好/厌氧条件下反硝化细菌脱氮特性与功能基因
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摘要
从西安市金盆水库沉积物中分离筛选出1株高效反硝化细菌,经形态学观察和16S r DNA测序鉴定为施氏假单胞菌(Pseudomonas stutzeri),命名为KK99.探究了该菌株在好/厌氧条件下的脱氮特性以及nar G、nir S和nos Z功能基因的表达水平.结果表明,KK99菌株在好/厌氧条件下,均能进行反硝化作用,且在两种不同的条件下均具有高效的脱氮效率.经过24h的培养,其总氮(TN)去除率分别为85.08%和89.05%.胞内功能基因nos Z和nir S的表达量较高,nos Z在好氧条件下的反硝化发挥主要作用,nir S在厌氧条件下的反硝化发挥主要作用,nar G在两种条件下的表达均较低.同时该菌株还具有同步硝化反硝化性能,在好氧条件下,能同时去除硝酸盐氮和氨氮,24 h时,TN去除率为76%.施氏假单胞菌(P.stutzeri KK99)将为微污染水体富营养化控制和总氮削减工程应用提供菌源保障.
An efficient aerobic denitrifying bacterium was isolated from the sediments of the Jin Pen Reservoir in Xi'an. The strain was identified by morphological features and 16 S r DNA sequences as Pseudomonas stutzeri and named it KK99. The denitrification characteristics of the strain and the expression level of the functional genes nar G,nir S,and nos Z in aerobic/anaerobic conditions were investigated. The results showed that the strain can carry out both anaerobic and aerobic denitrification and has a high efficiency of denitrification,which occurs under both aerobic and anaerobic conditions; after 24 h,the removal rates of total nitrogen( TN) were85. 08% and 89. 05%,respectively. Under both the conditions,the expression levels of the functional genes nos Z and nir S are high.nos Z plays a vital role in denitrification in the aerobic pathway,nir S plays a vital role in denitrification in the anaerobic pathway,and nar G expression is low under both the conditions. At the same time,simultaneous nitrification and denitrification( SND) capacity of the strain was observed when nitrate and ammonium salts were the nitrogen sources,with the total nitrogen removal rate being 76%within 24 h in aerobic conditions. The P. stutzeri KK99 strain can be used for controlling eutrophication of micro-polluted water,and the application of total nitrogen reduction engineering.
An efficient aerobic denitrifying bacterium was isolated from the sediments of the Jin Pen Reservoir in Xi'an. The strain was identified by morphological features and 16 S r DNA sequences as Pseudomonas stutzeri and named it KK99. The denitrification characteristics of the strain and the expression level of the functional genes nar G,nir S,and nos Z in aerobic/anaerobic conditions were investigated. The results showed that the strain can carry out both anaerobic and aerobic denitrification and has a high efficiency of denitrification,which occurs under both aerobic and anaerobic conditions; after 24 h,the removal rates of total nitrogen( TN) were85. 08% and 89. 05%,respectively. Under both the conditions,the expression levels of the functional genes nos Z and nir S are high.nos Z plays a vital role in denitrification in the aerobic pathway,nir S plays a vital role in denitrification in the anaerobic pathway,and nar G expression is low under both the conditions. At the same time,simultaneous nitrification and denitrification( SND) capacity of the strain was observed when nitrate and ammonium salts were the nitrogen sources,with the total nitrogen removal rate being 76%within 24 h in aerobic conditions. The P. stutzeri KK99 strain can be used for controlling eutrophication of micro-polluted water,and the application of total nitrogen reduction engineering.
引文
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[13]Zhu L,Ding W,Feng L J,et al.Isolation of aerobic denitrifiers and characterization for their potential application in the bioremediation of oligotrophic ecosystem[J].Bioresource Technology,2012,108:1-7.
[14]Xu Y,He T X,Li Z L,et al.Nitrogen removal characteristics of Pseudomonas putida Y-9 capable of heterotrophic nitrification and aerobic denitrification at low temperature[J].Bio Med Research International,2017,2017:1429018.
[15]康鹏亮,张海涵,黄廷林,等.湖库沉积物好氧反硝化菌群脱氮特性及种群结构[J].环境科学,2018,39(5):2431-2437.Kang P L,Zhang H H,Huang T L,et al.Denitrification characteristics and community structure of aerobic denitrifiers from lake and reservoir sediments[J].Environmental Science,2018,39(5):2431-2437.
