Identification and in situ Detection of Gram-negative Filamentous Bacteria in Activated Sludge

Gram-negative filamentous bacteria are commonly observed in activated sludge and contribute to poor settlement of activated sludge flocs in secondary sedimentation tanks, a problem referred to as activated sludge bulking. However, the standard morphological identification system is of limited value for a high resolution, rapid monitoring of these bacteria. Therefore, specific 16S rRNA-targeted oligonucleotide probes were developed for Haliscomenobacter spp., Sphaerotilus spp., Leptothrix spp., Thiothrix spp., Leucothrix mucor and bacteria of the Eikelboom type 021N. Probe specificities were evaluated by nonisotopic dot blot hybridization to 145 reference strains representing a diverse collection of taxa. In situ hybridization with fluorescent probe derivatives was combined with scanning confocal laser microscopy (SCLM) for analyzing the three dimensional localization of the filaments inside the sludge flocs. Filaments could be localized even in the center of fixed flocs at a high resolution undisturbed by problems like autofluorescence.