【作者】 肖方竹；

【导师】 何淑雅；

【作者基本信息】 南华大学 ， 基础医学， 2020， 博士

【摘要】 随着核电项目的陆续重启和国家加快推进核电“走出去”战略,天然铀资源供应仍面临巨大压力。在铀资源开采、加工和生产应用过程中产生的含铀废水,若不及时有效处理,将会对周边区域的土壤、水等生态环境造成放射性污染,甚至会对人类生存环境构成一定的潜在威胁。因此,研究低浓度含铀废水中铀的分离富集新技术、新方法与新材料,对铀资源化和放射污染环境治理均具有重要的战略意义。微生物修复法以其修复效率高、经济成本低、操作简单且无二次污染物的优势而备受瞩目,目前研究逐渐认为利用微生物治理含铀废水是一种极具应用前景的方法。耐辐射奇球菌（Deinococcus Radiodurans,DR）是一种具有超强辐射抗性的微生物,其细胞壁具有至少六层的特殊组成和结构,最外层（S层）由规则排列的六角形蛋白亚基组成。由于该菌的超强极端抗性,兼有较好的基因转化和遗传操作性,DR菌被列为可用来构建放射性环境工程菌的优势菌种。耐辐射奇球菌在放射性环境污染治理领域,以耐辐射、抗干燥等多重抗性特点,越来越凸显其应用优势。硫酸盐还原菌（Sulfate-Reducing Bacteria,SRB）许多蛋白都参与将可溶性的六价铀还原为不可溶、易于回收的四价铀沉淀这一过程,异化型亚硫酸盐还原酶是参与该过程的关键酶之一,由dsrA还原基因控制表达。野生型硫酸盐还原菌在铀废水中的生存活性受限,增殖率不理想,进而影响其对铀的持续富集效率,需要增强对铀的耐受性能。本研究围绕耐辐射奇球菌在治理含铀废水中的应用开展实施。考察了野生型耐辐射奇球菌在培养条件下活体菌对铀的富集行为,并通过转录组表达谱差异分析了铀胁迫下DR菌的应激机制;以生物表面活性剂功能化修饰耐辐射奇球菌,构建了表面活化DR铀富集剂BSDR,进行铀富集实验,考察其对铀的富集性能与机理;将还原基因dsrA转入野生型大肠杆菌（Escherichia coli,E.coli）,构建重组大肠杆菌DH5α-dsrA,并完成铀还原富集实验,证实了dsrA基因可增强大肠杆菌DH5α铀富集效率;将dsrA还原基因转入耐辐射奇球菌,构建了多效基因工程菌Deino-dsrA,对含铀废水中的六价铀进行了生物还原富集,阐释其还原富集行为与机理,从而为实现铀废水生物治理技术提供了科学依据和实验基础理论,也为我国国防科技工业、铀资源化产业可持续健康发展提供重要的技术支撑。第一部分野生型耐辐射奇球菌对铀富集行为与铀胁迫应激机制研究目的:通过野生型耐辐射奇球菌对铀的富集实验以及转录组测序分析耐辐射奇球菌富集铀前后的基因表达差异,初步探索耐辐射奇球菌中铀胁迫应激过程中的关键基因。从溶液的p H值、温度、铀初始浓度和菌剂投加量等影响因素确定DR菌富集铀的最适宜条件。方法:将培养条件下的耐辐射奇球菌菌液投放到模拟含铀废水中进行铀富集实验,从溶液的p H值、温度、铀初始浓度和菌剂投加量等影响因素探索对铀的富集效率的最佳条件。同时监测铀富集过程中溶液p H值随时间的变化趋势。通过转录组测序分析耐辐射奇球菌富集铀前后的基因表达差异,筛选差异表达较大的基因,经GO分析和KEGG pathway富集分析,揭示差异基因参与的主要调控通路。使用扫描电镜表征铀富集后的DR菌表面形貌,使用EDS能谱仪对菌体沉淀做元素成分分析。结果:培养条件下的野生型耐辐射奇球菌,在p H=5、温度30℃、铀初始浓度为10 mg/L的情况下,投加16 m L的耐辐射奇球菌体富集剂,吸附时间为60 min,铀（Ⅵ）富集率可达91%;在铀富集过程中同时测量溶液p H的动态值,初始p H为5时,投加16 m L的耐辐射奇球菌体富集剂,富集终点p H值为6.1。转录组测序结果显示,DR菌中DR＿RS11290、DR＿RS03605、DR＿RS13480等基因表达水平上调,为参与铀胁迫应激的主要基因;DR＿RS10750、DR＿RS07335、DR＿RS04590等基因表达水平下调,受到铀胁迫抑制。经GO分析和KEGG pathway富集分析,预测表达差异基因参与了多种信号通路,主要涉及氧化磷酸化、各类糖代谢和氨基酸合成代谢等通路;细胞色素C氧化酶显著上调。小结:（1）DR菌在铀溶液中生存周期仍可达90h,提示DR菌具有超强抗性。（2）耐辐射奇球菌多个关键基因参与了铀胁迫应激过程,DR＿RS11290、DR＿RS03605、DR＿RS13480等环境刺激应答蛋白、运动动力蛋白、DNA复制解旋酶等基因表达水平上调,而DR＿RS10750、DR＿RS07335、DR＿RS04590等与肽聚糖结合的膜效应因子相关基因下调,上下调基因中有多个基因为功能未知的DR菌独有基因。（3）差异表达显著的基因可能参与氧化磷酸化、各类糖代谢和氨基酸合成代谢等通路;细胞色素C氧化酶显著上调,在氧化磷酸化通路中起到重要的催化作用。（4）DR菌铀富集最适条件,p H为5,温度为30℃,铀初始浓度为10 mg/L,菌剂投加量为16m L。DR菌的铀富集过程会通过氧化还原反应,反向调节p H值,使其趋向中性。（5）扫描电镜结果显示DR菌富集铀前后其表面形貌发生了较大变化,能谱图和元素分析结果均显示富集铀之后元素成分中新增铀的含量达到了4.3%,证实铀富集到DR菌表面。第二部分生物表面活性剂功能化修饰DR菌及其对铀的富集性能与机理研究目的:以生物表面活性剂功能化修饰DR菌菌体表面,构建表面活化DR铀富集剂BSDR。探索溶液温度、p H值、铀初始浓度和菌剂用量等因素对表面活化DR铀富集剂BSDR富集铀的性能影响,并得到最适宜的富集条件。方法:经堆肥发酵培养制备含生物表面活性剂的发酵液,再以生物表面活性剂功能化修饰耐辐射奇球菌菌体,构建表面活化DR铀富集剂BSDR。将其投放入低浓度铀溶液中,研究溶液温度、p H值、铀初始浓度和菌剂用量等因素对表面活化DR铀富集剂BSDR富集铀的性能影响。使用红外光谱（FT-IR）、扫描电镜（SEM）、能谱分析仪（EDS）分别测试表面活化DR铀富集剂BSDR富集铀后的表面形貌及元素成分。并使用HCl和Na OH、EDTA作解吸剂,考察表面活化DR铀富集剂BSDR的再生利用性能。结果:表面活化DR铀富集剂BSDR富集铀的最佳温度为25℃,最佳p H值为4.5,最佳铀初始浓度为20 mg/L,富集剂BSDR的最佳剂量为10 mg,对铀的富集率可达到93.3%,单位铀富集量为45.4 mg/g。扫描电镜及EDS能谱分析结果显示,铀已被表面活化DR铀富集剂BSDR成功富集。表面活化DR铀富集剂BSDR,用HCl、Na OH和EDTA解吸回收循环6次,富集效率仍可达到60%,具有较理想的可再生性。小结:（1）成功构建一种表面活化DR铀富集剂BSDR。6次解吸后富集率仍可达60%,重复利用性较高。（2）表面活化DR铀富集剂BSDR最佳铀富集条件,温度为25℃,p H值为4.5,铀初始浓度为20 mg/L,富集剂BSDR的最佳剂量为10 mg,富集率可达到90-93.3%。耐辐射奇球菌是表面活化DR铀富集剂BSDR基体的适宜选择。（3）红外光谱分析结果表明表面活化DR铀富集剂BSDR发生了铀富集的过程。扫描电镜分析结果,表面活化DR铀富集剂BSDR富集铀后表面附着了大量颗粒,形貌发生较大改变。能谱结果显示富集铀后BSDR的元素成分中新增铀的含量达到了9.27%,证明铀已被表面活化DR铀富集剂BSDR富集至表面。