Alkylotrophic methanogenesis forms biogenic methane with δ13C values comparable to thermogenic gas

Methane, a primary component of natural gas, is mainly derived from biogenic and thermogenic origins. Biogenic methane exhibits lower δ13C-CH4 (<-55‰)(1, 2) than thermogenic methane. However, secondary microbial methane (SMM), an important biogenic gas formed through the methanogenic degradation of petroleum, was speculated to have δ13C-CH4 from -55‰ to -35‰ (3) overlapping with thermogenic. Conventional methanogenetic pathways cannot adequately explain this deviation, indicating alternative methanogenesis producing methane with less 13C depletion. Alkylotrophic methanogenesis differs from previous methanogenic metabolism and is widespread in oil reservoirs (4, 5). Therefore, we incubated the enrichment culture of alkylotrophic methanogens with the eicosane and several crude oils as substrates (δ13C-values from -35 to -27‰). The accumulated CH4 exhibited δ13C values of −48‰ to −41‰, CO2 from −7‰ to +14‰. It exhibited smaller carbon isotopic fractionation than other pathways, ranging from -10 to -17‰. Since about 1/3 of methane derives from CO2 reduction, the only likely process with substantial fractionation, the overall fractionation of alkylotrophic methanogenesis will be rather low. Its δ13C values depart from canonical methanogenesis and plots in the interface between thermogenic and SMM. Given the prevalence of alkylotrophic methanogenesis in subsurface reservoirs, biogenic methane in reservoirs has been previously underestimated. This oversight not only calls for re-evaluating methane inventories in reservoir systems but also implies that subsurface microbial processes could be a more critical component of the global carbon cycle than previously recognized.