一株安全高效的好氧反硝化菌Pseudomonas stutzeri DZ11的生物安全性及脱氮性能研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-1084

摘要/Abstract

摘要：

对中山市民众镇水产养殖场底泥及污水中分离筛选出的好氧反硝化细菌进行研究，旨在为开发养殖废水的脱氮工艺奠定基础。从样品中筛选出高性能菌DZ11，对其进行形态学观察，生理生化测定以及16S rDNA基因序列分析，确定菌株的种属；进行药敏试验和生物安全性检测，确认菌株的安全性；从碳源种类、温度、pH值和碳氮比这4个因素对脱氮效率的影响进行研究，提高菌株的脱氮效率。鉴定结果表明，DZ11为假单胞菌属施氏假单胞菌，并命名为Pseudomonas stutzeri DZ11。经药敏试验和生物安全性检测发现该菌株无明显耐药性并具有较高的生物安全性。单因素优化的结果为：菌株DZ11的最佳碳源为柠檬酸钠，最适温度35.0℃，最佳pH为7，最佳的C/N为10。在分别以氨氮，硝态氮和亚硝态氮为唯一底物时的转化率最高可分别达到84.39%，99.0%和97.65%，说明菌株具有良好的脱氮作用。菌株DZ11具有较高的生物安全性和高效的脱氮性能，在养殖废水的脱氮工艺中具有较大的应用潜力。

关键词: 好氧反硝化菌, 假单胞菌, 生物脱氮, 生物安全性

Abstract:

The aerobic denitrifying bacteria separated and screened from the bottom sludge and sewage of the aquaculture farm in Minzhong town，Zhongshan city，were studied，aiming to lay the foundation for the development of the denitrification process of aquaculture wastewater. The high-performance bacterium DZ11 was selected from the samples，and its morphological observation，physiological and biochemical determination and 16S rDNA gene sequence analysis were performed to determine the species of the strain. Besides，drug susceptibility test and biological safety test were conducted to confirm the safety of strains. Then the influences of carbon source types，temperature，pH value and carbon-nitrogen ratio on the nitrogen removal efficiency were studied to improve the denitrification efficiency of strains’ performance. The identification results showed that the strain DZ11 was identified as Pseudomonas stutzeri and named Pseudomonas stutzeri DZ11. Drug susceptibility test and biological safety test demonstrated that the strain had no obvious drug resistance and had high biological safety. The result of single factor optimization is that the optimal carbon source of strain DZ11 was sodium citrate，the optimal temperature was 30.0-35.0℃，the optimal pH was 7，and the optimal C/N was in the range of 10-20. When NH₄-N，NO₃-N，and NO₂-N were the only substrates，the transformation rates were 84.39%，99.0% and 97.65%，respectively，indicating that the strain had a good denitrification effect. In sum，the strain DZ11 has high biological properties and high-efficiency denitrification performance，and has great application potential in the denitrification process of aquaculture wastewater.

Key words: aerobic denitrifying bacterium, Pseudomonas, biological removal of nitrogen, bio-safety

赵洋, 孙慧明, 林浩澎, 罗娉婷, 朱雅婷, 陈琼华, 舒琥. 一株安全高效的好氧反硝化菌Pseudomonas stutzeri DZ11的生物安全性及脱氮性能研究[J]. 生物技术通报, 2022, 38(10): 226-234.

ZHAO Yang, SUN Hui-ming, LIN Hao-peng, LUO Ping-ting, ZHU Ya-ting, CHEN Qiong-hua, SHU Hu. Biosafety and Nitrogen Removal Performance of a Safe and Efficient Aerobic Denitrifying Pseudomonas stutzeri DZ11[J]. Biotechnology Bulletin, 2022, 38(10): 226-234.