第三部分还原基因dsrA转化及其对大肠杆菌还原富集铀的性能影响目的:利用硫酸盐还原菌（SRB）关键还原基因dsrA将六价可溶性的铀还原为四价不可溶、易于回收沉淀的铀,将其转入大肠杆菌中构建含还原基因的重组大肠杆菌DH5α-dsrA,对比dsrA基因转入大肠杆菌DH5α前后对铀的富集效率,研究还原基因dsrA对异源微生物大肠杆菌还原富集铀的性能影响。方法:利用前期构建的载体,提取质粒p RADK-dsrA,经酶切后电泳检测鉴定其表达情况,菌落PCR鉴定及Blast比对分析还原基因dsrA在重组大肠菌内表达情况。将含还原基因dsrA的重组大肠杆菌DH5α-dsrA投入低浓度铀溶液中,摇瓶培养,绘制生存曲线获得含还原基因的重组大肠杆菌DH5α-dsrA在铀溶液中的生存活性情况;考察溶液p H值、铀初始浓度、还原富集时间、菌剂量和溶液温度等各因素影响下的对铀还原富集效率,研究该重组大肠杆菌DH5α-dsrA对铀的还原行为;并使用液相色谱与ICP-MS检测该重组大肠杆菌DH5α-dsrA还原富集铀沉淀物的元素价态,以分析其对铀的还原富集效率。结果:PCR鉴定及Blast比对结果显示还原基因载体p RADK-dsrA在重组大肠DH5α-dsrA中表达正常。含还原基因的重组大肠杆菌DH5α-dsrA在铀溶液中的生长大约在10h后进入对数生长期,18h左右进入稳定期,其生存活性随着铀浓度的增加而降低,该重组大肠杆菌DH5α-dsrA在高浓度含铀废水中会提前进入衰亡期。含还原基因的重组大肠杆菌DH5α-dsrA还原富集铀的最佳时间为40 min,菌剂量0.4 g,最佳温度为37℃,最佳p H值为4,最佳铀初始浓度为5 mg/L,富集率最高达到95.83%,富集量为43.4 mg/g。液相色谱与ICP-MS分析沉淀物元素价态含量,结果显示该重组大肠杆菌DH5α-dsrA富集铀后,沉淀物中U（IV）和U（III）总含量约为34,348.9 mg/kg。小结:（1）成功构建含还原基因的重组大肠杆菌DH5α-dsrA。（2）重组大肠杆菌DH5α-dsrA在铀溶液中的生存活性受到抑制,生长周期为27h,需要增强其多重抗性。（3）重组大肠杆菌DH5α-dsrA对铀的富集率达到95.12%-95.83%,提示还原基因dsrA的转化可增强野生型大肠杆菌对铀的富集效率。（4）重组菌DH5α-dsrA和野生菌DH5α最佳铀还原富集条件,p H值为4,铀初始浓度为5 mg/L,还原富集时间约为40min,菌剂量为0.4g,温度为37℃。还原产物U（IV）和U（III）总含量约为34348.9mg/kg。第四部分多效基因工程菌Deino-dsrA的构建及其还原富集铀的行为与机理研究目的:利用耐辐射奇球菌超强的辐射抗性,结合硫酸盐还原菌的强还原能力,将硫酸盐还原菌的关键还原基因dsrA转入到耐辐射奇球菌中,构建抗辐射、高还原性能的多效基因工程菌Deino-dsrA,以期对含铀废水进行持续性高效处理。方法:将前期已构建的p RADK-dsrA质粒,转化入DR菌感受态细胞中,筛选阳性克隆,PCR鉴定及Blast比对显示dsrA基因在DR菌内正常表达。将构建的新型基因工程菌投放入低浓度铀溶液中,摇培生存曲线法检测野生型DR与重组菌Deino-dsrA在铀溶液中的生存活性情况;调整时间、温度、p H值、菌剂量及铀初始浓度各因素,研究该基因工程的富集行为及富集效率;使用FTIR分析研究铀富集前后Deino-dsrA多效基因工程菌表面的基团变化;使用SEM和EDS分析富集铀后的Deino-dsrA多效基因工程菌表面形貌及元素成分;使用液相色谱与ICP-MS检测Deino-dsrA多效基因工程菌富集铀沉淀物的元素价态,以分析其对铀的还原效率。结果:PCR鉴定及Blast比对结果显示dsrA基因在DR菌内正常表达,含dsrA基因的DR重组菌Deino-dsrA构建成功。18-20 h后野生型DR与重组菌Deino-dsrA进入对数生长期,菌体数量均迅速上升,且生存周期比较长,重组菌增长速率滞后于野生型菌。重组菌Deino-dsrA还原富集铀的最佳时间为60 min,最佳温度为30℃,最佳p H值为5,最适菌剂量为35 mg,最佳铀初始浓度为10 mg/L,铀富集率达到93.5%,单位铀富集量为43.4 mg/g;该重组菌适应的温度范围及p H范围比较宽,经选育获得多效基因工程菌Deino-dsrA。FTIR、SEM和EDS分析结果提示,铀富集后Deino-dsrA多效基因工程菌表面已结合铀离子。液相色谱与ICP-MS检测结果显示,Deino-dsrA多效基因工程菌铀富集后,沉淀物中U（IV）和U（III）总含量约为48,293.4 mg/kg。小结:（1）成功构建了抗辐射、高还原性能的多效基因工程菌Deino-dsrA。（2）Deino-dsrA多效基因工程菌在铀溶液中生存周期较长,具备理想的极端抗性,增值速率略滞后于野生型DR菌。（3）Deino-dsrA多效基因工程菌还原富集铀的最佳时间为60 min,最佳温度为30℃,最佳p H值为5,最适菌剂量为35 mg,最佳铀初始浓度为10 mg/L,富集率达到93.5%,富集量为43.4 mg/g,其还原富集效率优于野生型DR菌;在酸性条件下,也优于文献报道硫酸盐还原菌的富集率。（4）红外光谱分析结果Deino-dsrA多效基因工程菌出现了UO₂²⁺的特征峰,扫描电镜结果证明,富集前后菌体表面发生较大变化。EDS能谱分析结果证明铀已被Deino-dsrA多效基因工程菌富集至表面。液相色谱与ICP-MS检测结果显示,Deino-dsrA多效基因工程菌铀富集后,沉淀物中U（IV）和U（III）总含量约为48,293.4 mg/kg。结论1.耐辐射奇球菌多个关键基因参与了铀胁迫应激过程,DR＿RS11290、DR＿RS03605、DR＿RS13480等环境刺激应答蛋白、运动动力蛋白、DNA复制解旋酶等基因表达水平上调,而DR＿RS10750、DR＿RS07335、DR＿RS04590等与肽聚糖结合的膜效应因子相关基因下调,上下调基因中有多个基因为功能未知的DR菌独有基因。2.构建表面活化DR铀富集剂BSDR。BSDR最佳铀富集条件是温度为25℃,p H值为4.5,铀初始浓度为20 mg/L,投加菌的最佳剂量为10 mg,富集率可达到93.3%,并具有较好的再生性能及应用条件拓宽。3.构建含还原基因的重组大肠杆菌DH5α-dsrA。重组菌DH5α-dsrA和野生菌DH5α最佳铀还原富集条件,p H值为4,铀初始浓度为5 mg/L,菌剂量为0.4g,温度为37℃,还原富集时间约为40min,还原富集率最高分别可达95.83%和88.23%,差异具有显著意义。4.成功构建获得多效基因工程菌Deino-dsrA。Deino-dsrA多效基因工程菌还原富集铀的最佳时间为60min,最佳温度为30℃,最佳p H值为5,最适菌剂量为35 mg,最佳铀初始浓度为10 mg/L,富集率91%-99%,富集量为43.4 mg/g,在铀溶液中生存周期可达90h,其还原富集效率优于野生型DR菌,差异具有显著意义。更多还原

