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 -3 to +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 similar to 3 mol ATP/mol glucose (except 0.8 g-glucose/L, which had similar to 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.