Characterization of a p-nitrophenol-degrading mixed culture with an improved methodology of fluorescence in situ hybridization and confocal laser scanning microscopy

Background: Confocal laser scanning microscopy (CLSM) provides critical information of the presence of autofluorescence signals inherent to the samples under study, which may interfere with probe fluorescence. Therefore, it is important to determine the emission spectrum of the biomass autofluorescence under study in order to avoid, as much as possible, this wavelength range when selecting specific probe emissions and to modify, if necessary, the original fluorescence in situ hybridization (FISH) protocol in order to enhance probes signals. Results: Microbial characterization of a p-nitrophenol-degrading activated sludge through a conventional FISH protocol was not possible because the fluorescence signal of the unlabelled cells was similar to or even higher than the signal emitted by hybridized cells, impeding the separation of the two signals and therefore, preventing biomass characterization. An optimized but simple FISH approach was developed in order to ease observation of the p-nitrophenol-degrading bacteria studied in this work. The final results allowed identification of Arthrobacter sp. (Micrococcaceae family) and genus Acinetobacter (Moraxellaceae family) in the p-nitrophenol-degrading activated sludge, whereas no hybridization was found for Pseudomonas spp. Conclusions: The hypothesis of a mixed culture following, at the same time, different degradation pathways has been confirmed through the microbial characterization of enriched p-nitrophenol-degrading activated sludge. © 2011 Society of Chemical Industry.