Nonequilibrium clumped isotope signals in microbial methane

Methane is a key component in the global carbon cycle with a wide range of anthropogenic and natural sources. Although isotopic compositions of methane have traditionally aided source identification, the abundance of its multiply-substituted “clumped” isotopologues, e.g., ¹³CH₃D, has recently emerged as a proxy for determining methane-formation temperatures; however, the impact of biological processes on methane’s clumped isotopologue signature is poorly constrained. We show that methanogenesis proceeding at relatively high rates in cattle, surface environments, and laboratory cultures exerts kinetic control on ¹³CH₃D abundances and results in anomalously elevated formation temperature estimates. We demonstrate quantitatively that H₂ availability accounts for this effect. Clumped methane thermometry can therefore provide constraints on the generation of methane in diverse settings, including continental serpentinization sites and ancient, deep groundwaters.