Optimal microbial pathway variants can be determined by large-scale bioenergetic evaluation in syntrophic propionate oxidation

The complete understanding of microbial propionate oxidation in syntrophy with hydrogenotrophic methanogenesis remains elusive due to uncertainties in pathways and mechanisms for interspecies electron transfer (IET). Possible pathway variants differ in their intermediate metabolites, on which electron carriers are involved and in which steps are coupled to (and to how many) proton translocations. In this work, a systematic methodology was developed (based on sound biochemical, physiological and bioenergetic principles) to evaluate the feasibility and net ATP yield of large sets of pathway variants under different physiological and environmental conditions. A pathway variant is deemed feasible under given conditions only if all pathway reaction steps have non-positive Gibbs energy change and if all the metabolite concentrations remain within an acceptable physiological range (10 −6 to 10 −2 M). Several million combinations of pathway variants and parameters/conditions were evaluated for propionate oxidation, providing an unprecedented mechanistic insight into its biochemical and bioenergetic landscape. Propionate oxidation via lactate appeared as the most ATP yielding pathway under most of the conditions evaluated. Results under typical methanogenic conditions indicate that syntrophic propionate oxidation can sustain life only at hydrogen partial pressures within the range of 1.2 to 4 Pa. These extremely low concentrations constitute a kinetic impossibility and strongly suggest for IET mechanisms other than dissolved hydrogen. Importance In this work an original methodology was developed that quantifies the bioenergetically and physiologically feasible net ATP yields for large numbers of microbial metabolic pathways and their variants under different conditions. This ensures global optimality in finding the pathway variant(s) leading to the highest ATP yield. The methodology is especially relevant to hypothesise which microbial pathway variants are most likely to prevail in microbial ecosystems under high selective pressure for efficient metabolic energy conservation. Syntrophic microbial oxidation of propionate to acetate has extremely low energy available and requires very high metabolic efficiency in order to sustain life. Our results bring mechanistic insights into the optimum pathway variants and the impact of environmental conditions on the ATP yields and other metabolic bottlenecks. Additionally, our results conclude that IET mechanisms other than hydrogen must exist to simultaneously sustain the growth of both propionate oxidisers and hydrogenotrophic methanogens.