本文關(guān)鍵詞：鄰苯二甲酸二丁酯降解菌株的篩選及相關(guān)降解特性的研究　出處：《江蘇大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 鄰苯二甲酸酯 鄰苯二甲酸二丁酯 植物內(nèi)生菌 生物降解 青菜

【摘要】：鄰苯二甲酸酯類(phthalate esters,PAEs)簡(jiǎn)稱PAEs,是一類非常重要的有機(jī)化合物,被廣泛應(yīng)用于塑料、化妝品、香料以及涂料等工業(yè)生產(chǎn)中。由于PAEs的廣泛應(yīng)用,目前我國(guó)大部分地區(qū)土壤、大氣以及水體中均檢測(cè)到了一定濃度的PAEs,其中鄰苯二甲酸二丁酯(DBP)的污染尤為嚴(yán)重。由于部分地區(qū)土壤中PAEs的污染嚴(yán)重超標(biāo),因此植物在生長(zhǎng)的過程中體內(nèi)富集了大量的塑化劑。例如,有報(bào)道指出人們?nèi)粘Ｋ车娜~菜類青菜中70%以上PAEs含量超標(biāo),這對(duì)農(nóng)產(chǎn)品的安全帶來(lái)極大的隱患。植物內(nèi)生菌是指在其生活史的一定階段或全部階段,生活在健康植物的各種器官和組織的細(xì)胞內(nèi)或細(xì)胞間隙的細(xì)菌。它可以有效的定殖到植物體內(nèi),而且不容易受到外界環(huán)境的影響。因此,利用具有DBP降解特性的植物內(nèi)生菌加速青菜體內(nèi)殘留的DBP的代謝與降解,對(duì)未來(lái)塑化劑的防治與控制具有重要的意義。本研究以采自江蘇省農(nóng)業(yè)科學(xué)研究院試驗(yàn)大田的韭菜為供試植物,采用表面消毒研磨法,以DBP為單一碳源的無(wú)機(jī)鹽培養(yǎng)基連續(xù)多代培養(yǎng)篩選DBP降解內(nèi)生菌,并分別對(duì)其在植物體外與體內(nèi)的DBP降解特性進(jìn)行分析,采用生理生化分析測(cè)定并結(jié)合16SrDNA序列分析對(duì)所分離的內(nèi)生細(xì)菌進(jìn)行菌種鑒定,同時(shí)對(duì)其在植物體外與體內(nèi)的降解途徑進(jìn)行了探討對(duì)比,得出該菌株降解DBP的一般途徑。為進(jìn)一步利用植物內(nèi)生菌調(diào)節(jié)植物體內(nèi)PAEs殘留等持久性有機(jī)污染物的降解機(jī)理的研究建立了一定的基礎(chǔ),研究的結(jié)果可為利用植物內(nèi)生菌調(diào)控農(nóng)產(chǎn)品的塑化劑殘留提供一定的理論指導(dǎo),同時(shí)提供一些新的思路。主要研究結(jié)果如下:1.從韭菜體內(nèi)分離出一株解淀粉芽孢桿菌亞種(Bacillus amyloliquefaciens subsp.)植物內(nèi)生菌JR20,其對(duì)初始濃度為5 mg/L的DBP 10天的降解率為98.06%。菌株JR20對(duì)溫度具有廣譜耐受性,在30~40℃范圍內(nèi),對(duì)DBP的降解率基本無(wú)差異。在pH7.0~8.0范圍內(nèi)對(duì)DBP的降解率大于pH為6.0時(shí)。因此,菌株JR20降解DBP的最佳培養(yǎng)條件為37℃、pH為7.0、初始濃度為5 mg/L。通過質(zhì)譜分析,在DBP的降解產(chǎn)物中檢測(cè)到了鄰苯二甲酸(PA),隨著DBP濃度的不斷降低,PA的濃度在不斷地增加,到第5天PA的累積量達(dá)到了最大值。因此,菌株JR20對(duì)DBP的代謝途徑為先將鄰苯二甲酸正二丁酯脫去一個(gè)酯基生成鄰苯二甲酸單丁酯,之后再脫去一個(gè)酯基生成鄰苯二甲酸,因此,鄰苯二甲酸即為菌株JR20降解DBP的最終產(chǎn)物。2.從韭菜根部分離篩選的內(nèi)生菌株JR20,除了在體外對(duì)DBP有一定的降解特性外,還可以通過定殖轉(zhuǎn)接入青菜體內(nèi)促進(jìn)青菜中DBP的降解。通過梯度馴化JR20,逐步提高平板中利福平濃度,篩選出能在含有100μg/m L利福平的平板上穩(wěn)定生長(zhǎng)而且生理生化特性與原始菌株相一致的突變體菌株�？估Ｆ綐�(biāo)記菌株JR20可以通過高梗白在生長(zhǎng)過程中吸收營(yíng)養(yǎng)液途徑進(jìn)入到其體內(nèi),達(dá)到定殖結(jié)果,并在第8天左右達(dá)到定殖數(shù)量峰值,之后由于高梗白生長(zhǎng)到了晚期,組織開始老化,因此菌株在根、莖、葉中的數(shù)量有所降低,直至最后為零。試驗(yàn)得出,DBP在營(yíng)養(yǎng)液中可以進(jìn)行自降解,且當(dāng)初始濃度為1 mg/L時(shí),基本20天內(nèi)可以被完全降解。3.將抗利福平標(biāo)記菌株JR20定殖到青菜體內(nèi)后,檢測(cè)結(jié)果顯示,根、莖葉中DBP的濃度均呈下降趨勢(shì),但接入菌株JR20的試驗(yàn)組降解速率明顯大于未接入菌株JR20的對(duì)照組。到第20天左右,試驗(yàn)組根中DBP的降解率達(dá)95.13%,然而對(duì)照組根中DBP降解率為89.52%,試驗(yàn)組莖葉中DBP降解率為90.45%,對(duì)照組莖葉中DBP降解率為80.61%,試驗(yàn)組中DBP濃度明顯低于對(duì)照組。因此,菌株JR20的接入加速了青菜體內(nèi)DBP的降解速率,使其濃度明顯低于同期未接入菌株JR20的植株。試驗(yàn)組與對(duì)照組營(yíng)養(yǎng)液中DBP的濃度也基本呈下降的趨勢(shì),到第20天左右,試驗(yàn)組與對(duì)照組營(yíng)養(yǎng)液中DBP含量均已接近于零。因此,轉(zhuǎn)入菌株JR20的青菜,由于其體內(nèi)DBP濃度的迅速降解,為了達(dá)到平衡,會(huì)促使其更加快速的吸收環(huán)境中的DBP。菌株JR20在高梗白根中和莖葉中均能降解DBP,且降解DBP的最終產(chǎn)物為PA,降解途徑與在體外無(wú)機(jī)鹽培養(yǎng)基中相同。4.菌株JR20的定殖對(duì)青菜體內(nèi)DBP的轉(zhuǎn)移因子以及根部對(duì)營(yíng)養(yǎng)液中DBP的吸附因子在青菜生長(zhǎng)前期影響不大,但在中后期會(huì)促進(jìn)青菜對(duì)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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：X172

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