【作者】 宋立超；

【导师】 张玉龙；

【作者基本信息】 沈阳农业大学 ， 土壤学， 2011， 博士

【摘要】 我国陆上许多油田位于盐碱土地区,随着石油工业的发展,油田周边土壤受到多环芳烃(PAHs)污染越来越严重；此外,最近频发的海上石油泄漏事故也使滨海地区盐土PAHs污染事件常有发生。为此,盐碱土壤PAHs污染问题已越来越受到人们的关注。微生物降解被认为是去除环境中PAHs的主要途径,但盐碱土壤苛刻的生境条件和芳香族化合物危害的双重胁迫,使普通微生物难于在其中存活生长,致使盐碱土壤微生物修复PAHs污染变得困难。本文从长期受石油污染的天津大港油田滨海盐土中筛选分离出耐盐碱PAHs高效降解菌,采用液体培养的方法,系统研究了不同盐分含量、酸碱度以及PAHs浓度等生境条件,对菌株降解PAHs的影响；并以大港油田盐土为供试土壤,采用人工室内模拟的方法,对高效菌株固定化、以及向污染土壤添加表面活性剂等生物强化方式降解PAHs进行了系统的研究；在此基础上,通过测定细菌Pantoea sp.TJB5降解PAHs酶活性及降解酶相关基因的表达,从酶学和分子水平上阐释了细菌Pantoea sp.TJB5对PAHs降解机制。结果表明,菌株具有较强降解PAHs的能力,用于盐土PAHs修复是可行的。所得主要研究结果如下。1.以菲、芘混合样品为唯一碳源和能源,在滨海盐土中分离出5株细菌、3株真菌。效率测定结果表明,分离出的8个菌株均能降解PAHs,但降解能力不同。分离出的5株细菌分别编号为TJB1、TJB2、TJB3、TJB4和TJB5,3株真菌编号为TJF1、TJF2、TJF3。TJB1为叶杆菌属(Phyllobacterium sp.)、TJB2和TJB3为假单胞菌属(Pseudomonassp.)、TJB4为盐单胞属(Halomonas sp.), TJB5为泛菌属(Pantoea sp.); TJF1为青霉属(Penicillium sp.), TJF2为双曲孢属(Sigmoidea sp.), TJF3为胶孢炭疽属(Colletotrichum sp.)。2.选取降解能力较强的细菌Pantoea sp.TJB5、真菌Penicillium sp.TJF1和8株菌混合液(TJM)为供试菌株进行液体培养,以典型PAHs—菲和芘为研究对象,系统分析了pH、盐分含量、PAHs初始浓度以及葡萄糖供应水平对供试菌株降解PAHs效率的影响。结果表明,随着PAHs初始浓度增加,供试菌株对PAHs的降解效率逐渐降低,但以Pantoea sp.TJB5对菲、TJM对芘的降解效果较好；Pantoea sp.TJB5、Penicillium sp.TJF1和TJM具有很强的耐盐碱特性,在菲、芘初始浓度均为50 mg/L、盐浓度2%、pH 8.6的液体条件下,它们对菲、芘的降解率分别达到93.9%、41.3%、56.6%和20.1%、27.9%、52%；加入适量葡萄糖作为共代谢底物可促进对PAHs的降解,其中Pantoea sp.TJB5、Penicillium sp.TJF1和JTM对菲、芘降解率分别提高了1.2%、5.5%和22.5%、29.3%及35.5%、13.8%。3.对Pantoea sp.TJB5降解PAHs功能酶进行定位测定,结果表明该酶为胞内酶。红外图谱分析结果表明,胞内酶含有—OH、—NH、C=O、C—O、C=S等基团,属于某些酶蛋白质和糖类的基团,在对PAHs羟基化,脱羧等开环反应中发挥着一定作用,使PAHs水溶性增强,易被生物所降解。邻苯二酚-2,3-双加氧酶(C230)是PAHs开环裂解关键胞内酶,并从降解菌Pantoea sp.TJB5中扩增得到C230的部分基因序列,克隆测序获得534 bp,证明了PAHs降解过程中C230关键酶的存在。4.降解菌Pantoea sp.TJB5、Penicillium sp.TJF1和菌群TJM在固定化、添加表面活性剂等处理条件下对菲和芘污染的盐土修复效果明显；实验结果表明对微生物做固定化处理和向污染土壤中添加表面活性剂,供试菌株对盐土中的菲、芘污染物具有较高的去除率,证明这在污染盐土的修复技术上是可行的。供试菌株固定化和添加表面活性剂处理50天对芘的降解率均较添加游离菌的对照处理高出20%以上；其中,固定化处理的混合菌效果最好,20天时盐土中的菲接近背景值,50天芘的去除率达到97.4%、高出对照处理游离菌的25.8%。5.固定化菌株和固定化与鼠李糖脂联合处理修复盐土PAHs污染效果显著；添加固定化菌株和鼠李糖脂5个月后,土壤中4环PAHs的去除率为94.9%,高于游离菌处理。更多还原

