A two-stage hydrogen/methane fermentation process has emerged as a feasible engineering system to recover bio-energy from wastewater. Hydrogen-producing bacteria (HPB) generate hydrogen from readily available carbohydrates, and organic acids produced during the hydrogen fermentation step can be degraded to generate methane in the following step. Three strong acids, HCl, H2SO4, and HNO3, were tested to determine the appropriate pre-treatment method for enhanced hydrogen production. The hydrogen production rates of 230, 290, and 20 L/kg-glucose/day was observed for the sludge treated with HCl, H2SO4, and HNO3, respectively, indicating that the acid pre-treatment using either HCl or H2SO4 resulted in a significant increase in hydrogen production. The fluorescent in situ hybridization method indicated that the acid pre-treatment selectively enriched HPB including Clostridium sp. of cluster I from inoculum sludge. After hydrogen fermentation was terminated, the sludge was introduced to a methane fermentation reactor. This experiment showed methane production rates of 100, 30, and 13 L/kg-glucose/day for the sludge pre-treated with HCl, H2SO4, and HNO3, respectively, implying that both sulfate and nitrate inhibited the activity of methane-producing bacteria. Consequently, the acid pre-treatment might be a feasible option to enhance biogas recovery in the two-stage fermentation process, and HCl was selected as the optimal strong acid for the enrichment of HPB and the continuous production of methane.

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