[16]黄廷林,张丽娜,张海涵,等.一株贫营养异养硝化-好氧反硝化菌的筛选及脱氮特性[J].生态环境学报,2015,24(1):113-120.Huang T L,Zhang L N,Zhang H H,et al.Screening and nitrogen removal characteristics of a heterotrophic nitrificationaerobic denitrification strain[J].Ecology and Environmental Sciences,2015,24(1):113-120.
[17]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 Q H,Yu D S,Zhang P Y,et al.Identification and nitrogen removal characteristics of a heterotrophic nitrification-aerobic denitrification strain isolated from marine environment[J].Environmental Science,2016,37(2):647-654.
[18]López-Gutiérrez J C,Henry S,Hallet S,et al.Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR[J].Journal of Microbiological Methods,2004,57(3):399-407.
[19]Braker G,Fesefeldt A,Witzel K P.Development of PCR primer systems for amplification of nitrite reductase genes(nir K and nir S)to detect denitrifying bacteria in environmental samples[J].Applied and Environmental Microbiology,1998,64(10):3769-3775.
[20]Scala D J,Kerkhof L J.Nitrous oxide reductase(nos Z)genespecific PCR primers for detection of denitrifiers and three nos Z genes from marine sediments[J].FEMS Microbiology Letters,1998,162(1):61-68.
[21]Harms G,Layton A C,Dionisi H M,et al.Real-Time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant[J].Environmental Science&Technology,2003,37(2):343-351.
[22]Huang T L,Guo L,Zhang H H,et al.Nitrogen-removal efficiency of a novel aerobic denitrifying bacterium,Pseudomonas stutzeri strain ZF31,isolated from a drinking-water reservoir[J].Bioresource Technology,2015,196:209-216.
[23]国家环境保护总局.水和废水监测分析方法[M].(第四版).北京:中国环境科学出版社,2002.
[24]Ji B,Yang K,Wang H Y,et al.Aerobic denitrification by Pseudomonas stutzeri C3incapable of heterotrophic nitrification[J].Bioprocess and Biosystems Engineering,2015,38(2):407-409.
[25]肖继波,江惠霞,褚淑祎.不同氮源下好氧反硝化菌Defluvibacter lusatiensis str.DN7的脱氮特性[J].生态学报,2012,32(20):6463-6470.Xiao J B,Jiang H X,Chu S Y.Denitrification characteristics of an aerobic denitrifying bacterium Defluvibacter lusatiensis str.DN7 using different sources of nitrogen[J].Acta Ecologica Sinica,2012,32(20):6463-6470.
[26]Baggs E M.A review of stable isotope techniques for N2O source partitioning in soils:recent progress,remaining challenges and future considerations[J].Rapid Communications in Mass Spectrometry,2008,22(11):1664-1672.
[27]颜薇芝,张汉强,余从田,等.1株异养硝化好氧反硝化不动杆菌的分离及脱氮性能[J].环境工程学报,2017,11(7):4419-4428.Yan W Z,Zhang H Q,Yu C T,et al.Isolation of Acinetobacter sp.YN3 and its heterotrophic nitrification-aerobic denitrification characters[J].Chinese Journal of Environmental Engineering,2017,11(7):4419-4428.
[28]颜薇芝,郝健,孙俊松,等.拉乌尔菌sari01的分离及其异养硝化好氧反硝化特性[J].环境科学,2016,37(7):2673-2680.Yan W Z,Hao J,Sun J S,et al.Isolation of Raoultella sp.sari01 and its heterotrophic nitrification-aerobic denitrification characteristics[J].Environmental Science,2016,37(7):2673-2680.
[29]Sun Y L,Li A,Zhang X N,et al.Regulation of dissolved oxygen from accumulated nitrite during the heterotrophic nitrification and aerobic denitrification of Pseudomonas stutzeri T13[J].Applied Microbiology and Biotechnology,2015,99(7):3243-3248.
[30]Bell L C,Richardson D J,Ferguson S J.Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha:the periplasmic enzyme catalyzes the first step in aerobic denitrification[J].FEBS Letters,1990,265(1-2):85-87.
