过表达亚硝酸盐还原酶的重组大肠杆菌辅助污水短程硝化
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摘要
【目的】为了体现并突出亚硝酸盐还原酶在污水脱氮以及短程硝化中的重要性,对过表达亚硝酸盐还原酶的大肠杆菌进行了污水脱氮的研究。【方法】通过转化带有亚硝酸盐还原酶基因的重组质粒,将亚硝酸盐还原酶在大肠杆菌中过表达,通过分析重组大肠杆菌的产物研究了该酶的表达及还原亚硝酸盐的情况,通过将该重组菌与已报道的硝化-反硝化细菌或生活污水进行混合培养,研究重组菌用于辅助氨氮去除的短程硝化能力。【结果】重组大肠杆菌能正确表达亚硝酸盐还原酶,OD600=2.0的菌悬液在2 h内还原约1 mmol/L的亚硝酸盐,并产生几乎等量的一氧化氮;重组大肠杆菌与Acinetobacter sp.YF14菌株等比例混合时,12 h能够提高氨氮脱氮效率约(36.0±7.4)%,且在4 h时,最大亚硝酸盐的积累量减少37%;重组大肠杆菌(OD600=1.0)12 h内能够提高污水厂活性污泥的脱氮效率约(31.0±5.7)%,且未检测到亚硝酸盐和硝酸盐的积累;溶氧水平对于亚硝酸盐还原酶重组菌辅助脱氮具有明显的影响,中等溶氧量[(6.4?0.7)mg/L]时脱氮效果最好。【结论】过表达亚硝酸盐还原酶的大肠杆菌可以提高污水脱氮的短程硝化能力。
[Objective] To highlight the significance of nitrite reductase in wastewater denitrification and shortcut nitrification, we studied wastewater denitrification by Escherichia coli expressing nitrite reductase. [Methods] Nitrite reductase was overexpressed by transformed a recombinant plasmid with nir gene in E. coli. Expression of nitrite reductase and metabolism of nitrite were studied by detecting the products in the whole cell of recombinant E. coli. To evaluate the function of recombinant E. coli used for shortcut nitrification, we measured the denitrification efficiency of the mixture consisting of the recombinant E. coli and YF14 that was a nitrifying and denitrifying bacterium. [Results] The recombinant E. coli could express nitrite reductase, reduced about 1 mmol/L nitrite within 2 hours and produced almost equal amounts of nitric oxide(OD600=2.0 bacterial suspension). When the recombinant E. coli and YF14 were mixed in equal proportions, the ammonia nitrogen removal efficiency increased by about(36.0±7.4)% within 12 hours, and the maximum accumulated amount of nitrite decreased by about 37%. Recombinant E. coli(OD600=1.0) significantly improved the ammonia nitrogen removal efficiency of activated sludge by about(31.0±5.7)% within 12 hours, and no nitrite and nitrate were detected. Oxygen supply affected ammonia nitrogen removal with optimal DO of(6.4?0.7) mg/L. [Conclusion] Recombinant E. coli with nitrite reductase can contribute to the shortcut nitrification capacity of wastewater.
[Objective] To highlight the significance of nitrite reductase in wastewater denitrification and shortcut nitrification, we studied wastewater denitrification by Escherichia coli expressing nitrite reductase. [Methods] Nitrite reductase was overexpressed by transformed a recombinant plasmid with nir gene in E. coli. Expression of nitrite reductase and metabolism of nitrite were studied by detecting the products in the whole cell of recombinant E. coli. To evaluate the function of recombinant E. coli used for shortcut nitrification, we measured the denitrification efficiency of the mixture consisting of the recombinant E. coli and YF14 that was a nitrifying and denitrifying bacterium. [Results] The recombinant E. coli could express nitrite reductase, reduced about 1 mmol/L nitrite within 2 hours and produced almost equal amounts of nitric oxide(OD600=2.0 bacterial suspension). When the recombinant E. coli and YF14 were mixed in equal proportions, the ammonia nitrogen removal efficiency increased by about(36.0±7.4)% within 12 hours, and the maximum accumulated amount of nitrite decreased by about 37%. Recombinant E. coli(OD600=1.0) significantly improved the ammonia nitrogen removal efficiency of activated sludge by about(31.0±5.7)% within 12 hours, and no nitrite and nitrate were detected. Oxygen supply affected ammonia nitrogen removal with optimal DO of(6.4?0.7) mg/L. [Conclusion] Recombinant E. coli with nitrite reductase can contribute to the shortcut nitrification capacity of wastewater.
