Clumped isotope evidence for microbial alteration of thermogenic methane in terrestrial mud volcanoes

Methane in oil reservoirs originates mostly from thermogenic sources, yet secondary microbial methane production from petroleum biodegradation is known to be pervasive. The conventional approach for identifying this secondary microbial methane commonly relies on geochemical characteristics of other gas molecules such as the carbon isotopic composition of carbon dioxide and propane. This information is sometimes obscured by isotopic variations in source material and may not be available in certain geological reservoirs. To better constrain the presence of secondary microbial methane, we studied the clumped isotopologue compositions of methane in terrestrial Azerbaijanian mud volcanoes, which support the occurrence of secondary microbial gas. Here, a deficit in Δ12CH2D2 of thermogenic methane occurs due to different δD of hydrogen sources that contribute to the formation of methane molecules (i.e., combinatorial effect). The Δ12CH2D2 is expected to move toward equilibrium as thermal maturity increases. More importantly, both Δ13CH3D and Δ12CH2D2 values of methane approach low-temperature thermodynamic equilibrium in most gases, suggesting that the original thermogenic methane has been altered by newly formed microbial methane in addition to isotope exchange among methane molecules catalyzed by the methyl-coenzyme M reductase enzyme. We conclude that methane clumped isotopes provide a unique proxy for identifying secondary microbial methane and understanding the exact evolution stages for natural gases.