| 2,4-dichlorophenoxyacetic acid?2,4-D?classified as the phenoxyalkanoic acid herbicides is a systemic and broad-spectrum foliar herbicide that is used in a wide range of wheat,rice,corn and sugar cane to control of chenopodium album,amaranthus retroflexu broadleaf weeds and bud gramineous weeds.Due to a long term largely use,2,4-D has resulted in considerable pollution to the water and soil and thus threatened to ecological environment and human health.Therefore,there is an increasing concern about microbial bioremediation of herbicide residual contamination.In this study,a bacteria strain T1 capable of degrading 2,4-D was isolated from soil,and morphological,genetics identification,principal degradation characteristics and influence factors of strain T1,degradation pathway of 2,4-D were investigated,and microorganism agent of strain T1 was also studied.The results were summarized as follows:A bacterial strain capable of utilizing 2,4-D as a sole source of carbon and energy was isolated from soil of soybean field contaminated by herbicides such as 2,4-D.The isolated strain T1 was identified as Cupriavidus gilardii based on morphology,physiological and biochemical properties,16 S rDNA sequence analysis.The optimal growth temperature and pH of C.gilardii in LB?Lysogeny Broth?medium are 30 ?,and 8.0.The degradation percentages of 2,4-D by the strain C.gilardii in MSM is relevant to initial concentration of 2,4-D,incubation temperature,pH and initial inoculating amounts.The half-lives of 2,4-D at concentrations of 10.0,50.0 and 100.0 mg?L-1 were measured to 9.3 h,12.8 h and 23.5 h,respectively under 0.5 mL of initial inoculating amounts?OD600=0.6?,7.0 of pH,30 ? of incubation temperature.The degradation percentages of 50.0 mg?L-1 of 2,4-D were 17.0%,26.6%,49.0%,45.9%,1.8% when it was incubated for 10 h at 20 ?,25 ?,30 ?,37 ?,42 ? under pH 7.0 condition,respectively.Approximately 96.3%,97.93%,98.9%,95.8%,and 83.5% of 50.0 mg?L-1 of 2,4-D degraded within 24 h at five different pH regime?5.0,6.0,7.0,8.0,9.0?under 30? condition,respectively.The degradation percentages were 7.2%,11.9%,14.4%,48.9% 100.0%,when initial inoculating amounts were 0.2,0.5,1,2,4 mL,respectively.These results indicated that the optimal degradation temperature of C.gilardii is the range of from 30 to 37 ?,high degradation percentage can be achieved at the pH range of from 5.0 to 8.0,and the degradation percentage increased significantly with the initial inoculating amounts increased.C.gilardii can degrade other pesticides such as 2-methyl-4-chlorophenoxyacetic acid,2,4-D sodium salt and MCPA-2-ethylhexyl ester.The degradation percentages of these herbicides at 50.0 mg?L-1 of initial concentration were 99.7%,100.0%,37.4%,respectively.C.gilardii can not degrade 4-?2,4-dichlorophenoxy?butyric acid,2-?2,4-dichlorophenoxy?propionic acid,2,4,6-trichlorophenol and isooctyl-2,4-dichlorophenoxyacetate.A probable degradation pathway of 2,4-D were explored by GC-MS/MS and UPLC-MS/MS.The results indicated that the possible degradation pathway of 2,4-D by C.gilardii as follows: sloughing the acetic acid side chain of 2,4-D into 2,4-dichlorophenol firstly,then the ortho position of hydroxy on benzene ring of 2,4-dichlorophenol was hydroxylated to 3,5-dichloropyrocatechol rapidly.C.gilardii is capable to degrade 10.0 mg?L-1 of 2,4-dichlorophenol and 3,5-dichloropyrocatechol completely when incubated for 6 h and16 h,respectively.Sodium alginate and CaCl2 were chose as embedding medium and fixing agent for immobilizing of strain C.gilardii respectively in this study.Concentration of CaCl2,concentration of sodium alginate,wet weight of the bacteria and immobilizing time were chose as factors of the orthogonal test for the microorganism agent preparation of C.gilardii.The optimum immobilization conditions were as follows: the concentrations of sodium alginate and CaCl2 were 4% and 1%,wet weight of the bacteria was 3.0 g,and immobilizing time was 8 h. |
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