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

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 Δ₁₈/Δ¹³CH₃D 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 Δ¹³CH₃D 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 (Δ¹³CH₃D and Δ¹²CH₂D₂) 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.