污水生物处理中纳米颗粒对典型亚硝酸菌的毒性效应与机制

Adverse effects of nanoparticles (NPs) on municipal wastewater treatment processes have attracted growing attention recently due to NPs' potential toxicity to microbial communities at cellular, subcellular, and gene levels. However, the interaction mechanisms between microbial cells and NPs are not clearly elucidated. In this innovative proposal, we will comprehensively investigate the impacts of three common nanoparticles (TiO2, ZnO, and CeO2) on a representative and toxin sensitive chemolithoautotrophic ammonia oxidizing bacterium, Nitrosomonas europaea with the combination of environmental engineering, microbial physiology and molecular biology information. The NPs risks to N. europaea performance will be assessed by correlation of NPs stress at different NPs concentrations, exposure time and dissolved oxygen conditions in the chemostat reactor. The morphology, physiology, biochemical metabolism and the microbial recovery capacities of injured N.europaea cells will be examined. The relationships between N.europaea physiological and biochemical characteristics and the transcriptional information of nitrogen redox reaction functional genes or NPs stress response genes will be discussed based on our previous studies of N.europaea nitrogen transformation metabolism and the genome-wide expression analysis. The impact factors, cellular response pathways and mechanisms of NPs cytotoxicity and genotoxicity will be explored. Microbial self-regulation and repair mechanisms after NPs exposure will be revealed. In addition, the NPs toxicity biomarkers will be developed to help establish early warning system for NPs threatened wastewater treatment plants. In summary, the study will provide the nesessary theoretical guidance and technical supports for NPs risk assesment and emergency regulation of biological nitrogen removal processes upon potential NPs contamination.

纳米颗粒对污水处理工艺菌群结构与活性具有潜在毒性影响并导致工艺处理性能下降，但相关毒性作用机制尚未明确。本课题从环境工程学、微生物生理学和分子生物学层面，开展纳米颗粒对生物脱氮工艺中毒性敏感指示细菌-欧洲亚硝化单胞菌（Nitrosomonas europaea）的毒性影响综合研究。课题基于对该细菌氮循环生化途径的研究基础、借助全基因组表达谱学、并结合细胞宏观生理生化特性变化规律，揭示典型纳米颗粒（TiO2，ZnO和CeO2）毒性胁迫下细胞的应激反应基因及影响其转录表达水平的关键因素与作用规律，阐释纳米颗粒毒性影响下细菌的自我应激调节机制，掌握纳米颗粒对该细菌的生物毒性作用途径与机理，探讨受损菌群生理生化功能修复性能以及自我调节和修复机制，并在此基础上确立可用于污水生物脱氮系统纳米颗粒毒性预警监控的生物标志物，为污水生物脱氮工艺中纳米颗粒的环境风险评价与应急调控提供必要的理论指导和技术支撑。

纳米颗粒（NPs）在市政污水生物处理系统中的归趋会对其中的菌群结构与活性产生潜在毒性影响并导致工艺处理性能的下降。本课题从环境工程学、微生物生理学和分子生物学层面，开展了纳米金属氧化物（n-TiO2、n-ZnO和n-CeO2）对生物脱氮工艺中毒性敏感指示细菌Nitrosomonas europaea的毒性影响综合研究。三种NPs短期作用即可对N.europaea产生与NPs投加浓度呈正相关性的生物胁迫影响，相同剂量条件下n-ZnO 生物毒性效应最强、n-TiO2最弱。受NPs的带电性、溶解度和吸附性能影响，n-TiO2与n-CeO2 或n-ZnO2共存可导致胁迫拮抗效应，而n-CeO2与n-ZnO共存则产生胁迫协同效应。低浓度（1 mg/L或10 mg/L）NPs长期胁迫对细菌不产生显著影响。受高浓度（50 mg/L）n-ZnO持续胁迫的细菌在7d内即因生化活性受抑制程度不断增加而死亡。氨氧化性能受n-ZnO抑制达25%的细菌经恢复培养后其各项生理生化指标仅能维持现有水平；受抑制达50%和75%的细胞虽经恢复培养，但其生化活性仍会持续下降。而受50 mg/L n-CeO2或n-TiO2长期胁迫的细菌最终分别可稳定维持其减弱的氨氧化性能和恢复其至接近初始状态。但受50 mg/L n-CeO2长期胁迫至氨氧化活性抑制率分别达10%和25%的受损菌群经过恢复培养后各生化指标均能显著上升。高DO环境有利于提高细菌抗NPs冲击负荷能力以及受损菌群生化活性的恢复，但不同DO浓度条件下NPs对细菌的长期胁迫影响程度相近。结合细菌全基因组表达谱图分析发现细胞膜形态结构破坏是NPs重要的生物毒性作用途径，细胞膜生物起源和翻译后蛋白修饰或转换相关基因表达受NPs抑制最显著，故可干扰细胞膜功能、破坏膜上功能蛋白酶结构、影响呼吸链电子传递和能量代谢、胁迫细胞内遗传物质和功能蛋白；但氨基酸代谢过程和能量转换过程所涉及应激反应蛋白和修复蛋白相关基因表达明显上调。此外，金属离子应激反应基因merA、merP、merT仅在受n-ZnO胁迫试验组显著差异表达，而蛋白修饰伴随基因groES、groEL、dnaK和ClpB仅在受n-TiO2胁迫下显著下调表达。受NPs胁迫的菌群可通过刺激自身的氧化磷酸化作用增强对能源或氮源的利用，或刺激核糖体蛋白的过表达来修复转录翻译过程中功能酶活性以达到自我修复目的。