【作者】 夏颖；

【导师】 闵航；

【作者基本信息】 浙江大学 ， 微生物学， 2004， 博士

【摘要】 多环芳烃(Polycyclic aromatic hydrocarbon，PAHs)在环境中分布广泛，极易在环境中累积并可通过食物链传递，对人类健康和生态环境具有很大危害性。菲因具有独特的化学结构，成为PAHs研究中的模式化合物，菲污染的研究不仅有利于污染环境中菲的去除，还可为其它PAHs污染的防治提供有价值的资料。 本文全面探讨了菲污染对淹水稻田土壤的微生物生态毒性效应并分离到两株菲降解细菌，对其进行了鉴定以及系统发育研究，同时进行了降解特性和降解基因克隆及表达方面的研究，为建立有效的菲污染预警指标体系和菲降解微生物的有效利用提供有益的参考。 本研究所获主要结论如下： 1、以杭州临平镇附近农田水稻土为材料，采用室内培养方法系统研究了不同程度的菲污染对淹水稻田土壤可培养微生物种群以及酶活性动态变化过程的影响。研究结果表明，不同浓度的菲对好氧细菌的生长和脲酶都具有刺激作用，并且刺激作用随菲浓度的升高而有所加强；不同浓度的菲对真菌和脱氢酶则在整个培养过程中都具有毒性作用，特别在高浓度菲处理土样中，这一表现更为明显；各浓度的菲都对放线菌、厌氧固氮菌和产甲烷细菌具有急性毒性效应，并且100mg／kg干土的浓度一直表现出对放线菌的毒性效应。在传统微生物污染生态研究基础上，运用PCR-DGGE方法，对菲污染条件下稻田土壤真细菌种群的基因多样性进行了初步研究。通过对菲处理后的淹水稻田土壤的DGGE图谱和多样性指数分析，发现各处理间的土壤微生物多样性出现了一定的差异，并且菲浓度越高与对照土样的多样性指数相似性越低。 2、从石油污染土壤中筛选分离到2株菲降解菌株，分别命名为ZX4和EVA17菌株。通过细菌形态学观察，生理生化测试，碳源利用实验(Biolog)，G+Cmol％含量分析，全细胞脂肪酸分析以及16SrDNA和ITS序列同源性分析，证明两株菲降解细菌应属于鞘氨醇单胞菌属细菌。其中菌株ZX4被鉴定为少动鞘氨醇单胞菌的又一菌株，推测菌株EVA17应当是不同于sPh功gomonas属现有各成员的一新种。 3、2株菲降解菌都具有良好的pH和菲含量适应性，菌株ZX4可以转化叫噪产生靛蓝。底物试验结果和降解酶(水杨酸轻化酶和邻苯二酚2，3一双加氧酶)活性的测定表明，2菌株可能都是通过水杨酸途径来降解菲。本文结果也显示GST酶与多环芳烃的降解有关，并且2菌株的GsT酶都具有与cDNB结合的活性。菌株ZX4的菲降解实验说明，初始pH 7.0更利于菌株zx4对环境的适应和菲的彻底降解.初始pH为7.0时，在所试菲含量范围内，菌株ZX4的菲降解速率随菲含量的增加而加快。与硝酸按相比，氮源为硫酸钱时菌株ZX4对菲的降解更快。有机物酵母膏、蛋白陈及葡萄糖的加入可不同程度加快菌株Zx4对菲的降解。Tween一80的加入也对菲降解速率有显著促进作用。与菲的单基质培养相比，多环芳烃蔡、菲的共基质培养不仅促进了ZX4菌株的生长，且提高了其对菲的降解速率;与ZX4菌株的纯培养相比，菲降解菌株ZX4、EVA17的混培虽然对整体生物量的影响不大，却可明显提高培养基中菲的降解速率。 4、根据己知序列设计引物在菌株EVA17中扩增得到芳香环轻化双加氧酶铁硫蛋白a亚基编码基因，该基因全长1368bp，编码455个氨基酸。蛋白质三维结构显示其具有Rieske一型[2Fe一251中心和单核铁原子结合域这两个在酶的催化功能上具有重要作用的保守结构‘并且其氨基酸序列与来自助内功g口mon山sP.PZ的铁硫蛋白a亚基同源性最高。 5、通过对降解菌株ZX4降解基因的克隆转化以及序列分析，得到间位裂解操纵子的部分基因及其邻近基因，并探知它们基因排列顺序依次为编码谷胧甘肤转移酶基因、编码2-轻粘糠酸半醛水解酶基因以及邻苯二酚2，3一双加氧酶基因。ZX4间位裂解基因及邻近基因排列与S一type更为接近，说明本文得到的间位裂解操纵子应为S一卿e的。本文得到的2-轻粘糠酸半醛水解酶基因全长852bP，编码283个氨基酸，编码蛋白蛋白具有ser，09，Asp232，HisZ，9催化三联体结构和oly一As卜ser-Phe一。一y(oly一xaa-ser一xaa-Gly一oly)motif，说明其应属于可p水解酶家族。邻苯二酚2，3一双加氧酶编码基因全长924bP，编码306个氨基酸残基，序列分析结果表明，该基因与来自菌株助八功即胡on二少口月口j七侧口eBI和助八切gom口nassP.P2的砂lE基因(编码产物均为c230)具有较高的核酸序列同源性，并且本文得到的基因推导蛋白在246一2“位置之间具有外断裂双加氧酶模式结构。将邻苯二酚2，3一双加氧酶编码基因在E. coliBLZI进行了成功表达，初步检测结果显示，重组菌株的酶表达量要明显高于出发菌株。 6、根据己知序列设计引物，自菌株EVA17中克隆得到谷肤甘肤S一转移酶基因，该基因片段全长6o6bP，编码201个氨基酸残基，基因核酸序列与助八ing口monas sP.PZ和助入王刀gomonas Paucimobilis EPA505的GsT编码基因序列同源性最高。该GsT蛋白质序列N端具有保守丝氨酸残基，其在GST的催化功能中的作用尚有待进一步研究。 对来自EvA17的谷胧甘肤S一转移酶编码基因在大肠杆菌中进行表达，SDS一PAGE分析表明，出现与预期大小一致的明显条带，并随着时间更多还原

