Bio-hydrogen production from mixed culture fermentation (MCF) of glucose was studied by conducting a comprehensive product measurement and detailed mass balance analysis of their contributions to the final H-2 yield. The culture used in this study was enriched on glucose at 60 A degrees C through a sequential batch operation consisting of daily glucose feeds, headspace purging and medium replacement every third day in serum bottles for over 2 years. 2-Bromoethanesulfonate (BES) was only required during the first three 3-day cycles to permanently eliminate methanogenic activity. Daily glucose feeds were fully consumed within 24 h, with a persistent H-2 yield of 2.7 +/- A 0.1 mol H-2/mol glucose, even when H-2 was allowed to accumulate over the 3-day cycle. The measured H-2 production exceeded by 14 % the theoretical production of H-2 associated with the fermentation products, dominated by acetate and butyrate. Follow-up experiments using acetate with a C-13-labelled methyl group showed that the excess H-2 production was not due to acetate oxidation. Chemical formula analysis of the biomass showed a more reduced form of C5H11.8O2.1N1.1 suggesting that the biomass formation may even consume produced H-2 from fermentation.