A batch test and continuous operation were performed to identify the effect of lactate on hydrogen production at pH 4.5. When the initial lactic acid concentration was increased from 0 to 8 g/L in the batch test, the hydrogen yield also increased from 1.41 to 1.72 mol-H(2)/mol-glucose. The system exhibited a long lag time and an insignificant hydrogen yield with 16 g-lactic acid/L A continuous stirred tank reactor (CSTR) was operated at different organic loading rates (OLRs: 10, 15,20 and 40 g/L/day) and hydraulic retention times (HRTs: 6, 12 and 24 h). At an OLR of 20 g-glucose/L/day and 12 h of HRT, the hydrogen yield was 1.2 mol-H(2)/mol-glucose. The yield decreased with a 24 h HRT. Even though lactate was one of the major constituents of volatile fatty acids (VFAs), hydrogen production was feasible throughout the operation. Clostridium sp. was the dominant hydrogen-producing bacteria in the system. (C) 2011 Elsevier Ltd. All rights reserved.