【Abstract】 Polycyclic aromatic hydrocarbons are widespread pollutants and can be released to atmosphere, soils, foods and water. Exposure to PAHs would be hazardous to human health and environments, and therefore, their elimination is considered a priority in many countries. Phenanthrene is one of the simplest aromatic hydrocarbons, that contains a "bay-region" and a "K-region", which are of importance in terms of reactivity and carcinogeniciry. So it has been taken as a model compound for studies on PAHs. Work on phenanthrene biodegradation not only is beneficial to eliminate the contamination of phenanthrene from environments, but also can be valuable in understanding the biodegradation mechanism and feasibility of other PAHs, providing control measurements for bioremediation of soil and other environment contaminated by PAHs.In this thesis, soil microbial physiological and ecological characteristics in submerged paddy soil contaminated by phenanthrene were studied using traditional incubation. This thesis also deals with the isolation and identification of two phenanthrene-degrading bacteria from soil contaminated by oil, the factors influencing growth of phenanthrene-degrading bacteria and degradation of phenanthrene, the routes of phenanthrene degradation and the cloning and expression of phenanthrene degrading gene of two strains. The results will certainly supply useful reference for building up alert index systems in paddy soil polluted by phenanthrene, for environmental quality evaluation and for the using of phenanthrene-degrading bacteria strain. Here are presented the main results of this study:1. Influence of phenanthrene on change processes of culturable microbial populations, and the enzyme activities in submerged paddy soil were studied using traditional incubation method. The results showed that various concentrations of phenanthrene could all stimulate the growth of aerobic bacteria and the activity of urease; but appeared toxic, especially higher concentrations togrowth of fungi and the activity of dehydrogease during whole treatment. All concentrations of phenanthrene showed a short term toxicity on actinomycetes, anaerobic nitrogen-fixing bacteria and methanogenic bacteria during 14days of treatment and the concentration of l00mg/kg dried soil could inhibit the growth of actinomycetes during whole treatment. The effect on diversity of eubacteria in submerged paddy soil with phenanthrene was also studied using the denaturing gradient gel electrophoresis (DGGE) technique. The result showed that phenanthrene had the impact on the microbial community structures, what’s more the effect would be enhanced with the increase of concentrations of phenanthrene.2. Two strains which could use phenanthrene as sole carbon source were isolated from oil-contaminated soil. Both strains were considered to belong to Sphingomonas based on morphology, physio-biochemical identification, whole cell fatty acid analysis and the phylogenetic analysis of 16S rDNA and ITS sequences. Strain ZX4 was identified as Sphingomonas paucimobilis, while strain EVA17 was presumed as a new species of the genus, due to 93%~98% homology with other all species in the genus.3. These two strains had wide pH range for growth and degradation, and could tolerate high concentration of phenanthrene, indicating that the strain should be a potential one used for bioremediation of oil-contaminated environments. Furthermore, strain ZX4 could transform indole to blue indigo, suggesting it could also be an engineering bacterium used to produce indigo. The growth on salicylic acid and catechol showed that the strain degrades phenanthrene via salicylate pathway. The salicylate hydroxylase and catechol 2, 3-dioxygenase were detected in cell-free lysates, suggesting the pathway for phenanthrene catabolism furthermore. The measurement of phenanthrene degradation by strain ZX4 indicated that the rate of phenanthrene degradation was the highest at the initial pH 7 in medium, and the degradation rate of phenanthren was larger when (NH4)2SCX4 as nitrogen sour更多还原