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

Abstract

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 from 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 for P. putida showed reduced rRNA content and cell size (relative to that in a batch culture), indicating that the P. putida population was 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 was responsible for a significant part (65%) of the toluene degraded by the entire community.

Biased 16S rDNA PCR amplification caused by interference from DNA flanking the template region

“…The four bacterial species used in the present investigation were all isolated from a waste gas bio¢lter [10]. They constitute part of a model microbial consortium that we study in our laboratory, and they were chosen for the present study because we wished to develop a fast molecular method to monitor changes in species compositions in our microbial consortia.…”

“…The strains of Pseudomonas putida, Microbacterium arborescens, Nocardioides simplex, and the Lsubgroup proteobacterium used in the present investigation were all isolated from a bio¢lter treating toluene-containing waste gas [10]. Cultivations were performed in Luria-Bertani broth (LB) [11].…”

Bacterial dynamics in steady-state biofilters: beyond functional stability

“…First, this high diversityeven in the compartments without gaseous contaminantsmay result from the proliferation of saprophytes using products from cell lysis, the extracellular polymeric substances (EPS) matrix or packing material (Stoffels et al, 1998;Borin et al, 2006). In some BF, saprophytes dominate (Juteau et al, 1999;Song & Kinney, 2000;Hekmat et al, 2004), whereas the active primary-degraders can represent only 4% of the community (Møller et al, 1996). Secondly, the high diversity can be interpreted as the expression of species coexistence through niche differentiation (Fernandez et al, 1999;Siripong & Rittmann, 2007).…”

Study of a hexane-degrading consortium in a biofilter and in liquid culture: biodiversity, kinetics and characterization of degrading strains

“…In addition, most microbiological studies involved isolation techniques, which allowed the identification of new species (Reichert et al, 1998), but not the characterization of entire populations, because most organisms are not cultivable in the standard media used for isolation (Amann et al, 1995). Although molecular techniques have already been used to describe the population of a toluene-degrading biofilter (Moller et al, 1996) and of a phosphate-removing biofilm (Gieseke et al, 2001), little is known about population dynamics in biofilters in relation to changing operating conditions.…”