基于LAMP的好氧反硝化菌筛选及其反硝化性能  

Screening of Aerobic Denitrifying Bacteria Based on Loop-mediated Isothermal Amplification and Denitrification Performance

作　　者：李秋芬 刘启臣[1,2] 徐爱玲 张艳[2] 宋志文[1] LI Qiu-fen;LIU Qi-chen;XU Ai-ling;ZHANG Yan;SONG Zhi-wen(Institute of Environment and Municipal Engineering,Qingdao University of Technology,Qingdao 266033;Yellow Sea FisheriesResearch Institute,Chinese Academy of Fishery Sciences,Qingdao 266071)

机构地区：[1]青岛理工大学环境与市政工程学院,青岛266033 [2]中国水产科学研究院黄海水产研究所,青岛266071

出　　处：《生物技术通报》2019年第9期210-217,共8页Biotechnology Bulletin

基　　金：水科院基本科研业务费课题(2017HY-ZD0502,2017HY-ZD1003);国家自然科学基金项目(31170113)

摘　　要：为了快捷、精确的筛选海水好氧反硝化菌株,应用专用软件设计了 nirS 基因、nirK 基因和 nosZ 基因的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)引物,利用特异性的 LAMP 反应体系,对候选好氧反硝化菌株进行初筛,再用 PCR 法、产气检测法进行复筛,并对筛选出的反硝化菌株进行了反硝化性能测试。结果显示,通过 LAMP 体系在 60℃下反应60 min、GeneFinderTM 核酸染料直接染色法对扩增产物进行观察,从好氧培养条件下分离的候选菌株中筛选出 KFDX1、KFDX2、KFDX3、KFDX4、KWDX1、FJ3-1 和 FJ3-2 等 7 株同时含 nirS 基因或 nirK 基因和 nosZ 基因的好氧反硝化菌株初选菌株;PCR 的筛选结果与 LAMP 体系筛选结果一致,经产气检测,其中有 6 株产气,最终筛选出 KFDX1、KFDX2、KFDX3、KWDX1、FJ3-2 5 株可产 N2 的好氧反硝化菌。反硝化性能测试结果表明,24 h 后,这 5 株菌对 NO2-N 和 NO3-N 的去除率分别达到 99%和 90%以上,最大去除速率分别可高达 4.9 mg/(L·h)和 4.03 mg/(L·h)。这对改进反硝化菌株的筛选方法和推动其在废水处理中的应用具有重要意义。In order to quickly and accurately screen aerobic denitrifying bacterial strains,a specific software Primer Explorer V4 was applied to design the LAMP(loop-mediated isothermal amplification technology)primers for the gene nirS,nirK and nosZ. The strains were firstly screened by the LAMP system after accumulation and isolation on suitable medium in aerobic condition,and further screened by PCR and gas-producing detection. Then,the denitrification performance of the selected strains was detected. The results indicated that 7 denitrifying bacteria strains containing genes nirS or nirK gene and nosZ,including KFDX1,KFDX2,KFDX3,KFDX4,KWDX1,FJ3-1 and FJ3-2, were firstly screened,after reacting for 60 min at 60℃ in LAMP system and GeneFinderTM staining direct observation. The result of PCR was consistent with the screening result of LAMP,and finally,6 strains produced gas after gas-generating detection. Finally,5 aerobic denitrifying strains producing N2,i.e.,KFDX1,KFDX2,KFDX3,KWDX1,and FJ3-2 were screed out. The results of denitrifying capacity test indicated that,the removal rates of NO2-N and NO3-N by the 5 selected strains were over 99% and 90% after 24 h,respectively. The maximum removal rates were up to 4.9 mg/(L·h)and 4.03 mg/(L·h). The result of this study is of significance for improving the screening method of denitrifying bacterial strains and promoting its application in wastewater treatment.

关 键 词：好氧反硝化菌 环介导等温扩增 NOSZ 基因 快速筛选 

分 类 号：X172[环境科学与工程—环境科学] X703