【Abstract】 With the resumption of nuclear power projects and the accelerated"going global"strategy of nuclear power,the supply of natural uranium resources is still under great pressure.If the uranium-bearing wastewater generated in the mining,processing,production and application of uranium resources is not treated in a timely and effective manner,it will cause radioactive pollution to the surrounding soil,water and other ecological environment,and even pose a certain potential threat to human living environment.Therefore,it is of great strategic significance to study the new technology,new method and new material for uranium separation and enrichment in low-concentration uranium-containing wastewater for uranium recycling and environmental treatment of radioactive pollution.Due to its high efficiency,low economic cost,simple operation and no secondary pollutants,it is gradually considered that using microorganism to treat uranium-bearing wastewater is a promising method.Deinococcus radiodurans（DR）is a kind of bacteria with super radiation resistance.Its cell wall has at least six layers of special composition and structure,and the outermost layer（S layer）is composed of regular hexagonal protein subunits.Due to its super extreme resistance,good gene transformation and genetic operability,DR is listed as the dominant strain that can be used to construct radioactive environmental engineering bacteria.In the field of radioactive environmental pollution control,the radiation-resistant and drought-resistant cocci have become more and more prominent in their application advantages.Many proteins of Sulfate-Reducing Bacteria（SRB）are involved in the soluble hexavalent uranium reduction for tetravalent unsolvable uranium deposits,which are easy to recycle,dissimilatory sulfite reductase（DSR）is one of the key enzymes,controlled expression by the dsr A gene.However,the proliferation rate of common indigenous microorganisms SRB in uranium wastewater is relatively low,thus affecting its continuous enrichment efficiency of uranium.In this study,with Deinococcus radiodurans（DR）as the main research object,enrichment of uranium by living bacteria of wild-type DR under culture conditions was investigated,and stress mechanism of DR under uranium stress was analyzed by transcriptome expression spectrum difference.A new functional enrichment agent BSDR was constructed by functionalizing radiosurviridae,and uranium enrichment experiments were carried out to investigate its uranium enrichment performance and mechanism.The reductive gene dsr A was transferred into Escherichia coli（E.coli）,and recombinant Escherichia coli DH5α-dsr A was constructed.Uranium reduction enrichment experiment was conducted to study its enrichment efficiency and reduction efficacy.Turn dsr A reduction genes resistant to radiation,aureus,build the gene engineering bacteria Deino-dsr A multiple effect,the enrichment of uranium in uranium wastewater biological reduction,explain its enrichment behavior and mechanism of the reduction to achieve uranium wastewater biological treatment technology has provided the scientific basis and theory basis of experiment,also for China’s defense science,technology and industry,uranium resource sustainable healthy development of the industry to provide important technical support.Part Ⅰ:Study on uranium enrichment and stress mechanism of wild-type Deinococcus radioduransObjective:To analyze the gene expression differences of DR before