Resistance To Uranium Stress Process Of Deinococcus Radiodurans And Effect Of Uranium Enrichment After Dsra Gene Transformed Into Dr

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.