【作者】 刘娜；

【导师】 宋东辉；

【作者基本信息】 天津科技大学 ， 海洋生物学， 2020， 硕士

【摘要】 随着经济的快速发展,芳香族化合物污水排放已严重影响了人类的健康生活,其中邻苯二酚、苯甲酸为苯类化合物代谢过程中的两种中间代谢产物,而壬基酚(NP)作为一种致突变性的环境内分泌干扰物,更容易在海洋生物食物链富集而威胁海洋生物的健康。因此,环境中这三种污染物的生物去除已刻不容缓。但有关苯类污染物对菌株代谢组学的研究很少,因此本文研究的NP对菌株的代谢响应对环境修复具有很大的价值。本试验利用课题组前期分离纯化出来的高效石油烃降解菌Acnetobacter sp.Tust-DM21,研究了菌株对邻苯二酚、苯甲酸钠、NP的降解特性和以NP为碳源时菌株的代谢变化。本试验先研究了高效降解石油中芳香烃组分的菌株Tust-DM21对邻苯二酚、苯甲酸钠的降解特性,结果表明,菌株可以耐受700 mg/L邻苯二酚,其降解邻苯二酚的最佳条件为:接种量5%、pH 6.0、温度35℃,在24 h内对600 mg/L邻苯二酚降解率达92%以上。菌株可以耐受4 500 mg/L苯甲酸钠,其降解苯甲酸钠的最佳条件为:接种量3%、pH 8.0、温度35℃,在24 h内对1 500 mg/L苯甲酸钠的降解率达95%以上。此外对菌株中邻苯二酚1,2-双加氧酶(C12O)的粗酶特性研究表明,菌株是通过C12O酶催化苯环邻位开环来实现邻苯二酚降解的,C12O的最适pH值为8.0,最适温度为35℃,其动力学参数Km=25.68μmol/L,Vmax=0.13 mol/(L·min)。此外还测定了菌株中苯甲酸1,2-双加氧酶的酶活,结果表明该酶的酶活为3.53 U。菌株对NP的降解特性表明,菌株能够耐受150 mg/L NP,其降解NP的最佳条件为:pH 8.0、温度35℃、葡萄糖浓度为70 mg/L,其降解率最高可以达到73.9%。然后利用正交试验分析表明,NP浓度对菌株降解NP的影响最大,然后依次是温度、葡萄糖浓度和pH,其中在NP浓度为90 mg/L、pH 8.0、温度35 ℃、葡萄糖浓度为30 mg/L的条件下菌株对NP的降解率可达81.2%,优化后的体系对NP的降解率提高了 7.3%。利用代谢组学技术,研究了菌株对NP的代谢机制。结果表明,共筛选出178个差异代谢物,其中显著上调的差异代谢物和下调的差异代谢物分别有24、154个,发现大多数的差异代谢物属于芳香族化合物和脂类化合物,其余的为有机氧化合物、有机氮化合物和酮类化合物等。然后对这些差异代谢物进行KEGG代谢路径分析,发现大多数差异代谢物参与的代谢通路主要为脂肪酸的生物合成、氨基酸的代谢、芳香族化合物的降解以及次级代谢物的生物合成等。最后从代谢组学数据中筛选出9种与NP降解途径有关的代谢物,在此基础上确定菌株降解NP的途径是从NP烷基链末端开始的,烷基链末端碳原子先发生羟化反应,然后逐步氧化为羧基,最后发生脱羧反应,依次循环。本文研究表明,本菌株对邻苯二酚、苯甲酸钠、NP具有很好的去除效果,对这些污染物的去除具有很好的应用前景,而代谢组学技术的应用,为NP对菌株代谢调控的研究提供了理论依据。更多还原

【Abstract】 With the rapid development of the economy,organic aromatic hydrocarbon pollutants discharged from industrial wastewater have seriously affected human health.Catechol and benzoic acid are two intermediate metabolites in the metabolism of benzene compounds,and are also common environment pollutants in industry wastewater.Nonylphenol(NP)as an environmental endocrine disruptor,can be enriched by food chain and eventually threaten marine life health.So we have to remove these environment pollutants immediately.However,there were few studies on the metabolomics of benzene pollutants by strains.The metabolic response of the strain to nonylphenol was important for environment repair.In this paper,the degradation characteristics of catechol and sodium benzoate by the strain which had a capability of highly effective degrading aromatic compounds were studied.The maximum tolerated concentration of the strain Tust-DM21 on catechol was 700 mg/L.The optimum conditions for the strain to degrade 600 mg/L of catechol were as follows:5%of inoculumvalue,pH 6.0 and temperature 35℃,respectively.Under the optimal condition,the degradation rate of catechol could reach 92%.The maximum tolerated concentration of the strain on sodium benzoate was 4 500 mg/L.The optimum conditions for the strain to degrade 1 500 mg/L of sodium benzoate were as follows:3%of inoculum value,pH 8.0 and temperature 35℃,respectively.Under the optimal condition,the degradation rate of sodium benzoate could reach 95%.In addition,the crude enzyme characteristics of catechol 1,2-dioxygenase(C12O)in the strain showed that the C12O enzyme catalyzed the ortho-ring opening of the benzene ring to achieve catechol degradation.The optimal conditions for the enzymatic reaction of C12O were as follows:pH 8.0 and temperature 35℃.The crude enzyme kinetic parameters of C12O were as follows:Km=25.68 mol/L,Vmax=0.13 mol/(mL·min).In addition,the enzyme activity of benzoic acid 1,2-dioxygenase was measured,and the enzyme activity of benzoic acid 1,2-dioxygenase was 3.53 U.The degradation characteristics of NP by the strain were studied.The maximum tolerated concentration of the strain Tust-DM21 on NP was 150 mg/L.The optimum conditions for the strain to degrade 90 mg/L NP were as follows:pH 8.0,temperature 35℃,70 mg/L glucose concentration.When the glucose concentration was 70 mg/L,the strain had the best degradation effect on nonylphenol with a degradation rate of 73.9%.Then orthogonal experiments results that showed the optimal conditions for removing NP were as follows:NP concentration had the greatest effect on NP degradation by strain,then followed by temperature,glucose concentration and pH.When the NP concentration was 90 mg/L,pH 8.0,temperature 35℃,and glucose concentration 30 mg/L,the NP degradation rate by the strain could reach 81.2%.Under the same treatment time,NP degradation rate could increase 7.3%.Using metabolomics technology,the metabolic mechanism of NP was studied.The results showed as fllows:a total of 178 differential metabolites were detected by HPLC-MS.There were 24 significantly up-regulated metabolites and 154 down-regulated metabolites.Most of differential metabolites were aromatic compounds and lipid compounds,and the rest were organic oxygen compounds,organic nitrogen compounds and ketone compounds.The KEGG metabolic pathway analysis of these differential metabolites showed as follows:most of the metabolic pathways of differential metabolites were fatty acid biosynthesis,amino acid metabolism,aromatic compound degradation,and secondary metabolite biosynthesis.Finally we screened 9 metabolites which were related to the NP degradation pathway.So nonylphenol degradation pathway of this strain started from the nonylphenol alkyl chain end within a cycle of hydroxylation,subsequent and decarboxylation.Acinetobacter sp.Tust-DM21 as a petroleum-degrading strain could highly degrade catechol,sodium benzoate and NP.The results showed that the Tust-DM21 strain had a good application prospect for removing these pollutants.The application of metabolomics technology provided a theoretical basis for the study of stain metabolic regulation to NP.更多还原