植物内生菌降解二甲四氯的菌株筛选及其降解特性研究

发布时间：2018-05-15 17:48

本文选题：除草剂 + 二甲四氯　；参考：《广西大学》2017年硕士论文

【摘要】：二甲四氯(MCPA)是一种选择性、内吸传导型和激素型的苯氧乙酸类除草剂。主要用于防除小粒谷物、水稻、豌豆和草坪作物中的多种一年生和多年生双子叶宽叶杂草以及某些单子叶杂草。该除草剂在广西甘蔗田大量使用,对套种和后茬作物可能产生药害。本试验以二甲四氯为研究对象,主要针对二甲四氯降解微生物的筛选及其降解特性展开研究,从茅草、飞机草和番石榴等植物体内筛选出对二甲四氯有降解作用的菌株,为利用微生物降解二甲四氯及其在土壤修复上的应用提供科学依据。主要研究结果如下:1.二甲四氯的降解菌的筛选采集茅草、飞机草和番石榴等植物样本,通过平板涂布法和摇瓶富集法等手段,从植物体内筛选出对二甲四氯有降解作用的菌株5株。其中细菌3株,分别命名为E47、E67和E69号,真菌2株,分别命名为E41号和E68号。选取效果较好的内生真菌E41号和E68号作为本课题主要研究对象,对其降解特性进行研究。2.降解菌的鉴定运用形态学和分子生物学的手段对分离的菌株进行了鉴定。对细菌E47、E67和E69号16S rDNA序列分析,分别鉴定为芽孢杆菌属(Bacillus sp.)、短小杆菌属(Curtobacterium sp.)和短小杆菌属(Curtobacterium sp.)。对真菌E41和E68进行ITS、TUB和LUS rDNA序列分析,结合真菌形态特征,分别鉴定为拟茎点霉属(Phomopsis sp.)和炭角菌属(Xylaria sp.)。3.降解菌的降解特性研究利用液相色谱法检测降解菌对二甲四氯的降解效果,对菌株E41和E68降解二甲四氯的条件(最适温度、pH、营养源)进行优化和降解动力学进行研究。结果表明,以50.0 mg/L为起始浓度,E41在pH值为5.0-7.0、温度为20.0-35.0℃和二甲四氯为唯一碳源时具有较好的降解效果,降解率最高为89.21%。降解动力学试验结果表明,培养条件在28.0 ℃和pH=7.0时半衰期为1.7 d。以50.0 mg/L为起始浓度,E68在pH值为4.0-5.0、温度为20.0-30.0 ℃和添加0.5%的葡萄糖时具有较好的降解效果,降解率最高为95.93%。降解动力学试验结果表明,以50.0 mg/L为起始浓度,培养条件在28.0 ℃和pH=5.0时,半衰期为3.0 d。4.二甲四氯降解产物鉴定使用气质联用仪和液质联用仪,鉴定E41和E68号降解菌对二甲四氯的降解产物,并与标准品进行比对,结果表明降解产物均为4-氯-2-甲基苯酚(4-Chloro-2-methylphenol)。对该降解产物进行定量分析,发现经5 d培养,E41和E68号降解菌的降解产物含量分别为12.18 mg/L和1.41 mg/L,随后其含量逐渐下降。5.降解菌在土壤中生物修复应用在室内条件下模拟研究降解菌对受二甲四氯污染土壤的修复效果,结果表明二甲四氯在未灭菌土壤、未灭菌土壤+E41和未灭菌土壤+E68号降解菌的三种处理中半衰期分别为12.84、2.07和4.62 d,在灭菌土壤、灭菌土壤+E41和灭菌土壤+E68号降解菌的半衰期分别为27.73、2.73和11.00d,在灭菌和不灭菌土壤中,添加降解菌的处理显著提高土壤中二甲四氯的降解速度。以小白菜为靶标生物,利用光照培养箱盆栽试验,进行植物药害研究,发现接种E41或E68号降解菌处理的植株生长情况均好于不接种降解菌的处理。以水稻为靶标生物,利用光照培养箱培养皿水培试验进行降解液毒性研究,E41和E68号降解液毒性低于原始药液毒性。
[Abstract]:Two a four chlorine (MCPA) is a selective, endogenous and hormonal phenoxy herbicide. It is used mainly for the prevention of a variety of annual and perennial dicotyledonous weeds and some monocotyledon weeds in small grain, rice, pea, and turf crops. This herbicide is widely used in Guangxi sugarcane fields and is used for interplanting and after cropping. In this experiment, two a four chlorine was used as the research object, mainly aimed at the screening and degradation characteristics of two a four chlorine degrading microorganism. From the plants of thatch, aeroplane grass and guava, the biodegradation strains of two a four chlorine were screened out for the degradation of two a, four chlorine and the soil remediation by microorganism. The main research results are as follows. The main results are as follows: 1. the screening of two a four chlorine degrading bacteria from plant samples such as thatch, aeroplane grass and guava, screened 5 strains of two a four chlorine by plate coating method and shake flask enrichment. Among them, 3 bacteria were named E47, E67 and E69, respectively. 