图/表 10

图1 DZ11菌落形态图

Fig.1 Colony morphology of DZ11

图2 菌株DZ11扫描电镜图

Fig.2 Scanning electron micrograph of strain DZ11

表1 菌株DZ11生理生化鉴定结果

Table 1 Physiological and biochemical identification of strain DZ11

项目 Item	结果 Result	项目 Item	结果 Result
氧化酶反应Oxidase reaction	+	反硝化Denitrification	+
明胶液化Gelatin liquefaction	-	蔗糖发酵Sucrose fermentation	-
淀粉水解Starch hydrolysis	+	D-（+）-葡萄糖发酵 D-（+）-Glucose fermentation	+
吲哚生产Indole production	-	乳糖发酵Lactose fermentation	-
甲基红试验Methyl red	-	D-（+）-麦芽糖发酵 D-（+）-Maltose fermentation	+
V-P试验Vogs-Proskauer text	-	甘油Glycerol	-
Tween80	+	肌酸Creatine	₊
4℃生长Growth at 4℃	-	甘露醇Mannitol	-
41℃生长Growth at 41℃	+	D-半乳糖发酵D-Galactose fermentation	-

图3 基于16S rDNA构建菌株DZ11的系统发育树

Fig.3 Constructed phylogenetic tree of strain DZ11 based on 16S rDNA

表2 菌株DZ11对不同药品的耐药性结果表

Table 2 Resistance results of strain DZ11 to different drugs

药品 Drug	抑菌环大小 Size of inhibition ring/mm	敏感等级 Sensitive level	药品 Drug	抑菌环大小 Size of inhibition ring/mm	敏感等级 Sensitive levels
诺氟沙星 Norfloxacin	44±1.73	S	环丙沙星Ciprofloxacin	41±1.73	S
氯霉素 Chloramphenicol	19.67±0.58	S	头孢他啶Ceftazidime	24.33±0.57	S
硫酸庆大霉素Gentamicin sulfate	23.67±0.58	S	左氧氟沙星Levofloxacin	32±1	S
盐酸四环素Tetracycline hydrochloride	24±1	S	链霉素 Streptomycin	19.33±0.58	S

图4 菌株DZ11浸泡处理后斑马鱼存活率

Fig.4 Survival rate of zebrafish soaked with strain DZ11

图5 C/N比对菌株DZ11生长和脱氮性能的影响

Fig.5 Effects of C/N ratio on the growth and denitrification performance of strain DZ11

图6 pH对菌株DZ11生长和脱氮性能的影响

Fig.6 Effects of pH on the growth and denitrification performance of strain DZ11

图7 温度对菌株DZ11生长和脱氮性能的影响

Fig.7 Effect of temperature on the growth and denitrifica-tion performance of strain DZ11

图8 C源对菌株DZ11生长和脱氮性能的影响

Fig.8 Effects of source C on the growth and denitrification performance of strain DZ11

