AbstractSerpentinization involves the hydration and alteration of ultramafic rocks, which produces hydrogen (H2) and methane (CH4) and results in distinctive groundwater chemistries. As reacted fluids mix with recharging surface water, gradients in chemistry and microbiology develop in the subsurface. We present a comprehensive analysis of biogeochemical gradients in the water column of a serpentinite‐hosted well, CSW1.1, at the Coast Range Ophiolite Microbial Observatory (CROMO) in California, USA. Samples for geochemistry, 16S rRNA gene sequencing, and metagenomics were collected at four discrete depths from the top of the well corresponding to 100%, 50%, 15%, and 0% of atmospheric oxygen (O2) levels, and from the well base at 19.5 m depth. Gibbs energy calculations assessed the energy available for a suite of reactions coupled to O2, sulfate (SO42−), and nitrate (NO3−). Metagenomic data from the profile was used to construct metagenome assembled genomes (MAGs) to evaluate the completeness of biochemical pathways and compare the relative abundance of key diagnostic genes. Bioenergetic data point to the favorability of CH4 oxidation reactions despite little genetic evidence for this. Amplicon sequencing results highlight the abundance of key taxa affiliated with the genera Truepera, Serpentinomonas, and Dethiobacter. Although concentrations of NO3− and H2 are low, genes for NO3− reduction and oxidation of H2 and carbon monoxide (CO) were found in high abundance. Conceptual modeling results demonstrate the net depletion of H2 and CO in the groundwater, the consumption of CO2 and O2, and the potential for CH4 emission into the atmosphere at this terrestrial site of serpentinization.
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