2 strains of fungi named E41 and E68 respectively. Select the best endophytic fungi E41 and E68 as the main object of the study. The degradation characteristics of the bacteria were identified by the identification of the morphology and molecular biology of the.2. degrading bacteria. The sequence of E47, E67 and E69 16S rDNA sequence of the bacteria was identified. Analysis, respectively identified as Bacillus sp., Curtobacterium sp. and Curtobacterium sp.. ITS, TUB and LUS rDNA sequence analysis of fungal E41 and E68, combined with the morphological characteristics of fungi, respectively, to identify the degradation bacteria of Mycobacterium tumefaciens and carbonaceous fungi. The degradation effect of degrading bacteria to two a four chlorine was detected by liquid chromatography. The optimization and degradation kinetics of the strain E41 and E68 degradation of two a four chlorine (the optimum temperature, pH, nutrient source) were studied. The results showed that the initial concentration of 50 mg/L, E41 at pH value 5.0-7.0, temperature 20.0-35.0 and two a four chlorine When one carbon source has good degradation effect, the highest degradation rate is 89.21%. degradation kinetics test, which shows that the half life of the culture condition at 28 and pH=7.0 is 1.7 D. to 50 mg/L, E68 has a good degradation effect when pH value is 4.0-5.0, the temperature is 20.0-30.0 and 0.5% glucose, and the highest degradation rate is 9. The results of 5.93%. degradation kinetics show that the degradation products of E41 and E68 degrading bacteria to two a and four chlorine are identified with 50 mg/L as the initial concentration and the incubation conditions at 28 and pH=5.0 and 3 d.4. two a four chlorine degradation products to identify the degradation products of E41 and E68 degrading bacteria to four chlorine and compare with the standard products. The results show degradation The products were all 4- chloro -2- methyl phenol (4-Chloro-2-methylphenol). The degradation products were quantitatively analyzed. It was found that the degradation products of E41 and E68 degrading bacteria were 12.18 mg/L and 1.41 mg/L respectively after 5 d culture, and then the content of the degradation bacteria decreased gradually and the bioremediation of the bacteria in the soil was simulated under indoor conditions to simulate the degradation bacteria. The results of the remediation of two a four chlorine contaminated soil showed that the half-life of three treatments of two a four chlorine in the non sterilized soil, the non sterilized soil +E41 and the non sterilized soil +E68 degrading bacteria were 12.84,2.07 and 4.62 D respectively. In the sterilized soil, the half-life of the +E68 degrading bacteria in the sterilized soil +E41 and the sterilized soil were 27.73,2.73 and 11.00d, respectively. In the sterilized and non sterilized soil, the degradation rate of two a four chlorine in the soil was significantly improved. The plant drug damage was studied by the potted experiment of the light culture box. The results showed that the growth of the plants treated with E41 or E68 degrading bacteria was better than that of the non degrading bacteria. In order to target organisms, the toxicity of degradation liquid was studied using light culture box culture dish. The toxicity of E41 and E68 degradation liquor was lower than that of the original liquid.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172;X592

【参考文献】