融合菌株对多环芳烃（PAHs）的降解途径及机理研究

Though a large number of bacteria capable of biodegrading PAHs have been isolated from the natural environment, there are still some problems. The bacteria utilizing one kind of PAHs as the sole carbon source isolated from environment usually have high selectivity for compounds of PAHs. In addition, the high-efficiency bacteria can be constructed by mixing the dominant bacteria simply. However, the optimum combination of a variety of bacteria is a very complex and difficult subject. For example, it is difficult to achieve the maximum efficiency because of the interspecific inhibition. In this study, we aimed to study the degrading kinetics, PAHs metabolites (PAHm) and degradation pathway of F14 for degrading mixing PAHs, and demonstrate the interaction mechanisms between various PAHs. The quantitative, toxicity and kinetics of PAHm produced in degradation process were investigated. Furthermore, cell surface properties of strain F14 were characterized by AFM. PAHs and fusion induced protein were identified and investigated according two-dimensional electrophoresis and mass spectrometry techniques. Mechanisms of degrading PAHs by F14 were discussed in molecular level, which provide the possible application and scientific foundation for risk assessment of F14 in soil.

迄今从自然分离得到的多环芳烃 (PAHs) 高效降解菌大多是以某一种单一的PAH为唯一碳源，而将几种不同PAHs降解菌种简单地混合在一起构建高效菌群则很难实现作用最大化。本项目拟通过前期以原生质体融合技术构建的融合菌F14为研究对象，研究F14对各种混合PAHs的降解特性、动力学及降解的中间产物（PAHm）和代谢途径。探明各种PAHs之间的相互作用机制；对降解过程中产生的代谢产物进行定量化、毒性以及动力学研究；运用原子力显微镜（AFM）考察F14细胞表面性质的变化特征，并通过蛋白组学方法探求PAHs诱导性蛋白和融合特异性蛋白；从微生物自身层面探讨F14降解PAHs的机理。为F14进行PAHs污染土壤的修复及评价F14的生态风险提供科学依据和理论指导。

获得高效降解PAHs的、作用底物范围广、环境适应性更强、具有积累少甚至不积累有毒中间代谢产物降解途径的菌株是目前生物修复多环芳烃（PAHs）的关键，将有助于PAHs污染环境的生物修复。. 融合菌株F14 是以芘降解菌-假单胞菌Pseudomonas Sp.GP3A和菲降解菌-鞘氨醇单胞菌Sphingomonas sp.GY2B为亲本，通过原生质体融合技术构建的一株多环芳烃（PAHs）高效降解菌。F14 不仅能够比亲本高效的降解菲和芘，经过实验证明，F14 对各种混合的PAHs 都有一定的降解能力：对二环的萘、三环的菲和四环的芘的混合，四种三环芳烃（苊、蒽、菲和芴）的混合，七种多环芳烃的混合都有一定的降解效果。. 1-羟基-2-萘酸和2-羟基-1-萘酸都是菲降解过程中的产物，其EC50值分别为23.22 mg/L和24.29 mg/L。2-羟基-1-萘酸是F14菌降解菲的主要代谢产物，通过比较两者的EC50值可知，2-羟基-1-萘酸的毒性小于母体菲的毒性，说明F14在刚开始降解菲的过程中毒性逐渐减弱；随后的代谢产物水杨酸和邻苯二酚的15 min-EC50值分别为53.84 mg/L和40.28 mg/L，邻苯二酚的毒性比水杨酸大，说明在随后菲的降解过程中，毒性并不是一直在减小。F14降解其他多环芳烃产生的代谢产物毒性结果：9-芴醇的EC50为22.44 mg/L；1-二氢茚酮的EC50为22.83 mg/L；蒽酮的EC50为15.91 mg/L。. 处于对数期以及稳定期的F14表面疏水率分别达到了69.7%，65.2%，具有较高的表面疏水性。这对污染物的去除有很强的促进作用。. 实验研究了F14 对土壤中100 mg/L菲的降解效率大约为30%左右。为了提高土壤中芘的降解效果，分别采用冷冻解冻法，改进的PVA—硼酸法和“煤渣吸附-包埋-交联”相结合的固定化方法对融合菌株F14进行了固定化，结果表明固定化菌比游离菌对土壤中芘的去除效率显著提高。