邻苯二甲酸二甲酯对荧光假单胞菌的损伤研究
|
摘要
为探究邻苯二甲酸二甲酯(Dimethyl phthalate,DMP)对细菌的毒理作用,选择荧光假单胞菌(Pseudomonas fluorescens)为受试菌株,采用平板计数试验、对数生长数学模型、细菌表面Zeta电位、荧光偏振法、透射电镜和超高效液相色谱法,测定了P.fluorescens的比生长速率、代时、细胞表面电势、细胞膜流动性、细胞损伤程度和DMP在细胞内外分布,研究DMP对P.fluorescens的细胞毒性。结果表明:经0、10、20、40 mg·L~(-1)的DMP暴露后,细菌比生长速率减小,而代时增加;细菌表面Zeta电位从-12.21±0.56mV逐渐降低为-18.13±0.67 mV,同时DMP增加了细菌细胞膜的流动性,导致细胞明显变形、质壁分离严重并伴有内容物的泄露;在细胞内,DMP累积浓度从0.11±0.10 mg·L~(-1)增加到6.78±0.25 mg·L~(-1);在细胞外,DMP累积浓度从8.44±0.37 mg·L~(-1)增加到30.52±0.88 mg·L~(-1)。研究表明,DMP破坏了P.fluorescens的细胞壁和细胞膜,影响了细菌正常生长。
Dimethyl phthalate(DMP)is a ubiquitous pollutant that is very harmful to microorganisms.Pseudomonas fluorescens(P.fluorescens)is one of the most important beneficial bacteria in the environment.To explore the toxicological effects of DMP on bacteria,P.fluorescens was selected and investigated using several bio-techniques,such as the polarization method,transmission electron microscopy,and ultra-high performance liquid chromatography.The results showed that the specific growth rate of P.fluorescens was slower,but the generation time was extended under the 10,20 mg·L~(-1),and 40 mg·L~(-1)DMP treatments.The zeta potential on the surface of P.fluorescens decreased gradually from-12.21±0.56 mV to-18.13±0.67 mV under the 0,10,20 mg·L~(-1),and 40 mg·L~(-1)DMP treatments.Meanwhile,the mobility of the cell membrane increased by the DMP treatment and resulted in obvious cell deformation,severe plasmolysis,and leakage of dissoluble matter.The cumulative concentration of DMP inside the cells increased from 0.11±0.10 mg·L~(-1)to 6.78±0.25 mg·L~(-1),and the cumulative concentration outside the cells increased from 8.44±0.37 mg·L~(-1)to 30.52±0.88 mg·L~(-1),under the 10,20 mg·L~(-1),and 40 mg·L~(-1)DMP treatments.Therefore,we conclude that DMP destroys the cell wall and the cell membrane of P.fluorescens and strongly affects the normal growth of P.fluorescens.
Dimethyl phthalate(DMP)is a ubiquitous pollutant that is very harmful to microorganisms.Pseudomonas fluorescens(P.fluorescens)is one of the most important beneficial bacteria in the environment.To explore the toxicological effects of DMP on bacteria,P.fluorescens was selected and investigated using several bio-techniques,such as the polarization method,transmission electron microscopy,and ultra-high performance liquid chromatography.The results showed that the specific growth rate of P.fluorescens was slower,but the generation time was extended under the 10,20 mg·L~(-1),and 40 mg·L~(-1)DMP treatments.The zeta potential on the surface of P.fluorescens decreased gradually from-12.21±0.56 mV to-18.13±0.67 mV under the 0,10,20 mg·L~(-1),and 40 mg·L~(-1)DMP treatments.Meanwhile,the mobility of the cell membrane increased by the DMP treatment and resulted in obvious cell deformation,severe plasmolysis,and leakage of dissoluble matter.The cumulative concentration of DMP inside the cells increased from 0.11±0.10 mg·L~(-1)to 6.78±0.25 mg·L~(-1),and the cumulative concentration outside the cells increased from 8.44±0.37 mg·L~(-1)to 30.52±0.88 mg·L~(-1),under the 10,20 mg·L~(-1),and 40 mg·L~(-1)DMP treatments.Therefore,we conclude that DMP destroys the cell wall and the cell membrane of P.fluorescens and strongly affects the normal growth of P.fluorescens.
