稳定同位素与宏基因组学技术联合研究陕北黄土石油污染原位生物修复机制

The loess is seriously polluted by petroleum in the north region of Shaanxi Province, where remediation technology is in urgent demand. Previous studies show that in-situ bioremediation is the best choice for the application in large petroleum contamination area. However, the petroleum degrading bacteria screened out through traditionally environmental pressing method accounts for no more than 1% of the total degrading bacteria in the real environment. Besides, the accuracy of petroleum compounds analysis were influenced by both various environmental factors and the limitation of incomplete extraction, which leads to an inaccurate evaluation on the bioremediation process. Therefore, this project will focus on: (1) Investigation on the effects of environmental factors on various in-situ bioremediation technologies under the loess plateau climate conditions. In order to define critical environmental factors and optimize the remediation technology. (2) Combining stable isotope probing and metagenomics technology to identify the genomic sequence of in-situ uncultivated petroleum degrading microorganisms under the loess plateau climate conditions. In order to identify the population structure of the functional microorganism and to determine gene sequence of the key enzyme and its mechanism. (3) combining isotope and chemical analysis technology to establish precise quantitative analysis method for petroleum biodegradation and biotransformation in the complicated soil environment. In order to scientifically evaluate the process of petroleum compounds bioremediation. As a result, the theoretical studies and technology systems for in-situ bioremediation of petroleum contamination soil will be established.

陕北地区黄土石油污染严重，亟待适宜性技术加以修复。以往研究表明，原位生物修复是解决大面积土壤石油污染的最佳实用技术。然而，目前基于环境压力筛选的方法，只能获得环境中不到实际1%的石油降解菌。同时，常规石油污染物分析受到复杂环境因素的干扰及萃取不完全的局限，为生物修复过程科学评价造成一定困扰。为解决上述难题，本项目拟研究：（1）针对陕北黄土高原气候特点，研究环境因素对不同原位生物修复技术的影响，明确限制性环境因素，确定最优修复技术与条件；（2）通过稳定同位素探针与宏基因组学技术融合，原位探寻适宜黄土高原气候特点的未可培养石油降解微生物基因组序列，探明未可培养功能微生物种群结构，揭示关键酶基因序列及相关酶学机理；（3）通过稳定同位素与化学分析技术结合，建立复杂土壤环境中石油生物降解与转化的精确定量分析方法，科学准确地评价石油污染生物修复过程。最终构建土壤石油污染原位生物修复研究理论和技术体系。

项目开展了适于陕北黄土的石油污染土壤生物修复研究，结果如下：.（1）采用石油指纹图谱联合稳定碳同位素技术，调查了陕北黄土石油污染概况，采用分子生物学及生物信息技术，调查了污染土壤的生物多样性与环境因子。结果表明调查区域土壤和河道底泥存在石油高浓度持续输入，水土流失加剧了污染的迁移与扩散，当地土壤可为石油降解功能菌筛选提供丰富的菌源，营养失衡和含水率低为生物修复的限制性环境因子。.（2）以石油污染黄土为菌源，通过富集培养法筛选了7株可培养的石油降解优势菌，并用于生物强化及生物刺激联合生物强化修复污染土壤研究，结果表明外加菌群能显著提高正构烷烃和PAHs的生物降解，且氮源添加对污染物去除有促进作用。采用DNA-SIP和RNA-SIP技术首次识别出能降解菲、芘的新菌种分别为16和7种，为适于区域环境特点的高效降解菌剂开发提供了理论支持。.（3）联合稳定碳同位素、环境化学和分子生物学分析技术探索了典型石油污染物的生物降解机理。利用稳定氢同位素在生物化学过程中的分馏效应，基于Lord Rayleigh方程，构建了生物降解模型，确定了菲、n-C16和n-C21在NA和BA修复中的生物降解率，证明了生物降解在污染物去除中起主导作用，且生物强化优于自然衰减，揭示PAH-RHDαGP基因对菲降解起关键作用，AlkB和AlmA基因对n-C16和n-C21降解起关键作用。