Fig. 3
Phylum-level overall metabolic activities, the thermodynamics-based H2 thresholds of the activities, and expression of individual pathways. a For each phylum, the number of species clusters, average number of protease, glycosyl hydrolase, and lipase families expressed across species are shown (normalized to maximum observed average among phyla). Likewise, the average number of sugar-, AA-, and FA-degradation pathways expressed across species is shown. AA degradation pathways are split into those that are H2-tolerant (HT) and H2-sensitive (HS) based on panel b. b The maximum H2 concentration that each degradation pathway can tolerate is shown (i.e., ∆Greaction + x*∆GATPsynthesis = 0, where x is the amount of ATP synthesized per substrate degraded). The ATP yield for each pathway was based on the sum of (i) the ATP consumption/generation in the main carbon transformation pathway and (ii) vectorial H+ translocation associated with membrane-based electron transfer (e.g., Rnf, Hyb, Fdn), assuming the shortest electron flow route from substrate oxidation to H2/formate generation that involves electron bifurcation and reverse electron transport where possible; all of this was based on pathways that were observed to be expressed in this study. Reactions that would either lose much energy as heat (e.g., cytosolic Fdred-oxidizing H2 generation) or require energy input under in situ conditions (e.g., cytosolic NADH-oxidizing H2 generation) were not considered. For substrates whose degradation proceeds through pyruvate or acetyl-CoA, maximum H2 concentrations for oxidation to acetate are shown (see Supplementary Table S1 for a list of reactions). Note that fermentation pathways (e.g., acetyl-CoA reduction to butyrate) would increase the maximum H2 but reduce ATP yield. The Gibbs free energy yield at standard conditions and pH 7 (∆G°’) and estimated ATP yields are also shown. See Fig. 1 for details for calculating ATP yield and maximum tolerable H2 concentration. For each pathway, ∆Greaction was calculated assuming 300 μM acetate, 10 μM for other FAs, 1 mM NH4+, 50 kPa CH4, 50 mM HCO3-, 37 °C, 3.9 × 10-4 atm H2S, and 0.1 μM for all other compounds. ∆GATPsynthesis was assumed to be 60 kJ/mol. *Although more exergonic alternative pathways exist for these HS AA degradation pathways (e.g., through butyrate fermentation), species only expressing the HS pathway(s) were identified in situ, indicating that HS metabolism of these AAs is relevant in situ. 1For isovalerate degradation, an ATP synthase ATP:H+ ratio of 1:4 was assumed. 2For H2-oxidizing CO2-reducing methanogenesis, two H2 concentrations for two ATP yields assuming different ATP synthase ATP:H+ ratios. 3For propionate and acetate degradation, an ATP synthase ATP:H+ ratio of 1:5 was assumed. †Pathways whose directionality cannot be determined by sequence data alone. c For each phylum, the percentage of species expressing individual degradation pathways are shown