In situ characterization of Nitrospira-like nitrite oxidizing bacteria active in wastewater treatment plants

Uncultivated Nitrospira-like bacteria in different biofilm and activated-sl udge samples were investigated by cultivation-independent molecular approac hes. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Su bsequently, a phylogenetic consensus tree of the Nitrospira phylum includin g all publicly available sequences was constructed. This analysis revealed that the genus Nitrospira consists of at least four distinct sublineages. B ased on these data, two 16S rRNA-directed oligonucleotide probes specific f or the phylum and genus Nitrospira, respectively, were developed and evalua ted for suitability for fluorescence in situ hybridization (FISH). The prob es were used to investigate the in situ architecture of cell aggregates of Nitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, c onfocal laser scanning microscopy, and computer-aided three-dimensional vis ualization. Cavities and a network of cell-free channels inside the Nitrosp ira microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by Nitrospi ra-like bacteria within their natural habitat under different incubation co nditions was studied by combined FISH and microautoradiography. Under aerob ic conditions, the Nitrospira-like bacteria in bioreactor samples took up i norganic carbon (as HCO3- or as CO2) and pyruvate but not acetate, butyrate , and propionate,, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the Nitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or a naerobic conditions.