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 cultureindependent 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 ␤-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 NO 3 ؊ -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 ␤-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 ␤-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.Biological denitrification is an important process for nitrogen removal in wastewater treatment. Published reports suggest that 10 to 90% of bacteria in the activated-sludge system are capable of denitrification (16,33,44). However, the system is often confronted with the problem that the efficiency of nitrogen removal decreases due to low availability of organic matter as the reducing power for denitrification. To overcome this problem, a simple organic compound, such as methanol or acetate, is added intentionally as an electron donor to the denitrification process (16). In recent years, a new biotechnology of nitrogen removal using solid biopolymer as the electron donor has been developed (7). This type of nitrogen removal process, called here the solid-phase denitrification process, may have some advantages, e.g., a constant supply of reducing power, no secondary organic pollution, and ease of operation. A promising solid substrate for denitrification is the bacterial polyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) (6, 30, 40, 52), which serves as the source of biodegradable plastic (4,17,34,50). The biodegradability of PHBV in natural environments has been extensively studied, and a number of PHBV-degrading bacteria have been isolated and characterized (1, 11, 38, 39, 41-43, 45, 55). The asso...

A laboratory-scale solid-phase denitrification process for nitrogen removal was constructed by acclimating sewage activated sludge with poly(e-caprolactone) (PCL) as the sole substrate under denitrifying conditions. The sludge thus acclimated exhibited a nitrogen removal rate of 6-7 mg NO3-N g-1 h-1 with PCL as the sole source of reducing power. The microbial community and the distribution of denitrifying bacteria in this process were studied by rRNA-targeted fluorescence in situ hybridization, quinone profiling and standard cultivation methods. The culture-independent molecular and biomarker approaches demonstrated that members of the class Alphaproteobacteria predominated and those of Betaproteobacteria were the second most abundant group of bacteria in the process. The plate counts of denitrifying bacteria with a non-selective agar medium accounted for 6% of the total count and 10% of the direct viable count on average. The most probable number (MPN) obtained with PCL-containing medium under denitrifying conditions was one order of magnitude lower than the plate count. Most of the denitrifying isolates from the MPN enrichment tubes and 10% of the predominant denitrifying bacteria isolated by the plate-counting method were capable of degrading PCL. 16S rRNA gene sequence comparisons showed that the greater majority of the predominant denitrifiers were members of the genera Comamonas, Diaphorobacter and Paracoccus. All of the PCL-degrading denitrifying strains isolated were assigned to a previously unknown species of the genus Comamonas. The results of this study suggest that, apart from their PCL-degrading capacity, members of Alphaproteobacteria and Betaproteobacteria are mainly responsible for nitrogen removal in the PCL-acclimated denitrification process.

Two laboratory-scale solid-phase denitrification (SPD) reactors, designated reactors A and B, for nitrogen removal were constructed by acclimating activated sludge with pellets and flakes of poly(3-hydoxybutyrate-co-3-hydroxyvalerate) (PHBV) as the sole added substrate under denitrifying conditions, respectively. The average denitrification rate in both reactors was 60 mg NO3 − -N g −1 (dry wt) h −1 under steady-state conditions, whereas washed sludge taken from the reactors showed an average denitrification rate of 20 mg NO3-N g −1 (dry wt) hwith fresh PHBV as the sole substrate. The difference in the denitrification rate between the two might be due to the bioavailability of intermediate metabolites as the substrate for denitrification, because acetate and 3-hydroxybutyrate were detected in the reactors. Most of the predominant denitrifiers isolated quantitatively by the platecounting method using non-selective agar medium were unable to degrade PHBV and were identified as members of genera of the class Betaproteobacteria by studying 16S rRNA gene sequence information. nirS and nosZ gene clone library-based analyses of the microbial community from SPD reactor A showed that most of the nirS and nosZ clones proved to be derived from members of the family Comamonadaceae and other phylogenetic groups of the Betaproteobacteria. These results suggest that the efficiency of denitrification in the PHBV-SPD process is affected by the availability of intermediate metabolites as possible reducing-power sources as well as of the solid substrate, and that particular species of the Betaproteobacteria play the primary role in denitrification in this process.