UNDERSTANDING STABLE ISOTOPIC FRACTIONATION DURING MICROBIAL METHANOGENESIS

Microbial methane (CH₄) production (methanogenesis) occurs mainly by reducing CO₂ with H₂ (hydrogenotrophy) or by fermenting acetate. Isotopic fractionation during hydrogenotrophic methanogenesis, dominating methanogenic activity in marine sediments, depletes CH₄ of the heavier ¹³C and ²H isotopes. Furthermore, recent advances in analytical methods revealed that biological ¹³CH₃D isotopologue content may deviate significantly from its equilibrium composition. These isotopic signatures are highly variable within both natural and laboratory culture setups and are often used to assess CH₄ reservoirs origins and its formation temperature. It is believed the isotope fractionation in multistep enzymatic processes, such as methanogenesis, depend on the reversibility degree of enzymatically catalyzed reactions, and not only on the isotopic equilibrium effects, thus allowing variation to occur. The mechanistic reasons for this phenomenon are not entirely understood.

We developed a bio-isotopic model describing the metabolism and isotopic fractionation of stable carbon and hydrogen isotopes and clumped methane isotopologues in hydrogenotrophic methanogenesis. We solved the model using a set of kinetic and thermodynamic parameters under a steady state assumption, yielding the reversibility of each reaction in the pathway. We later predicted the net isotopic fractionation during methanogenesis using a novel set of equilibrium fractionation factors and assigned kinetic fractionation factors. We finally compared our results to previous culture studies of methanogens.

Our model can generate the isotopic fractionation trend related to the actual Gibbs free energy of the overall reaction (ΔG_r). We show that kinetic isotope effects (KIE) of the last enzymes in the pathway control isotopic fractionation when ΔG_r tends to zero, and KIE of the upstream enzymes control fractionation in highly negative ΔG_r. Thus our model provides a possible mechanism for the relation of isotope fractionation and ΔG_r of methanogenesis, further elucidating the factors governing the isotopic signature of CH₄ in the environment.