The maximum propionate removal rate was 13.7 g/L-reactor/day at the organic loading rate of 66.4 kg-CODcr/m(3)-reactor/day (HRT, 4.75 h); however, the removal efficiency was very low. Clone library analysis and quantification by real-time PCR using 16S rRNA gene revealed that the population of methanogenic archaea in the biofilm fraction that developed on the packed bed was higher than that in the liquid fraction. The clone, which is related to Methanosarcina, was detected only in the biofilm fraction. The clones closely related to Pelotomaculum, which is capable of degrading propionate, and the hydrogenotrophic methanogen Methanothermobactor were also detected only in the biofilm fraction in the acetate and propionate-fed reactor. The experimental results indicate that the packed-bed design can maintain a sufficiently high density of methanogenic microorganisms within the system even at reduced HRTs as well as facilitate an efficient degradation of propionate and acetate, possibly through syntrophic reactions. (c) 2007 Elsevier Ltd. All rights reserved.