and after uranium enrichment through uranium enrichment experiments and transcriptome sequencing,in order to preliminarily explore the key genes of DR in the process of uranium stress.The optimum conditions for enrichment of uranium by DR were determined from the factors such as p H value of solution,temperature,initial concentration of Uranium and dosage of bacteria.Methods:The uranium enrichment experiment was carried out on the simulated uranium containing wastewater in the culture condition,and the optimum conditions for uranium removal rate were explored from the factors such as p H value of solution,temperature,initial concentration of Uranium and dosage of bacteria.At the same time,observing the trend of the p H value of the solution changes with time during uranium enrichment.Transcriptome sequencing was used to analyze the gene expression differences of DR before and after uranium enrichment,and the genes with higher differential expression were screened.Through GO analysis and KEGG pathway enrichment analysis,to find the main regulatory pathways.which the different genes were involved.Scanning electron microscope was used to characterize the surface morphology of DR after uranium enrichment,and EDS was used to analyze the composition of the bacteria precipitation.Results:The wild-type DR under the culture condition,in the p H=5,temperature 30℃,initial concentration of 10 mg/L,dosage of DR was 16 m L,adsorption time for 60min,uranium（Ⅵ）adsorption rate was 91%;In the enrichment process,the dynamic p H value was measured at the same time.When the initial p H was 5,16 m L of the DR enrichment agent was added,and the end p H value of the enrichment was 6.1.The results of transcriptome sequencing showed that DR＿RS11290、DR＿RS03605、DR＿RS13480 and other genes were up-regulated,which were the main genes involved in uranium stress,the expression level of DR＿RS10750、DR＿RS07335、DR＿RS04590 and other genes were down regulated,which were inhibited by uranium stress.The GO analysis and KEGG pathway enrichment analysis showed that the predicted differential genes were involved in a variety of signaling pathways,mainly involving oxidative phosphorylation,various glucose metabolism and amino acid synthesis metabolism,etc.Cytochrome C oxidase was significantly up-regulated.Conclusions:（1）The survival period of DR in uranium solution was still relatively long.The results of the combination suggested that DR were less inhibited under uranium stress and had super extreme resistance.（2）Several key genes of Deinococcus radiodurans are involved in uranium stress,uranium stress inhibited the expression levels of DR＿RS10750、DR＿RS07335、DR＿RS04590,and upregulated the expression levels of DR＿RS11290、DR＿RS03605、DR＿RS13480 etc.The expression level was mostly down-regulated,suggesting that the uranium solution inhibited the stress of DR to a certain extent,and the survival rate of DR decreased with the increase of uranium concentration.（3）It was predicted that differentially expressed genes were involved in a variety of signaling pathways,mainly involving oxidative phosphorylation,various glucose metabolism,amino acid synthesis and other pathways;Cytochrome C oxidase was significantly up-regulated and plays an important catalytic role in oxidative phosphorylation pathway.