石油污染土壤中芘、荧蒽降解菌的筛选鉴定及降解特性研究
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摘要
多环芳烃(Polycyclic aromatic hydrocarbons,PAHs)在环境中分布广泛,极易在环境中累积并可通过食物链传递,对人类健康和生态环境具有很大危害性。特别是高分子量PAHs(四环及四环以上的芳烃)具有潜在的致畸、致癌和致突变效应。芘和荧蒽因具有独特的化学结构,常被作为四环PAHs降解研究的模式化合物。芘和荧葸降解菌的筛选和降解特性研究是PAHs污染土壤生物修复工作的关键,也能够为进一步探索高分子量PAHs降解的分子机制奠定基础。
本研究采用选择性富集培养方法和双层平板筛选模型,从沈抚灌区石油污染土壤中分离、筛选到一株芘高效降解菌(B31)和一株荧葸高效降解菌(Y34),根据菌体形态特征、生理生化特征以及16S rDNA基因序列分析,将菌株B31鉴定至假单胞菌(Pseudomonas sp.);菌株Y34鉴定为产碱假单胞菌(Pseudomonas alcaligenes)。
菌株B31有较强的降解芘的能力,经过6天的培养,可将液体培养基中的芘(25mg·L~(-1))降解83.50%。在摇瓶中菌株B31最佳生长和芘降解条件为:初始pH 8.0、培养温度为30℃、芘浓度25 mg·L~(-1)、250 mL三角瓶培养基装量为30 mL。添加葡萄糖、水杨酸、邻苯二酚和邻苯二甲酸能够促进菌株B31对芘的降解。
菌株Y34有较强的降解荧葸的能力,经过6天的培养,可将液体培养基中的荧蒽(50mg·L~(-1))降解80.75%。在摇瓶中菌株Y34最佳生长和荧葸降解条件为:初始pH 7.0、培养温度为35℃、荧葸浓度50 mg·L~(-1)、250 mL三角瓶培养基装量为30 mL。添加水杨酸、邻苯二酚和邻苯二甲酸能够促进菌株Y34对荧蒽的降解。
两株菌均能够利用多种链烷烃、环烷烃、单环和多环芳烃,具有很好的底物多样性。两株菌都能产生生物表面活性剂,可以减少水的表面张力,提高PAHs在水中的生物可利用度,从而提高PAHs的降解效率。两株菌混合培养对芘的降解率比菌株B31单独作用提高14.25%,但抑制了菌株对荧蒽的降解作用,降解率比菌株Y34单独作用降低20.88%。
酶活测定实验显示菌株B31具有芘诱导的水杨酸羟化酶和邻苯二酚2,3-双加氧酶活性,菌株Y34具有荧蒽诱导的水杨酸羟化酶和吲哚双加氧酶活性。利用PCR扩增技术,从菌株B31中扩增出了邻苯二酚2,3-双加氧酶基因;从菌株Y34中扩增出了吲哚双加氧酶基因和龙胆酸1,2-双加氧酶基因。
Polycyclic aromatic hydrocarbons are widespread pollutants and can be released to atmosphere,soils,foods and water.Exposure to PAHs would be hazardous to human health and environments.The high molecular weight(HMW) PAHs(four or more fused rings) are of particular environmental concern because of their potential toxicity,mutagenicity and carcinogenicity.Pyrene and fluoranthene are generally taken as the model compounds of tetranuclear aromatic hydrocarbons for the study of PAHs degradation.Isolation and characteristics of pyrene- and fluoranthene-degrading bacteria is the key step for biodegradation of petroleum contaminated soil.Also,it is the basis for further analysis on the molecular mechanisms of HMW-PAHs degradation.
In this study,one strain of pyrene-degrading bacteria and one strain of fluoranthene-degrading bacteria were isolated from Shenfu irrigation area petroleum contaminated soil, using selective enrichment culture and screening model of overlayer techniques.They were identified as Pseudomonas sp.B31 and Pseudomonas alcaligenes Y34 using physiological-biochemical determination and 16S rDNA sequence analysis.
The degradation rate of pyrene(25 mg·L~(-1)) of strain B31 was 83.50%after 6 days.The optimum condition for pyrene degardation by strain B31 in liquid culture was:the initial pH of the culture was 8.0,the incubation temperature was 30℃,pyrene concentrations was 25 mg·L~(-1),and 30 mL liquid culture in 250 mL flask.Addition of glucose,salicylic acid,catechol and phthalic acid could promote pyrene degradation by strain B31 to different degree.
The degradation rate of fluoranthene(50 mg·L~(-1)) of strain Y34 was 80.75%after 6 days. The optimum condition for fluoranthene degardation by strain Y34 in liquid culture was:the initial pH of the culture was 7.0,the incubation temperature was 35℃,fluoranthene concentrations was 50 mg·L~(-1),30 mL liquid culture in 250 mL flask.Addition of salicylic acid,catechol and phthalic acid could promote fluoranthene degradation of strain Y34 to different degree.
Both strains could grow on paraffin hydrocarbon,cycloparaffinic hydrocarbons, monocycle and some other polycyclic aromatic hydrocarbons as sole source of carbon.Both strains could produce surface active agent,which could reduce the surface tension of water, and increase the bioavailability of PAHs in water,thus,increase the PAHs degadation rate. The pyrene degradation rate increased 14.25%by using mixed cultures of B31 and Y34, compared with single isolate B31,but fluoranthene degradation rate decreased 20.88%, compared with single isolate Y34.
