An outbreak of nonflocculating catabolic populations caused the breakdown of a phenol-digesting activated-sludge process

Activated sludge was fed phenol as the sole carbon source, and the phenol-l oading rate was increased stepwise from 0.5 to 1.0 g liter(-1) day(-1) and then to 1.5 g liter(-1) day(-1). After the loading rate was increased to 1. 5 g liter(-1) day(-1), nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial populatio n structure of the activated sludge was analyzed by temperature gradient ge l electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragment s. We found that the population diversity decreased as the phenol-loading r ate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. T he R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increa sed to 1.5 g liter(-1) day(-1). A total of 41 bacterial strains with differ ent repetitive extragenic palindromic sequence PCR patterns were isolated f rom the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequen ced. Some bacterial isolates could be associated with major TGGE bands by c omparing the 16S rDNA sequences. All of the bacterial strains affiliated wi th the R6 population had almost identical 16S rDNA sequences, while the gyr B phylogenetic analysis divided these strains into two physiologically dive rgent groups; both of these groups of strains could grow on phenol, while o ne group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which s pecific gyrB sequences were used as the primers showed that a population sh ift from R6F to R6T occurred following the increase in the phenol-loading r ate to 1.5 g liter-l day-l. The R10 population corresponded to nonflocculat ing phenol-degrading bacteria. Our results suggest that an outbreak of nonf locculating catabolic populations caused the breakdown of the activated-slu dge process. This study also demonstrated the usefulness of gyrB-targeted f ine population analyses in microbial ecology.