基于模式细菌E.coli及其单基因敲除突变菌株的纳米零价铁细胞毒理关键过程研究

The increasing use of nanoscale zero valent iron (nZVI) for in situ soil and groundwater remediation caused raising concerns about its potential adverse effects on the local environment and indigenous ecosystem. Nevertheless, the toxicity mechanism of nZVI is far from well understood till now. In this project, the typical Gram negative bacteria E. coli would be adopted as the model organism, to investigate the mechanism of nZVI toxicity toward microorganisms. First, the E. coli parental strain BW25113 and its single-gene knockout mutants with different extracellular polymeric substance (EPS) secretion would be used, to explore the effect of EPS on direct nZVI toxicity, including adherence of nZVI by bacterial cells and the redox reactions between nZVI and bacterial cells. The iron ion released by nZVI and the production of reactive oxygen species (ROSs) would be analyzed to unveil the ROSs mediated indirect toxicity route of nZVI. Second, iron intake regulation deficient E. coli mutants and ROSs fluorescent probe substances would be introduced to investigate the bacterial iron intake and subsequent ROSs production inside the cell during nZVI treatment. The DNA repair gene knockout mutant would be used to study the DNA damage caused by nZVI. The intracellular toxicity mechanism via iron intake and endogenous ROSs can therefore be elucidated. Finally, the cell damage caused by nZVI such as membrane disruption and metabolism decrease would be investigated. The reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) would be performed to see the impact of nZVI on E. coli DNA transcription, and two-dimensional gel electrophoresis (2-DE) analysis would be performed to reveal the protein damage and responses of E. coli to the nZVI stress. This project would deepen our understanding about the nZVI cellular toxicity, as well as the ecological safety assessment of nZVI remediation technology.

纳米零价铁（nZVI）广泛用于污染地下水及土壤修复，其生态环境安全性日益引发关注。本项目以E. coli为模式生物，研究nZVI细胞毒理的关键过程：利用单基因敲除E. coli，研究胞外聚合物分泌量对nZVI吸附、氧化还原等直接毒理过程的影响；考察nZVI非接触毒性，鉴定主要致毒活性氧物种（ROSs）并测定其浓度，披露ROSs介导的nZVI间接毒理细节；利用铁摄入调控及DNA损伤修复等缺陷菌株，研究nZVI对细菌铁摄入、胞内ROSs反应及DNA损伤的影响，揭示nZVI的细胞内毒理；考察细胞结构损伤和代谢活性，用逆转录定量PCR和差异蛋白质组学技术研究E. coli基因转录及蛋白表达水平的变化，揭示nZVI胁迫下E. coli的损伤和应激机制。本研究对于深入理解nZVI的毒理过程、合理评价nZVI生态安全性具有显著意义。

近数十年来，纳米零价铁（nZVI）被广泛用于地下水和土壤的污染修复，但其生态环境毒性也引发关注。nZVI能够显著致死多种微生物如细菌、病毒等，但其具体毒性机制仍不清晰。本项目以E.coli为模式微生物，采用单基因突变菌株的手段，对nZVI毒性作用的胞外及胞内作用途径和机理进行了深入研究。胞外毒理过程主要包括nZVI在细菌外表面的吸附进而导致细菌细胞膜发生物理损伤，此过程伴随着nZVI的氧化腐蚀，一方面nZVI转化为低毒或者无毒的铁氧化物或铁氢氧化物而逐渐脱毒，另一方面，nZVI在有氧腐蚀过程中会活化分子氧生成活性氧物种，对细胞造成氧化损伤。相对而言，细胞膜物理损伤不是细菌灭活的直接原因，在营养充足的培养条件下，膜损伤甚至可以部分修复；活性氧物种介导的氧化损伤应该是细菌灭活的主要途径。细菌EPS组分在这些毒理过程中起到如下作用：一是淬灭体系中产生的活性氧物种，从而保护细胞免受氧化损伤；二是EPS 能够增加nZVI的吸附，并促进其氧化腐蚀，更快到向无毒的铁氧化物转化，从而降低零价铁的毒性，但同时会导致细胞膜损伤增加。总体而言，EPS分泌量增加会提升细菌对nZVI的抗性。胞内毒性作用途径为：nZVI易于吸附在细菌的表面，并造成细胞膜的物理损伤，使得其通透性增加，同时nZVI氧化释放的铁离子经损伤的细胞膜注入细胞内，导致细胞内游离态铁离子浓度上升，引发胞内Fenton反应等致使胞内活性氧物种浓度升高，后者对胞内组分如DNA等造成氧化损伤并致死细菌。该胞内毒理途径对细菌灭活的贡献度可达30%-70%. 这些研究结果更为清晰地描绘了纳米零价铁灭活细菌的机理图景，对于深入理解零价铁毒性、评估零价铁的环境风险具有重要意义。最后，我们利用零价铁的毒性，开发其作为杀菌剂在水消毒方面的应用潜力，制备了一种具有高效消毒性能的非晶零价铁材料，实现了水中细菌的快速灭活和同步物理去除，并厘清了其相关的反应机制。