The strain B31 showed salicylate hydroxylase and catechol 2,3-dioxygenase activity induced by pyrene.The strain Y34 showed salicylate hydroxylase and indole dioxygenase activity induced by fluoranthene.The gene of catechol 2,3-dioxygenase was cloned from strain B31 by PCR technology.The gene of indole dioxygenase and gentisate 1, 2-dioxygenase was cloned from strain Y34 by PCR technology.
本研究采用选择性富集培养方法和双层平板筛选模型,从沈抚灌区石油污染土壤中分离、筛选到一株芘高效降解菌(B31)和一株荧葸高效降解菌(Y34),根据菌体形态特征、生理生化特征以及16S rDNA基因序列分析,将菌株B31鉴定至假单胞菌(Pseudomonas sp.);菌株Y34鉴定为产碱假单胞菌(Pseudomonas alcaligenes)。
菌株B31有较强的降解芘的能力,经过6天的培养,可将液体培养基中的芘(25mg·L~(-1))降解83.50%。在摇瓶中菌株B31最佳生长和芘降解条件为:初始pH 8.0、培养温度为30℃、芘浓度25 mg·L~(-1)、250 mL三角瓶培养基装量为30 mL。添加葡萄糖、水杨酸、邻苯二酚和邻苯二甲酸能够促进菌株B31对芘的降解。
菌株Y34有较强的降解荧葸的能力,经过6天的培养,可将液体培养基中的荧蒽(50mg·L~(-1))降解80.75%。在摇瓶中菌株Y34最佳生长和荧葸降解条件为:初始pH 7.0、培养温度为35℃、荧葸浓度50 mg·L~(-1)、250 mL三角瓶培养基装量为30 mL。添加水杨酸、邻苯二酚和邻苯二甲酸能够促进菌株Y34对荧蒽的降解。
两株菌均能够利用多种链烷烃、环烷烃、单环和多环芳烃,具有很好的底物多样性。两株菌都能产生生物表面活性剂,可以减少水的表面张力,提高PAHs在水中的生物可利用度,从而提高PAHs的降解效率。两株菌混合培养对芘的降解率比菌株B31单独作用提高14.25%,但抑制了菌株对荧蒽的降解作用,降解率比菌株Y34单独作用降低20.88%。
酶活测定实验显示菌株B31具有芘诱导的水杨酸羟化酶和邻苯二酚2,3-双加氧酶活性,菌株Y34具有荧蒽诱导的水杨酸羟化酶和吲哚双加氧酶活性。利用PCR扩增技术,从菌株B31中扩增出了邻苯二酚2,3-双加氧酶基因;从菌株Y34中扩增出了吲哚双加氧酶基因和龙胆酸1,2-双加氧酶基因。
Polycyclic aromatic hydrocarbons are widespread pollutants and can be released to atmosphere,soils,foods and water.Exposure to PAHs would be hazardous to human health and environments.The high molecular weight(HMW) PAHs(four or more fused rings) are of particular environmental concern because of their potential toxicity,mutagenicity and carcinogenicity.Pyrene and fluoranthene are generally taken as the model compounds of tetranuclear aromatic hydrocarbons for the study of PAHs degradation.Isolation and characteristics of pyrene- and fluoranthene-degrading bacteria is the key step for biodegradation of petroleum contaminated soil.Also,it is the basis for further analysis on the molecular mechanisms of HMW-PAHs degradation.
In this study,one strain of pyrene-degrading bacteria and one strain of fluoranthene-degrading bacteria were isolated from Shenfu irrigation area petroleum contaminated soil, using selective enrichment culture and screening model of overlayer techniques.They were identified as Pseudomonas sp.B31 and Pseudomonas alcaligenes Y34 using physiological-biochemical determination and 16S rDNA sequence analysis.
The degradation rate of pyrene(25 mg·L~(-1)) of strain B31 was 83.50%after 6 days.The optimum condition for pyrene degardation by strain B31 in liquid culture was:the initial pH of the culture was 8.0,the incubation temperature was 30℃,pyrene concentrations was 25 mg·L~(-1),and 30 mL liquid culture in 250 mL flask.Addition of glucose,salicylic acid,catechol and phthalic acid could promote pyrene degradation by strain B31 to different degree.
The degradation rate of fluoranthene(50 mg·L~(-1)) of strain Y34 was 80.75%after 6 days. The optimum condition for fluoranthene degardation by strain Y34 in liquid culture was:the initial pH of the culture was 7.0,the incubation temperature was 35℃,fluoranthene concentrations was 50 mg·L~(-1),30 mL liquid culture in 250 mL flask.Addition of salicylic acid,catechol and phthalic acid could promote fluoranthene degradation of strain Y34 to different degree.
Both strains could grow on paraffin hydrocarbon,cycloparaffinic hydrocarbons, monocycle and some other polycyclic aromatic hydrocarbons as sole source of carbon.Both strains could produce surface active agent,which could reduce the surface tension of water, and increase the bioavailability of PAHs in water,thus,increase the PAHs degadation rate. The pyrene degradation rate increased 14.25%by using mixed cultures of B31 and Y34, compared with single isolate B31,but fluoranthene degradation rate decreased 20.88%, compared with single isolate Y34.
The strain B31 showed salicylate hydroxylase and catechol 2,3-dioxygenase activity induced by pyrene.The strain Y34 showed salicylate hydroxylase and indole dioxygenase activity induced by fluoranthene.The gene of catechol 2,3-dioxygenase was cloned from strain B31 by PCR technology.The gene of indole dioxygenase and gentisate 1, 2-dioxygenase was cloned from strain Y34 by PCR technology.
引文
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