【Abstract】 Many oil fields in China are located in the salt-alkali soil, with the development of prtroleum industry, the pollution of nearby soil by polycyclic aromatic of oil fields is getting more serious; In addition, the frequent happening of oil spills recently at coastal area also leads to the PAHs contamination of salt-alkali soil. Thus, the PHAs pollution of salt-alkali soil is getting more and more attention. Microbial degradation is considered to be the major approach for the removal of PAHs in the environment. However, dual stresses of the salt-alkali environment and the aromatic compounds make it difficult for the microbes to survive and grow, which result in the difficulty of the microbial rehabilitation of PAHs pollution in salt-alkali soil. We isolated some PAHs degrading bacteria from the long-standing Oil-polluted coastal salt-alkali soil of Dagang field in Tianjin.The infuluence of different salt concentration, pH, PAHs concentration and other factors are studied systematically using liquid culture method, The salt soil of Dagang oil field was taken as the experimental soil to study systematically the degradation of PAHs after the immobilization of degrading strain and the addition of surface active agen to the polltuted soil. On this basis, by measuring the degrading enzyme activity of PAHs-degrading bacteria Pantoea sp.TJB5 and expression of related genes, we drew some conclusions on the degradation mechanism of the PAHs on enzymetic and molecular level. The results showed that this strain has a strong activity on PAHs degradation and is possible to be applied on salt-alkali soil rehabilitation. The main results obtained are as follows.1. Taking mixed samples of pehnanthrene and pyrene as sole carbon and energy source, we isolated 5 strains of bacteria and 3 strains of fungi from the salt-alkali soil. It was showed that all the 8 strains isolated could degrade PAHs, but the degradation capacities were different. The 5 strains of bacteria isolated were named as TJB1、TJB2、TJB3、TJB4 and TJB5, the 3 strains of fungi were named as TJF1、TJF2、TJF3. TJB1 was leaf spp(Phyllobacterium sp.), TJB2 and TJB3 were Pseudomonas (Pseudomonas sp.), TJB4 was the salt unit cell(Halomonas sp.), TJB5 was pan-genus(Pantoea sp.); TJF1 was Penicillum(Penicillium sp.), TJF2 was Hyperbolic sp(Sigmoidea sp.), TJF3 was anthrax spores for the plastic case (Colletotrichum sp.).2. Bacteria Pantoea sp.TJB5 and fungus Penicillium sp. TJF1 with high degradation ability and 8 strains mixture (TJM) were tested using the liquid culture, Taking PAHs-phenanthrene and pyrene as the research object, the effect of pH, salt content, the initial concentration of PAHs and suppling levels of glucose on the strains’s degradation rate on PAHs. The results showed that the degradation efficiency of the tested strains on PAHs decreased with increasing initial concentration of PAHs, but Pantoea sp.TJB5 had better effect on pehnanthrene, and TJM had better effect on pyrene; Pantoea sp.TJB5, Penicillium sp. TJF1 and TJM were more tolerant to salt-alkali. When the initial concentration of phenanthrene and pyrene was 50 mg/L, salt concentration was 2%, pH was 8.6, the degradation rate on phenanthrene of them reached 93.9%、41.3%、56.6% and 20.1%、27.9%、52% respectively; Addition of appropriate amount of glucose as co-metabolic substrate can promote the degradation of PAHs, for example, the degradation rate of Pantoea sp.TJB5, Penicillium sp.TJF1 and TJM on phenanthrene and pyrene increased by 1.2%、5.5% and 22.5%、29.3% and 35.5%、13.8%.3. The functional degrading enzyme of Pantoea sp.TJB5 on PAHs was showed to be intracellular enzyme through positioning measurement. The IR spectra analysis showed that the intracellular enzymes containing groups as OH、NH、C=O、C—O、and C=S, these groups were connected with some enzymes and Carbohydrates, and played some roles in the oxidation, decarboxylation and reduction, and thus make PAHs more soluble and easy degrading. Catechol-2,3-dioxygenase (C230) is a key enzyme in the ring cleavage of PAHs, we amplified C230 partial gene sequence of 543 bp from Pantoea sp.TJB5, indicating that the key enzyme-- C230 of PAHs degrading exists.4. Degrading effect of bacterium Pantoea sp.TJB5, Penicillium sp. TJF1 and flora TJM on phenantheren and pyrene was significant after immobilization or addition of the surface-active agent in the salt-alkali soil remediation. The results showed that the tested strains has a high removal rate of pollutants on phenanthren, pyrene in salt-alkali soil after addition of immobilized micro-organisms and surface-active agent to the contaminated soil, which proved that it’s practical in the repair of contaminanted alkali soil. Immobilization of the tested strains, and addition of surfactrant for 50 days could improve the degradation rate of pyrene by more than 20% compared with control of sole bacteria; The effect of immobilized bacteria mixture was best, the phenanthrene content in salt-alkali soil was close to the background value after 20 days, the removal rate of pyrene 50 days was 97.4%, higher than the control treatment of free bacteria’s 25.8%.5. The remediation effect of immobilized strain and rhamnolipid treated immobilized strain was remediation of PAHs in salt soil; Removal rate of 4 rings PAHs in soil was 94.9% 5 months after the addition of rhamnolipid, higher than in the treatment of free bacteria.更多还原