An approach of acidification was examined on formation of hydrogen-producing granules and biofilms in upflow column-shaped reactors. The reactors were fed with synthetic glucose wastewater and operated at 37 degrees C and pH 5.5. The acclimated anaerobic culture was inoculated in four reactors designated R1, R2, R3 and R4, with R3 and R4 filled with granular activated carbon as support medium. To unveil the roles of acidification, microbial culture in R2 and R3 was subject to an acid incubation for 24 h by shifting the culture pH from 5.5 to 2.0. The experimental results suggested that the acidification substantially accelerated microbial granulation, but not biofilm formation. Microbial activities were inhibited by the acid incubation for about 78 h, resulting in the retarded formation of biofilms of the acidified Culture. Reducing culture pH resulted in improvement in cell surface physicochemical properties favoring microbial adhesion and immobilization. Zeta potential increased from -25.3 mV to 11.9 mV, hydrophobicity in terms of contact angle improved from 31 degrees to 38 degrees and production of extracellular polymers increased from 66 mg/g-VSS to 136 mg/g-VSS. As a result of the formation of granules and biofilms, high hydrogen production rates of 6.98 and 7.49 L/L h were achieved in granule-based and biofilm-based reactors, respectively. It is concluded that acid incubation is an efficient means to initiate the rapid formation of granules by regulating the surface characteristics of microbial culture. The use of support media as starting nuclei may result in rapid formation of biofilms without the acidification. (C) 2008 International Association for Hydrogen Energy, Published by Elsevier Ltd. All rights reserved.