参考文献 33

[1]	第二次全国污染源普查公报[J]. 环境保护, 2020, 48(18):8-10.
	The second national pollution source census bulletin[J]. Environ Prot, 2020, 48(18):8-10.
[2]	周晓黎, 孙迎雪, 沈丹丹, 等. 一株施氏假单胞菌Pseudomonas stutzeri DN-LWX19的脱氮性能[J]. 环境工程学报, 2015, 9(1):247-252.
	Zhou XL, Sun YX, Shen DD, et al. Denitrification performance of Pseudomonas stutzeri DN-LWX19[J]. Chin J Environ Eng, 2015, 9(1):247-252.
[3]	孙庆花, 于德爽, 张培玉, 等. 1株海洋异养硝化-好氧反硝化菌的分离鉴定及其脱氮特性[J]. 环境科学, 2016, 37(2):647-654.
	Sun QH, Yu DS, Zhang PY, et al. Identification and nitrogen removal characteristics of a heterotrophic nitrification-aerobic denitrification strain isolated from marine environment[J]. Environ Sci, 2016, 37(2):647-654.
[4]	孙庆花, 于德爽, 张培玉, 等. 海洋菌株y3的分离鉴定及其异养硝化-好氧反硝化特性[J]. 环境科学, 2016, 37(3):1089-1097.
	Sun QH, Yu DS, Zhang PY, et al. Isolation, identification and nitrogen removal characteristics of a heterotrophic nitrification-aerobic denitrification strain y3 isolated from marine environment[J]. Environ Sci, 2016, 37(3):1089-1097. doi: 10.1021/es0261168 URL
[5]	朱晓明, 赵东华, 阮晓红. 含水层介质中1株异养硝化-好氧反硝化菌的分离鉴定与脱氮性能[J]. 环境工程, 2020, 38(5):43-48.
	Zhu XM, Zhao DH, Ruan XH. Identification and nitrogen removal characteristics of a heterotrophic nitrification-aerobic denitrifying strain isolated from unconfined aquifer porous media[J]. Environ Eng, 2020, 38(5):43-48.
[6]	李文甫, 杜柳冰, 刘思莹, 等. 一株高效好氧反硝化细菌的分离鉴定及脱氮性能研究[J]. 生物技术通报, 2019, 35(9):202-209. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0339
	Li WF, Du LB, Liu SY, et al. Isolation and identification of an efficient aerobic denitrifying bacterium[J]. Biotechnol Bull, 2019, 35(9):202-209.
[7]	田雪雪, 程玉立, 张圆圆, 等. 一株异养硝化-好氧反硝化功能菌的分离鉴定及其脱氮特性[J]. 环境工程学报, 2017, 11(2):1269-1275.
	Tian XX, Cheng YL, Zhang YY, et al. Isolation, identification and nitrogen removal characteristics of a heterotrophic nitrification-aerobic denitrification bacterium[J]. Chin J Environ Eng, 2017, 11(2):1269-1275.
[8]	王田野, 魏荷芬, 胡子全, 等. 一株异养硝化好氧反硝化菌的筛选鉴定及其脱氮特性[J]. 环境科学学报, 2017, 37(3):945-953.
	Wang TY, Wei HF, Hu ZQ, et al. Isolation and identification of a heterotrophic nitrifying and aerobic denitrifying strain and its denitrification characteristics[J]. Acta Sci Circumstantiae, 2017, 37(3):945-953.
[9]	陈军昌, 徐欣, 吕晓民, 等. 腐败假单胞菌人工感染河蟹的毒性试验及药物敏感性实验[J]. 水产科学, 1999, 18(2):18-20
	Chen JC, Xu X, Lu XM, et al. Experimental toxicity of Pseudomonas putrefacienst to Eriocheir Scinensis and its susceptibility test to antibiotics[J]. Fish Sci, 1999, 18(2):18-20
[10]	Balasubramanian D, Schneper L, Kumari H, et al. A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence[J]. Nucleic Acids Res, 2013, 41(1):1-20. doi: 10.1093/nar/gks1039 pmid: 23143271
[11]	郭静文. 好氧反硝化细菌的筛选及菌藻联合对养殖废水的处理[D]. 广州: 广州大学, 2020:1-68.
	Guo JW. Isolation of aerobic denitrification bacterium and treatment of aquaculture wastewater by combination of bacteria and algae[D]. Guangzhou: Guangzhou University, 2020:1-68.