（4）The optimal conditions for uranium enrichment of DR were determined,p H was 5,temperature was 30℃,initial concentration of uranium was 10 mg/L,and dosage of DR was 16 m L.In the uranium solution with acidic initial p H value,the uranium enrichment process of DR will produce water through peroxidation reduction reaction,and dilute H⁺concentration to a certain extent.（5）The results of SEM and EDS analysis showed that the surface morphology of DR changed greatly before and after uranium enrichment,and the surface of DR before uranium enrichment was not smooth,the surface arrangement was irregular and there were many pores,after uranium enrichment,the surface voids are obviously reduced,and a certain degree of agglomeration appeared.The results of energy spectrum and element analysis showed that the surface of the bacteria did not contain uranium before uranium enrichment,and the content of uranium in the elements reached 4.3%after uranium enrichment.Part Ⅱ:Functional modification of DR by biosurfactant and its enrichment performance and mechanism for uraniumObjective:To construct a new functional DR by functionally modified DR with biological surfactant.The optimum conditions such as temperature of solution,p H value,initial concentration of Uranium and dosage of bacteria were explored,and the enrichment efficiency was obtained.Methods:Biosurfactant bacteria solution was prepared by compost fermentation culture,and then a new functional DR enrichment agent was constructed by functionalizing the DR with biosurfactant bacteria solution.The enrichment efficiency was studied by temperature of solution,p H value,initial concentration of Uranium and dosage of bacteria.FT-IR spectrum、Scanning electron microscope（SEM）and energy spectrum analyzer（EDS）were used to test the surface morphology and element composition after enrichment.Results:The optimum temperature and p H value for uranium enrichment of the novel functional DR were 25℃,4.5 p H value,20mg/L initial concentration of uranium,dosage of bacteria 10 mg,enrichment rate was 93.3%and enrichment was 45.4mg/g.Scanning electron microscopy and EDS analysis results showed that uranium was successfully enriched by the novel functionalized modified DR.The biosurfactant functionalized DRl colonies,with ideal reusability,desorption and recycling cycles for 6 times,and the enrichment efficiency could still reach 60%.Conclusions:（1）A new biosurfactant functionalized modifier for DR was successfully constructed by using biological Surfactant as a functional modified strain,and it has good reusability.（2）The optimal uranium enrichment conditions for the novel functionalized DR were obtained through experiments.The temperature was25℃,the p H value was 4.5,the initial concentration of Uranium was 20mg/L,dosage of bacteria was 10mg,and the enrichment rate was 90-93.3%.It has been verified by experiments that DR are an appropriate choice for the enrichment matrix.