引文
[1]Hao XD,Zhang ZJ.Nitrogen pollution of water and its control.Environmental Pollution&Control,1990,12(4):25–27,47.(in Chinese)郝晓地,张自杰.水体氮污染及其防治对策.环境污染与防治,1990,12(4):25–27,47.
[2]Li XY,Xu F.Progress of biological treatment technology of micro-polluted water with low concentration of ammonia-nitrogen.Pollution Control Technology,2012,25(4):31–34.(in Chinese)李学尧,徐富.微污染水体中低浓度氨氮的生物处理技术进展.污染防治技术,2012,25(4):31–34.
[3]Wang H,Li HB.Study and development of biotechnology of ammonia removal in wastewater.Journal of Henan Normal University(Natural Science),2008,36(5):97–100,103.(in Chinese)王洪,李海波.氨氮废水生物处理工艺及研究进展.河南师范大学学报(自然科学版),2008,36(5):97–100,103.
[4]Yuan LJ,Peng DC,Wang ZY.Biological nitrogen removal by shortcut nitrification-denitrification.China Water&Wastewater,2000,16(2):29–31.(in Chinese)袁林江,彭党聪,王志盈.短程硝化-反硝化生物脱氮.中国给水排水,2000,16(2):29–31.
[5]Zhu GB,Peng YZ,Guo JH.Biological nitrogen removal with nitrification and denitrification via nitrite pathway.Journal of Harbin Institute of Technology,2008,40(10):1552–1557.(in Chinese)祝贵兵,彭永臻,郭建华.短程硝化反硝化生物脱氮技术.哈尔滨工业大学学报,2008,40(10):1552–1557.
[6]Liu CX,Hu HY,Peng DC,Wang ZY,Qian Y.Short-cut nitrification and denitrification for treatment of high ammonia content wastewater from coking plant.China Water&Wastewater,2003,19(8):11–14.(in Chinese)刘超翔,胡洪营,彭党聪,王志盈,钱易.短程硝化反硝化工艺处理焦化高氨废水.中国给水排水,2003,19(8):11–14.
[7]Shang HL,Peng YZ,Zhang JR,Wang SY.The effect of temperature on short-cut nitrification and denitrification.Acta Scientiae Circumstantiae,2009,29(3):516–520.(in Chinese)尚会来,彭永臻,张静蓉,王淑莹.温度对短程硝化反硝化的影响.环境科学学报,2009,29(3):516–520.
[8]Zhi XH,Wang HW,Ding F,Peng YZ,Ma LM.The study of shortcut nitrification-denitrification at normal temperature.Research of Environmental Sciences,2006,19(1):26–29.(in Chinese)支霞辉,王红武,丁峰,彭永臻,马鲁铭.常温条件下短程硝化反硝化生物脱氮研究.环境科学研究,2006,19(1):26–29.
[9]Ma Y,Li J,Wu XL,Peng YZ.A/O pilot-scale nitrogen removal process treating domestic wastewater III.The study of nitritation process control.Acta Scientiae Circumstantiae,2006,26(5):716–721.(in Chinese)马勇,李军,吴学蕾,彭永臻.A/O生物脱氮工艺处理生活污水中试(三)短程硝化过程控制的研究.环境科学学报,2006,26(5):716–721.
[10]Li JY,Lin WT,Luo JF,Tian GL.Community structure of microbes involved in nitrification and denitrification in typical shrimp-farming water.Acta Microbiologica Sinica,2012,52(4):478–488.(in Chinese)李敬源,林炜铁,罗剑飞,田国梁.典型对虾养殖水体中参与硝化与反硝化过程的微生物群落结构.微生物学报,2012,52(4):478–488.
[11]He X,Lv J,He YL,Zhao B,Li CJ.Study progress on the mechanism of heterotrophic nitrification.Acta Microbiologica Sinica,2006,46(5):844–847.(in Chinese)何霞,吕剑,何义亮,赵彬,李春杰.异养硝化机理的研究进展.微生物学报,2006,46(5):844–847.
[12]Xin YF,Zhao TY,Qu XH.Distribution,structure and sequence alignment,and metagenomics analysis of two nitrite reductases with NO forming.Acta Microbiologica Sinica,2017,57(3):597–608.(in Chinese)辛玉峰,赵天颖,曲晓华.两类产NO的亚硝酸盐还原酶的分布,结构与序列比较及宏基因组分析.微生物学报,2017,57(3):597–608.
