Restriction Pattern Analysis by High-performance Liquid Chromatography of PCR-amplified 16S rDNA Fragments from Scum-forming Bacteria in Activated Sludge

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High-performance liquid chromatography (HPLC) with an anion-exchange nonporous column was used to analyze restriction fragment length polymorphism (RFLP) of the 16S ribosomal DNA (rDNA) of scum-forming actinomycetes found in three different activated sludge plants. 16S rDNA fragments from washed scum samples were amplified by PCR and cloned into plasmid vectors. The 16S rDNA inserts in the plasmids were reamplified in vitro, cut with restriction endonucleases, and separated directly by HPLC. Compared to conventional agarose electrophoresis, the HPLC system used allowed better separation of fragments up to 500bp. The HPLC-RFLP patterns of the scum clones were compared with those of PCR-amplified 16S rDNAs from authentic strains and with computer-predicted restriction patterns of the available 16S rDNA data on the actinomycetes. 16S rDNA-RFLP comparisons indicated that the predominat scum-forming bacteria were Gordona amarae in two activated sludge plants and Rhodococcus sp. in the remaining one. These findings agreed well with results of lipoquinone profiling of the scum performed concurrently. The results of this study indicated that a combination of 16S rDNA-RFLP analysis and quinone profiling should provide a more powerful non-cultivating tool for the identification of scum-forming bacteria in activated sludge.