Recognizing the pathways of microbial methanogenesis through methane isotopologues in the subsurface biosphere

Abstract Microbial methanogenesis is a significant component in the global carbon cycle, a driver of greenhouse warming from atmospheric methane, and contributes to natural gas resources. There are several metabolic pathways of methanogenesis, and it is challenging to discriminate between them and quantify their relative contributions in natural settings. Here we measure and compile four rare isotopologues of methane (13CH4, 12CH3D, 13CH3D and 12CH2D2) from the Qaidam Basin (China) and a large set of previous data across the world to identify distinctive fingerprints of two principal sources of subsurface microbial methane — hydrogenotrophic and methylotrophic methanogenesis — and use those fingerprints to deconvolve the budgets of microbial methane in the Qaidam Basin. Our data show that biogenic methanes in the Qaidam Basin have equilibrium Δ 18 / Δ 13 CH3D values respect to reservoir temperature without anaerobic methane oxidation. Our results suggest that methylotrophic methanogenesis produces methane with large deficits in 13CH3D and 12CH2D2 relative to that controlled by homogeneous equilibrium among methane isotopologues at ambient environmental temperatures, whereas hydrogenotrophic methanogenesis produces methane with 13CH3D abundance near equilibrium and relatively subtle 12CH2D2 deficits. We find a good linear correlation between the Δ 13 CH3D value of natural biogenic methane and independent estimates of the fraction of hydrogenotrophic (methylotrophic) methanogenesis, based on established interpretations of hydrogen isotope data. These findings are consistent with studies of laboratory cultures, which also show methylotrophic methane is more depleted in clumped isotopologues (13CH3D and 12CH2D2) than hydrogenotrophic methane, though both forms of cultured (hydrogenotrophic vs. methylotrophic) methane exhibit strong deficits in both isotopic species. The sensitivity of clumped isotopologues to methanogenesis pathways in natural settings provides a powerful tool for monitoring the activity of methanogenic microbial communities in the subsurface. Anomalies of both studied clumped isotopologues ( Δ 13 CH3D and Δ 12 CH2D2) decrease over time in wells that have been re-sampled repeatedly, suggesting that such measurements are capable of detecting and quantifying shifts in proportions of these two metabolic pathways over the timescales of gas production history.