The biological sludge from an animal wastewater treatment plant was treated to enrich hydrogen-producing mixed bacteria, and effects on hydrogen yield were investigated during anaerobic fermentation at 55 degrees C. Enrichment of hydrogen-producing bacteria was conducted at pH adjustment of inocula to 3 and 5 with and without additional heat treatment (NHT and HT). The enriched mixed bacteria were cultivated at initial pHs of 5, 6, and 7 with synthetic organic wastewater containing different levels of nitrogen (2.0 and 0.8 g/l as total nitrogen) under static batch conditions. The main effects of heat treatment and enrichment pH were significant on hydrogen production. There was no significant effect of different nitrogen concentrations on hydrogen production. The methane-free biogas contained hydrogen levels of up to 64% for a fermentative condition that showed maximum hydrogen evolution (at culture pH 5 after enrichment at pH 5 with HT). The dominating intermediate metabolites were acetate, n-butyrate, and ethanol. Yields of produced hydrogen were significantly dependent upon levels of n-butyrate.