[13]Zumft WG.Cell biology and molecular basis of denitrification.Microbiology and Molecular Biology Reviews,1997,61(4):533–616.
[14]Xin YF,Qu XH,Yuan MD,Jing YD.Isolation and identification of a heterotrophic nitrifying and aerobic denitrifying Acinetobacter sp.YF14 and its denitrification activity.Acta Microbiologica Sinica,2011,51(12):1646–1654.(in Chinese)辛玉峰,曲晓华,袁梦冬,荆延德.一株异养硝化-反硝化不动杆菌的分离鉴定及脱氮活性.微生物学报,2011,51(12):1646–1654.
[15]Tiso M,Schechter AN.Nitrate reduction to nitrite,nitric oxide and ammonia by gut bacteria under physiological conditions.PLo S One,2015,10(3):e0119712.
[16]Ma Y,Wang SY,Zeng W,Peng YZ,Zhou L.A/O pilot-scale nitrogen removal process treating domestic wastewaterⅠ.The study of short-cut nitrification and denitrification.Acta Scientiae Circumstantiae,2006,26(5):703–709.(in Chinese)马勇,王淑莹,曾薇,彭永臻,周利.A/O生物脱氮工艺处理生活污水中试(一)短程硝化反硝化的研究.环境科学学报,2006,26(5):703–709.
[17]Qu Y,Zhang PY,Yu DS,Guo SS,Yang RX.Bioaugmentation for shortcut nitrification in SBR treating for sewage containing sea water by nitrification-aerobic denitrification bacteria.Environmental Science,2010,31(10):2376–2384.(in Chinese)曲洋,张培玉,于德爽,郭沙沙,杨瑞霞.异养硝化/好氧反硝化菌生物强化含海水污水的SBR短程硝化系统初探.环境科学,2010,31(10):2376–2384.
[18]Pan D,Huang QY,Chen WL.Screening and identification of two heterotrophic nitrifying bacteria and characterization of their capacity for nitrogen removal.Acta Microbiologica Sinica,2011,51(10):1382–1389.(in Chinese)潘丹,黄巧云,陈雯莉.两株异养硝化细菌的分离鉴定及其脱氮特性.微生物学报,2011,51(10):1382–1389.
[19]Sun XM,Li QF,Zhang Y,Liu HD,Zhao J,Qu KM.Phylogenetic analysis and nitrogen removal characteristics of a heterotrophic nitrifying-aerobic denitrifying bacteria strain from marine environment.Acta Microbiologica Sinica,2012,52(6):687–695.(in Chinese)孙雪梅,李秋芬,张艳,刘淮德,赵俊,曲克明.一株海水异养硝化-好氧反硝化菌系统发育及脱氮特性.微生物学报,2012,52(6):687–695.
[20]Zhang GY,Chen MC,Han RY,Min H.Isolation,identification and phylogenetic analysis of a heterotrophic nitrifier.Acta Microbiologica Sinica,2003,43(2):156–161.(in Chinese)张光亚,陈美慈,韩如旸,闵航.一株异养硝化细菌的分离及系统发育分析.微生物学报,2003,43(2):156–161.
[21]Hong YG,Li M,Gu JD.Bacterial anaerobic ammonia oxidation(Anammox)in the marine nitrogen cycle—a review.Acta Microbiologica Sinica,2009,49(3):281–286.(in Chinese)洪义国,李猛,顾继东.海洋氮循环中细菌的厌氧氨氧化.微生物学报,2009,49(3):281–286.
[22]Zhao YG,Zheng P.Composition,structure and function of anammoxosome—a review.Acta Microbiologica Sinica,2016,56(1):8–18.(in Chinese)赵弋戈,郑平.厌氧氨氧化体的组成、结构与功能.微生物学报,2016,56(1):8–18.
[23]Wang H,Pei WZ,Li XD,Hao C,Zeng KM.Removing nitrogen from low-C/N-piggery-wastewater using shortcut nitrification/denitrification-ANAMMOX.Environmental Science,2009,30(3):815–821.(in Chinese)王欢,裴伟征,李旭东,郝纯,曾抗美.低碳氮比猪场废水短程硝化反硝化-厌氧氨氧化脱氮.环境科学,2009,30(3):815–821.
