The start-up period for fermentative hydrogen production was investigated using a continuous-flow stirred-tank reactor (CSTR) inoculated with heat-treated anaerobic sludge. To germinate endospores of H(2)-producing bacteria (HPB), continuous feeding was postponed until H(2) production from the initial batch feeding reached a given level. In the first trial, continuous feeding started when the initial H(2) production was equivalent to 0.5 mol H(2)/mol hexose(added). After the switchover from batch to continuous operation, H2 production was sustained for 12 h; however, it decreased with propionate production as the operation continued. We hypothesized that propionic acid bacteria (PAB), whose abundance had been reduced by heat-shock but not completely eliminated, grew during the initial batch operation mode and resulted in the failure of the following continuous mode. PAB such as Selenomonas spp. were detected only in an unsuccessful start-up based on 16S rDNA analysis by PCR denaturing gradient gel electrophoresis, which agrees with our hypothesis. To avoid H(2)-consuming propionate production, an earlier switchover from batch to continuous operation, at 0.2 mol H(2)/mol hexose(added), was tested in the second trial. In this case, H(2) production fluctuated during the 10 days of continuous operation but eventually stabilized at 0.8 mol H(2)/mol hexose(added). Alcohol production data showed that the early fluctuation in the second trial resulted from a selfdetoxification mechanism of HPB. (c) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.