[12]	中华人民共和国国家质量监督检验检疫总局. 中华人民共和国出入境检验检疫行业标准:动物及其制品中细菌耐药性的测定纸片扩散法 SN/T 1944—2007[S]. 北京: 中国标准出版社, 2007.
	Commodity Inspection Standard of the People’s Republic of China. Detection of antimicrobial resistance of the bacteria in animal and animal products-Disk diffusion testing. SN/T 1944—2007[S]. Beijing: China Standard Press, 2007.
[13]	国家环境保护总局. 中华人民共和国环保行业标准:环保用微生物菌剂环境安全评价导则 HJ/T 415—2008[S]. 北京: 中国环境科学出版社, 2008.
	State Environmental Protection Administration of the People’s Republic of China. Environmental Protection Standard of the People’s Republic of China:Guide of safety-assessment on application of microbial blends in the environmental protection. HJ/T 415—2008[S]. Beijing: China Environment Science Press, 2008.
[14]	Ji B, Yang K, Zhu L, et al. Aerobic denitrification:a review of important advances of the last 30 years[J]. Biotechnol Bioprocess Eng, 2015, 20(4):643-651. doi: 10.1007/s12257-015-0009-0 URL
[15]	张苗, 黄少斌, 肖先念. C/N和pH值对高温好氧反硝化菌产N₂O的影响研究[J]. 环境工程学报, 2012, 6(1):275-279.
	Zhang M, Huang SB, Xiao XN. Effect of C/N ratio and pH on nitrous oxide production of themophic aerobic denitrifier[J]. Chin J Environ Eng, 2012, 6(1):275-279.
[16]	闫苗苗, 张海涵, 钊珍芳, 等. 生物脱氮技术中好氧反硝化细菌的代谢及应用研究进展[J]. 环境科学研究, 2020, 33(3):668-676.
	Yan MM, Zhang HH, Zhao ZF, et al. Research progress of metabolism and application of aerobic denitrifying bacteria in biological denitrification technology[J]. Res Environ Sci, 2020, 33(3):668-676.
[17]	赵林, 付贵萍, 武金发, 等. 一株耐盐的卓贝儿氏菌(Zobellella sp. )的分离鉴定及其硝酸盐还原能力[J]. 微生物学通报, 2020, 47(5):1354-1365.
	Zhao L, Fu GP, Wu JF, et al. Isolation, identification and nitrate reduction ability of a salt-tolerant strain of Zobellella sp[J]. Microbiol China, 2020, 47(5):1354-1365.
[18]	安健, 宋增福, 杨先乐, 等. 好氧反硝化细菌YX-6特性及鉴定分析[J]. 中国水产科学, 2010, 17(3):561-569.
	An J, Song ZF, Yang XL, et al. Characteristics of aerobic denitrifying strain YX-6 and identification[J]. J Fish Sci China, 2010, 17(3):561-569.
[19]	Du DL, Zhang CY, Zhao KX, et al. Effect of different carbon sources on performance of an A_2N-MBR process and its microbial community structure[J]. Front Environ Sci Eng, 2017, 12(2):1-10.
[20]	Shan J, Yang PP, Shang XX, et al. Anaerobic ammonium oxidation and denitrification in a paddy soil as affected by temperature, pH, organic carbon, and substrates[J]. Biol Fertil Soils, 2018, 54(3):341-348. doi: 10.1007/s00374-018-1263-z URL
[21]	李思琦, 杨静丹, 刘琳, 等. 好氧反硝化菌Achromobacter sp. L16的脱氮特性[J]. 生物技术通报, 2020, 36(6):93-101. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0978
	Li SQ, Yang JD, Liu L, et al. Denitrification characteristics of aerobic denitrifying bacteria Achromobacter sp. L16[J]. Biotechnol Bull, 2020, 36(6):93-101.
[22]	朱云, 龚望宝, 谢骏, 等. 好氧反硝化细菌的鉴定及其脱氮特性研究[J]. 水生生物学报, 2020, 44(4):895-903.
