Study On Screening,Degradation Performance And Product Toxicity Of Tetracycline Degrading Strains

Antibiotics is a kind of anti-pathogenic substances,and can interfere with development action of other living cell.There are a great variety of antibiotic being widely used in the treatment of diseases caused by non-viral infections and in the disease prevention of aquaculture and animal husbandry.In recent decades,it is reported that residue of antibiotics is frequently detected in the environment as a result of severe abuse of antibiotics,even the emergence of superbug in some patients.With regard to the residual status of antibiotics in the environment and the residual hazards,the paper proposes a microbial degradation method to solve the problem.The main contents include isolating strains of degrading tetracycline function from the soil,which is fetched from a antibiotic production workshop of a local pharmaceutical factory,and acclimating the degradation performance of the strains.In addition,carrying out a test on the cytotoxicity before and after the degradation process respectively.In the second chapter,the target strains were screened by the select culture medium,and the TCD1,TCD2 and TCD3 with higher degradation rate were selected by increasing the concentration of tetracycline in the culture medium.The initial degradation rates of TCD1,TCD2 and TCD3 were 20.50%,26.32%and 24.97%respectively.The results of genetic identification showed that TCD1 and TCD2 belonged to Klebsiella in taxonomy,and named them with Klebsiella sp.TCD1 and Klebsiella sp.TCD2,while TCD3 belonged to Serratia,named it with Serratia sp.TCD3.In the third chapter,the degradation performance of the target strains was optimized,including the optimization of the culture conditions and the degradation conditions.The optimized results showed that the optimum carbon source for TCD1,TCD2 and TCD3 were carbon-free,1%glucose and carbon-free,respectively;the optimal nitrogen source for all is 0.5%urea;and among the additional trace elements,Cu2+ and Fe2+ have obvious effect of promoting the degradation of the three strains.The optimal degradation time for TCD1,TCD2 and TCD3 are all 5d;the optimal temperatures are 30 ?,30 ?and 35?,respectively;the optimum initial pH is 5,8 and 5 respectively.The optimum rotate speed of the incubator were 150rpm,150rpm and 130rpm,respectively;the optimal inoculation amount was 1%for all;and the cytotoxicity increased with the substrate concentration,thus,the worse degradation effect.The substrate concentration there was determined at 100mg/L.After optimization process,the degradation rates of TCD1,TCD2 and TCD3 increased from the initial 20.50%,26.32%and 24.97%to 84.63%,90.49%and 83.71%,respectively.In the fourth chapter,cytotoxicity test of tetracycline was carried out.MTT assay showed that when the concentration of tetracycline reached 300mg/L,the survival rate of HSF cell was only 47.46%,and the toxicity was positively correlated with the concentration of tetracycline;and the cell apoptosis and cell cycle tests both operated by flow cytometry further confirmed the result of MTT assay.After degraded by target strains,the tetracycline product caused the HSF cell survival rate rise compared with the original tetracycline drug,and survival rate was increased from 49.48%to 68.36%,69.18%and 77.71%respectively.All of the cytotoxicity test further confirmed the feasibility and safety of the method.