Two biofilm-based column reactors with walnut shell (WS) as carrier media were applied for fermentative hydrogen production using glucose as substrate by mixed microbial cultures at the temperature of 35 degrees C. Pure hydrogen producing bacteria Ethanoligenens harbinense B49 was supplemented into the reacting system periodically or continuously to enhance hydrogen production ability at the startup period. The results showed that the bioreactors supplemented with E. harbinense B49 performed better than the reactor without bacteria addition. Continuous addition mode was recommended, since the hydrogen production performance was better and the operation was easily to be accomplished. The optimal addition amount of pure bacteria was also investigated. The optimal bacterial addition amount was found to be 2.5% which led a better hydrogen production rate. In addition, the bioreactor supplemented with pure bacteria continuously presented a high hydrogen production ability as the specific hydrogen production rate (SHPR) maximized at 1.36 L/g-VSS.d; whereas, the bioreactor without bacteria addition obtained a maximum specific hydrogen production rate of 1.10 L/g-VSS.d. The addition of E. harbinense B49 favored the transformation to ethanol type fermentation in the bioreactor. Thereby, the startup period had been accelerated remarkably. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.