Isolation, Metabolism And Soil Bioremediation Application Of Ethametsulfuron-Methyldegrading Strain SW4

Ethametsulfuron-Methyl（ESM）,methyl-2-[（4-ethoxy-6-methylamino-1,3,5-triazin-2-yl） carbamoylsulfamoyl]benzoate,is a sulfonylurea herbicide that is used to control broad-leaved weeds and some grasses in cereal crops at very low application rates i.e.2-15 g ha^-1.Because of their high phytotoxicity and the likelihood of their transportation in surface runoff,there is concern about their possible impact on aquatic ecosystems.Because of its low cost and high efficiency,ESM has been used worldwide,ultimately resulting into more serious environment pollution.Microbial activity has been deemed to be the most influential and significant cause of sulfonylurea herbicide removal and biotransformation.Microbial remediation has been deemed to be much more advantageous method than the others.So it has been an important research item for us to exploit and utilize microbial resource to remove environment pollution.The effects of a persistent sulfonylurea herbicide,Ethametsulfuron-Methyl（ESM）, on soil microecosystem were studied in garden soil samples with a short-term treatment of ESM at different concentrations.The culturable bacteria（plate counts）, soil enzyme activities;and-changes in microbial community structure were used to assess biological community in garden soil contaminated by ESM.Moreover,three bacterial strains capable of degrading ESM were isolated,identified and phylogenetically analyzed.The metabolic pathway of ESM by strain SW4 was detected and inferred basically.The results will be valuable to build up alert index systems in acetamiprid-contaminated upland soil,environmental quality evaluation and virtual utilization of acetamiprid-degrading bacterium in upland soil.The main results of this study are as follows:The influences of ESM on the cultural microorganisms in Yellow-brown earths, using traditional selective plating and direct viable counts methods,and soil enzymes activities were investigated.The results showed that the bacteria differed markedly in their response to ESM.The concentration of ESM applied is an important factor affecting populations of various microorganisms,except those characteristics of ESM itself.When the concentration of ESM was higher than 10μg kg^-1 dry soil（d.w.）,the total number of bacteria in the soil samples polluted by ESM was significantly lower than that in the control group.The resumed rates of bacterial number were significantly slower than that the control group during incubation.Actinomycete and aerobic nitrogen-fixing bacteria in the soil samples polluted by ESM were inhibited evidently during incubation.The fungi growth in the treatment soils was stimulated by the ESM at the concentration higher than 20μg kg^-1 dry soil（d.w.） within the first week,and then recovered to the same level as the control group.The soil respiration was inhibited after two weeks of ESM treatment,and the higher the applied concentration was,the stronger the inhibition was observed.The results of enzymological studies indicated that the ESM had a great effect on urease activity in the soil samples planted with vegetable.In the first week,the urease activity was inhibited by ESM with low concentration,and then resumed to the same level as the control group,but it was stimulated at first and then restrained by ESM with high concentration.In the first week,the catalase activity was stimulated by the ESM at the concentration below safe dose,20μg kg^-1 dry soil（d.w.）,but inhibited by increasing the concentration of ESM.In addition,dehydrogenase activity was inhibited slightly after the first week of ESM treatment.From the second week,the activity of dehydrogenase was stimulated and then resumed to the normal level subsequently.However,phosphatase and sucrase were significantly restrained by ESM at all of the applied concentrations throughout the test,and there were dose-effect regression relationship at the 7th day.Three soil ESM-degrading bacteria,strain SW1,SW2 and SW4,were isolated from sludge collected from the bottom soil of a workshop in an herbicide factory. They can use ESM as sole nitrogen resource rather than sole carbon resource.Based on physiological characteristics,biochemical tests and a partial analysis of the 16S rRNA gene sequence,the strain SW1 and SW4 were identified as pseudomonas sp. and the strain SW2 was identified as Arthrobacte sp..Among three strains,SW4 shows the highest ESM-degrading ability.DNA-DNA hybridization showed that,SW4 has a homology of 93.79%with the type strain Pseudomonas nitroreducens IAM1439, so it was identified as Pseudomonas nitroreducens SW4.In addition,Strain SW4 showed the resistance to SXT,AMP,CAZ and STR.This bacterium could degrade 84.8%of 100mg L^-1 ESM within 7 days.Biological properties of SW4 were also studied.The optimal growth temperature and initial pH are 30℃and 7.0,respectively. The aeration had a positive effect on the growth of strain SW4.The optimum carbon sources for SW4 were glucose,sucrose and maltose,and the optimal nitrogen source was organic nitrogen,and the most optimal C/N（mol atom） ratio was 8/1.It grew well under conditions of 25℃-37℃and pH 8.0.Its resting cells degraded ESM well at 25℃-30℃and pH6-10.Besides ESM,SW4 could also grow on Nicosulfuron,Pyrazosulfuron-Ethyl,Metsulfuron-methyl,and Thifensulfuronmethy using as sole nitrogen sources.The degradation products of ESM in the culture medium extracts were isolated and identified by LC/MS.Five metabolites of ESM degradation were detected.Based on the identified products,strain SW4 seemed to degrade ESM following two separate and different pathways:One route includes the cleavage of the sulfonylurea bridge,yielding the corresponding sulfonamide and heterocyclic amine.The other route implicates the loss of the alkyl from the triazine portion of ESM and yielding the N-desethyl Ethametsulfuron-Methyland N-desmethyl-O-desethyl Ethametsulfuron-Methyl,the later was further transformed to methyl 2-[[[[[amino[（aminocarbonyl）imino]methyl] amino]carbonyl]amino]sulfonyl]benzoate due to the cleavage oftriazine ring.The ESM-degrading crude enzyme was extracted from SW4 and analysis showed that it was an intracellular enzyme.It was a constitutive and non inductive enzyme. The best reaction system was as following:incubate 30μL crude enzyme in 3 mL PBS with a pH value 7.0 for 60 min at 30℃.The effect of temperature and pH on the crude enzyme were determined,the optimal condition of enzymatic degradation activity is 30℃and pH8.0.Metal ion and surfactants affect the activity of crude enzyme.Hg²⁺,Ca²⁺ and Cu²⁺ intensively inhibits the activity of enzyme,while the Mg²⁺,Mn²⁺,Fe³⁺ and Li⁺ promote the activity of enzyme in some degree.SDS inhibits the catalytic efficiency of enzyme upto 30.9%.Chelant EDTA would inhibit enzyme activity if the adding concentration exceeds 10mM.Bioassay of ESM has been conducted under laboratory conditions,with the indicator of growth inhibition of maize roots.Growth inhibition of maize roots in water samples was correlative with ESM’s residual concentration,and the relationship between them in water sample is y=4.1399+0.9694x,R=0.9902,and in soil sample, it is y=3.8692+1.4437x,R=0.9687,（y means the probit of the toxicity,x means the logarithmic form of the ESM）.The conclusion was applied to a case study of ESM polluted soil to provide primary evidence for the bioremediation of ESM by SW4.The inoculation of strain SW4 to soil treated with ESM resulted in a higher degradation rate than in non-inoculated soil regardless of the soil is sterilized or non-sterilized. Organic carbon,opportune soil moisture and neutral or week alkalescent condition can stimulate strain SW4 to degrade ESM in soil sample.The ESM degradation of strain SW4 was restrained in flood condition or the soil with moisture less than 5%.In the optimum laboratory condition,the ESM-polluted soil resumed to the safe level after 15 days of inoculation with strain SW4.Finally,we believe strain SW4 could be applied to bioremediation of environment,water body or soil,polluted by ESM or some other sulfonylurea herbicide after further investigation.