An electron-flow model can predict complex redox reactions in mixed-culture fermentative bioH2: microbial ecology evidence.

We developed the first model for predicting community structure in mixed-culture fermentative biohydrogen production using electron flows and NADH2 balances. A key assumption of the model is that H2 is produced only via the pyruvate decarboxylation-ferredoxin-hydrogenase pathway, which is commonly the case for fermentation by Clostridium and Ethanoligenens species. We experimentally tested the model using clone libraries to gauge community structures with mixed cultures in which we did not pre-select for specific bacterial groups, such as spore-formers. For experiments having final pHs 3.5 and 4.0, where H2 yield and soluble end-product distribution were distinctly different, we established stoichiometric reactions for each condition by using experimentally determined electron equivalent balances. The error in electron balancing was only 3% at final pH 3.5, in which butyrate and acetate were dominant organic products and the H2 yield was 2.1 mol H2/mol glucose. Clone-library analysis showed that clones affiliated with Clostridium sp. BL-22 and Clostridium sp. HPB-16 were dominant at final pH 3.5. For final pH 4.0, the H2 yield was 0.9 mol H2/mol glucose, ethanol, and acetate were the dominant organic products, and the electron balance error was 13%. The significant error indicates that a second pathway for H2 generation was active. The most abundant clones were affiliated with Klebsiella pneumoniae, which uses the formate-cleavage pathway for H2 production. Thus, the clone-library analyses confirmed that the model predictions for when the pyruvate decarboxylation-ferredoxin-hydrogenase pathway was (final pH 3.5) or was not (final pH 4.0) dominant. With the electron-flow model, we can easily assess the main mechanisms for H2 formation and the dominant H2-producing bacteria in mixed-culture fermentative bioH2.