海洋兼性厌氧石油烃降解菌的降解特性及其在环境中的行为特征

Petroleum hydrocarbon contamination is becoming a major widespread environmental problem, especially in coastal areas subjected to high anthropogenic inputs, such as oil spills, urban runoff, shipping and industrial activities. The high hydrocarbon concentrations accumulated in surface and subsurface ocean pose serious concerns for the environmental and human health. The low levels of dissolved oxygen in the ocean make the anaerobic or facultative anaerobic degrading bacteria the most important functional group. However, few data have been presented about the anaerobic or facultative anaerobic petroleum hydrocarbon degrading bacteria from the marine environment, especially the autecological properties of the pure bacterial strains. To this end, Green Fluorescent Protein (GFP) tagged pure facultative anaerobic bacterial strains isolated in our lab will be investigated for their abilities and mechanisms to degrade petroleum hydrocarbons under both the aerobic and anaerobic conditions with the combination of interdisciplinary mixed methods of marine microbiology, autecology, biogeochemistry and molecular physiology. The study of the pure bacterial strains under controlled mesocosm systems will lead to an appreciation of their physical and chemical requirements for growth, reproduction and geographical distributions and provide autecological information to explain their behavior. This study also will reveal the relationship between the GFP tagged bacterial strains and the indigenous bacteria and the different degrees of requirements of various nutritional elements during the dagradation process. The insights and theories of this study will increase our understanding of the aerobic and anaerobic degradation process and mechanisms of marine facultative bacteria and provide fundamental evidences for in situ bioremediation of petroleum hydrocarbon pollutions in the marine ecosystem.

由于海上溢油事故频发及陆源含油废水大量输入海洋，石油烃已成为海洋环境的主要污染物之一。海洋环境低溶氧的特性使得厌氧降解菌成为海上石油污染生物降解的主要功能菌群。然而，对海洋厌氧或兼性厌氧降解菌生理特性及其在好氧或是厌氧环境中的行为特征的研究尚不清楚。本研究从实验室分离得到的高效海洋兼性厌氧石油烃降解菌入手，借助传统的微生物学、分子生物学及分子标记等研究方法，通过分析降解产物、功能基因和功能酶，研究兼性厌氧降解菌在好氧或是厌氧环境中对石油烃的降解机理；通过绿色荧光蛋白分子标记兼性厌氧降解菌株，追踪降解菌在厌氧或是好氧微宇宙培养体系中存活、繁殖、发展和空间分布等行为特征。旨在揭示海洋兼性厌氧石油烃降解菌在有氧或厌氧环境中的作用机制，明晰其在环境中与土著菌或是氮、磷营养元素之间的关系，为指导相关的微生物修复活动提供相应的理论依据。

日益频繁的海上溢油事故以及陆源含油废水的输入，对海洋生态环境造成了严重的污染。本研究从渤海分离到的高效海洋兼性厌氧石油烃降解菌Acinetobacter sp. HC8-3S入手，通过微生物学、微生物个体生态学、分析化学以及分子生物学多学科交叉的分析方法，揭示海洋兼性厌氧石油烃降解菌的降解特性及其在环境中的行为特征。Acinetobacter sp. HC8-3S在5天内降解烷烃83.7%，并且在pH（5.6-8.6）和高达70 g/L NaCl浓度条件下，降解活性几乎不受影响。将棉花纤维作为吸附剂和载体，对该菌株进行固定化研究，发现固定化细菌比游离态细菌降解烷烃效率高出30%。基因组学分析显示，Acinetobacter sp. HC8-3S菌株中参与烷烃代谢的基因分散在基因组中，并不聚集成簇或在一个操纵子上。Acinetobacter sp. HC8-3S菌株利用末端氧化途径降解烷烃，烷烃单加氧酶alkM起始该反应，烷烃降解基因的转录调控因子是AraC-XylS类型。基于iTRAQ技术的差异蛋白质组学探究Acinetobacter sp. HC8-3S菌株对石油烃胁迫的响应。结果表明，石油诱导前后72个蛋白表达上调，15个蛋白表达下调，并且脂肪酸降解、香叶醇降解、已内酰胺降解、柠檬烯和蒎烯降解和苯酸盐代谢等与石油降解有关的代谢途径有显著变化。利用基因重组技术，构建绿色荧光蛋白标记菌株Acinetobacter sp. HC8-3S-9。通过荧光显微镜观察，绿色荧光蛋白能够在该菌株中长期稳定表达。模拟原位环境构建了室内微宇宙培养体系。通过高通量测序技术，发现Acinetobacter sp. HC8-3S-9能够与土著菌厚壁菌门（Firmicutes）、绿弯菌门（Chloroflexi）、拟杆菌门（Bacteroidete）协同作用，石油烃降解率高达89.5%。在门水平上，变形菌门（Proteobacteria）与水体和沉积物中的总石油烃都呈现明显的正相关关系。高效石油烃降解菌剂被研发并成功应用到到渤海石油污染海域的生物修复工程中。修复完成后污染海域沉积物样品中的石油烃含量下降，对发光细菌的急性毒性和遗传毒性都显著降低。本研究利用优良降解菌株建立海洋石油污染生物修复技术，并开展修复中的分子生态学研究，揭示石油烃及其降解产物的环境效应，具有重要的生态效益和经济效益。