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

生物反硝化脱氮是污水处理中的重要研究课题之一。近年来，已经考虑了使用可生物降解的固体聚合物作为碳源和能量源的用于脱氮的新系统，称为固相脱氮工艺，因为使用固体基质与常规工艺相比具有几个优点，例如恒定供应的还原功率，二次有机污染的风险较小，并且易于操作。然而，这方面的研究才刚刚开始，哪些微生物参与了这一过程尚不清楚。本研究的主要目的是开发一种固相反硝化过程中使用适当的生物聚合物，并阐明这些过程中涉及的微生物。首先，采用多相分类法研究了活性污泥中聚羟基丁酸-羟基戊酸共聚物（PHBV）降解菌的分布和系统发育关系， ...更多信息 方法，包括非培养物依赖性生物标志物和分子分析以及培养方法。16 S rRNA基因序列比较表明，大多数分离物被确定为科丛枝菌科，β-变形菌的主要群体的成员。这些分离物中的一些已被建议分类为丛枝孢科的一个新分类群，Diaphoracetriroreducens gen.nov.，在实验室规模的反应器中，不同植物来源的污泥在PHBV的驯化条件下，硝酸盐的去除率在前4周内呈线性增长，在稳定状态下达到20 mg NO_3 ~--N·h·<-1>g·<-1>干污泥。通过rRNA靶向荧光原位杂交和醌分析，监测了驯化过程中实验室规模污泥中细菌群落的变化。这两种方法都表明，无论其来源如何，实验室污泥中的β变形菌种群在驯化过程中急剧增加。16 S rRNA，nirS和nosZ基因克隆文库分析表明，在这个过程中检测到的大多数克隆与丛枝藻科的成员分组。我们的多相方法的结果表明，β-变形菌，特别是那些的丛枝藻科，是主要的PHBV降解活性污泥中的澄清剂。以聚ε-己内酯（PCL）为唯一基质驯化污水活性污泥，构建了实验室规模的固相反硝化脱氮工艺。驯化后的污泥，以PCL为唯一还原力源时，脱氮速率为6- 7mgNO_3·N·g·<-1>h··<-1>。非培养物依赖的分子和生物标志物方法表明，在该过程中，α变形菌纲的成员占主导地位，而β变形菌纲的成员是第二丰富的细菌群。非选择性琼脂培养基中的微生物平板计数平均占总计数的6%，占直接活菌计数的10%。在纯化条件下用含PCL的培养基获得的最可能数（MPN）比平板计数低一个数量级。从MPN富集管中分离的大多数降解菌和平板计数法分离的10%的优势降解菌能够降解PCL。16 S rRNA基因序列比较表明，占主导地位的发酵菌的大部分成员的属Comamonas，Diaphorococcus和Paracoccus。所有分离的PCL降解菌株被分配到一个以前未知的种属丛毛单胞菌。本研究的结果表明，除了他们的PCL降解能力，Alphaproteobacteria和Betaproteobacteria的成员主要负责在PCL驯化反硝化过程中的氮去除。少

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