Batch tests were carried out to investigate the effects of heat-pretreated inocula on the fermentative hydrogen production characteristics of various types of substrates. A total of 8 different inocula and 4 different substrates (starch, glycerol, oil and peptone) were used. Heat pretreatment of the inocula was conducted in order to harvest spore-forming clostridial bacteria. Significant hydrogen production potentials were observed from starch (20.5-174.4 ml H(2)/9-COD(starch)) and glycerol (11.5-38.1 ml H(2)/g-COD(glycerol)); however, almost no hydrogen was produced from oil and peptone. When starch was used as a substrate, two different fermentation patterns were observed, according to the inocula: butyric acid-type and ethanol-type fermentation. Polymerase chain reaction combined with denaturing gradient gel electrophoresis (PCR-DGGE) analysis was conducted to compare the bacterial structures cultivated on the starch medium. Different species of clostridial bacteria were observed between the butyric acid-type and ethanol-type fermentation cultures. When glycerol was used as a substrate, 1,3-propanediol was the main by-product with each inoculum. The results of the present study suggest that simultaneous production of ethanol or 1,3-propanediol in addition to hydrogen is a more promising strategy than conventional hydrogen production in acidogenesis. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.