引文
[1]韩文辉,赵颖,党晋华,等.汾河流域邻苯二甲酸酯的分布特征及生态风险评价[J].环境化学,2017,36(6):1377-1387.HAN Wen-hui,ZHAO Ying,DANG Jin-hua,et al.Distribution and ecological risk evaluation of phthalate esters in Fenhe River basin[J].387Environmental Chemistry,2017,36(6):1377-1387.
[2]Jin D,Kong X,Cui B,et al.Biodegradation of di-n-butyl phthalate by a newly isolated halotolerant Sphingobium sp.[J].International Journal of Molecular Sciences,2013,14(12):24046-24054.
[3]Wang Z G,Hu Y L,Xu W H,et al.Impacts of dimethyl phthalate on the bacterial community and functions in black soils[J].Frontiers in Microbiology,2015,6:405.
[4]Wang P,Wang S L,Fan C Q.Atmospheric distribution of particulateand gas-phase phthalic esters(PAEs)in a Metropolitan City,Nanjing,east China[J].Chemosphere,2008,72(10):1567-1572.
[5]Zheng X,Zhang B T,Teng Y.Distribution of phthalate acid esters in lakes of Beijing and its relationship with anthropogenic activities[J].Science of the Total Environment,2014,476/477:107-113.
[6]吴山,李彬,梁金明,等.汕头市蔬菜产区土壤-蔬菜中邻苯二甲酸酯(PAEs)污染分布特征研究[J].农业环境科学学报,2015,34(10):1889-1896.WU Shan,LI Bin,LIANG Jin-ming,et al.Distribution characteristics of phthalic acid esters in soils and vegetables in vegetable producing areas of Shantou City,China[J].Journal of Agro-Environment Science,2015,34(10):1889-1896.
[7]张海光,孙国帅,孙磊,等.典型覆膜作物土壤中邻苯二甲酸酯污染的初步研究[J].中国环境监测,2013,29(4):60-63.ZHANG Hai-guang,SUN Guo-shuai,SUN Lei,et al.Preliminary study on phthalic add esters pollution of typical plastic mulched crops soils[J].EnvironmentaI Monitoring in China,2013,29(4):60-63.
[8]朱振宇,姬亚芹,赵静波,等.天津市大气PM10与降尘中邻苯二甲酸酯的污染特征及相关性分析[J].环境污染与防治,2015,37(12):24-28.ZHU Zhen-yu,JI Ya-qin,ZHAO Jing-bo,et al.Pollution characteristics and correlation analysis of PAEs in atmospheric PM10 and dust in Tianjin[J].Environmental Pollution and Prevention,2015,37(12):24-28.
[9]高莉,赵开楼,田义静.黄河兰州断面半挥发性有机污染物分布特征研究[J].华东地质,2008,29(4):315-318.GAO Li,ZHAO Kai-lou,TIAN Yi-jing.Study on distribution characteristics of semi-volatile organic pollutants in Lanzhou section of the Yellow River[J].East China Geology,2008,29(4):315-318.
[10]张道来,刘娜,朱志刚,等.青岛市典型海岸带表层沉积物中邻苯二甲酸酯的组成、分布特征及生态风险评价[J].海洋环境科学,2016,35(5):652-657.ZHANG Dao-lai,LIU Na,ZHU Zhi-gang,et al.Composition,distribution characteristics and ecological risk assessment of phthalate esters in surface sediments of typical coastal zones in Qingdao[J].Marine Environmental Science,2016,35(5):652-657.
