Isolation And Identification Of Prometryn-degrading Bacteria,and Its Microbial Degradation

Prometryn(2-methylthio-4,6-bis(isopropylamino)-s-triazine)classified as the triazines herbicides is a high efficiency,low toxicity and selective herbicide.Prometryn has been used widely for control some annual grasses and broad-leaved weeds in corn,cotton,sugarcane,peanut,rice and soybeans.Due to its low adsorption,chemical stability and long half-life in environment,prometryn has high potential to contaminate surface water,groundwater and soil.Bioremediation has more and more extensive application in dealing with the pollution of herbicides in soil and water because of its low cost and good effect and no secondary pollution.In this study,a new bacterial strain JW-1 of prometryn-degrading was isolated from active sludge,and physiological,biochemical and genetics identification,and growth characteristic of the species along with biodegradatio n characteristics and biodegradation pathway of prometryn were researched.The results of this study are as follow:A novel bacteria strain JW-1 capable of degrading prometryn as sole source of carbon and energy in pure culture was isolated from active sludge in a prometryn manufacture plant of Binzhou City.The isolate strain JW-1 was identified as Leucobacter sp.based on morphology characteristics,physiological and biochemical characteristics and 16 S r RNA gene sequence analysis.The optimal growth temperature and pH of Leucobacter sp.JW-1 in beef extract-peptone medium are 30 ?,and 7.5.The biodegradation rate of prometryn by strain JW-1 in MSM is relevant to pH,temperature,initial concentration of prometryn and inoculums bacterial biomass,while its degradation function was accorded with the first-order kinetics equation.The degradation conditions of strain JW-1 were optimized by the single factor controlled variable method,the results are as follow: the half-lives of prometryn at concentration of 50 mg L-1were measured to be 4.51,4.50,2.19,2.11 and 2.80 hours at pH 5.0,6.0,7.0,8.0,and 9.0,respectively under 0.3 mL of initial inoculating biomass(O D600=0.6,1.2×109 cfu/mL),30 ?.The half-lives of 50 mg L-1 of prometryn,p H 8.0 and inoculation condition of 0.3 mL(OD600=0.6,1.2×109 cfu/mL)were 11.37,4.25,2.85,1.38,1.26,and 1.89 h,respectively.The half-lives of 200 mg L-1 of prometryn were 21.30,3.76,0.86,1.08,and 1.12 h,respectively(p H 8.0,37 ?),when the mediums were inoculated with 0.1,0.3,1.0,2.0,and 4.0 mL of bacterial cells suspensions(OD600=0.6,1.2×109 cfu/mL).The half-lives of prometryn at concentration of 50,200,400 mg L-1 and inoculation condition of 0.3 mL(OD600=0.6,1.2×109 cfu/mL)were measured to be 0.97,3.71,and 5.54 h,respectively(pH 8.0,37 ?).The ANOVA analysis(p?0.05)suggested that the optimal degradation p H of Leucobacter sp.JW-1 is the range of from 7.0 to 8.0,and that the optimum temperature range is among 37 and 42 ? for prometryn degradation in pure culture,and the optimum inoculums biomass for degradation 200 mgL-1 of prometryn is 1.0 mL of bacterial cells suspensions(OD600=0.6,1.2×109 cfu/mL).Four identified metabolites through GC,HPLC and LC-HRMS analysis were: 2-hydroxypropazine(2-hydroxy-4,6-bis(isopropylamino)-s-triazine),methanethiol,dimethyl disulfide,N-isopropylammelide(2,4-dihydroxy-6-isopropylamino-s-triazine)and cyanuric acid(2,4,6-trihydroxy-s-triazine).The possible biodegradation pathway of prometryn by Leucobacter sp.JW-1 as follow: prometryn underwent de-methylthio hydrolysis reaction to give the 2-hydroxypropazine and methanethiol,methanethiol is oxidized to dimethyl disulfide,and then subsequent deaminize hydrolysis reactions of this intermediate may lead to complete elimination of substituent to finally give cyanuric acid.This pathway is a new discovery of the current research on the pathway of the biodegradation of prometryn.Leucobacter sp.JW-1 can degrade other triazines herbicides,the determination of degrading spectrum of bacteria strain JW-1 suggest,at an initial concentration of 50 mg L-1,the isolate strain JW-1 can degraded 100.0 % of ametryn,99.4 % of simetryne,51.6 % of atrazine,46.6 % of propazine,29.6 % of simazine,15.0 % of terbutylhylazine,14.5 % of prometon,13.1 % of atraton after incubation for 4 d,respectively.The strain JW-1 can not degraded terbumeton,metribuzin,cyanuric acid.The results suggest Leucobacter sp.JW-1 can degrade various triazines herbicides such as methylthio group(-SCH3),chlorine group(-Cl)and methoxyl(-OCH3).The prometryn-degrading enzymes produced by Leucobacter sp JW-1 are an intracellular enzyme.The degradation rate o f intracellular enzyme on prometryn was 99.9 % at pH 7.0,37 ? in the enzymatic reaction system with prometryn of 50 mg L-1.The activity of intracellular crude enzymes increased significantly with increase of the temperature,and the activity of intracellular crude enzymes decreased when the temperature exceeded the optimum temperature.The optimum temperature of intracellular crude enzymes are 42 ? for prometryn,it was consistented with the optimum prometryn-degrading temperature of strain JW-1.