Identification of Denitrifying Bacteria and Nitrite Reductase (nirS) Gene Quantification in Nitrogen Removal Fluidized Bed Reactor
Identification of Denitrifying Bacteria and Nitrite Reductase (nirS) Gene Quantification in Nitrogen Removal Fluidized Bed Reactor
复制标题
除氮流化床反应器中反硝化细菌的鉴定和亚硝酸盐还原酶 (nirS) 基因定量
DOI：
10.2965/jswe.27.791
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
H. Harada
中科院分区：
文献类型：
--
作者：
Y. Tsukamoto;N. Araki;A. Nagano;Takashi Yamaguchi;H. Harada
采用分子生物学方法研究了床内反硝化细菌的鉴定和计数。FBR作为一个从生活污水处理厂流出物中脱除氮的装置持续运行，并补充供应甲醇作为碳源。通过变性梯度凝胶电泳(DGGE)和16S核糖体DNA序列分析，表明生物膜中的反硝化细菌中以Thauera群占优势。用于荧光原位杂交(FISH)的寡核苷酸探针THA155是新设计的针对Thauera组的寡核苷酸探针。THA155能够从生物膜污泥中检测到Thauera细胞，但只有0.9-5.7%的DAPI染色的细胞被杂交。针对反硝化过程的关键酶亚硝酸还原酶基因的基因序列，采用实时聚合酶链式反应(PCR)对包括Thauera群在内的反硝化细菌细胞进行了定量。生物膜污泥中NIRS基因的数量与生物反应器中反硝化菌的活性之间存在很好的相关性。因此，实时荧光定量检测NIRS基因可以作为脱氮反应器中反硝化细菌监测的一种合适工具。
Molecular approaches were applied to study the identification and enumeration of denitrifying bacteria in a fluidized bed reactor (FBR). The FBR was continuously operated as a unit for the removal of nitrogen from domestic sewage treatment plant effluents, with a supplementary supply of methanol as a carbon source. By denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S ribosomal DNA, it was revealed that the Thauera group was dominant among the denitrifying bacteria in the biofilms obtained from the FBR. The oligonucleotide probe THA155 for fluorescence in situ hybridization (FISH) was newly designed for specifically targeting the Thauera group. THA155 was able to detect Thauera cells from the biofilm sludge, but only 0.9-5.7 percent of DAPI-stained total cells was hybridized. The real-time polymerase chain reaction (PCR) targeting of the gene sequences of nitrite reductase gene, a key enzyme of denitrification processes, was performed to quantify denitrifying bacteria cells including the Thauera group. An excellent correlation was found between the number of nirS genes in the biofilm sludge and the activity of denitrifiers in the FBR. Therefore, the quantification of the nirS gene by real-time PCR can be an appropriate tool for monitoring denitrifying bacteria in nitrogen removal reactors.