基于多组学的铬和四环素类抗生素单一/复合污染下的微生物驯化机制研究

Acclimatization is an important method to obtain highly active microorganisms in environmental microbial engineering systems. To further develop and improve environmental microbial processes, it is therefore very important and necessary to investigate the response mechanisms of microorganisms to pollutants during the process of acclimatization. Though many studies have reported the short-term stress effects of pollutants or adverse environmental factors to microorganisms, there is still a lack of clear understanding of microbial response to long-term stress of pollutants, i.e. the mechanism of acclimatization. In this project, we will use the strains which are able to degrade tetracyclines or transform Cr(VI) as the main targets. Then, we will develop different acclimatization methods and carry out long-term acclimation culture in the presence of one of Cr and tetracyclines or both. The conversion of these pollutants will also be studied during the acclimatization, and microbial samples will be carefully collected. Three omics technologies, which are genomics and transcriptomics based on high-throughput sequencing, and proteomics based on two-dimensional electrophoresis or bio-mass spectrometry, will be the main technologies employed to study the microorganisms. Using these omics technologies, we will be able to explore how microorganisms response to single and mixed pollutants during different stages of acclimatization with different methods. Based on the investigations on microorganisms from gene mutation, gene transcription and protein expression levels, we aim to illuminate the mechanisms of acclimatization for microorganisms treating Cr or/and tetracyclines. Furthermore, the risks of harmful mutations caused by different pollutants will be evaluated. The results obtained from this research project will definitely help people to optimize existing environmental microbial technologies, to develop new environmental microbial technologies and to prevent and control the harmful microorganisms from contaminated environments.

驯化是环境微生物工程中获得高活性微生物的重要手段，因此，研究微生物在驯化过程中对污染物胁迫的应答机制对开发和改进环境微生物工艺是十分重要且必要的。本项目拟以具有四环素类抗生素降解、Cr(VI)转化能力的菌株为主要对象，制定不同驯化策略，对其在Cr、四环素类抗生素的单一胁迫或共胁迫环境中进行驯化培养，监测系统中污染物的转化情况，收集合适微生物样品，主要利用基于高通量测序的基因组学和转录组学技术、以及基于二维电泳或生物质谱的蛋白质组学技术，从基因组突变、基因转录、蛋白质表达等多层次探索菌株在不同驯化策略下的不同驯化阶段对单一和混合污染物胁迫的应答机制，从而深入、全面地阐述处理Cr或（和）四环素类抗生素的微生物的驯化机制，并评估污染物可能引起环境微生物有害变异的风险，从而为环境微生物工程技术的优化和开发以及污染环境微生物危害的防控提供理论支撑。

驯化是提高微生物耐受和转化污染物能力的重要手段，利用多组学技术可以从基因组突变、基因转录、蛋白质表达等方面阐明微生物驯化的机制，以便于改进和开发环境微生物工艺。本研究的主要目的为基于多组学技术探究驯化微生物强化耐受和转化Cr或抗生素能力的机制。在项目执行期，本项目研究团队科学分工协作开展了一系列研究工作，首先以具有高Cr(VI)还原潜力的细菌希瓦氏菌MR-1为研究对象，比较了MR-1在Cr(VI)浓度递增环境下胁迫40天、80天和120天与对照组LB培养环境下生长40天、80天和120天的表型变异情况，并利用全基因组重测序技术对基因变异进行了系统研究，阐明了微生物对铬的分子适应机制和抗性机制；在蛋白质组层面，研究了在Cr(VI)浓度递增的环境中驯化120天的希瓦氏菌MR-1在长期Cr(VI)压力下的适应机制，为评估铬污染对环境微生物的长期影响提供了数据支撑，也为环境工程领域污染物的高效转化提供理论依据；还开展了恒定Cr(VI)和Cr(III)浓度的持续胁迫对微生物的影响机制研究、构建微生物电化学系统驯化微生物和利用纳米材料降解新兴污染物的研究以及种间底物竞争对微生物胞外电子传递的影响机制研究等研究工作。本研究取得了预期的研究成果，相关研究结果已发表期刊论文11篇，发表于National Science Review、Biosensors and Bioelectronics、Chemical Engineering Journal、Bioresource Technology、Environment International、Frontiers of Environmental Science & Engineering、Environmental Science and Pollution Research、Progress in Chemistry、中国环境科学等国内外学术期刊，申请了关于驯化希瓦氏菌MR-1以提高其对六价铬抗性的发明专利1项，参加国内会议10人次，做口头报告4人次，墙报2人次，研究成果得到了广泛传播和国内外同行的广泛关注。