Antioxidant Mechanism Of Di-n-butyl Phthalate Degrading Bacteria DNB-S1 And Optimization Of Culture Conditions

With the rapid development of the plastics industry, di-n-butyl phthalate (DBP) is widely used as plasticizers and employed in food enterprise, agriculture, industry, etc.. As DBP has reproductive toxicity, genetic toxicity and potential carcinogenic effects, the environmental problems caused by it can not be ignored. Due to the microorganism can forecast the change of environmental pollution in the earlier stadge, the gram-positive bacteria Bacillus subtilis B7 (B. subtilis B7), the gram-negative bacteria Escherichia coli K12 (E. coli K12)and the DBP degrading bacteria Novosphingobium aromaticivorans (DNB-S1) which screened from the DBP contaminated soil were used to investigate the changes of development, morphology, lipid peroxidation in membrane and antioxidant enzyme actives including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) at 0 mg/kg DBP and 40 mg/kg DBP in this research. The toxicological effects of DBP on the typical microorganisms were discussed, and the antioxidant mechanism of DNB-S1 on DBP was illustrated. The cheap culture medium for DNB-S1 was developed and optimized, which will lay the foundation for the practical application of the DBP degrading strain DNB-S1. The main results were as follows:(1) pared the control group with the 40mg/kg DBP stressed group, the B. subtilis B7 and E. coli K12 under DBP stress showed an increased OD. Compared with the blank ones, the quantity of DNB-S1 dramatically increased at 40 mg/kg DBP condition. According to transmission electron microscope (TEM), the submicroscopic structure of B. subtilis B7 and E. coli K12 showed that the cell wall structure of control strain was integrity and the cell membrane was clearly visible. Moreover, the ribosomes and chromatin were evenly distributed in the cell. However, the B. subtilis B7 and E. coli K12 under 40 mg/kg DBP stress showed that cellular structure was damaged with incompleted cell wall structure, distorted cell membrane fold and ribosomes and chromatin unevenly distribution. These facts reveal that although the growth rising under the DBP stress of the concentration at 40 mg/kg, the cell structure was damage by DBP as well. According to TEM, the structure of DNB-S1 showed normal similarity to the control, with integrated cell wall, clearly visible cell membrane, evenly distributed ribosomes and chromatin in cells. Under DBP stress, the content of MDA in the cells was 2 times of that in the blank group, indicate that DBP could produce lipid peroxidation damage to B. subtilis B7 and E. coli K12. The content of MDA in DNB-S1 cells under DBP stress was only 0.1nmol/mgprot more than the control one. It illustrated that the DBP stress caused little lipid peroxidation damage.(2) Analysis the SOD?CAT and POD activities in B. subtilis B7.E. coli K12 and DNB-Sl, the enzyme activity was significantly increased under DBP stress at 4-8 h, which showed that the B. subtilis B7?E. coli K12 and DNB-S1 was sensitive to DBP stress. The POD activity changed little under DBP stress condition, while the SOD and CAT activities were obviously increased at 4-8 h under DBP stress compare to the blank one. It indicated that SOD and CAT involved in E. coli K12 had self-protection function under DBP stress. DBP stress may produce excessive free radical to damage the cell wall, their self-protection achieve by improving the antioxidant enzymes activity to clear free radicals.The comparative analysis of the antioxidant mechanisms in different bacterial responses to DBP stress, it was found that DNB-S1 has a strong tolerance to DBP. In order to apply the DBP degrading bacteria DNB-S1 in the field repair widely, the green and cheap agricultural products were used as raw materials for the medium to develop a cheap culture medium for DNB-S1 that would reduce production costs and provide a higher strain production, and lay the foundation for the application of contaminated soil bioremediation.(3) Combined single factor test, Plackett-Burman test and central composite test, and nonlinear model based on quadratic polynomial regression equations, the medium formula was optimized as follows:Corn flour 23.9 g, soybean powder 37.5 g, K2HPO4-3H2O 0.8 g? MgSO4·7H2O 0.4 g?NaCl 0.4 g?FeCl3·6H2O 0.0428 g. CaCl2·2H2O 0.1 g?distilled water 1000ml, the culture time 24h. The optimized medium has many advantages, such as shorter training period, higher strain yield, lower cost, high application prospect and better economic benefit.