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The effect of pH on the conversion of glucose to hydrogen by a mixed culture of fermentative bacteria was evaluated. At 36 °C, six hours hydraulic retention, over 90% of glucose was degraded at pH ranging 4.0–7.0, producing biogas and an effluent comprising mostly fatty acids. At the optimal pH of 5.5, the biogas comprised 64±2% of hydrogen with a yield of 2.1±0.1 mol-H₂/mol-glucose and a specific production rate of 4.6±0.4 l-H₂/(g-VSS day). The effluent was composed of acetate (15.3–34.1%) and butyrate (31.2–45.6%), plus smaller quantities of other volatile fatty acids and alcohols. The diversity of microbial communities increased with pH, based on 16S rDNA analysis by denaturing gradient gel electrophoresis (DGGE).