Optimal conditions for acid pre-treatment were investigated for the enrichment of hydrogen-producing bacteria (HPB) in a mixed culture using three strong acids: HCl, HNO(3), and H(2)SO(4). HCl was selected as a suitable acid for the enrichment of HPB in the fermentation process. The volume of bio-hydrogen produced when the mixed culture was pre-treated using HCl at pH 2 was 3.2 times higher than that obtained without acid pre-treatment. Changes in the microbial community during acid pre-treatment were monitored using images obtained by the fluorescent in situ hybridization (FISH) method and the Live/Dead cell viability test. The tests clearly indicated that the Clostridium species of cluster 1 were the predominant strains involved in bio-H(2) fermentation, and could be selectively enriched by HCl pre-treatment. (c) 2008 Elsevier Ltd. All rights reserved.