PHYLOGENETIC ANALYSIS AND IN-SITU IDENTIFICATION OF BACTERIA IN ACTIVATED-SLUDGE

The bacterial community structure of activated sludge of a large munic ipal wastewater treatment plant was investigated by use of the rRNA ap proach. Almost-full-length genes coding for the small-subunit rRNA (rD NA) were amplified by PCR and subsequently cloned into the pGEM-T vect or. 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,vith fluorescently labeled, rRNA-targeted oligonucleoti de probes and found to be in general agreement. Twenty-five 16S rDNA c lones were fully sequenced, 11 were almost fully (>80%) sequenced, and 27 were partially sequenced. By comparative sequence analyses, the ma jority of the examined clones (35 of 67) could be affiliated with the beta subclass of the class Proteobacteria. The gamma and alpha subclas ses of Proteobacteria were represented by 13 and 4 clones, respectivel y. Eight clones grouped with the epsilon group of Proteobacteria, and five clones grouped with gram-positive bacteria with a low DNA G + C c ontent, The 16S rDNA of two clones showed similarity with 16S rDNA gen es of members of the phyla Chlamydiae and Planctomyces. 16S rRNA-targe ted oligonucleotide probes were designed and used for the enumeration of the respective bacteria. Interestingly, potentially pathogenic repr esentatives of the genus Arcobacter were present in significant number s (4%) in the activated sludge sample examined. Pairs of probes target ed to the 5' and 3' regions were used for detection of chimeric sequen ces by in situ hybridization. Two clones could be identified as chimer a by applying such a pair of probes.