Low Δ12CH2D2 values in microbialgenic methane result from combinatorial isotope effects

Methane generated by microorganisms is most often depleted in the doubly substituted isotopologue 12CH2D2 relative to the stochastic reference distribution. To constrain the controls on depleted Δ12CH2D2 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 Δ12CH2D2 values, but little to no change in Δ13CH3D values. We reproduced these observations with a model that considers only the combinatorial isotope effect. The variation in Δ12CH2D2 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 Δ12CH2D2 values, and must be considered for low temperature, abiotic or biotic systems where methane hydrogen is derived from multiple reservoirs.