Activity and three-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 biofilter for waste gas treatment, Pseudomonas putida was investigated with a 16S rRNA targeting probe, The three-dimensional distribution of P. putida was visualized in the biofilm matrix by scanning confocal laser microscopy. demonstrating that P. putida was present throughout the biofilm. Acridine orange staining revealed a very heterogeneous structure of the fully hydrated biofilm, with cell-free channels extending 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. Furthermore, measurements of growth rate-related parameters fur P. putida showed reduced I RNA content and cell size (relative to that ill a batch culture), indicating that the P. putida population mas not degrading toluene at a maximal rate in the biofilm environment, Assuming that the rRNA content reflected the cellular activity, a lower toluene degradation rate for P. putida present in the biofilm could be estimated, This calculation Indicated that P. putida mas responsible for a significant part (65%) of the toluene degraded by the entire community.