大连新港石油污染海域沉积物中厌氧微生物种群和功能基因多样性与氮源响应规律研究

Bioremediation of marine petroleum pollution is world-wide technical issue to be tackled. With the characteristics of environmental friendly and high efficiency, microbial degradation of crude oil was major practices highly valued by governments and researchers of the world. Our lab focus on studying the microbial remediation of crude oil pollution of marine sediments continuously since the incident of petroleum leakage happened on 16th, July, 2010. Thereafter, we carried out monitoring the petroleum contaminated marine area year by year. Our results indicated the rate of petroleum biodegradation presented higher efficiency in anaerobic microbes than in aerobic ones. In the meanwhile, the biodegradation ratio showed various results under different nitrogen sources. So it was presumed that under anaerobic condition, nitrogen sources may regulate the microbial diversities and their abundancy, through which manipulate petroleum biodegradation. In this project, we use petroleum contaminated marine sediments of Dalian Xingang as materials, our first intention is to discover the impact of different nitrogen sources on diversities of microbial communities and functional genes of petroleum biodegradation, via methods like amplicon sequencing and metagenomics sequencing. Then to elucidate the relationships among nitrogen sources, microbial diversity, and functional genes diversity; and define their effect on the ratio of petroleum biodegradation at preliminary level, and possible functional genes in crude oil degradation in anaerobic condition. At the same time, construction a library of anaerobic bacteria with the crude oil degrading function will be accomplished to enrich and develop novel technologies of microbial remediation.

海洋石油污染生物修复一直是世界性技术难题，作为环保高效的微生物修复技术日益受到各国政府和研究者重视。2010年以来，项目组对大连新港“7.16”石油泄漏污染海域进行了持续监控和微生物修复技术研究。其中研究发现，厌氧环境下微生物石油降解性能高于有氧环境，且不同氮源对微生物石油降解性能影响显著。进而推测,厌氧环境下氮源可能参与调控微生物种群多样性及丰度，并参与调控石油降解代谢。因此，项目拟以大连新港石油污染海域沉积物为研究对象，采用扩增子和宏基因组测序等技术，探索厌氧环境下，不同氮源富集样品中微生物种群多样性、石油烃降解基因多样性及其变化规律。阐明氮源、微生物种群多样性和功能基因多样性三者间关系，及其对石油烃降解性能的影响规律；推测厌氧环境下可能的石油降解功能基因；同时构建大连新港石油污染海域石油降解厌氧微生物菌种资源库，为丰富和开发石油污染微生物修复新技术奠定理论和实验基础。

海洋石油污染生物修复一直是一项世界性技术难题，作为环保高效的微生物修复技术日益受到各国政府和研究者重视。海洋沉积物处于微氧/厌氧、寡养、低温、黑暗等恶劣条件，厌氧微生物的修复技术显得更为重要。不同C/N比例是直接决定微生物生长繁殖条件之一，本项目系统分析了不同氮源对海洋石油污染沉积物样品中厌氧微生物菌群多样性及丰度的影响，明确了有机氮源对石油烃降解的正调控作用，并获得了一种高效石油烃降解富集培养基YH，对污染程度不同的样品进行富集后，降解率均高达70%以上；基于扩增子测序技术，揭示了石油烃降解优势厌氧微生物菌群为Proteobacteria，Fusobacteria和Bacteroidetes，且研究发现三种菌群的比例关系显著影响石油烃降解效果；基于宏基因组测序技术，系统分析了有机氮源、无机氮源及混合氮源的差异富集基因、富集代谢通路、参与石油烃降解的功能基因多样性与丰度及潜在的调控途径。基于宏基因组数据分析结果表明，有机氮源富集样品DD有利于增加ASS，BSS，ACCT和SD/FR酶的编码基因拷贝数，特别是ASS和BSS基因，混合氮源富集样品YH次之。此外，混合氮源组样本富集结果显示其促进了琥珀酰辅酶A的转移，促进了乙酰辅酶A的合成，增强了β-oxidation途径，进而增强其石油烃降解效率；基于培养组学研究，搭建了石油烃降解厌氧微生物资源研究平台，获得了405株厌氧微生物菌株，成功构建了厌氧微生物菌种资源库。已鉴定的75株菌株属于细菌域（Bacteria）下3个门，7个纲，9个目，15个科，20个属。其中，有30株新物种资源，在新发现物种中占比40.5％，对其进行了系统分类学鉴定，建立了多个新属、新种等新分类单元；新菌株将为我们提供新基因、新代谢途径及新型降解代谢机制。该研究结果将为今后石油污染厌氧微生物修复治理提供指导，为深入探索海底沉积物的石油污染治理方法，厌氧微生物相关机制的研究，高效石油污染降解菌剂的研发提供宝贵资源。

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