重金属抗性细菌CR3的抗铜分子机制及其处理含铜CMP废水的效能与特征

Heavy metal resistant bacteria have a distinguish advantage over the ordinary bacteria in the removal of low concentration heavy metal ions from industry wastewater due to their native resistance to heavy metals. However, the molecular mechanism of bacteria resistance to heavy metal is not completely understood, which hinders its practical application in waster water treatment. Currently, the knowledge about mechanisms of bacteria resistance to heavy metal mostly stem from plasmid-bearing bacteria in which heavy metal resistance genes locate plasmid. The isolated Cupriavidus gilardii CR3 is a unique intrinsic heavy metal resistance bacterium, since it is plasmid-free. The heavy metal resistance genes of strain CR3 locate in the chromosomes. The proposal will utilize the strain CR3 as a representative to study its mechanism of resistance to copper. The aims of the proposal focus on three layers, including the extracellular polymeric substances adsorption copper, the cell wall adsorption copper and copper transport system. On the basis of interpretation of the mechanism of resistance to heavy metals, we will establish experiment system of fix paced column, and employ the biofilm of strain of CR3 to remove the copper from chemical mechanical planarization (CMP) wastewater, which usually comes from semiconductor fabrication. The results from this study will contribute to the deep insight into the mechanism of resistance to copper for a kind of bacteria, in which their resistance genes locate in chromosomes. Moreover, the results will provide new technology and new idea to treat low concentraion copper ions from the CMP wastewater by growing resistant bacteria.

重金属抗性微生物具有抵抗重金属的特性，在低浓度重金属工业废水处理中显示出独特的优势。然而，人们对重金属微生物抗性机制尚缺乏完整的认识，这是制约微生物吸附技术实用化的重要因素。Cupriavidus gilardii CR3是具有天然铜抗性的细菌，其新颖之处在于抗性基因定位于染色体。目前，C. gilardii的铜抗性机制尚未见报道。本项目拟以CR3为模式菌，采用现代微量分析和分子生物学技术，定性、定量研究细菌胞外聚合物、菌细胞本身吸附铜的作用机制，通过胞内铜抗性基因的差异表达，解析铜抗性基因与抗性水平的相关性，阐明CR3的铜抗性分子机制，为完善和发展微生物重金属抗性分子机制提供理论依据。基于上述理论，设计和构建工艺放大体系，确定关键工艺运行条件，研究CR3生物膜去除CMP废水中低浓度铜离子的效能与特征，为基于抗性细菌生物膜处理含铜废水研究提供新的思路。

重金属抗性微生物是一类对重金属具有耐受能力的微生物，在低浓度重金属工业废水处理中显示了独特的优势。微生物对重金属抗性机制是其处理重金属工业废水的理论基础。然而，人们对重金属微生物抗性机制尚缺乏完整的认识，这是制约微生物吸附技术实用化的重要因素之一。目前，有关细菌对重金属抗性机制研究绝大数为仅针对重金属外排系统中的单个基因或某个基因簇。细胞如何从全局调控相关基因保持胞内重金属离子处于内稳态从而达到内部解毒还鲜见报道。.本项目采用具有铜抗性的细菌Cupriavidus gilardii CR3为模型，从细胞全局性角度出发，从胞外聚合物、细胞壁和胞内三个层面定性、定量地研究了细菌的铜抗性机制。1 mM 铜离子可刺激胞外聚合物中蛋白的分泌量增加64%，增强铜离子的生物吸附作用。C. gilardii CR3对铜吸附符合Langmuir模型（R2= 0.99），最大吸附量为18.25 mg/g。0.5和1.5 mM铜胁迫可导致菌株CR3基因组中310和413基因显著上调表达，还刺激了细胞的T3SS分泌系统、Fe-S装配系统、硫代谢和谷胱甘肽生物合成基因的显著表达。其中，T3SS分泌系统是首次发现铜胁迫可诱导其显著表达。基于上述理论和菌株CR3的对铜胁迫的耐受性能，确定CMP废水中铜离子去除的关键条件，菌细胞对初始浓度为0.5~1.5 mM模拟CMP废中铜离子的去除率大于90%，其去除作用由胞外吸附（胞外聚合物和细胞壁）和胞内累积作用共同完成，但胞内累积起主要作用。对初始浓度为1.5 mM铜离子的模拟CMP废水而言，胞内累积对于铜去除贡献率占比为92％。.本项目阐明了菌株CR3的铜抗性分子机制，认为细菌重金属抗性机制不仅依靠自身特异的铜抗性基因系统，而且还受到胞内多个代谢途径协同调控。首次发现铜胁迫对胞内T3SS分泌系统的诱导效应。本项目研究结果加深和拓展对细菌重金属抗性机制的认识，对于推动微生物在重金属废水处理中的应用具有重要意义。