High carbon isotope values (δ13CCH4 > −40‰) have widely been used as evidence that methane in alkaline rock-hosted fluids was formed abiotically, particularly in serpentinizing systems. However, isotope fractionation during microbial methanogenesis is relatively understudied at high pH. We isolated a hydrogenotrophic Methanobacterium sp. from hyperalkaline subsurface fluids in the Samail ophiolite to assess how carbon and hydrogen isotope values of CH4 varied depending upon pH and carbonate mineral source (NaHCO3 or CaCO3). The hydrogen isotope fractionation αH20/CH4 (1.46–1.66) did not vary across pH. In contrast, the expressed carbon isotope fractionation, αCO2/CH4, ranged from 1.028 to 1.089. Carbon isotope fractionation increased with pH, reaching a maximum 13C depletion of −85‰. However, the 13C depletion significantly diminished at pH ≥ 9 for CaCO3-amended experiments, generating δ13CCH4 as high as −28‰. To evaluate the large variability in δ13CCH4, we developed a steady-state model to assess how the rates of carbonate dissolution, cellular uptake of CO2 and irreversible CH4 production can affect the net isotope fractionation during methanogenesis. Methanobacterium sp. can produce highly depleted δ13CCH4 in simulated alkaline serpentinizing fluids when dissolved inorganic carbon levels are high and methanogenesis rates are slow. However, small carbon isotope fractionation occurs when rates of carbonate dissolution are slower than cellular uptake, leading to relatively high δ13CCH4 values (>∼−35‰) that are traditionally interpreted to be purely “abiotic”. Thus, microbial CH4 can be produced in carbon-limited mafic and ultramafic rock-hosted environments on Earth and potentially other planetary bodies, but it may be difficult to isotopically identify biogenic methane when mineral carbonates are the dominant carbon source.

The Samail ophiolite in Oman is undergoing modern hydration and carbonation of peridotite and may host a deep subsurface biosphere. Previous investigations of hyperalkaline fluids in Oman have focused on fluids released at surface seeps, which quickly lose their reducing character and precipitate carbonates upon contact with the O2/CO2-rich atmosphere. In this work, geochemical analysis of rocks and fluids from the subsurface provides new insights into the operative reactions in serpentinizing aquifers. Serpentinite rock and hyperalkaline fluids (pH > 10), which exhibit millimolar concentrations of Ca2+, H2 and CH4, as well as variable sulfate and nitrate, were accessed from wells situated in mantle peridotite near Ibra and studied to investigate their aqueous geochemistry, gas concentrations, isotopic signatures, mineralogy, Fe speciation and microbial community composition.The bulk mineralogy of drill cuttings is dominated by olivine, pyroxene, brucite, serpentine and magnetite. At depth, Fe-bearing brucite is commonly intermixed with serpentine, whereas near the surface, olivine and brucite are lost and increased magnetite and serpentine is detected. Micro-Raman spectroscopy reveals at least two distinct generations of serpentine present in drill cuttings recovered from several depths from two wells. Fe K-edge X-ray absorption near-edge spectroscopy (XANES) analysis of the lizardite shows a strong tetrahedral Fe coordination, suggesting a mixture of both Fe(II) and Fe(III) in the serpentine. Magnetite veins are also closely associated with this second generation serpentine, and 2-10 μm magnetite grains overprint all minerals in the drill cuttings. Thus we propose that the dissolved H2 that accumulates in the subsurface hyperalkaline fluids was evolved through low temperature oxidation and hydration of relict olivine, as well as destabilization of pre-existing brucite present in the partially serpentinized dunites and harzburgites. In particular, we hypothesize that Fe-bearing brucite is currently reacting with dissolved silica in the aquifer fluids to generate late-stage magnetite, additional serpentine and dissolved H2. Dissolved CH4 in the fluids exhibits the most isotopically heavy carbon in CH4 reported in the literature thus far. The CH4 may have formed through abiotic reduction of dissolved CO2 or through biogenic pathways under extreme carbon limitation. The methane isotopic composition may have also been modified by significant methane oxidation. 16S rRNA sequencing of DNA recovered from filtered hyperalkaline well fluids reveals an abundance of Meiothermus, Thermodesulfovibrionaceae (sulfate-reducers) and Clostridia (fermenters). The fluids also contain candidate phyla OP1 and OD1, as well as Methanobacterium (methanogen) and Methylococcus sp. (methanotroph). The composition of these microbial communities suggests that low-temperature hydrogen and methane generation, coupled with the presence of electron acceptors such as nitrate and sulfate, sustains subsurface microbial life within the Oman ophiolite.

In hyperalkaline ((Formula presented.)) fluids that have participated in low-temperature (<150 (Formula presented.)) serpentinization reactions, the dominant form of C is often methane ((Formula presented.)), but the origin of this (Formula presented.) is uncertain. To assess (Formula presented.) 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 ((Formula presented.) D and (Formula presented.)) relative abundances less than equilibrium, consistent with substantial microbial (Formula presented.) production. Furthermore, we observed an inverse relationship between dissolved inorganic C concentrations and (Formula presented.) 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 (Formula presented.) is evidenced by the presence of (Formula presented.) -bearing fluid inclusions in the Samail Ophiolite and our measurement of high (Formula presented.) values of ethane and propane, which are similar to those reported in studies of (Formula presented.) -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 (Formula presented.) in the ophiolite may further enrich (Formula presented.) in 13C. We conclude that substantial microbial (Formula presented.) is produced under varying degrees of C limitation and mixes with abiotic (Formula presented.) released from fluid inclusions. This study lends insight into the functioning of microbial ecosystems supported by water/rock reactions.