[2]曾明正,黄廷林,邱晓鹏,等.我国北方温带水库——周村水库季节性热分层现象及其水质响应特性[J].环境科学,2016,37(4):1337-1344.Zeng M Z,Huang T L,Qiu X P,et al.Seasonal stratification and the response of water quality of a temperate reservoir-Zhoucun Reservoir in north of China[J].Environmental Science,2016,37(4):1337-1344.
[3]周石磊,黄廷林,白士远,等.贫营养好氧反硝化菌群的水库原位投菌脱氮特性[J].微生物学报,2016,56(4):590-602.Zhou S L,Huang T L,Bai S Y,et al.Nitrogen removal characteristics of mixed aerobic denitrification bacteria under insitu biological inoculation[J].Acta Microbiologica Sinica,2016,56(4):590-602.
[4]张春华,黄廷林,方开凯,等.周村水库主库区热分层初期氮素降低的驱动因子分析[J].环境科学,2016,37(11):4187-4193.Zhang C H,Huang T L,Fang K K,et al.Analysis of driving factors on the nitrogen decrease in the early stage of the thermal stratification in main area of Zhoucun Reservoir[J].Environmental Science,2016,37(11):4187-4193.
[5]Robertson L A,Kuenen J G.Thiosphaera pantotropha gen.nov.sp.nov.,a facultatively anaerobic,facultatively autotrophic sulphur bacterium[J].Microbiology,1983,129(9):2847-2855.
[6]Ji B,Yang K,Zhu L,et al.Aerobic denitrification:a review of important advances of the last 30 years[J].Biotechnology and Bioprocess Engineering,2015,20(4):643-651.
[7]Zheng M S,He D,Ma T,et al.Reducing NO and N2O emission during aerobic denitrification by newly isolated Pseudomonas stutzeri PCN-1[J].Bioresource Technology,2014,162:80-88.
[8]Guo L J,Zhao B,An Q,et al.Characteristics of a novel aerobic denitrifying bacterium,Enterobacter cloacae Strain HNR[J].Applied Biochemistry and Biotechnology,2016,178(5):947-959.
[9]Su J F,Zhang K,Huang T L,et al.Denitrification characteristics of a newly isolated indigenous aerobic denitrifying bacterium under oligotrophic conditions.[J].Water Science&Technology,2015,72(7):1082-1088.
[10]姜怡,曹艳茹,赵立兴,等.超声波处理土样分离放线菌[J].微生物学报,2010,50(8):1094-1097.Jiang Y,Cao Y R,Zhao L X,et al.Ultrasonic treatment of soil samples for actinomycete isolation[J].Acta Microbiologica Sinica,2010,50(8):1094-1097.
[11]李小义,王丽萍,赖其良,等.一株卓贝尔氏菌F13-1好氧反硝化特性及其反硝化基因的初步研究[J].应用海洋学学报,2016,35(1):122-129.Li X Y,Wang L P,Lai Q L,et al.Preliminary study on aerobic denitrification characteristics of Zobellella sp.F13-1[J].Journal of Applied Oceanography,2016,35(1):122-129.
[12]牟东阳,靳鹏飞,彭永臻,等.1株异养硝化-好氧反硝化细菌DK1的分离鉴定及其脱氮特性[J].环境科学,2017,38(11):4763-4773.Mu D Y,Jin P F,Peng Y Z,et al.Identification and nitrogen removal characteristics of the heterotrophic nitrification and aerobic denitrification bacterial strain DK1[J].Environmental Science,2017,38(11):4763-4773.
[13]Zhu L,Ding W,Feng L J,et al.Isolation of aerobic denitrifiers and characterization for their potential application in the bioremediation of oligotrophic ecosystem[J].Bioresource Technology,2012,108:1-7.
[14]Xu Y,He T X,Li Z L,et al.Nitrogen removal characteristics of Pseudomonas putida Y-9 capable of heterotrophic nitrification and aerobic denitrification at low temperature[J].Bio Med Research International,2017,2017:1429018.
[15]康鹏亮,张海涵,黄廷林,等.湖库沉积物好氧反硝化菌群脱氮特性及种群结构[J].环境科学,2018,39(5):2431-2437.Kang P L,Zhang H H,Huang T L,et al.Denitrification characteristics and community structure of aerobic denitrifiers from lake and reservoir sediments[J].Environmental Science,2018,39(5):2431-2437.
