邻苯二甲酸二丁酯降解菌DNB-S1的筛选和其酶制剂的初步应用

发布时间：2018-03-26 20:03

  本文选题：邻苯二甲酸二丁酯　切入点：降解动力学　出处：《东北农业大学》2015年硕士论文

【摘要】：DBP(邻苯二甲二丁酯)是一种PAEs(邻苯二甲酸酯)化合物,作为一种塑化剂被广泛使用在生产生活中。目前,DBP在环境中普遍存在,对人体具有“三致”毒性。环境中自发光降解、水降解及微生物降解不能有效的分解DBP,因此筛选高效降解DBP菌株才是有效降低PAEs浓度的途径。本研究以DBP做为唯一的碳源从哈尔滨市某荒废污染土壤中分离出一种具有高效DBP降解能力的菌株DNB-S1,采用形态学、生理生化指标、16Sr DNA鉴定菌种,并对其生长特性、降解特性进行了研究。对DNB-S1的降解酶进行定位,对其最适条件进行了探究,采用海藻酸钠固定该酶,并用室内盆栽试验初步验证了固定化酶的降解效果。(1)分离获得一株DBP高效降解菌,经鉴定为假单胞菌(Pseudomonas sp.),命名为DNB-S1,该菌株在透射电镜下呈杆状,长1721nm宽709nm,以极生鞭毛运动,无细胞核。菌落为白色、边缘为锯齿状、中心凸起、圆形、无芽抱。革兰氏染色呈阴性,并且为严格耗氧菌,能以DBP为唯一碳源生长。(2)DNB-S1菌株的最适生长条件为,DBP浓度500mg/L,温度35℃,p H为7.0,接菌量为3%,摇床转速设置为125r/min,高浓度的DBP(2000mg/L)含量会对DNB-S1的生长具有一定抑制作用。在该条件下培养菌株48h,测定发现,DNB-S1对500mg/L DBP的降解率为90%,得到DNB-S1一级动力学方程ln c?6.444?0.032t(R2=0.9837),降解半衰期为20.9小时。(3)DNB-S1的降解酶为胞内酶。酶促反应最适条件为:35℃,p H=7.0,该条件下反应24h后,可将500mg/L的DBP降解85.6%。(4)选用海藻酸钠为固定化材料对DNB-S1胞内酶进行固定化处理,最佳固定条件为:固定化体系(18ml)中粗酶液最佳添加量为900ug,海藻酸钠的质量分数为1.0%,包埋比1:2,Ca Cl2溶液质量分数为2.0%。在24h内固定化酶小球可以将500mg/L的DBP降解到98.5mg/L,降解率可以达到80.3%。(5)分别设置空白处理、游离酶处理、固定化酶处理3个不同的修复组,进行室内盆栽模拟修复试验,初始土壤DBP含量为25mg/kg,在第0、7、14、21、28天取样检测剩余DBP含量。同空白处理比较,添加降解酶之后,DBP的含量明显降低,经过28天的处理之后,游离酶降解率为63.2%,效果显著。固定化酶的修复效果更加出色,28天的DBP降解率为91.01%,DBP含量降低至2.2mg/kg,远远低于美国土壤污染的治理标准8.1mg/kg,到达修复目的。
[Abstract]:DBP (phthalic dibutyl ester) is a kind of PAes (phthalate) compound, which is widely used in production and life as a plasticizer. At present, DBP is ubiquitous in the environment and has "tritoxic" toxicity to human body. Water degradation and microbial degradation can not effectively decompose DBP, so screening high efficient degradation of DBP strains is the effective way to reduce the concentration of PAEs. In this study, DBP is the only carbon source isolated from a waste polluted soil in Harbin. The strain DNB-S1, which has the ability to degrade DBP efficiently, was characterized by morphology. The physiological and biochemical index, 16Sr DNA, was used to identify the strain, and its growth and degradation characteristics were studied. The optimum conditions of DNB-S1 degradation enzyme were studied, and the enzyme was immobilized by sodium alginate. A strain of Pseudomonas sp. Sp., named DNB-S1, was isolated and identified as Pseudomonas sp. (1) the strain was identified as Pseudomonas sp. sp. and named DNB-S1. The strain was rod shaped under transmission electron microscope with a long 1721nm width of 709nmand moved to polar flagella. Without nucleus. Colony is white, margin is serrated, central protuberance, round, no bud clasping. Gram staining negative, and strictly oxygen consuming bacteria, The optimum growth conditions of DNB-S1 strain can be grown with DBP as the sole carbon source: the concentration of DNB-S1 is 500 mg / L, the temperature is 35 鈩，

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