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

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

Abstract

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

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.

References

[1] 孙雪梅, 李秋芬, 张艳, 等. 一株海水异养硝化-好氧反硝化菌系统发育及脱氮特性[J]. 微生物学报, 2012, 52(6):687-695.
[2] 梁丽华, 左剑恶. 反硝化功能基因——检测反硝化菌种群结构的分子标记[J]. 微生物学通报, 2009, 36(4):627-633.
[3] 陈茂霞, 王欢, 周后珍, 等. 异养硝化-好氧反硝化菌HN-02的筛选及其特性[J]. 应用与环境生物学报, 2013, 19(4):688-693.
[4] 连红民, 邱忠平, 何昆明, 等. 一株好氧反硝化-异养硝化菌的筛选及脱氮特性研究[J]. 生物技术通报, 2015, 31(6):138-143.
[5] 魏利, 马放, 苏俊峰, 等. 基于nirS基因的反硝化细菌快速定量检测[J]. 哈尔滨工业大学学报, 2008, 40(10):1563-1565.
[6] 杨平, 杨迎伍, 陈伟, 等. 食品中4种致病微生物的多重PCR快速检测技术研究[J]. 西南大学学报:自然科学版, 2007(05):90-94.
[7] Notomi T, Okayama H, Masubuchi H, et al.Loop-mediated isothermal amplification of DNA.[J]. Nucleic Acids Research, 2000, 28(12):E63.
[8] Wang Y, Wang Y, Lan R, et al.Multiple endonuclease restriction real-time loop-mediated isothermal amplification[J]. The Journal of Molecular Diagnostics, 2015, 17(4):392-401.
[9] 付振芳, 汪小福, 徐俊锋, 等. 环介导等温扩增终端产物检测及结果判断的研究进展[J]. 浙江农业科学, 2016, 57(5):725-729.
[10] 曹以诚, 石磊, 陈洵, 等. 基于环介导等温扩增技术的嗜肺军团菌基因快速诊断试剂盒及其检测方法:中国, CN101654706 A[P].2008-11-12.
[11] 陈云娇, 陈莉敏, 美亮, 等. 核酸染料Genefinder使用方法的比较[J]. 生物技术, 2011, 21(5):66-68.
[12] 黄鹤, 李应国, 肖进文, 等. 羊痘病毒环介导等温扩增(LAMP)检测方法的建立[J]. 中国畜牧兽医, 2012, 39(6):72-75.
[13] Yano A, Ishimaru R, Hujikata R.Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by Loop-Mediated Isothermal Amplification[J]. Journal of Microbiological Methods, 2007, 68(2):414-420.
[14] Zhong Q, Li K, Chen D, et al. Rapid detection and subtyping of human papillomaviruses in condyloma acumination using loop-mediated isothermal amplification with hydroxynaphthol blue dye[J]. British Journal of Biomedical Science, 2018, 75(3)1-6.
[15] Ferrara M, Perrone G, Gallo A, et al.Development of loop-mediated isothermal amplification(LAMP)assay for the rapid detection of Penicillium nordicum in dry-cured meat products.[J]. International Journal of Food Microbiology, 2015, 202:42-47.
[16] Throbäck IN, Enwall K, Jarvis A, et al.Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE[J]. Fems Microbiology Ecology, 2010, 49(3):401-417.
[17] 孔庆鑫, 李君文, 王新为, 等. 一种新的好氧反硝化菌筛选方法的建立及新菌株的发现[J]. 应用与环境生物学报, 2005, 11(2):95-98.
[18] 杨浩锋, 唐佳玙, 胡安辉, 等. 一株反硝化细菌的分离鉴定及其反硝化特性[J]. 环境工程学报, 2014, 8(1):366-371.
[19] Braker G, Fesefeldt A, Witzel K P.Development of PCR primer systems for amplification of nitrite reductase genes(nirK and nirS)to detect denitrifying bacteria in environmental samples[j]. Applied & Environmental Microbiology, 1998, 64(10):3769-3775.
[20] 周丹丹, 马放, 王弘宇, 等. 关于好氧反硝化菌筛选方法的研究[J]. 微生物学报, 2004, 44(6):837-839.
[21] 梁贤, 任勇翔, 杨垒, 等. 异养硝化-好氧反硝化菌YL的脱氮特性[J]. 环境科学, 2015, 36(5):1749-1756.
[22] 冯雅丽, 李洪珊, 李浩然. 一株深海反硝化菌的分离鉴定及反硝化特性研究[J]. 环境污染与防治, 2016, 38(12):27-30.
[23] 熊焰, 刘力源, 罗睿, 等. 1株亚硝酸盐降解菌的筛选、鉴定、降解条件及效果[J]. 中国水产科学, 2010, 17(6):1264-1271.
[24] 刘星彤, 林柏荣, 廖金铃, 等. 环介导等温扩增法(LAMP)检测最短尾短体线虫[J]. 华中农业大学学报, 2018, 37(06):23-30.
[25] Dhama K, Karthik K, Chakraborty S, et al.Loop-mediated isothermal amplification of DNA(LAMP):a new diagnostic tool lights the world of diagnosis of animal and human pathogens:a review.[J]. Pak J Biol Sci, 2014, 17(2):151-166.
[26] Pai SL, Chong NM, Chen CH.Potential applications of aerobic denitrifying bacteria as bioagents in wastewater treatment[J]. Bioresource Technology, 1999, 68(2):179-185.
[27] Kong QX, Wang XW, Jin M, et al.Development and application of a novel and effective screening method for aerobic denitrifying bact- eria[J]. FEMS Microbiol Lett, 2006, 260(2):150-155.
[28] 张峥, 黄家富, 覃华静, 等. 一株好氧反硝化菌的筛选及其脱氮性能研究[J]. 广西科技大学学报, 2018, 29(4):50-56, 61.
[29] Nagamine K, Hase T, Notomi T.Accelerated reaction by loop-mediated isothermal amplification using loop primers.[J]. Mol Cell Probes, 2002, 16(3):223-229.
[30] Fu S, Qu G, Guo S, et al.Applications of loop-mediated isothermal DNA amplification.[J]. Applied Biochemistry and Biotechnology, 2011, 163(7):845-850.