[24]Yang YN,Yang DH.Review on mechanism and application status of bioaugmentation technique in wastewater treatment.Journal of Anhui Agricultural Sciences,2015,43(1):230–233.(in Chinese)杨懿宁,杨殿海.生物强化技术在污水处理中的作用机理及应用现状.安徽农业科学,2015,43(1):230–233.
[2]Li XY,Xu F.Progress of biological treatment technology of micro-polluted water with low concentration of ammonia-nitrogen.Pollution Control Technology,2012,25(4):31–34.(in Chinese)李学尧,徐富.微污染水体中低浓度氨氮的生物处理技术进展.污染防治技术,2012,25(4):31–34.
[3]Wang H,Li HB.Study and development of biotechnology of ammonia removal in wastewater.Journal of Henan Normal University(Natural Science),2008,36(5):97–100,103.(in Chinese)王洪,李海波.氨氮废水生物处理工艺及研究进展.河南师范大学学报(自然科学版),2008,36(5):97–100,103.
[4]Yuan LJ,Peng DC,Wang ZY.Biological nitrogen removal by shortcut nitrification-denitrification.China Water&Wastewater,2000,16(2):29–31.(in Chinese)袁林江,彭党聪,王志盈.短程硝化-反硝化生物脱氮.中国给水排水,2000,16(2):29–31.
[5]Zhu GB,Peng YZ,Guo JH.Biological nitrogen removal with nitrification and denitrification via nitrite pathway.Journal of Harbin Institute of Technology,2008,40(10):1552–1557.(in Chinese)祝贵兵,彭永臻,郭建华.短程硝化反硝化生物脱氮技术.哈尔滨工业大学学报,2008,40(10):1552–1557.
[6]Liu CX,Hu HY,Peng DC,Wang ZY,Qian Y.Short-cut nitrification and denitrification for treatment of high ammonia content wastewater from coking plant.China Water&Wastewater,2003,19(8):11–14.(in Chinese)刘超翔,胡洪营,彭党聪,王志盈,钱易.短程硝化反硝化工艺处理焦化高氨废水.中国给水排水,2003,19(8):11–14.
[7]Shang HL,Peng YZ,Zhang JR,Wang SY.The effect of temperature on short-cut nitrification and denitrification.Acta Scientiae Circumstantiae,2009,29(3):516–520.(in Chinese)尚会来,彭永臻,张静蓉,王淑莹.温度对短程硝化反硝化的影响.环境科学学报,2009,29(3):516–520.
[8]Zhi XH,Wang HW,Ding F,Peng YZ,Ma LM.The study of shortcut nitrification-denitrification at normal temperature.Research of Environmental Sciences,2006,19(1):26–29.(in Chinese)支霞辉,王红武,丁峰,彭永臻,马鲁铭.常温条件下短程硝化反硝化生物脱氮研究.环境科学研究,2006,19(1):26–29.
[9]Ma Y,Li J,Wu XL,Peng YZ.A/O pilot-scale nitrogen removal process treating domestic wastewater III.The study of nitritation process control.Acta Scientiae Circumstantiae,2006,26(5):716–721.(in Chinese)马勇,李军,吴学蕾,彭永臻.A/O生物脱氮工艺处理生活污水中试(三)短程硝化过程控制的研究.环境科学学报,2006,26(5):716–721.
[10]Li JY,Lin WT,Luo JF,Tian GL.Community structure of microbes involved in nitrification and denitrification in typical shrimp-farming water.Acta Microbiologica Sinica,2012,52(4):478–488.(in Chinese)李敬源,林炜铁,罗剑飞,田国梁.典型对虾养殖水体中参与硝化与反硝化过程的微生物群落结构.微生物学报,2012,52(4):478–488.
[11]He X,Lv J,He YL,Zhao B,Li CJ.Study progress on the mechanism of heterotrophic nitrification.Acta Microbiologica Sinica,2006,46(5):844–847.(in Chinese)何霞,吕剑,何义亮,赵彬,李春杰.异养硝化机理的研究进展.微生物学报,2006,46(5):844–847.
[12]Xin YF,Zhao TY,Qu XH.Distribution,structure and sequence alignment,and metagenomics analysis of two nitrite reductases with NO forming.Acta Microbiologica Sinica,2017,57(3):597–608.(in Chinese)辛玉峰,赵天颖,曲晓华.两类产NO的亚硝酸盐还原酶的分布,结构与序列比较及宏基因组分析.微生物学报,2017,57(3):597–608.
[13]Zumft WG.Cell biology and molecular basis of denitrification.Microbiology and Molecular Biology Reviews,1997,61(4):533–616.
