AbstractIn order to lower the costs for second generation bioethanol from lignocellulosic biomass anaerobic digestion of the effluent from ethanol fermentation was implemented using an upflow anaerobic sludge blanket (UASB) reactor system in a pilot‐scale biorefinery plant. Both thermophilic (53°C) and mesophilic (38°C) operation of the UASB reactor was investigated. At an OLR of 3.5 kg‐VS/(m3 day) a methane yield of 340 L/kg‐VS was achieved for thermophilic operation (53°C) while 270 L/kg‐VS was obtained under mesophilic conditions (38°C). For loading rates higher than 5 kg‐VS/(m3 day) the methane yields were, however, higher under mesophilic conditions compared to thermophilic conditions. The conversion of dissolved organic matter (VSdiss) was between 68% and 91%. The effluent from the ethanol fermentation showed no signs of toxicity to the anaerobic microorganisms. However, a high content of suspended matter reduced the degradation efficiency. The retention time of the anaerobic system could be reduced from 70 to 7 h by additional removal of suspended matter by clarification. Implementation of the biogas production from the fermentation effluent accounted for about 30% higher carbon utilization in the biorefinery compared to a system with only bioethanol production. Biotechnol. Bioeng. 2010;107: 59–64. © 2010 Wiley Periodicals, Inc.