Phylogenetic analysis and in situ identification of bacteria in activated sludge

The bacterial community structure of activated sludge of a large municipal wastewater treatment plant was investigated by use of the rRNA approach. Almost-full-length genes coding for the small-subunit rRNA (rDNA) were amplified by PCR and subsequently cloned into the pGEM-T vector. Clones were screened by dot blot hybridization with group-specific oligonucleotide probes. The phylogenetic affiliations of clones were compared with the results obtained with the original sample by in situ hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes and found to be in general agreement. Twenty-five 16S rDNA clones were fully sequenced, 11 were almost fully (> 80%) sequenced, and 27 were partially sequenced. By comparative sequence analyses, the majority of the examined clones (35 of 67) could be affiliated with the beta subclass of the class Proteobacteria. The gamma and alpha subclasses of Proteobacteria were represented by 13 and 4 clones, respectively. Eight clones grouped with the epsilon group of Proteobacteria, and five clones grouped with gram-positive bacteria with a low DNA G+C content. The 16S rDNA of two clones showed similarity with 16S rDNA genes of members of the phyla Chlamydiae and Planctomyces. 16S rRNA-targeted oligonucleotide probes were designed and used for the enumeration of the respective bacteria. Interestingly, potentially pathogenic representatives of the genus Arcobacter were present in significant numbers (4%) in the activated sludge sample examined. Pairs of probes targeted to the 5' and 3' regions were used for detection of chimeric sequences by in situ hybridization. Two clones could be identified as chimera by applying such a pair of probes.