抗辐射耐高温高效铀铬还原工程菌株的构建及其治理核废料前景的探索

China will vigorously develop nuclear power during the twelfth five-year plan. Proper disposal of nuclear waste will be an important issue. Hexavalent uranium (U(VI)), a major component of nuclear waste,is highly toxic and water-soluble. Microorganisms can reduce U(VI) to tetravalent uranium (U(IV)) which has low toxicity and water solubility, and therefore immobilize uranium in the nuclear waste. This process has a great potential to remediate nuclear waste. Compared with other methods, microbial remediation is more cost-effective and rarely introduces secondary contamination. Microbes in nuclear waste environment will suffer radiation and heat stresses. However,most microbial strains currently used for nuclear waste treatment are not radiation/heat resistant, thus their reducing ability is greatly decreased. We will use Deinococcus radiodurans R1 which can tolerate high temperature and high radioactivity as host strain to express the highly efficient U(VI)/Cr(VI)-reducing protein and the U(VI)/Cr(VI)-reducing operon from the sulfate-reducing bacteria Desulfovibrio desulfuricans G20. The engineered strain might be able to grow under nuclear waste condition and reduce U(VI)/Cr(VI). We will measure the engineered strain's resistance to radiation and heat as well as its U(VI)/Cr(VI)-reducing ability in pure culture. The strain will also be inoculated into the soil sample to test its in situ suvival and U(VI)/Cr(VI) reducing ability.This will be the first attempt to construct an engineered microorganism for microbial nuclear waste remediation.

我国将在十二五期间大力发展核能,如何安全处置核废料是一个待解决的重要问题。六价铀是核废料的主要组成部分，某些微生物可以将高毒性、高水溶性的六价铀还原成低毒性、低水溶性的四价铀。利用微生物的这种特性来治理核废料是一种极具发展潜力的方法。在核废料环境下，微生物将受到高温、高辐射等逆境胁迫。但目前国际上用于治理核废料研究的微生物尚无抗辐射、耐高温的能力，导致其实际治理效果降低。本项目拟在抗辐射耐高温菌株Deinococcus radiodurans R1中表达来源于硫酸还原细菌Desulfovibrio desulfuricans G20的高效铀铬还原蛋白和铀铬还原操纵子，从而得到可在核废料环境中正常生长的铀铬还原菌株。工程菌株耐辐射与高温能力及还原铀铬的能力将会被鉴定。工程菌株也将被接种至土壤样品中以检测其实际生存与还原铀铬的能力。本项目将是国际上首次尝试构建工程菌株的方法来治理核废料的研究。

甘肃北山一带作为我国核废料处置库重点候选地带，有其不可比拟的优势。本项目利用将具有U(Ⅵ)，Cr(Ⅵ)处理能力相关的菌株与Deinococcus radiodurans抗辐照能力结合起来，构建了兼具铀铬处理能力与抗辐照能力的工程菌株（含有重组质粒pRADK-chrR、pRADK-yieF、pRADK-phoN的D.radiodurans），以更好的处置高放核废料，在核废料填埋地周围形成一道生物防护屏障，保护周边的生态环境乃至人民的身体健康。.核能产生的放射性废料和重金属严重污染当地的环境。构建能够在核废料地区修复核废料和重金属的抗辐射菌株将是一个很好的解决方案。我们构建ChrR（编码六价铬还原酶）来自恶臭假单胞杆菌和PhoN(编码非特异性酸性磷酸酶)来自肠道沙门氏菌到抗辐射奇球菌R1中进行超表达。构建的菌株Deino-ChrR表达22-kDa的ChrR蛋白并且有高还原六价铬的能力，能够在48小时内还原0.5mM的六价铬。同时构建的Deino-PhoN菌株表达了PhoN蛋白并且在48小时内沉淀90% 1mM的硝酸双氧铀。构建的菌株在照射6 kGy 60Co射线后仍具有很高的六价铬还原和铀的沉淀能力。我们建立了一个曝气生物反应器来扩大构建菌株的修复应用。构建的Deino-ChrR 和 Deino-PhoN菌株可以在30天内分别还原90%的六价铬和沉淀95%铀。总之Deino-ChrR 和 Deino-PhoN 提供了一个有效和环境友好的方法来修复核废料中的六价铬和铀。