[16]黄廷林,张丽娜,张海涵,等.一株贫营养异养硝化-好氧反硝化菌的筛选及脱氮特性[J].生态环境学报,2015,24(1):113-120.Huang T L,Zhang L N,Zhang H H,et al.Screening and nitrogen removal characteristics of a heterotrophic nitrificationaerobic denitrification strain[J].Ecology and Environmental Sciences,2015,24(1):113-120.
[17]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 Q H,Yu D S,Zhang P Y,et al.Identification and nitrogen removal characteristics of a heterotrophic nitrification-aerobic denitrification strain isolated from marine environment[J].Environmental Science,2016,37(2):647-654.
[18]López-Gutiérrez J C,Henry S,Hallet S,et al.Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR[J].Journal of Microbiological Methods,2004,57(3):399-407.
[19]Braker G,Fesefeldt A,Witzel K P.Development of PCR primer systems for amplification of nitrite reductase genes(nir K and nir S)to detect denitrifying bacteria in environmental samples[J].Applied and Environmental Microbiology,1998,64(10):3769-3775.
[20]Scala D J,Kerkhof L J.Nitrous oxide reductase(nos Z)genespecific PCR primers for detection of denitrifiers and three nos Z genes from marine sediments[J].FEMS Microbiology Letters,1998,162(1):61-68.
[21]Harms G,Layton A C,Dionisi H M,et al.Real-Time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant[J].Environmental Science&Technology,2003,37(2):343-351.
[22]Huang T L,Guo L,Zhang H H,et al.Nitrogen-removal efficiency of a novel aerobic denitrifying bacterium,Pseudomonas stutzeri strain ZF31,isolated from a drinking-water reservoir[J].Bioresource Technology,2015,196:209-216.
[23]国家环境保护总局.水和废水监测分析方法[M].(第四版).北京:中国环境科学出版社,2002.
[24]Ji B,Yang K,Wang H Y,et al.Aerobic denitrification by Pseudomonas stutzeri C3incapable of heterotrophic nitrification[J].Bioprocess and Biosystems Engineering,2015,38(2):407-409.
[25]肖继波,江惠霞,褚淑祎.不同氮源下好氧反硝化菌Defluvibacter lusatiensis str.DN7的脱氮特性[J].生态学报,2012,32(20):6463-6470.Xiao J B,Jiang H X,Chu S Y.Denitrification characteristics of an aerobic denitrifying bacterium Defluvibacter lusatiensis str.DN7 using different sources of nitrogen[J].Acta Ecologica Sinica,2012,32(20):6463-6470.
[26]Baggs E M.A review of stable isotope techniques for N2O source partitioning in soils:recent progress,remaining challenges and future considerations[J].Rapid Communications in Mass Spectrometry,2008,22(11):1664-1672.
[27]颜薇芝,张汉强,余从田,等.1株异养硝化好氧反硝化不动杆菌的分离及脱氮性能[J].环境工程学报,2017,11(7):4419-4428.Yan W Z,Zhang H Q,Yu C T,et al.Isolation of Acinetobacter sp.YN3 and its heterotrophic nitrification-aerobic denitrification characters[J].Chinese Journal of Environmental Engineering,2017,11(7):4419-4428.
[28]颜薇芝,郝健,孙俊松,等.拉乌尔菌sari01的分离及其异养硝化好氧反硝化特性[J].环境科学,2016,37(7):2673-2680.Yan W Z,Hao J,Sun J S,et al.Isolation of Raoultella sp.sari01 and its heterotrophic nitrification-aerobic denitrification characteristics[J].Environmental Science,2016,37(7):2673-2680.
[29]Sun Y L,Li A,Zhang X N,et al.Regulation of dissolved oxygen from accumulated nitrite during the heterotrophic nitrification and aerobic denitrification of Pseudomonas stutzeri T13[J].Applied Microbiology and Biotechnology,2015,99(7):3243-3248.
[30]Bell L C,Richardson D J,Ferguson S J.Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha:the periplasmic enzyme catalyzes the first step in aerobic denitrification[J].FEBS Letters,1990,265(1-2):85-87.
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