance, anaerobic methanotrophs, indicating that microbial consumption of CH4 in the ophiolite may further enrich CH4 in 13C. We conclude that substantial microbial CH4 is produced under varying degrees of C limitation and mixes with abiotic CH4 released from fluid inclusions. This study lends insight into the functioning of microbial ecosystems supported by water/rock reactions.