固定化基因工程菌强化挥发修复砷污染土壤及其机制

Arsenic (As) is one of the main heavy metals contaminated in the soil. Due to more and more As pollution incidents occurred in China recently, The severe As-contaminated soil significantly poses health risk to local population. A method for cheap, high efficient and environment-friendly bioremediation of As-contaminated soil in situ is becoming research hotpot. The key points are adoption of bacteria with high As volatilization capacity and feasible bioaugmentation in situ. The object of this research is to study augmentation mechanism of the remediation of soil As through immobilized bacterium or plant-immobilized microorganism. The strain is genetic engineering bacterium with high As volatilized ability, obtained through synthetic biology. Natural plant materials or biochars from them are chosen as immobilized materials for bacteria. The augmentation mechanism by immobilized genetic bacterium was investigated by observation of absorption/descortion of As from the soil, changes of As species and their fates during the remediation process. In order to improve the efficiency of soil remediation, environmental parameters, key factors and rate limiting step of As biovolatilization during remediation were intensively investigated and optimized. The study will offer an important reference for perfecting bioremediation of heavy metals contaminated soil. The investigation will be significantly practical meaning for reducing As levels in the soil, lessening and decreasing human As exposure from the soil.

砷乃土壤中的优控重金属污染物。鉴于近年来砷污染事件频发，造成的严重土壤砷污染对人类健康产生了极大威胁，廉价、高效、环境友好的土壤原位修复技术研究成为近期的热点。其中，高效菌株的利用以及恰当的生物强化方法是土壤原位修复亟待解决问题。本研究拟采用合成生物学方法构建的具有极强砷挥发能力的基因工程菌，以自然植物残体或生物炭做为固定化载体材料，联合微生物侵染的植物进行土壤砷污染的原位修复。观察强化修复对土壤砷的吸附解吸规律，修复过程中砷的形态转化与归趋等环境行为，分析影响修复效率的限速步骤和关键作用因子，探讨固定化基因工程菌修复土壤砷污染并提高修复效率的相关机制。项目研究完善土壤重金属修复手段提供参考，为减少土壤中砷含量，降低土壤重金属污染造成的健康威胁，具有重要的现实意义。

本研究以稻田厌氧环境中普遍存在的微生物—硫酸盐还原细菌和产甲烷古菌为研究对象，阐述了厌氧微生物的砷甲基转移酶的作用机制以及生化功能特性，为详细阐明厌氧微生物的砷甲基化机制奠定基础。论文的主要研究内容及结果如下：（1）硫酸盐还原细菌Clostridium sp. BXM具有砷甲基化能力，可以将无机砷甲基化为单甲基砷（MMAs）和二甲基砷（DMAs）衍生物。克隆其甲基化基因并在大肠杆菌中表达，发现大肠杆菌获得了甲基化和挥发无机砷的能力。氨基酸定点突变研究表明，CsArsM酶的三个半胱氨酸（cysteine, Cys）位点，即Cys65、Cys153和Cys203，在砷的甲基化过程中起关键作用。（2）产甲烷古菌Methanosarcina acetivorans C2A M. acetivorans C2A能将无机砷甲基化为MMAs。从该古菌中克隆了一个arsM同源基因MaarsM，在合适条件下，表达MaarsM的大肠杆菌可将几乎100%的无机砷甲基化；也可将培养基中约0.4%的无机砷转化为挥发砷。氨基酸定点突变结果表明，古菌MaArsM酶有三个重要的半胱氨酸，即Cys62、Cys150和Cys200，在砷甲基化过程中起关键作用。在MaArsM酶的催化体系中添加适量亚硒酸钠（Se(IV)），对砷的甲基化有促进作用。多种巯基类化合物可以作为MaArsM酶的辅因子，与谷胱甘肽相比，辅酶M和高半胱氨酸对甲基化的作用效率更高。（3）本研究采用具有极强砷挥发能力的基因工程菌GE P. putida KT2440，以自然植物残体（秸秆）或生物炭（稻壳生物炭）做为固定化载体材料，进行土壤砷污染的修复。单独添加秸秆（5%）能显着增强土壤中砷的甲基化和挥发，增强幅度达到了几百倍，且在淹水条件下对砷挥发促进作用强于在未淹水条件下。与GE P. putida联用进一步促进了砷的甲基化和挥发，且在未淹水条件下对砷挥发的促进作用更强。生物炭对土壤中砷的甲基化和挥发没有显著作用。以上结果表明，秸秆与GE P. putida联用对土壤中砷甲基化和挥发促进作用明显，表明了生物刺激和生物强化共同促进土壤中砷的甲基化和挥发，在修复砷污染土壤中是有潜在实用价值的。