Low Δ<SUP>12</SUP>CH₂D₂ values in microbialgenic methane result from combinatorial isotope effects

Methane generated by microorganisms is most often depleted in the doubly substituted isotopologue (CH2D2)-C-12 relative to the stochastic reference distribution. To constrain the controls on depleted Delta(CH2D2)-C-12 values, we experimentally isolated the root cause with microorganisms that produce methane from methylphosphonate via the C-P lyase pathway. This mechanism of methane production preserves the three hydrogens from methylphosphonate and adds one hydrogen from water. When maintaining the same methylphosphonate source, but varying the D/H composition of growth medium water, we observed significant shifts in methane Delta(CH2D2)-C-12 values, but little to no change in Delta(CH3D)-C-13 values. We reproduced these observations with a model that considers only the combinatorial isotope effect. The variation in Delta(CH2D2)-C-12 values of product methane resulted from the differences in D/H between reactants water and methylphosphonate. This work validates the hypothesis that combinatorial effects can strongly influence methane Delta(CH2D2)-C-12 values, and must be considered for low temperature, abiotic or biotic systems where methane hydrogen is derived from multiple reservoirs. (C) 2020 Published by Elsevier Ltd.