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The potential for molecular hydrogen () generated via serpentinization to fuel subsurface microbial ecosystems independent from photosynthesis has prompted biogeochemical investigations of serpentinization‐influenced fluids. However, investigations typically sample via surface seeps or open‐borehole pumping, which can mix chemically distinct waters from different depths. Depth‐indiscriminate sampling methods could thus hinder understanding of the spatial controls on nutrient availability for microbial life. To resolve distinct groundwaters in a low‐temperature serpentinizing environment, we deployed packers (tools that seal against borehole walls during pumping) in two‐deep, peridotite‐hosted wells in the Samail Ophiolite, Oman. Isolation and pumping of discrete intervals as deep astobelow ground level revealed multiple aquifers that ranged in pH from 8 to 11. Chemical analyses and 16S rRNA gene sequencing of deep, highly reactedgroundwaters bearing up to,methane () andsulfate () revealed an ecosystem dominated by Bacteria affiliated with the class Thermodesulfovibrionia, a group of chemolithoheterotrophs supported byoxidation coupled toreduction. In shallower, oxidizedgroundwaters, aerobic and denitrifying heterotrophs were relatively more abundant. Highandof(up toand, respectively) indicated microbialoxidation, particularly inwaters with evidence of mixing withwaters. This study demonstrates the power of spatially resolving groundwaters to probe their distinct geochemical conditions and chemosynthetic communities. Such information will help improve predictions of where microbial activity in fractured rock ecosystems might occur, including beyond Earth.

In hyperalkaline () fluids that have participated in low‐temperature (<150) serpentinization reactions, the dominant form of C is often methane (), but the origin of thisis uncertain. To assessorigin 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 (D and) relative abundances less than equilibrium, consistent with substantial microbialproduction. Furthermore, we observed an inverse relationship between dissolved inorganic C concentrations andacross 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 ofis evidenced by the presence of‐bearing fluid inclusions in the Samail Ophiolite and our measurement of highvalues of ethane and propane, which are similar to those reported in studies of‐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 ofin the ophiolite may further enrichin¹³C. We conclude that substantial microbialis produced under varying degrees of C limitation and mixes with abioticreleased from fluid inclusions. This study lends insight into the functioning of microbial ecosystems supported by water/rock reactions.