[11]Wallace R L,Hirkala D L,Nelson L M.Postharvest biological control of blue mold of apple by Pseudomonas fluorescens during commercial storage and potential modes of action[J].Postharvest Biology&Technology,2017,133:1-11.
[12]Kai M,Effmert U,Berg G,et al.Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani[J].Archives of Microbiology,2007,187(5):351-360.20197
[13]Mayak S,Tirosh T,Glick B R.Plant growth-promoting bacteria that confer resistance to water stress in tomatoes and peppers[J].Plant Science,2004,166(2):525-530.
[14]Manaf H H,Zayed M S.Productivity of cowpea as affected by salt stress in presence of endomycorrhizae and Pseudomonas fluorescens[J].Annals of Agricultural Sciences,2015,60(2):219-226.
[15]Wang Z,Liu S,Xu W,et al.The microbiome and functions of black soils are altered by dibutyl phthalate contamination[J].Applied Soil Ecology,2016,99:51-61.
[16]王志刚,由义敏,徐伟慧,等.黑土微生物丰度和多样性对邻苯二甲酸二丁酯污染的响应[J].生态环境学报,2015,24(10):1725-1730.WANG Zhi-gang,YOU Yi-min,XU Wei-hui,et al.Response of the abundance and diversity of microbes in black soil to di-n-butyl phthalate contamination[J].Ecology and Environmental Sciences,2015,24(10):1725-1730.
[17]王志刚,胡云龙,徐伟慧,等.邻苯二甲酸二甲酯污染对黑土微生物丰度和多样性的影响[J].生态与农村环境学报,2015,31(5):779-783.WANG Zhi-gang,HU Yun-long,XU Wei-hui,et al.Impacts of dimethyl phthalate contamination on abundance and diversity of microbes in black soil[J].Journal of Ecology and Rural Environment,2015,31(5):779-783.
[18]Liu G,Zhang S,Yang K,et al.Toxicity of perfluorooctane sulfonate and perfluorooctanoic acid to Escherichia coli:Membrane disruption,oxidative stress,and DNA damage induced cell inactivation and/or death[J].Environmental Pollution,2016,214:806-815.
[19]窦京娇.北极海冰细菌脂肪酸系统在生境适应性中的作用研究[D].济南:齐鲁工业大学,2013.DOU Jing-jiao.A researsh on the role of arctic sea-ice bacteria fatty acid system in environment adaptability[D].Jinan:Qilu University of Technology,2013.
[20]魏玉利,邹宁.光合细菌的透射电镜观察[J].生命科学仪器,2007,5(5):16-18.WEI Yu-li,ZOU Ning.Observation of photosynthetic bacteria by transmission electron microscopic[J].Life Science Instruments,2007,5(5):16-18.
[21]徐国发,陈平,张建博,等.RP-HPLC法测定PC12细胞内外液中甜味剂赤藓糖醇的含量变化[J].中国食品添加剂,2016(1):133-136.XU Guo-fa,CHEN Ping,ZHANG Jian-bo,et al.Determination of erythritol content change in intracellular and extracellular fluid of PC12 cells by RP-HPLC[J].China Food Additives,2016(1):133-136.
[22]Cavaliere B,Macchione B,Sindona G,et al.Tandem mass spectrometry in food safety assessment:The determination of phthalates in olive oil[J].Journal of Chromatography A,2008,1205(1/2):137-143.
[23]Wang Z,Wang C,You Y,et al.Response of Pseudomonas fluorescens to dimethyl phthalate[J].Ecotoxicology and Environmental Safety,2019,167:36-43.
[24]向思琴.细菌细胞壁肽聚糖的结构与作用[J].技术与市场,2017(1):169.XIANG Si-qin.Structure and function of peptidoglycan in bacterial cell wall[J].Technology and Market,2017(1):169.
