The distribution and phylogenetic affiliations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria in activated sludge were studied by a polyphasic approach including culture-independent biomarker and molecular analyses as well as cultivation methods. A total of 23 strains of PHBV-degrading denitrifiers were isolated from activated sludges from different sewage treatment plants. 16S ribosomal DNA (rDNA) sequence comparisons showed that 20 of the isolates were identified as members of the family Comamonadaceae, a major group of β-Proteobacteria. When the sludges from different plants were acclimated with PHBV under denitrifying conditions in laboratory scale reactors, the nitrate removal rate increased linearly during the first 4 weeks and reached 20 mg NO3−-N h−1 g of dry sludge−1 at the steady state. The bacterial-community change in the laboratory scale sludges during the acclimation was monitored by rRNA-targeted fluorescence in situ hybridization and quinone profiling. Both approaches showed that the population of β-Proteobacteria in the laboratory sludges increased sharply during acclimation regardless of their origins. 16S rDNA clone libraries were constructed from two different acclimated sludges, and a total of 37 clones from the libraries were phylogenetically analyzed. Most of the 16S rDNA clones were grouped with members of the family Comamonadaceae. The results of our polyphasic approach indicate that β-Proteobacteria, especially members of the family Comamonadaceae, are primary PHBV-degrading denitrifiers in activated sludge. Our data provide useful information for the development of a new nitrogen removal system with solid biopolymer as an electron donor.

Members of the Family Comamonadaceae as Primary Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)-Degrading Denitrifiers in Activated Sludge as Revealed by a Polyphasic Approach

Application of polyhydroxyalkanoates (PHAs) and related biodegradable polymers has gained momentum in various areas of biotechnology. A promising application that started appearing in the past decade is the use of PHAs as the solid substrate for denitrification of water and wastewater. This type of denitrification, termed here "solid-phase denitrification", has several advantages over the conventional system supplemented with liquid organic substrate. PHAs serve not only as constant sources of reducing power for denitrification but also as solid matrices favorable for development of microbial films. In addition, in contrast to conventional processes, the use of PHAs has no potential risk of release of dissolved organic carbon with the resultant deterioration of effluent water quality. If the production cost of PHAs can be brought down, its application to the denitrification process will become economically more promising. A number of PHA-degrading denitrifying bacteria have been isolated and characterized from activated sludge and continuous flow-bed reactors for denitrification with PHAs. Most of these isolates have been assigned phylogenetically to members of beta-Proteobacteria, especially those of the family Comamonadaceae. The metabolic and regulatory relationships between PHA degradation and denitrification, and the interactive relationship between PHA-degrading cells and the solid surface structure are important subjects awaiting future studies, which would provide a new insight into our comprehensive understanding of the solid-phase denitrification process.

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Population density and enzyme activities of heterotrophic bacteria in sewer biofilms and activated sludge