Biohydrogen production process from glucose using extreme-thermophilic H-2-producing bacteria enriched from digested sewage sludge was investigated for five cycles of repeated batch experiment at 70 degrees C. Heat shock pretreatment was used for preparation of hydrogen-producing bacteria comparing to an untreated anaerobic digested sludge for their hydrogen production performance and responsible microbial community structures. The results showed that the heat shock pretreatment completely repressed methanogenic activity and gave the maximum hydrogen production yield of 355-488 ml H-2/g COD in the second cycle of repeated batch cultivation with more stable gas production during the cultivation when compared with control. Hydrogen production was accompanied by production of acetic acid. The average specific hydrogen in five cycles experiment ranged from 150 to 200 ml H-2/g VSS. PCR-DGGE profiling showed that the extreme-thermophilic culture predominant species were closely affiliated to Thermoanaerobacter pseudethanolicus. Copyright (C) 2010, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.