邻苯二甲酸二丁酯降解菌株的筛选及相关降解特性的研究

发布时间：2017-12-27 00:14

本文关键词：邻苯二甲酸二丁酯降解菌株的筛选及相关降解特性的研究　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：邻苯二甲酸酯类(phthalate esters,PAEs)简称PAEs,是一类非常重要的有机化合物,被广泛应用于塑料、化妆品、香料以及涂料等工业生产中。由于PAEs的广泛应用,目前我国大部分地区土壤、大气以及水体中均检测到了一定浓度的PAEs,其中邻苯二甲酸二丁酯(DBP)的污染尤为严重。由于部分地区土壤中PAEs的污染严重超标,因此植物在生长的过程中体内富集了大量的塑化剂。例如,有报道指出人们日常所食的叶菜类青菜中70%以上PAEs含量超标,这对农产品的安全带来极大的隐患。植物内生菌是指在其生活史的一定阶段或全部阶段,生活在健康植物的各种器官和组织的细胞内或细胞间隙的细菌。它可以有效的定殖到植物体内,而且不容易受到外界环境的影响。因此,利用具有DBP降解特性的植物内生菌加速青菜体内残留的DBP的代谢与降解,对未来塑化剂的防治与控制具有重要的意义。本研究以采自江苏省农业科学研究院试验大田的韭菜为供试植物,采用表面消毒研磨法,以DBP为单一碳源的无机盐培养基连续多代培养筛选DBP降解内生菌,并分别对其在植物体外与体内的DBP降解特性进行分析,采用生理生化分析测定并结合16SrDNA序列分析对所分离的内生细菌进行菌种鉴定,同时对其在植物体外与体内的降解途径进行了探讨对比,得出该菌株降解DBP的一般途径。为进一步利用植物内生菌调节植物体内PAEs残留等持久性有机污染物的降解机理的研究建立了一定的基础,研究的结果可为利用植物内生菌调控农产品的塑化剂残留提供一定的理论指导,同时提供一些新的思路。主要研究结果如下:1.从韭菜体内分离出一株解淀粉芽孢杆菌亚种(Bacillus amyloliquefaciens subsp.)植物内生菌JR20,其对初始浓度为5 mg/L的DBP 10天的降解率为98.06%。菌株JR20对温度具有广谱耐受性,在30~40℃范围内,对DBP的降解率基本无差异。在pH7.0~8.0范围内对DBP的降解率大于pH为6.0时。因此,菌株JR20降解DBP的最佳培养条件为37℃、pH为7.0、初始浓度为5 mg/L。通过质谱分析,在DBP的降解产物中检测到了邻苯二甲酸(PA),随着DBP浓度的不断降低,PA的浓度在不断地增加,到第5天PA的累积量达到了最大值。因此,菌株JR20对DBP的代谢途径为先将邻苯二甲酸正二丁酯脱去一个酯基生成邻苯二甲酸单丁酯,之后再脱去一个酯基生成邻苯二甲酸,因此,邻苯二甲酸即为菌株JR20降解DBP的最终产物。2.从韭菜根部分离筛选的内生菌株JR20,除了在体外对DBP有一定的降解特性外,还可以通过定殖转接入青菜体内促进青菜中DBP的降解。通过梯度驯化JR20,逐步提高平板中利福平浓度,筛选出能在含有100μg/m L利福平的平板上稳定生长而且生理生化特性与原始菌株相一致的突变体菌株。抗利福平标记菌株JR20可以通过高梗白在生长过程中吸收营养液途径进入到其体内,达到定殖结果,并在第8天左右达到定殖数量峰值,之后由于高梗白生长到了晚期,组织开始老化,因此菌株在根、茎、叶中的数量有所降低,直至最后为零。试验得出,DBP在营养液中可以进行自降解,且当初始浓度为1 mg/L时,基本20天内可以被完全降解。3.将抗利福平标记菌株JR20定殖到青菜体内后,检测结果显示,根、茎叶中DBP的浓度均呈下降趋势,但接入菌株JR20的试验组降解速率明显大于未接入菌株JR20的对照组。到第20天左右,试验组根中DBP的降解率达95.13%,然而对照组根中DBP降解率为89.52%,试验组茎叶中DBP降解率为90.45%,对照组茎叶中DBP降解率为80.61%,试验组中DBP浓度明显低于对照组。因此,菌株JR20的接入加速了青菜体内DBP的降解速率,使其浓度明显低于同期未接入菌株JR20的植株。试验组与对照组营养液中DBP的浓度也基本呈下降的趋势,到第20天左右,试验组与对照组营养液中DBP含量均已接近于零。因此,转入菌株JR20的青菜,由于其体内DBP浓度的迅速降解,为了达到平衡,会促使其更加快速的吸收环境中的DBP。菌株JR20在高梗白根中和茎叶中均能降解DBP,且降解DBP的最终产物为PA,降解途径与在体外无机盐培养基中相同。4.菌株JR20的定殖对青菜体内DBP的转移因子以及根部对营养液中DBP的吸附因子在青菜生长前期影响不大,但在中后期会促进青菜对DBP的吸附。
[Abstract]:Phthalate esters (PAEs), referred to as PAEs, is a very important organic compound. It is widely used in plastics, cosmetics, spices and coatings, and other industrial production. Two With the wide application of PAEs, the soil, atmosphere and water in most parts of China were detected in a certain concentration of PAEs, dibutyl phthalate (DBP) which the pollution is particularly serious. Because of the serious pollution of PAEs in the soil in some areas, a large number of plasticizers are enriched in the process of plant growth. For example, it is reported that more than 70% of the PAEs content in leafy vegetables that people eat daily is exceeding the standard, which brings great potential for the safety of agricultural products. Endophyte is a bacteria that lives in the cells or interstitial cells of various organs and tissues of healthy plants at the stage or at all stages of their life history. It can be effectively colonized in the plant, and is not easily affected by the external environment. Therefore, the use of DBP endophytic bacteria with the characteristics of degradation of DBP to accelerate the metabolism and degradation of residual organic matter in vegetables is of great significance for the prevention and control of future plasticizers. In this study, collected from Jiangsu Academy of agricultural sciences field test of leek as tested plants, using surface disinfection grinding method, using DBP as the sole carbon source of the inorganic salt medium for screening DBP degradation of cultured endophytic bacteria, and analyze it in DBP degradation characteristics of plants in vitro and in vivo. Determination and 16SrDNA sequence analysis of the isolated endophytic bacteria strains were identified by physiological and biochemical analysis, and comparative study of the degradation pathway of plants in vitro and in vivo, the general approach to the degradation of DBP. Establish a base for further research the degradation mechanism of endophytic bacteria in plants PAEs regulating residues of persistent organic pollutants, the results of this research can provide some theoretical guidance for the use of plasticizer endophyte regulation of