微生物种群演替对胶州湾溢油的响应规律及其指示的石油降解菌群构建研究

This research is mainly on constructing degradative consortium based on the changing trends, which is the microbial reponse to the oil spill of the shorelines, and this is one of the key scientific problem in bioremediation research. The object of the study is the shoreline containmated by the oil spill. The changes of the bacterial community in the main stages of the biodegradation processes were monitored by high-throughput Illumina MiSeq sequencing. The sequencing data was to predict community dynamics and functions under variable environmental conditions. Analysis instruments, like TLC, GC-FID, GC-MS, etc., are used to detect the composition of the crude oil after the oil spill and these data was used to run the correlation analysis with the consortium succession. The predominant strains are determined by sequence analysis. And DNA probes are designed according to the analysis result. The biodegradation strains are screened firstly. The designed probes are hybridized with the DNA from the strains. So the dominant bacterias on the shoreline are conformed. The biodegradative consortium is constructed and the function will be evaluated. Our aim is to clarify the regularity of oil spill bioremediation and constructing degrading bacterial consortium under the degradation mechanism of crude oil. All of these studies would be the essential part in environmental remediation theory and desired to be an sparkle for the recovery of marine oil pollution control and ecological environment.

研究岸滩微生物群落对溢油污染的响应趋势，并以此为依据构建符合响应趋势的降解菌群，是生物修复实现的关键科学问题之一。本项目以黄岛输油管管爆炸后溢油污染岸滩为研究对象，通过Illumina MiSeq高通量测序，分析不同降解阶段岸滩微生物群落动态变化对溢油的响应趋势；利用TLC、GC-FID、GC-MS等仪器，分析降解过程中烷烃、芳烃、沥青等石油成分变化差异，与菌群变化数据进行相关性分析，确定原油降解不同阶段的主要功能菌群；以优势菌群特异序列为基础设计荧光探针，初步筛选石油烃降解菌，提取其DNA与探针杂交，从而进一步筛选在环境处于竞争优势的降解菌，并构建降解菌群，进行降解性能验证。本项目通过上述研究，揭示微生物群落受溢油污染后的响应趋势，构建符合其响应趋势的功能菌群，为海洋石油污染的治理和生态环境的恢复提供切实可行的技术支持和理论指导。

溢油生物修复技术易受环境条件影响，限制了其在海洋溢油污染清除中的应用。溢油降解菌群中丰度较高的优势菌是最适应该环境的菌株，在实际应用过程中会降低环境因素对生物修复的影响。本研究对功能微生物群落动态变化与石油降解过程关系，MiSeq高通量测序分析环境中微生物群落动态变化，分析主要功能菌株；通过分子杂交的方式将高通量测序技术与传统降解菌筛选相结合，筛选优势降解菌，构建高效降解菌群，研究其协同作用机制。采集了溢油120d岸滩沉积物样品分析了菌群的动态变化，在门分类水平内，含量最大的菌群分别为Proteobacteria和Bacteroidetes，Proteobacteria含量在46.3%和61.5%之间，含量变化不大。Bacteroidetes与Proteobacteria有较大差异，其丰度在26.3%-51.0%间；随着烷烃浓度降低Alkanindiges、Pseudospirillum和Leucothrix等烷烃降解菌株含量逐渐降低，芳烃降解菌Cycloclasticus的丰度逐渐增加。溢油对海底不同污染程度的沉积物菌群有较大差异，在油污浓度较高区域Alcanivorax、Lutibacter等烷烃或多环芳烃降解菌丰度较高。根据菌群多样性数据确定主要优势菌，设计荧光探针，筛选到一株具石油烃降解能力的优势菌为Alcanivorx sp. E4。环境因素影响模拟实验证明E4相对于非优势降解菌E3受环境因素的影响较小。以E4为基础构建降解菌群，其中菌株E3和E4所构建的降解菌群降解效率最高，研究了两者之间的协同作用。E3所分泌胞外物质能够促进E4的浓度的增加，提高了E4的降解效率。探索了以环保低成本的海带渣为载体固定化技术研究，以海带渣、海藻酸钠、高岭土和降解菌发酵液制备固定化菌剂，用于海洋沉积物的生物修复技术。