（3）The results of FT-IR spectrometer analysis show that the enrichment of uranium takes place in the surface-activated DR Uranium enrichment agent BSDR.The results of SEM showed that there were a lot of particles on the surface of Uranium enriched by surface-activated DR Uranium enrichment agent BSDR.The result of energy spectrum shows that the Uranium content of BSDR was 3.21%,which proves that Uranium has been enriched to the surface by surface activated DR Uranium Enrichment Agent BSDR.Part Ⅲ:Transformation of reducing gene dsr A and its effect on reduction and enrichment of uranium in Escherichia coliObjective:Using sulfate-reducing bacteria（SRB）key restore gene dsr A hexavalent soluble uranium compounds can be reduced into tetravalent unsolvable,easy to recycle the characteristics of uranium deposits,it moved to construct recombinant E.coli DH5α-dsr A,comparing to uranium enrichment efficiency before and after transformating dsr A gene into E.coli DH5α,to verify influence of reduction gene dsr A to the performance of different source microorganism enrichment of uranium.Methods:The plasmid p RADK-dsr A was extracted with the vector constructed by the research group.The expression was confirmed to be normal by electrophoresis after enzyme digestion.Colony PCR identification and Blast comparison showed no mutation in the reduced-gene recombinant coliform.The reconstructed reduced-gene recombinant coliform was put into low-concentration Uranium solution,and the survival activity of recombinant E.coli DH5α-dsr A in Uranium solution was obtained by shaking flask culture survival curve method.The reduction enrichment efficiency under the influence of p H value of solution,initial concentration of uranium,reduction enrichment time,bacterial dose and solution temperature on uranium reduction enrichment efficiency were explored to study the uranium reduction behavior of the recombinant strain.The reduction efficiency of uranium in recombinant E.coli DH5α-dsr A by ICP-MS and liquid phase was analyzed.Results:PCR identification and Blast comparison confirmed that the expression of p RADK-dsr A was normal in recombinant E.coli DH5α-dsr A.The growth of reduced-gene recombinant E.coli DH5α-dsr A in uranium solution enters the logarithmic growth stage after about 10 h,and enters the stable stage after about 20 h.Its survival activity decreases with the increase of uranium concentration,and the recombinant E.coli DH5α-dsr A will enter the decay stage in advance in high concentration uranium-containing wastewater.The optimal enrichment time of recombinant E.coli DH5α-dsr A was 40 min,the bacteria dose was 0.4 g,the optimal temperature was 37℃,the optimal p H value was 4,the optimal initial concentration of uranium was5mg/L,the highest enrichment rate was 95.83%,and the enrichment amount was 43.4 mg/g.ICP-MS and liquid phase were used to analyze the ionic valence content of the sediment.The results showed that the total U（IV）and U（III）contents in the sediment were about 34,348.9 mg/kg after the enrichment of recombinant E.coli DH5α-dsr A.Conclusions:（1）Construction of recombinant E.coli DH5α-dsr A containing reducing gene.（2）The survival activity of reduced-gene recombinant E.coli DH5α-dsr A in uranium solution was inhibited,and it was necessary to enhance its multiple resistance.