	Zhu Y, Gong WB, Xie J, et al. Identification and denitrification characteristics of the aerobic denitrifying bacteria Pseudomonas furukawaii zs1[J]. Acta Hydrobiol Sin, 2020, 44(4):895-903.
[23]	郭超, 尹辉, 范奎, 等. 一株好氧反硝化菌的分离鉴定及脱氮特性研究[J]. 安全与环境工程, 2020, 27(4):41-47.
	Guo C, Yin H, Fan K, et al. Isolation, identification and nitrogen removal characteristics of aerobic denitrifying bacteria[J]. Saf Environ Eng, 2020, 27(4):41-47.
[24]	Huang HK, Tseng SK. Nitrate reduction by Citrobacter diversus under aerobic environment[J]. Appl Microbiol Biotechnol, 2001, 55(1):90-94. pmid: 11234965
[25]	Yang XP, Wang SM, Zhou LX. Effect of carbon source, C/N ratio, nitrate and dissolved oxygen concentration on nitrite and ammonium production from denitrification process by Pseudomonas stutzeri D6[J]. Bioresour Technol, 2012, 104:65-72. doi: 10.1016/j.biortech.2011.10.026 URL
[26]	Zheng HY, Liu Y, Gao XY, et al. Characterization of a marine origin aerobic nitrifying-denitrifying bacterium[J]. J Biosci Bioeng, 2012, 114(1):33-37. doi: 10.1016/j.jbiosc.2012.02.025 URL
[27]	李静, 张璐, 王霖慧, 等. 一株石化废水中脱氮产微生物絮凝剂菌株的鉴定与性能[J]. 安全与环境学报, 2017, 17(3):1117-1124.
	Li J, Zhang L, Wang LH, et al. Identification and characterization of a nitrogen removal bacterial strain with the flocculate capability in the oil-refinery sewage[J]. J Saf Environ, 2017, 17(3):1117-1124.
[28]	何腾霞, 李振轮, 杨珊. 好氧反硝化细菌及其去除亚硝酸盐的作用机理研究进展[J]. 过程工程学报, 2015, 15(1):174-180.
	He TX, Li ZL, Yang S. Research progress in aerobic denitrifier and its removing mechanism of nitrite[J]. Chin J Process Eng, 2015, 15(1):174-180.
[29]	Ji B, Yang K, Zhu L, et al. Aerobic denitrification:a review of important advances of the last 30 years[J]. Biotechnol Bioprocess Eng, 2015, 20(4):643-651. doi: 10.1007/s12257-015-0009-0 URL
[30]	魏巍, 黄廷林, 苏俊峰, 等. 1株贫营养好氧反硝化菌的分离鉴定及其脱氮特性[J]. 生态环境学报, 2010, 19(9):2166-2171.
	Wei W, Huang TL, Su JF, et al. Isolation and identification of an oligotrophic and aerobic denitrification and its denitrification characteristics[J]. Ecol Environ Sci, 2010, 19(9):2166-2171.
[31]	何腾霞, 李振轮. 耐冷好氧亚硝酸盐型反硝化细菌的鉴定及脱氮特性研究[J]. 生物技术通报, 2015, 31(10):191-198. doi: 10.13560/j.cnki.biotech.bull.1985.2015.10.002
	He TX, Li ZL. Identification and denitrification characterization of a psychrotrophic and aerobic nitrite-bacterium[J]. Biotechnol Bull, 2015, 31(10):191-198. doi: 10.13560/j.cnki.biotech.bull.1985.2015.10.002
[32]	李誉琦, 马佩钰, 刘涵, 等. 一株耐高温亚硝酸盐型反硝化细菌的鉴定及脱氮特性[J]. 生物技术通报, 2019, 35(9):194-201. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0130
	Li YQ, Ma PY, Liu H, et al. Identification and denitrification characters of a high-temperature-resistant nitrite-denitrifying bacterium[J]. Biotechnol Bull, 2019, 35(9):194-201.
[33]	杨洋, 左剑恶, 沈平, 等. 温度、pH值和有机物对厌氧氨氧化污泥活性的影响[J]. 环境科学, 2006, 27(4):691-695. pmid: 16767989
	Yang Y, Zuo JE, Shen P, et al. Influence of temperature, pH value and organic substance on activity of ANAMMOX sludge[J]. Environ Sci, 2006, 27(4):691-695. pmid: 16767989