Solid-phase denitrification process using biodegradable polymer and population dynamics of microorganisms involved

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Biological denitrification for nitrogen removal is one of the most important subjects of research in wastewater treatment. In recent years, new systems for nitrogen removal using a biodegradable solid polymer as the carbon and energy source, known as the solid-phase denitrification process, have been considered, because the use of a solid substrate has several advantages over the conventional process, such as a constant supply of reducing power, less risk of secondary organic pollution, and ease of operation. However, research in this area has just begun, and which microorganisms are involved in the processes is not known. The main purposes of this study are to develop a solid-phase denitrification process using an appropriate biopolymer and to elucidate the microorganisms involved in these processes. First, the distribution and phylogenetic affiliations of poly(3-hydroxybutyrate-co-hydroxyvalerate)(PHBV)-degrading denitrifying bacteria in activated sludge were studied by a polyphasic … More approach including culture-independent biomarker and molecular analyses as well as cultivation methods. 16S rRNA gene sequence comparisons showed that most of the isolates were identified as being members of the family Comamonadaceae, a major group of Betaproteobacteria. Some of these isolates have been proposed to be classified as a new taxon of Comamonadaceae, Diaphorobacter niroreducens gen.nov., sp.nov. 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^<-1> dry sludge 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 the approaches showed that the population of Betaproteobacteria in the laboratory sludges increased sharply during the acclimation regardless of their origin. 16S rRNA,nirS, and nosZ gene clone library analyses showed that most of the clones detected in this process were grouped with members of the Comamonadaceae. The results of our polyphasic approach indicate that Betaproteobacteria, especially those of the Comamonadaceae, are primary PHBV-degrading denitrifiers in activated sludge. Another laboratory-scale solid-phase denitrification process for nitrogen removal was constructed by acclimating sewage activated sludge with poly(ε-caprolactone)(PCL) as the sole substrate under denitrifying conditions. The sludge thus acclimated exhibited a nitrogen removal rate of 6-7 mg NO_3-N g^<-1> h^<-1> with PCL as the sole source of reducing power. 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. Less