ACTIVITY AND 3-DIMENSIONAL DISTRIBUTION OF TOLUENE-DEGRADING PSEUDOMONAS-PUTIDA IN A MULTISPECIES BIOFILM ASSESSED BY QUANTITATIVE IN-SITU HYBRIDIZATION AND SCANNING CONFOCAL LASER MICROSCOPY

As a representative member of the toluene-degrading population in a bi ofilter for waste gas treatment, Pseudomonas putida was investigated w ith a 16S rRNA targeting probe, The three-dimensional distribution of P. putida was visualized in tile biofilm matrix by scanning confocal l aser microscopy. demonstrating that P. putida was present throughout t he biofilm. Acridine orange staining revealed a very heterogeneous str ucture of the fully hydrated biofilm, with cell-free channels extendin g ft om the surface Into the biofilm. This indicated that toluene may penetrate to deeper layers of the biofilm, and consequently P. putida may be actively degrading toluene in all regions of the biofilm. Furth ermore, measurements of growth rate-related parameters fur P. putida s howed reduced I RNA content and cell size (relative to that ill a batc h culture), indicating that the P. putida population mas not degrading toluene at a maximal rate in the biofilm environment, Assuming that t he rRNA content reflected the cellular activity, a lower toluene degra dation rate for P. putida present in the biofilm could be estimated, T his calculation Indicated that P. putida mas responsible for a signifi cant part (65%) of the toluene degraded by the entire community.