[25]叶淑娟,何占航,洪广峰,等.化学消毒剂对污水处理系统中游离细菌表面zeta电位的影响[J].中国消毒学杂志,2006,23(5):415-418.YE Shu-juan,HE Zhan-hang,HONG Guang-feng,et al.Influence of chemical disinfectant on zeta potential of free bacterial surface in sewage treatment system[J].Chinese Journal of Disinfection,2006,23(5):415-418.
[26]Learmonth R P.Membrane fluidity in yeast adaptation:Insights from fluorescence spectroscopy and microscopy[M].New York:Springer,2012:67-93.
[27]Brejchov?J,S?Kora J,Dlouh?K,et al.Fluorescence spectroscopy studies of HEK293 cells expressing DOR-Gi1αfusion protein;the effect of cholesterol depletion[J].Biochim Biophys Acta,2011,1808(12):2819-2829.
[28]Li J,Chikindas M L,Ludescher R D,et al.Temperature-and surfactant-induced membrane modifications that alter listeria monocytogenes nisin sensitivity by different mechanisms[J].Applied&Environmental Microbiology,2002,68(12):5904-5910.
[29]Sikkema J,De Bont J A,Poolman B.Interactions of cyclic hydrocarbons with biological membranes[J].Journal of Biological Chemistry,1994,269(11):8022-8028.
[30]Xie W,Ludewig G,Wang K,et al.Model and cell membrane partitioning of perfluorooctanesulfonate is independent of the lipid chain length[J].Colloids Surf B Biointerfaces,2010,76(1):128-136.
[31]赵晓松,王志刚,王春龙,等.采用光谱学技术分析邻苯二甲酸酯与DNA互作机制[J].生态毒理学报,2017,12(4):385-391.ZHAO Xiao-song,WANG Zhi-gang,WANG Chun-long,et al.Bonding mechanism between phthalic acid esters and DNA by spectroscopy technologies[J].Asian Journal of Ecotoxicology,2017,12(4):385-391.
[32]王志刚,胡影,崔竞文.邻苯二甲酸二甲酯对典型细菌生长和氧化应激酶系的影响[J].生态毒理学报,2015,10(3):297-303.WANG Zhi-gang,HU Ying,CUI Jing-wen.Impact of dimethyl phthalate on the activities of oxidative stress enzymes and growth of typical bacteria[J].Asian Journal of Ecotoxicology,2015,10(3):297-303.
[2]Jin D,Kong X,Cui B,et al.Biodegradation of di-n-butyl phthalate by a newly isolated halotolerant Sphingobium sp.[J].International Journal of Molecular Sciences,2013,14(12):24046-24054.
[3]Wang Z G,Hu Y L,Xu W H,et al.Impacts of dimethyl phthalate on the bacterial community and functions in black soils[J].Frontiers in Microbiology,2015,6:405.
[4]Wang P,Wang S L,Fan C Q.Atmospheric distribution of particulateand gas-phase phthalic esters(PAEs)in a Metropolitan City,Nanjing,east China[J].Chemosphere,2008,72(10):1567-1572.
[5]Zheng X,Zhang B T,Teng Y.Distribution of phthalate acid esters in lakes of Beijing and its relationship with anthropogenic activities[J].Science of the Total Environment,2014,476/477:107-113.
[6]吴山,李彬,梁金明,等.汕头市蔬菜产区土壤-蔬菜中邻苯二甲酸酯(PAEs)污染分布特征研究[J].农业环境科学学报,2015,34(10):1889-1896.WU Shan,LI Bin,LIANG Jin-ming,et al.Distribution characteristics of phthalic acid esters in soils and vegetables in vegetable producing areas of Shantou City,China[J].Journal of Agro-Environment Science,2015,34(10):1889-1896.
