Screening Of Triasulfuron Co-metabolic Degradation Bacteria MB-1,and Its Degradation Characteristics And Immobilization Cell

Triasulfuron was a kind of sulfonylurea herbicide.Sulfonylurea herbicides was the second largest category of herbicides in the world because of its less amount in use,high efficiency,being widely used,low toxicity,less residue,and high selectivity which significantly and it had promoted the development of agriculture.After the long time application of the sulfonylurea herbicides,the residual caused huge losses to agriculture due to its high selectivity and being persistent in soil.Microbial degradation had the advantages of low cost,small impact to environment and could maximum reduce the concentration of pollutant made it become an important way of degrading sulfonylurea herbicides.In this study,a bacterial strain named MB-1 which could degrade triasulfuron through co-metabolism using glucose as the growth source was isolated from the contaminated actived sludge collected from wastewater-treating system of a pesticide manufacturer.According to its morphological,physiological and biochemical analysis and the comparison of 16S rDNA gene sequence,the strain MB-1 was identified as Pseudomonas aeruginosa..Strain MB-1 was cultured in LB liquid medium,the growth curve showed that 4?10h was the logarithmic growth period.Ten hours later,strain's growth gradually become stable.After culturing for 12h,the strain had the maximum growth amount.Strain's optimum growing pH and temperature range were 6.0?9.0 and 35? respectively.The cell growth amount increased with the volume of liquid reduced and the growth of bacteria was better under the oscillation condition than under stationary condition.When the concentrationin of NaCl changed from 0 to 10g/L,the growth of strain MB-1 didn't have significant difference,while the concentrationin of NaCl ranged from 15-30g/L,the growth of strain was inhibited,and the amount of bacteria was reduced with NaCl growed.The bacteria strain MB-1 was sensitive to gentamicin and chloramphenicol and was resistant to the carbenicillin,tetracycline,streptomycin and ampicillinThe degradation ability of strain MB-1 which was treated by starving did not improved.The optimum co-metabolizing conditions for strain MB-1 on triasulfuron were glucose 2000mg/L,triasulfuron 200mg/L and cell adsorption effect of triasulfuron was excluded.Different Inoculum amount has no obvious effect on triasulfuron degradation.Other optimum conditions are sodium chloride 1.0g/L,ammonium sulfate500mg/L,initial pH 6.0 and temperature 35 ?,respectively..When the concentration for metal ions of Fe³⁺?Zn²⁺?Co²⁺ was 0.1mmol/L,degradation of triasulfuron would be promoted,and all the metal ions at 1.0mmol/L would inhibit the degradation.The co-metabolic products of triasulfuron were identified by LC-MS.There were three products which were 2-amino-4-methoxy-6-methyl-1,3,5-triazine,methyl {[2-?2-chloroethoxy?phenyl]sulfonyl}-carbamic acid tert-butyl ester and 4-[?{[2-?2-chloroethoxy?phenyl]sulfonyl}carbamoyl?amino]-6-methyl-1,3,5-triazine-2 ester.The results of LC-MS suggested that the hydrolysis of triasulfuron occured at first.This resulted in cleavage of two amino groups at the urea bridge and produced 2-amino-4-methoxy-6-methyl-1,3,5-triazine and an unstable material D.The material D was unstable and quickly converted to methyl {[2-?2-chloro-ethoxy yl?phenyl]sulfonyl }-carbamic acid tert-butyl ester.The methoxy group on the triazine ring was substituted by a hydroxy which reacted with carboxylic acid and produce 4-[?{[2-?2-chloroethoxy?phenyl]sulfonyl}carbamoyl?amino]-6-methyl-1,3,5-triazin-2-methyl ester.The carboxylic acid may be derived from the growth of strains.In this study,strain MB-1 was immobilized by polyvinyl alcohol and sodium alginate,respectively.Through orthogonal experiment,the optimum immobilization conditions of two immobilization beads were found.For immobilization by sodium alginate,sodium alginate 4%,CaCl₂ 2%,inoculum 1%and calcification time 4h were the optimal conditions;For immobilization by polyvinyl alcohol:polyvinyl alcohol 10%,sodium alginate 1%,inoculum 1%,CaCl₂ 2%,prior crosslinking time 1h,after crosslinking time 4h,and the concentration of Na₂SO₄ was 0.5mol/L were the optimal conditions.Triasulfuron degradation abilities of two immobilized beads that immobilized under the optimum conditions decreased with the incresed using frequency.