嗜盐菌与固氮微生物协同修复石油污染潮间带技术研究

During the past few years，marine oil spill were occurred frequently .Even most petroleum hydrocarbons were effective controled or recoveried , part of compounds found in intertidal zone.Under the hydrodynamic conditions and effects of marine ecosystem, oil concentration have periodical changes, which do much harm to the marine ecosystem . Biodegradation of petroleum compounds in saline environments seems intricate and needs more attention. In this study, we try to analyze the microbial community structure of petroleum-contaminated seawater and intertidal zone in Dalian bonded area by high throughput sequencing , then using marine medium, with crude oil as carbon source, under 3-8% salt concentration enrich and screen for halopile ,which could degrade oil effectively under saline condition; At the same time, we also study on diversity of nifH genes in oil-popullution area by PCR and gene clone library, in order to give more informations of nitrogen-fixation microorganism ,which tolerance to petroleum hydrocarbons. According to the foundmentals of mutual ecosystem of microorganism , seeking for the effective superiority crosses of halophile and nitrogen-fixation microorganism will carry out to enhance the ability of biodegradation and bioremediation. Those work are promising for bioremediation of marine ecosystem and providing special strain resources.

近年来，海洋溢油事故频发，绝大部分泄漏污染物得到有效控制或回收后，部分石油污染物会进入潮间带，在独特的水动力条件及生态系统影响下，引起近岸海域中油含量周期性超标，给海洋生态系统造成长期危害。本课题利用高通量测序技术分析大连保税区石油污染海域近海海水及潮间带微生物菌群特征，以原油为碳源，在3-8%盐浓度下的海水培养中定向富集、筛选适合潮间带生态环境的中度嗜盐石油降解菌种；同时利用兼并引物扩增，构建克隆文库分析石油污染区固氮酶基因nifH多样性，指导分离耐受石油烃的固氮微生物，探求嗜盐菌与固氮微生物在碳源利用、氮素固定方面的优势组合，利用微生物互生作用强化潮间带生物修复能力，为受到溢油损害海洋生态系统的恢复和重建提供特殊的菌种资源和技术支持。

了解并发现石油污染潮间带自然修复优势菌群，是从实验室研究向实际溢油生物修复应用转化的研究重点。分离中度嗜盐石油降解菌种，筛选耐受石油烃的固氮微生物，可克服高盐环境对降解效率影响，为改善环境C/N比例提供特殊的菌种资源和技术支持。 .本研究利用高通量测序技术对大连石油污染潮间带微生物区系进行分析，发现溢油事件发生地微生物多样性及群落结构差异明显，γ-变形菌纲和α-变形菌纲是生物修复的优势种群。分离获得适合潮间带修复的中度嗜盐菌32株，固氮菌41株，多样性分析表明与高通量测序结果有很好的呼应性。驯化实验筛选获得降解重质稠油的2株细菌和1组菌群，其中盐单胞菌(halomonas sp.)XZL41不仅可耐受25%以上盐浓度，且具有固氮酶活性。鉴定6个潜在的新种，并完成11株嗜盐和固氮菌基因组序列分析。本研究表明高通量测序技术能较客观反映生物多样性，是环境修复评价的有效技术，所获得的菌种资源为后续研究互生作用机制打下基础。