[7]张海光,孙国帅,孙磊,等.典型覆膜作物土壤中邻苯二甲酸酯污染的初步研究[J].中国环境监测,2013,29(4):60-63.ZHANG Hai-guang,SUN Guo-shuai,SUN Lei,et al.Preliminary study on phthalic add esters pollution of typical plastic mulched crops soils[J].EnvironmentaI Monitoring in China,2013,29(4):60-63.
[8]朱振宇,姬亚芹,赵静波,等.天津市大气PM10与降尘中邻苯二甲酸酯的污染特征及相关性分析[J].环境污染与防治,2015,37(12):24-28.ZHU Zhen-yu,JI Ya-qin,ZHAO Jing-bo,et al.Pollution characteristics and correlation analysis of PAEs in atmospheric PM10 and dust in Tianjin[J].Environmental Pollution and Prevention,2015,37(12):24-28.
[9]高莉,赵开楼,田义静.黄河兰州断面半挥发性有机污染物分布特征研究[J].华东地质,2008,29(4):315-318.GAO Li,ZHAO Kai-lou,TIAN Yi-jing.Study on distribution characteristics of semi-volatile organic pollutants in Lanzhou section of the Yellow River[J].East China Geology,2008,29(4):315-318.
[10]张道来,刘娜,朱志刚,等.青岛市典型海岸带表层沉积物中邻苯二甲酸酯的组成、分布特征及生态风险评价[J].海洋环境科学,2016,35(5):652-657.ZHANG Dao-lai,LIU Na,ZHU Zhi-gang,et al.Composition,distribution characteristics and ecological risk assessment of phthalate esters in surface sediments of typical coastal zones in Qingdao[J].Marine Environmental Science,2016,35(5):652-657.
[11]Wallace R L,Hirkala D L,Nelson L M.Postharvest biological control of blue mold of apple by Pseudomonas fluorescens during commercial storage and potential modes of action[J].Postharvest Biology&Technology,2017,133:1-11.
[12]Kai M,Effmert U,Berg G,et al.Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen Rhizoctonia solani[J].Archives of Microbiology,2007,187(5):351-360.20197
[13]Mayak S,Tirosh T,Glick B R.Plant growth-promoting bacteria that confer resistance to water stress in tomatoes and peppers[J].Plant Science,2004,166(2):525-530.
[14]Manaf H H,Zayed M S.Productivity of cowpea as affected by salt stress in presence of endomycorrhizae and Pseudomonas fluorescens[J].Annals of Agricultural Sciences,2015,60(2):219-226.
[15]Wang Z,Liu S,Xu W,et al.The microbiome and functions of black soils are altered by dibutyl phthalate contamination[J].Applied Soil Ecology,2016,99:51-61.
[16]王志刚,由义敏,徐伟慧,等.黑土微生物丰度和多样性对邻苯二甲酸二丁酯污染的响应[J].生态环境学报,2015,24(10):1725-1730.WANG Zhi-gang,YOU Yi-min,XU Wei-hui,et al.Response of the abundance and diversity of microbes in black soil to di-n-butyl phthalate contamination[J].Ecology and Environmental Sciences,2015,24(10):1725-1730.
[17]王志刚,胡云龙,徐伟慧,等.邻苯二甲酸二甲酯污染对黑土微生物丰度和多样性的影响[J].生态与农村环境学报,2015,31(5):779-783.WANG Zhi-gang,HU Yun-long,XU Wei-hui,et al.Impacts of dimethyl phthalate contamination on abundance and diversity of microbes in black soil[J].Journal of Ecology and Rural Environment,2015,31(5):779-783.
[18]Liu G,Zhang S,Yang K,et al.Toxicity of perfluorooctane sulfonate and perfluorooctanoic acid to Escherichia coli:Membrane disruption,oxidative stress,and DNA damage induced cell inactivation and/or death[J].Environmental Pollution,2016,214:806-815.
[19]窦京娇.北极海冰细菌脂肪酸系统在生境适应性中的作用研究[D].济南:齐鲁工业大学,2013.DOU Jing-jiao.A researsh on the role of arctic sea-ice bacteria fatty acid system in environment adaptability[D].Jinan:Qilu University of Technology,2013.
