The microbial composition and structure of a multispecies biofilm of a laboratory-scale trickle-bed bioreactor for the treatment of waste gas was examined. The model pollutant was a volatile organic compound-mixture of polyalkylated benzenes called Solvesso 100((R)). Fluorescent in-situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) were applied. Two new Solvesso 100((R))-degrading Pseudomonas sp strains were isolated from the multispecies biofilm. Corresponding isolate-specific oligonucleotide probes were designed and applied successfully. A major finding was that the fraction of Solvesso 100((R))-degrading bacteria in the biofilm was low (about 3-6% during long-term operation). The majority of the active cells were saprophytes which utilized intermediates and cell lysis products. The measured fraction of extracellular polymeric substances of the mature biofilm was 89-93% of the total biomass. The CLSM examinations of a 3-days-old approx 10mum thick biofilm revealed highly heterogeneous structures with distinguished three-dimensional matrix-enclosed microcolony bodies spread across the substratum surface. The 28-days-old 80-960mum thick biofilm exhibited voids, cell-free channels, and pores of variable sizes. In both cases, an even distribution of active cells and pollutant-degrading bacteria throughout the biofilm cross-section as well as through the biofilm depth was observed. (C) 2003 Society of Chemical Industry.