agricultural residues, also provided some new ideas. The main results are as follows: 1.. A plant endophyte JR20 isolated from leek (Bacillus amyloliquefaciens subsp.) was isolated from leek, and its degradation rate for DBP 10 days with an initial concentration of 5 mg/L was 98.06%. The strain JR20 has broad spectrum tolerance to the temperature, and the degradation rate of DBP is basically no difference within the range of 30~40. The degradation rate of DBP in the range of pH7.0~8.0 is greater than that of pH 6. Therefore, the optimum conditions for the degradation of DBP by strain JR20 are 37, 7, and 5 mg/L for the initial concentration. The concentration of DBP was detected in the degradation products of PA by mass spectrometry. With the decrease of DBP concentration, the concentration of PA increased continuously, and the accumulation of PA reached the maximum value on the fifth day. Therefore, the metabolic pathway of strain JR20 to DBP is to first remove the ester of ortho benzene two carboxylic acid, n-butyl ester, and form o-butyl benzoic acid two butyl ester, then remove one ester group to form o-benzoic acid two. Therefore, o-benzoic acid two is the final product of strain JR20 degrading DBP. 2., the endophytic strain JR20 isolated from the roots of Chinese chive can not only degrade DBP in vitro, but also promote the degradation of DBP in Chinese Cabbage by colonization and transfer to green vegetables. By gradient JR20 domestication and gradually increasing the concentration of rifampicin in the flat, we screened a mutant strain that could grow stably on the plate containing 100 g/m L rifampicin, and its physiological and biochemical characteristics were consistent with the original strain. Rifampin resistant marker strain JR20 by high white terrier in the growth process, absorb the nutrient solution way to enter into the body, achieve the colonization results, and reached the peak number of colonization in about eighth days, due to the high growth of White Terrier after the late start, tissue aging, so the number of strains in root, stem and leaf. Decreased, until the last is zero. The experiment shows that DBP can be self degraded in the nutrient solution and can be completely degraded within 20 days when the initial concentration is 1 mg/L. 3., when the rifampin labeled strain JR20 was colonized into the cabbage, the results showed that the concentration of DBP in roots and stems decreased. However, the degradation rate of the test group was significantly larger than that of the control group without JR20. The control group was JR20. After twentieth days, the degradation rate of DBP in the root of the test group was 95.13%, but the DBP degradation rate in the control group was 89.52%. The degradation rate of DBP in the stem and leaf of the test group was 90.45%, while the DBP degradation rate in the control group was 80.61%, and the DBP concentration in the test group was significantly lower than that in the control group. Therefore, the insertion of strain JR20 accelerated the degradation rate of DBP in the cabbage, and the concentration was significantly lower than that of the plant without the strain JR20. The concentration of DBP in the nutrient solution of the experimental group and the control group also showed a decreasing trend. Until about twentieth days, the content of DBP in the nutrient solution of the experimental group and the control group was all close to zero. In order to achieve the balance, the fast degradation of the DBP concentration in the strain JR20 will lead to a more rapid absorption of DBP in the environment. The strain JR20 can all degrade DBP in the white root and stem of the stem, and the final product of DBP is PA. The degradation pathway is the same as in the inorganic salt medium in vitro. The colonization of 4. strain JR20 had little effect on the transfer factor of DBP and the adsorption factor of DBP in the nutrient solution on the cabbage in the early stage of growth, but promoted the adsorption of DBP on vegetables in the later stage.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X172

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