Microbial community in thermophilic methane production from organic acids in a packed-bed reactor was investigated. 16S rDNA clone analysis showed that microbial populations change with hydraulic retention time (HRT) and are different in the fermentation broth and the biofilm that was developed on the surface of supporting materials. Thermoanaerobacteriaceae was mainly found in the biofilm with hydrogenotrophic methanogens whereas clones that are affiliated to Clostridiaceae were mainly found in the fermentation broth. It is also clarified that a certain amount of unknown or unidentified organisms were present in the microflora. Hydrogen evolution and consumption activity of the microflora differed between the biofilm and fermentation broth, implying that a differential in hydrogen partial pressures is generated across the interface between the biofilm and fermentation broth. It is suggested that methanogenesis in packed-bed reactor could be accomplished by the interrelated and complementary actions of the microorganisms in the biofilm and fermentation broth. (C) 2008 Elsevier Inc. All rights reserved.