The Construction Of Radiation-resistant Fusion Strains And Experimental Study On Its Biosorption Of Uranium

The radiation lethality of radionuclides uranium and its toxicity to organism,limited the development of biosorbent, therefore the treatment of waste water containing low concentration of uranium went into a bottleneck for a long time.We adopted single inactivated protoplast fusion technique in the fusion of Citrobacter freundii and Deinococcus radiodurans and got the radioresistant strains successfully. The screened fusants had been stably transferred for six generations.The condition of protoplast preparation and some influence factors of fusion process, stability, tolerance to uranium and biosorption capability of fusant were examined and compared in this paper.The optimal conditions of protoplast preparation for Citrobacter freundii was: lysozyme concentration of 1.5mg / L, hydrolysis time of 90min, hydrolysis temperature of 37℃. Under these conditions, formation rate of protoplasts was 93% and regeneration rates was 36%; while Deinococcus radiodurans was: lysozyme concentration of 2.0mg / L, hydrolysis time of 120min, hydrolysis temperature of 37℃, the formation rate 86%. With the lysozyme concentration, enzymolysis time increased and temperature rose, an upward trend of protoplast formation rate was showed, while regeneration rate was found gradually decreased in the test.The results of fusion influence factors orthogonal experiment indicated that the optimal condition was 40% PEG(6000) which induced protoplast for 10 minutes at 42℃, pH 8.0, and the concentration of PFG(6000) was main influence factor. The highest fusion rate could reach to2.74×10^-7 in this condition. Higher temperature and pH value could accelerate the fusion of protoplast. as the toxic effects of PEG on cell, the time of fusion should not be too long and the concentration of PEG(6000) should not be too high. Ca²⁺ and PO₄^3- could form insoluble complexes, as the calcium bridges between cells, which led to fuse.In the test of tolerance to uranium, the fusant showed good tolerance in the uranium concentration of 20,40,60,80 mg / L, while difficult grow in the 90mg / L and could not grow in the 100mg / L. The limit of sub-maximum tolerance was 85mg / L. In the uranium concentration 85mg / L of medium, Citrobacter freundii did not grow longer and eventually be killed in the medium with uranium concentration of 85 mg / L, but Deinococcus radiodurans could grow after the adaptation period for 45h, the fusant began to grow after adaptation period for 22 h, and growth were well.The adsorption rate and adsorption capacity of fusant was slightly higher than that of Citrobacter freundii and Deinococcus radiodurans in the same period in the biomass concentration above 2 g / L, its absorption rate and adsorption capacity was 0.9%, 0.1 mg / g higher than that of Citrobacter freundii. However, the adsorption rate and adsorption capacity was no substantial increase. that might be caused by the change of primer binding sites after recombinant fusion, or the genetic information of fusant obtained from the original strain was not entirely intergrated.The study work could provide a reference for constructing radiation-resistant genetic engineering strains and the applications of radiation-resistant strains in the heavy metal fusion, nuclear pollution bioremediation.