Continuous hydrogen production of auto-aggregative Ethanoligenens harbinense YUAN-3 was performed in a continuous stirred-tank reactor (CSTR) with glucose as substrate under non-sterile conditions. Cell counts of strain YUAN-3 was estimated in the reactor and effluent by real-time PCR. Diversity of invading bacteria was detected by denaturing gradient gel electrophoresis based on the V3 region of 16S rRNA genes. During 21 days of continuous operation, cell flocs formed quickly by aggregation in reactor. Hydrogen gas yield reached a maximum of 1.93 mol/mol-glucose at a dilution rate of 0.4 h(-1), and H(2) production rate reached a maximum of 32.2 mmol H(2)/L-medium/h at a dilution rate of 0.5 h(-1). The overflow rate was less than 0.1%, strain YUAN-3 retained well in the reactor. Some bacterial invaders appeared during continuous operation, and formed a stable community with YUAN-3. Dominant populations in continuous-flow reactor were affiliated with Firmicutes and Bacteroides, and the majority of dominant populations belonged to E. harbinense. The above results demonstrate that auto-aggregative bacteria can accomplish continuous hydrogen production. (c) 2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.