Fractionation of the methane isotopologues 13CH4, 12CH3D, and 13CH3D during aerobic oxidation of methane by Methylococcus capsulatus (Bath)

Aerobic oxidation of methane plays a major role in reducing the amount of methane emitted to the atmosphere from freshwater and marine settings. We cultured an aerobic methanotroph, Methylococcus capsulatus (Bath) at 30 and 37 degrees C, and determined the relative abundance of (CH4)-C-12, (CH4,)-C-13 (CH3D)-C-12, and (CH3D)-C-13 (a doubly-substituted, or clumped isotopologue of methane) to characterize the clumped isotopologue effect associated with aerobic methane oxidation. In batch culture, the residual methane became enriched in C-13 and D relative to starting methane, with D/H fractionation a factor of 9.14 ((D)epsilon/(13)epsilon) larger than that of C-13/C-12. As oxidation progressed, the Delta(CH3D)-C-13 value (a measure of the excess in abundance of (CH3D)-C-13 relative to a random distribution of isotopes among isotopologues) of residual methane decreased. The isotopologue fractionation factor for (CH3D)-C-13/(CH4)-C-12 was found to closely approximate the product of the measured fractionation factors for (CH4)-C-13/(CH4)-C-12 and (CH3D)-C-12/(CH4)-C-12 (i.e., C-13/C-12 and D/H). The results give insight into enzymatic reversibility in the aerobic methane oxidation pathway. Based on the experimental data, a mathematical model was developed to predict isotopologue signatures expected for methane in the environment that has been partially-oxidized by aerobic methanotrophy. Measurement of methane clumped isotopologue abundances can be used to distinguish between aerobic methane oxidation and alternative methane-cycling processes. (C) 2016 Elsevier Ltd. All rights reserved.