[14]Xin YF,Qu XH,Yuan MD,Jing YD.Isolation and identification of a heterotrophic nitrifying and aerobic denitrifying Acinetobacter sp.YF14 and its denitrification activity.Acta Microbiologica Sinica,2011,51(12):1646–1654.(in Chinese)辛玉峰,曲晓华,袁梦冬,荆延德.一株异养硝化-反硝化不动杆菌的分离鉴定及脱氮活性.微生物学报,2011,51(12):1646–1654.
[15]Tiso M,Schechter AN.Nitrate reduction to nitrite,nitric oxide and ammonia by gut bacteria under physiological conditions.PLo S One,2015,10(3):e0119712.
[16]Ma Y,Wang SY,Zeng W,Peng YZ,Zhou L.A/O pilot-scale nitrogen removal process treating domestic wastewaterⅠ.The study of short-cut nitrification and denitrification.Acta Scientiae Circumstantiae,2006,26(5):703–709.(in Chinese)马勇,王淑莹,曾薇,彭永臻,周利.A/O生物脱氮工艺处理生活污水中试(一)短程硝化反硝化的研究.环境科学学报,2006,26(5):703–709.
[17]Qu Y,Zhang PY,Yu DS,Guo SS,Yang RX.Bioaugmentation for shortcut nitrification in SBR treating for sewage containing sea water by nitrification-aerobic denitrification bacteria.Environmental Science,2010,31(10):2376–2384.(in Chinese)曲洋,张培玉,于德爽,郭沙沙,杨瑞霞.异养硝化/好氧反硝化菌生物强化含海水污水的SBR短程硝化系统初探.环境科学,2010,31(10):2376–2384.
[18]Pan D,Huang QY,Chen WL.Screening and identification of two heterotrophic nitrifying bacteria and characterization of their capacity for nitrogen removal.Acta Microbiologica Sinica,2011,51(10):1382–1389.(in Chinese)潘丹,黄巧云,陈雯莉.两株异养硝化细菌的分离鉴定及其脱氮特性.微生物学报,2011,51(10):1382–1389.
[19]Sun XM,Li QF,Zhang Y,Liu HD,Zhao J,Qu KM.Phylogenetic analysis and nitrogen removal characteristics of a heterotrophic nitrifying-aerobic denitrifying bacteria strain from marine environment.Acta Microbiologica Sinica,2012,52(6):687–695.(in Chinese)孙雪梅,李秋芬,张艳,刘淮德,赵俊,曲克明.一株海水异养硝化-好氧反硝化菌系统发育及脱氮特性.微生物学报,2012,52(6):687–695.
[20]Zhang GY,Chen MC,Han RY,Min H.Isolation,identification and phylogenetic analysis of a heterotrophic nitrifier.Acta Microbiologica Sinica,2003,43(2):156–161.(in Chinese)张光亚,陈美慈,韩如旸,闵航.一株异养硝化细菌的分离及系统发育分析.微生物学报,2003,43(2):156–161.
[21]Hong YG,Li M,Gu JD.Bacterial anaerobic ammonia oxidation(Anammox)in the marine nitrogen cycle—a review.Acta Microbiologica Sinica,2009,49(3):281–286.(in Chinese)洪义国,李猛,顾继东.海洋氮循环中细菌的厌氧氨氧化.微生物学报,2009,49(3):281–286.
[22]Zhao YG,Zheng P.Composition,structure and function of anammoxosome—a review.Acta Microbiologica Sinica,2016,56(1):8–18.(in Chinese)赵弋戈,郑平.厌氧氨氧化体的组成、结构与功能.微生物学报,2016,56(1):8–18.
[23]Wang H,Pei WZ,Li XD,Hao C,Zeng KM.Removing nitrogen from low-C/N-piggery-wastewater using shortcut nitrification/denitrification-ANAMMOX.Environmental Science,2009,30(3):815–821.(in Chinese)王欢,裴伟征,李旭东,郝纯,曾抗美.低碳氮比猪场废水短程硝化反硝化-厌氧氨氧化脱氮.环境科学,2009,30(3):815–821.
[24]Yang YN,Yang DH.Review on mechanism and application status of bioaugmentation technique in wastewater treatment.Journal of Anhui Agricultural Sciences,2015,43(1):230–233.(in Chinese)杨懿宁,杨殿海.生物强化技术在污水处理中的作用机理及应用现状.安徽农业科学,2015,43(1):230–233.