We developed the first model for predicting community structure in mixed-culture fermentative biohydrogen production using electron flows and NADH(2) balances. A key assumption of the model is that H-2 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 H-2 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 H-2 yield was 2.1 mol H-2/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 H-2 yield was 0.9 H-2/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 H-2 generation was active. The most abundant clones were affiliated with Klebsiella pneumoniae, which uses the formate-cleavage pathway for H-2 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 H-2 formation and the dominant H-2-producing bacteria in mixed-culture fermentative bioH,. Biotechnol. Bioeng. 2009;104: 687-697. (C) 2009 Wiley Periodicals, Inc.