Anaerobic dark fermentation biohydrogen production from xylose was investigated under mesophilic (35 °C) and thermophilic (55 °C) conditions at various initial pH (5.0–10.0) and substrate concentrations (2.5–12.5 g/L). In addition, the microbial community structure variations under different temperatures were analyzed. It was demonstrated that the maximum hydrogen yield (1.24 mol-H2/mol-xylose) was obtained with substrate concentration of 7.5 g/L and initial cultivation, pH 7.0, at 35 °C, with butyrate, acetate, and ethanol as the major byproducts. The increase of substrate concentration resulted in accumulation of volatile fatty acids (VFAs), especially propionate, and a decrease in final pH under mesophilic conditions. However, the hydrogen yield increased along with the increase of substrate concentration at 55 °C with butyrate and ethanol as the main metabolite. Stable pH of the system could be maintained even at high xylose concentration up to 12.5 g/L due to a low level of VFAs accumulation. A lower hydrogen yield of 1.14 mol-H2/mol-xylose was obtained at thermophilic condition, while a stable operation condition could be achieved and maintained more easily. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis showed that microbial community structures of both systems were dominated with bacterial species related to Clostridium, while the thermophilic system had fewer hydrogen-producing microbial species than that at mesothermal condition.
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