Thermodynamic evaluation on H₂ production in glucose fermentation

The normal maximum H₂ yield in mesophilic biohydrogen (bioH ₂) fermentation is ∼2 mol of H₂/(mol of glucose). Thermodynamics could be the most fundamental control for bioH₂ formation, since proton reduction is strongly energy consuming (+79.4 kJ/(mol of H₂)). However, most of the electron equivalents in glucose do not accumulate in H₂ but in a range of organic acids and alcohols. Thus, evaluating the hypothesis of thermodynamic control requires the full stoichiometry of the fermentation. We carried out batch bioH₂ reactions with a range of pH values that yielded H₂ yields from 0 to ∼2 mol of H₂/(mol of glucose). We constructed complete electron equivalent (e^- equiv) balances for high or low H₂ yield by measuring all e^- sinks. The highest H₂ yield occurred with pH ∼4 and was coincident with major butyrate accumulation; ethanol or lactate correlated to reduced H₂ yields at pH 7 and 10, respectively. Although the Gibb's free energies for all overall reactions were similar (-10.6 to -11.2 kJ/(e^- equiv)), thermodynamics controlled the H ₂-producing reaction coupled to ferredoxin; this reaction was favorable at acidic pH but thermodynamically blocked at pH 10. Also, butyrate formation was the most thermodynamically favorable reaction that produced ATP after glycolysis.