Study On Degradation Characterization And Metabolic Pathway Of Simazine Degrading Bacterias

Simazine appeared in 1955,because of its efficient herbicidal activity and inexpensive,had become one of the most popular herbicides in the world at some time in the past.However,the character of its slow degradation in the environmental media was easy to cause pollution of the environment and water bodies,and result in a serious threat to human health.So far the domestic research about biodegradation of simazine was reported rarely.Therefore,the workgroup had previous isolated two degrading bacterias?S-1 and S-3?were used to degrade simazine.The optimum growth conditions of two bacterias were investigated to use single factor and orthogonal experimental design by UV-visible spectrophotometry,the degradation ability of S-1 and S-3 were studied to determine the degradation rate of simazine by liquid chromatography?HPLC?,the degradation pathways of simazine were analysised by HPLC-MS and Ion Chromatography,the degrading enzymes of strain S-1 and S-3 were extracted by Ultrasonication and Osmotic Shock principle to determine the degradation activity of enzymes in different positions,and to study on their enzymatic properties.The main results are as follows:1.S-1 and S-3 belong to common bacterias,and the optimum conditions were 30?,pH 7.5,2.0 % salt concentration,2% inoculation amount,the liquid volume 50 mL,the rotation speed 150 r·min^-1,and the results of verification test were almost identical with the orthogonal test.2.The optimum conditions for the degradation reaction were 30?,pH 7.5,the degradation rate of 0.2 mg·L^-1 simazine was relatively faster and up to 80 % within 24 h by S-3,However,both S-1 and S-3 could reach more than 99% after 72 h.It indicated that two strains had a strong degradation ability,and the degradation effect of mixed bacterias was stronger than single bacteria,the optimal proportion of VS-1 and VS-3 was 1:1.The degradation rate of simazine was improved substantially after adding the ordinary carbon and nitrogen sources,Cu²⁺ and Cr³⁺ inhibited obviously on degradation reaction,and Zn²⁺ and Mg²⁺ had a certain promoting effect.3.The HPLC-MS results proved that simazine was converted to hydroxyl-simazine by the side-chain dechlorination,and molecular ion peak is m/z=184.4[M+H]+.The results of Ion chromatography showed that the final concentration of chloride ion were 1.86 mg·L^-1 and 1.78 mg·L^-1 after degrading 72 h by S-1 and S-3 in the degradation system,which was little difference between 10.0 mg·L^-1 simazine contained the chlorine ion 1.76 mg·L^-1.Further this conclusion explained that simazine had completely been converted to hydroxyl-simazine finally.The metabolic process successfully reduced the toxicity of simazine,but failed to mineralize simazine completely,The degradation reaction kinetics indicated that the degradation processes of S-1 and S-3 followed First-order Kinetics Model,the degradation rate decreased gradually with increasing concentration of simazine,Therefore,the concentration of simazine was too high not conducive to degrading.4.The results showed that the enzyme with degradation effect of two strains all were the endoenzyme and constitutive enzyme.Under the optimum enzymatic reaction conditions for 30??pH 7.5 and the same enzyme amount,obtained the Michaelis constant Km were 10.27 mg·L^-1 and 14.79 mg·L^-1.the maximum reaction rate Vmax were 0.0192 mg·L^-1·min^-1 and 0.0317 mg·L^-1·min^-1 by Lineweaver-Burk plot method.The Km value of S-3 degrading enzyme was smaller,and which indicated that S-3 had a greater affinity with simazine and degradation ability.