（3）The enrichment rate of uranium in recombinant E.coli DH5α-dsr A reached 95.12%-95.83%,which indicated that the transformation of dsr A could enhance the enrichment efficiency of uranium in wild-type E.coli.（4）The optimum conditions for uranium reduction and enrichment of recombinant E.coli DH5α-dsr A and wild-type E.coli DH5αwere obtained by orthogonal test,The p H value was 4,the initial concentration of uranium was 5 mg/L,the bacterial dose was 0.4 g,the temperature was 37°C and the time for uranium reduction and enrichment was about 40min.The reduction products U（IV）and U（III）of recombinant DH5α-dsr A containing the reduction gene were about 34,348.9 mg/kg.Part Ⅳ:Construction of a multi-functional genetically genetic engineering strain Deino-dsr A and its reduction and enrichment behavior and mechanism of uraniumObjective:Using strong radiation resistance of DR,the combination of sulfate-reducing bacteria（SRB）is strong reducing power,key reduction genes dsr A of sulfate-reducing bacteria was transferred into radiation resistant,staphylococcus aureus,build radiation resistance,high reduction performance of Deino-dsr A multi-functional genetically engineering strain,in order to process uranium waste water sustainably and efficiently.Methods:The preconstructed p RADK-dsr A plasmid was transformed into DR receptive cells,and positive clones were screened.PCR identification and Blast comparison showed that dsr A gene was normally expressed in DR.The newly constructed genetically engineered bacteria were put into low-concentration uranium solution,and the survival curve of wild-type DR and recombinant Deino-dsr A in uranium solution was tested by shaking culture.The uranium enrichment behavior and enrichment efficiency of the genetically engineered bacteria were studied by adjusting the time,temperature,bacteria dose and initial uranium concentration.FTIR analysis was used to study the group changes on the surface of Deino-dsr A multi-functional genetically engineering strain before and after uranium enrichment.Surface morphology and element composition of Deino-dsr A multi-functional genetically engineering strain after uranium enrichment were analyzed by SEM and EDS.The reduction efficiency of Deino-dsr A multi-functional genetically engineering strain to uranium was analyzed by ICP-MS and liquid phase.Results:PCR identification and Blast comparison results showed that dsr A gene was normally expressed in DR,and Deino-dsr A multi-functional genetically engineering strain was successfully constructed.After 18-20h,wild-type DR and recombinant Deino-dsr A entered the logarithmic growth stage,the number of bacteria increased rapidly and the life cycle was relatively long,but the growth rate of recombinant bacteria lagged behind that of wild-type bacteria significantly.The optimal time for uranium enrichment by reducing genetic engineering bacteria was 60min,the optimal temperature was 30℃,the optimal p H value was 5,the optimal dosage of bacteria was 35mg,the optimal initial concentration of uranium was 10 mg/L,the enrichment rate reached 93.5%,and the enrichment amount was 43.4 mg/g.FTIR、SEM and EDS analysis results indicated that the surface of Deino-dsr A multipotent genetic engineering bacteria was bound with uranium ions after uranium enrichment.ICP-MS and liquid phase analysis results showed that the total U（IV）and U（III）content in the sediment of Deino-dsr A multi-functional genetically engineering strain enriched with uranium was about 48,293.4mg/kg.Conclusions:（1）Deino-dsr A multi-functional genetically engineering strain with radiation resistance and high reduction performance were successfully constructed.