Evaluation of metabolism using stoichiometry in fermentative biohydrogen

We first constructed full stoichiometry, including cell synthesis, for glucose mixed-acid fermentation at different initial substrate concentrations (0.8-6 g-glucose/L) and pH conditions (final pH 4.0-8.6), based on experimentally determined electron-equivalent balances. The fermentative bioH ₂ reactions had good electron closure (-9.8 to +12.7% for variations in glucose concentration and -3to +2% for variations in pH), and C, H, and O errors were below 1%. From the stoichiometry, we computed the ATP yield based on known fermentation pathways. Glucose-variation tests (final pH 4.2-5.1) gave a consistent fermentation pattern of acetate + butyrate + large H ₂, while pH significantly shifted the catabolic pattern: acetate + butyrate + large H ₂ at final pH 4.0, acetate + ethanol + modest H ₂ at final pH 6.8, and acetate + lactate + trivial H ₂ at final pH 8.6. When lactate or propionate was a dominant soluble end product, the H ₂ yield was very low, which is in agreement with the theory that reduced ferredoxin (Fd _red) formation is required for proton reduction to H ₂. Also consistent with this hypothesis is that high H ₂ production correlated with a high ratio of butyrate to acetate. Biomass was not a dominant sink for electron equivalents in H ₂ formation, but became significant (12%) for the lowest glucose concentration (i.e., the most oligotrophic condition). The fermenting bacteria conserved energy similarly at ∼3 mol ATP/mol glucose (except 0.8 g-glucose/L, which had ∼3.5 mol ATP/mol glucose) over a wide range of H ₂ production. The observed biomass yield did not correlate with ATP conservation; low observed biomass yields probably were caused by accelerated rates of decay or production of soluble microbial products.