固定化降解菌Pseudomonas sp.DNB-S1对DBP污染土壤修复的研究

发布时间：2018-06-26 21:20

本文选题：固定化 + DBP　；参考：《东北农业大学》2015年硕士论文

【摘要】：土壤中酞酸酯(PAEs)污染来源十分广泛,农膜残留、农药和肥料施用、生活垃圾以及污水灌溉等都能够引起土壤PAEs污染。本研究运用固定化技术,对邻苯二甲酸二丁酯(DBP)降解菌进行包埋,对制备的固定化微球的性能进行表征,以及固定化微球对土壤中DBP的去除效果和生态修复效应等多方面考察固定化微球的修复效果。以固定化微球对DBP的降解率为考察目标,优化固定化成球条件。采用四因素三水平L9(34)的正交试验,选择不同的海藻酸钠/膨润土比例、Ca Cl2浓度、壳聚糖溶液浓度以及包菌量为组分。正交实验的结果表明固定化成球的最佳条件为海藻酸钠/膨润土为2:1,壳聚糖浓度为0.5%,氯化钙为1.5%,包菌量为10%。利用红外光谱、扫描电镜及比表面积分析仪等,对固定化微球进行性能表征。结果表明:膨润土的表面存在大量的-OH,海藻酸纳的羧基发生了离子化,海藻酸钙凝胶与膨润土之间形成了离子键,产生了微弱的静电引力。膨润土中的羟基,在水环境中与海藻酸钠能够产生氢键,从而限制了海藻酸钙凝胶的溶胀。固定化微球的载体材料海藻酸钠与膨润土的比例为2:1时,其具有较大的比表面积和大量的微孔,能够更好的实现菌体在其内部的存活以及对污染物DBP的有效降解。考察固定化微球在28 d内的实际修复效果以及特性。土壤修复过程中,14 d时,各处理组DBP残留量均在66%以上;在28 d时,固定化微球处理组中,DBP的残留量小于20%。固定化微球在三次重复使用后,对底物DBP的降解率仍然保持在20%以上。4℃条件下,28 d时,固定化微球处理组中,DBP的降解率为78.04%。采用PCR-DGGE技术考察修复后土壤微生物群落结构的变化情况,对DGGE凝胶上19个条带切胶后进行克隆,经测序鉴定,同源性比较,鉴定结果证明试验土壤中微生物主要分属于放线菌门、厚壁菌门和变形杆菌门(α-变形菌纲、β-变形菌纲及γ-变形菌纲)3个大类。受DBP污染的土壤样品和接种游离菌后微生物丰富度随着时间的推移,并未发生较大的波动。固定化微球处理和游离菌处理的多样性指数均呈现先上升后下降的趋势,空白处理组的变化不明显,在28d达到最高。各处理的微生物均匀度均没有发生显著性改变,其中空白处理微生物均匀度在后期呈现先下降的趋势。不同形态降解菌的接种以及DBP的投加均改变了土壤微生物群落结构。利用氯仿熏蒸提取法测定固定化微球对DBP污染土壤中微生物生物量碳、氮、磷随修复时间的动态变化。结果表明,修复过程中,固定化微球的加入可以有效的缓解DBP对土壤微生物活性的影响。研究表明,接种固定化微球能够有效的修复DBP污染的土壤,降低DBP对土壤的危害,同时为土壤中有机污染物的去除提供一定的理论基础以及技术支撑。
[Abstract]:Phthalic acid esters (PAEs) in soil are polluted from a wide range of sources, such as agricultural film residues, pesticide and fertilizer application, domestic waste and sewage irrigation can cause soil PAEs pollution. In this study, the immobilization technique was used to encapsulate the dibutyl phthalate (DBP) degrading bacteria, and the properties of the immobilized microspheres were characterized. The effects of immobilized microspheres on the removal of DBP and ecological remediation were investigated. The degradation rate of DBP by immobilized microspheres was taken as the objective to optimize the conditions of the immobilized microspheres. Four factors and three levels of L9 (34) orthogonal experiment were used to select different concentrations of sodium alginate / bentonite, concentration of chitosan solution and amount of bacteria. The results of orthogonal experiment showed that the optimum conditions for the formation of the pellets were as follows: sodium alginate / bentonite was 2: 1, chitosan concentration was 0.5, calcium chloride was 1.5 and the amount of bacteria was 100.The optimum conditions were as follows: (1) the concentration of sodium alginate / bentonite was 2: 1; The properties of the immobilized microspheres were characterized by IR, SEM and surface area analysis. The results show that there is a large amount of -OH on the surface of bentonite, the carboxyl group of sodium alginate is ionized, the ionic bond between calcium alginate gel and bentonite is formed, and the electrostatic force is weak. The hydroxyl group in bentonite and sodium alginate can form hydrogen bond in water environment, which limits the swelling of calcium alginate gel. When the ratio of sodium alginate to bentonite is 2:1, the carrier of immobilized microspheres has a large specific surface area and a large number of micropores, which can better realize the survival of bacteria in its interior and the effective degradation of pollutants DBP. The effect and characteristics of immobilized microspheres in 28 days were investigated. DBP residues in each treatment group were above 66% at 14 d during soil remediation, and less than 20% in immobilized microsphere treatment group at 28 d. After being reused for three times, the degradation rate of DBP in the immobilized microspheres was still above 20%, and the degradation rate of DBP in the immobilized microspheres group was 78.04 after 28 days. The changes of soil microbial community structure after restoration were investigated by PCR-DGGE technique. The 19 bands of DGGE gel were cloned and sequenced, and the homology was compared. The results showed that the microbes in the soil were mainly classified into three categories, namely Actinomycetes, Parthenomycetes and Proteus (伪 -Proteus, 尾 -Proteus and 纬 -Proteus). The soil samples contaminated by DBP and the microbial richness after inoculation of free bacteria did not fluctuate with time. The diversity index of immobilized microsphere treatment and free bacteria treatment increased first and then decreased, but the change of blank treatment group was not obvious and reached the highest level at 28 days. The microbial evenness of all treatments did not change significantly, and the microorganism evenness of blank treatment decreased first in the later stage. The structure of soil microbial community was changed by inoculation of different morphological degrading bacteria and addition of DBP. Chloroform fumigation extraction method was used to determine the dynamic changes of microbial biomass carbon, nitrogen and phosphorus in DBP contaminated soil with immobilized microspheres. The results showed that the effect of DBP on soil microbial activity could be effectively alleviated by immobilized microspheres during remediation. The results showed that inoculation with immobilized microspheres could effectively repair the soil contaminated by DBP, reduce the harm of DBP to soil, and provide some theoretical basis and technical support for the removal of organic pollutants in soil.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X53;X172

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