（2）Deino-dsr A multi-functional genetically engineering strain have ideal extreme resistance.They have a long life cycle in uranium solution,and their increment rate slightly lags behind that of wild-type DR.（3）The optimal time for reducing enrichment of uranium by Deino-dsr A multi-functional genetically engineering strain was 60min,the optimal temperature was 30℃,the optimal p H value was 5,the optimal dosage of bacteria was 35 mg,the optimal initial concentration of uranium was 10 mg/L,the enrichment rate reached 93.5%,the enrichment amount was 43.4 mg/g,and the reduction enrichment efficiency was better than the wild-type DR.（4）FTIR analysis showed that the characteristic peak of UO₂²⁺appeared in Deino-dsr A multi effect gene engineering bacteria.SEM results showed that the surface of bacteria changed greatly before and after enrichment.EDS analysis showed that uranium had been enriched to the surface by Deino-dsr A.The results of HPLC and ICP-MS showed that the total content of U（IV）and U（III）in the sediment was about 48293.4 mg/kg after enrichment.Conclusions1.Several key genes of Deinococcus radiodurans are involved in uranium stress,uranium stress inhibited the expression levels of DR＿RS10750、DR＿RS07335、DR＿RS04590,and upregulated the expression levels of DR＿RS11290、DR＿RS03605、DR＿RS13480 etc.The expression level was mostly down-regulated,suggesting that the uranium solution inhibited the stress of DR to a certain extent,and the survival rate of DR decreased with the increase of uranium concentration.2.BSDR,a surface activated uranium enrichment agent,was constructed.The optimum enrichment conditions of BSDR were as follows:temperature25℃,p H 4.5,initial concentration of uranium 20 mg/L,optimum dosage of uranium 10 mg.The enrichment rate can reach 93.3%.BSDR had good regeneration performance and wide application conditions.3.Construction of recombinant E.coli DH5α-dsr A containing reducing gene.The results showed that the optimal conditions of uranium reduction and enrichment were p H 4,initial uranium concentration 5 mg/L,microbial dosage 40 mg/L,temperature 37℃,reduction and enrichment time 40 min,and the maximum reduction and enrichment rates were 95.83%and 88.23%,respectively.4.Deino-dsr A multi-effect genetic engineering bacteria with radiation resistance and high reduction performance were successfully constructed.The results showed that the optimal time was 60 min,temperature was 30℃,p H value was 5,and initial concentration of uranium was 10 mg/L.The enrichment rate is 91%-99%,and the enrichment amount is 43.4 mg/g.The life cycle of Deino-dsr A was 90 h in uranium solution.The efficiency of Deino-dsr A was better than that of wild-type DR,the difference was significant.更多还原