[20]魏玉利,邹宁.光合细菌的透射电镜观察[J].生命科学仪器,2007,5(5):16-18.WEI Yu-li,ZOU Ning.Observation of photosynthetic bacteria by transmission electron microscopic[J].Life Science Instruments,2007,5(5):16-18.
[21]徐国发,陈平,张建博,等.RP-HPLC法测定PC12细胞内外液中甜味剂赤藓糖醇的含量变化[J].中国食品添加剂,2016(1):133-136.XU Guo-fa,CHEN Ping,ZHANG Jian-bo,et al.Determination of erythritol content change in intracellular and extracellular fluid of PC12 cells by RP-HPLC[J].China Food Additives,2016(1):133-136.
[22]Cavaliere B,Macchione B,Sindona G,et al.Tandem mass spectrometry in food safety assessment:The determination of phthalates in olive oil[J].Journal of Chromatography A,2008,1205(1/2):137-143.
[23]Wang Z,Wang C,You Y,et al.Response of Pseudomonas fluorescens to dimethyl phthalate[J].Ecotoxicology and Environmental Safety,2019,167:36-43.
[24]向思琴.细菌细胞壁肽聚糖的结构与作用[J].技术与市场,2017(1):169.XIANG Si-qin.Structure and function of peptidoglycan in bacterial cell wall[J].Technology and Market,2017(1):169.
[25]叶淑娟,何占航,洪广峰,等.化学消毒剂对污水处理系统中游离细菌表面zeta电位的影响[J].中国消毒学杂志,2006,23(5):415-418.YE Shu-juan,HE Zhan-hang,HONG Guang-feng,et al.Influence of chemical disinfectant on zeta potential of free bacterial surface in sewage treatment system[J].Chinese Journal of Disinfection,2006,23(5):415-418.
[26]Learmonth R P.Membrane fluidity in yeast adaptation:Insights from fluorescence spectroscopy and microscopy[M].New York:Springer,2012:67-93.
[27]Brejchov?J,S?Kora J,Dlouh?K,et al.Fluorescence spectroscopy studies of HEK293 cells expressing DOR-Gi1αfusion protein;the effect of cholesterol depletion[J].Biochim Biophys Acta,2011,1808(12):2819-2829.
[28]Li J,Chikindas M L,Ludescher R D,et al.Temperature-and surfactant-induced membrane modifications that alter listeria monocytogenes nisin sensitivity by different mechanisms[J].Applied&Environmental Microbiology,2002,68(12):5904-5910.
[29]Sikkema J,De Bont J A,Poolman B.Interactions of cyclic hydrocarbons with biological membranes[J].Journal of Biological Chemistry,1994,269(11):8022-8028.
[30]Xie W,Ludewig G,Wang K,et al.Model and cell membrane partitioning of perfluorooctanesulfonate is independent of the lipid chain length[J].Colloids Surf B Biointerfaces,2010,76(1):128-136.
[31]赵晓松,王志刚,王春龙,等.采用光谱学技术分析邻苯二甲酸酯与DNA互作机制[J].生态毒理学报,2017,12(4):385-391.ZHAO Xiao-song,WANG Zhi-gang,WANG Chun-long,et al.Bonding mechanism between phthalic acid esters and DNA by spectroscopy technologies[J].Asian Journal of Ecotoxicology,2017,12(4):385-391.
[32]王志刚,胡影,崔竞文.邻苯二甲酸二甲酯对典型细菌生长和氧化应激酶系的影响[J].生态毒理学报,2015,10(3):297-303.WANG Zhi-gang,HU Ying,CUI Jing-wen.Impact of dimethyl phthalate on the activities of oxidative stress enzymes and growth of typical bacteria[J].Asian Journal of Ecotoxicology,2015,10(3):297-303.