Rate Limits and Isotopologue Fractionations for Microbial Methanogenesis Examined with Combined Pathway Protein Cost and Isotopologue Flow Network Models

Microbial methanogenesis produces a range of isotope (C-13/C-12 and D/H) and isotopologue ((CH3D)-C-13 and (CH2D2)-C-12) fractionations. Differential reversibility of enzymatic reactions qualitatively explains the isotope and isotopologue fractionations observed in both laboratory cultures and environmental samples. We applied pathway thermodynamics and isotopologue flow network models to quantitatively describe C-13/C-12, D/H, (CH3D)-C-13, and (CH2D2)-C-12 fractionations during hydrogenotrophic methanogenesis. The model consists of the 10 enzymatic reactions of the methanogenesis pathway and tracks mass balance of isotopologues by taking into account the reaction symmetries of singly-and doublydeuterated isotopologues. Based on the thermodynamics and enzyme kinetic data, the model estimates the reversibilities of 8 reactions from predicted in vivo concentrations of 17 metabolites and cofactors. The isotopologue flow network model calculates the isotopologue composition of product methane as well as all intermediates as a function of reversibilities and prescribed fractionation factors.& nbsp;The model explains a number of observations for laboratory culture experiments, including the range of C-13/C-12 fractionation up to 80%o between CH4 and CO2, with increasing magnitudes while decreasing pH(2). Relatively constant D/H fractionations of 300 +/- 40 parts per thousand & nbsp;between methane and water can be explained when methane is produced from three near-equilibrium H in methyl-coenzyme M with the addition of one kinetic D-depleted H during the last step of methanogenesis. Abundances of the doubly substituted isotopologues, (CH3D)-C-13 and (CH2D2)-C-12, reflect kinetic and equilibrium end-members with additional complications due to non-linear mixing and/or combinatorial effect.& nbsp;Our model can make predictions for isotopologue fractionations under slow rates of methanogenesis in energy-limiting deep sedimentary environments, where a large quantity of methane is produced. Near-equilibrium isotopologue ratios, often observed in marine sedimentary environments, are produced when pH(2) is less than 10 Pa. Our model results indicate that methanogenesis does not occur or only proceeds at extremely slow rates at this low pH(2) because low concentration of methyl-tetrahydromethanopterin limits the rate and thermodynamic feasibility of methanogenesis. Accordingly, it is proposed that near-equilibrium methane isotopologue signals in deep marine sediments are produced by the catalytic reversibility of methyl-coenzyme M reductase, likely from anaerobic methanotrophic archaea performing either anaerobic methane oxidation or net methanogenesis. The pathway thermodynamics and isotopologue flow network model scheme presented herein can be applied and expanded to predict isotopologue fractionations for a range of metabolisms beyond methanogenesis. (C)& nbsp;2022 Elsevier Ltd. All rights reserved.