包气带微生物跨膜运输多环芳烃的动态过程及膜蛋白功能调控研究

Organic pollution in the aeration zone such as polycyclic aromatic hydrocarbons (PAHs) has been seriously contaminated and destroyed the groundwater environment. In order to provide scientific direction for the bioremediation project of PAHs contaminated and improve the PAHs biodegradation efficiency, the transport and metabolism process of PAHs and regulation abilities of microorganism should be re-recognized in the level of cell and membrane protein. According to the complex relations among the aeration zone, microorganism and PAHs, functional bacteria that we isolated from oil-contaminated soils were used as the target cells. Stable-isotope probing (SIP) 13C combined with Fluorescent In Situ Hybridization (FISH) technique were used to label the single PAHs and microorganism respectively. Laser Scanning Confocal Microscopy (LSCM) and Nano Secondary Ion Mass Spectrometry (Nano SIMS) were provided to observe and model the dynamic process of PAHs transmembrane transport across the microorganism. Technique iTRAQ was used to analyze the variation of functional regulation for membrane protein in the transport and metabolism process. Recognized the core links, key sits and steps of rate-control, functional regulation in the transport and metabolism processes. Meanwhile, improved the bioavailability of PAHs by regulating the environmental conditions of aeration zone. Reestablished the highly bioremediation system among the aeration zone, microorganism and PAHs contamination in the level of cell and membrane protein. The achievements will be used to provide the theoretical support and practical guidance in the in-suit bioremediation of organic pollution in the aeration zone and groundwater environment.

包气带多环芳烃等有机污染日趋严重，对地下水环境造成了严重的污染和破坏；为了科学指导包气带多环芳烃污染的微生物修复处理工程，有效提高多环芳烃的降解效率，需从细胞和膜蛋白水平上重新认识微生物运输代谢多环芳烃的过程和调控功能。针对包气带-微生物-多环芳烃修复体系之间的作用关系复杂，本项目以前期筛选的功能降解菌为靶细胞，利用稳定同位素13C与FISH技术对多环芳烃和微生物单体进行标记，通过激光扫描共聚焦显微镜结合纳米二次离子质谱技术对多环芳烃跨膜运输的动态过程进行观测和建模；利用iTRAQ技术分析细胞膜蛋白在运输代谢多环芳烃过程中功能调控能力的变化；识别微生物运输代谢多环芳烃的核心环节、关键速控点、速控步骤和调控功能。调控包气带环境以提高多环芳烃的生物可利用性，从细胞和膜蛋白分子水平上重新建立包气带-微生物-多环芳烃高效修复技术体系，为包气带/地下水有机污染的原位修复提供重要的理论依据和实践指导。

本项目以课题组筛选出的多环芳烃高效降解菌红球菌BAP-1、嗜麦芽寡养单胞菌W18和蜡样芽胞杆菌DQ-1作为研究靶微生物，以探究包气带微生物降解多环芳烃过程与膜蛋白调控机制为目标，通过实验研究，发现在红球菌BAP-1可以通过膜蛋白的变化促进荧蒽的跨膜运输过程，荧蒽的存在主要影响了红球菌BAP-1细胞膜上的与能量、转运相关的膜蛋白，并随时间推移，一部分转运蛋白表达量逐步提高；在Tween80的诱导下，嗜麦芽寡养单胞菌W18对荧蒽的降解效率显著提高，类NOD受体信号通路、核糖体通路以及鞭毛组装通路受影响较大；单细胞水平下观测W18对荧蒽的代谢过程，发现Tween80的加入使荧蒽在细胞膜上的含量显著增加；利用多组学技术研究蜡样芽孢杆菌DQ01降解荧蒽的代谢通路，发现具有新陈代谢功能的差异蛋白占比最多，细胞膜转运、群体感应、碳水化合物代谢以及能量代谢通路受影响较大，在此基础上对典型的差异膜蛋白的结构进行了模拟与分析。本项目从微观角度重新认识了微生物跨膜运输多环芳烃的模型与膜蛋白调控机制，为包气带多环芳烃污染的微生物修复技术提供重要的理论依据和实践指导。