基于LAMP的好氧反硝化菌筛选及其反硝化性能

doi:10.13560/j.cnki.biotech.bull.1985.2019-0555

摘要/Abstract

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、GeneFinder^TM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。

关键词: 好氧反硝化菌, 环介导等温扩增, nosZ基因, 快速筛选

Abstract: 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 GeneFinder^TM 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.

Key words: aerobic denitrifying bacteria, loop-mediated isothermal amplification, gene nosZ, quick screen

李秋芬, 刘启臣, 徐爱玲, 张艳, 宋志文. 基于LAMP的好氧反硝化菌筛选及其反硝化性能[J]. 生物技术通报, 2019, 35(9): 210-217.

LI Qiu-fen, LIU Qi-chen, XU Ai-ling, ZHANG Yan, SONG Zhi-wen. Screening of Aerobic Denitrifying Bacteria Based on Loop-mediated Isothermal Amplification and Denitrification Performance[J]. Biotechnology Bulletin, 2019, 35(9): 210-217.

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