Large carbon isotope variability during methanogenesis under alkaline conditions

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.