| Two bacterial strains capable of degrading MSM were isolated,identified and phylogenetically analyzed.The primary metabolic pathway of three sulfonylurea herbicides by strain XJ-412-1 was detected and inferred basically.Moreover,The effects of a persistent sulfonylurea herbicide,Metsulfuron-methyl(MSM),on soil microecosystem were studied in garden soil samples with a short-term treatment of MSM 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 MSM.The results will be valuable to build up alert index systems in MSM-contaminated upland soil,environmental quality evaluation and virtual utilization of MSM-degrading bacterium in upland soil.The main results of this study are as follows:Two efficient MSM-degrading bacteria XJ-412 and XJ-412-1 were isolated by continuous enrichment,screened from long MSM-treated soils.They were identified as Brevundimonas sp.and Ancylobacter sp.according to their morphological observation,physiological biochemical test,comparison sequences of 16S rDNA and phylogenetic analysis.The optimum temperature and pH value for their growth were all 30? and 7.0 respectively.Still they could all sustain on high salt concentration,and some antibiotics such as Ampicillin,Spectinomycin and Ceftazidime.They could grow well when using glucose or fructose as sole carbon sources,and peptone,beef cream or yeast extract as sole nitrogen sources.Studies indicate that the isolates could use MSM as the sole carbon or nitrogen source.Among two strains,XJ-412-1 showed the higher MSM-degrading ability.The strain could degrade 90.52%of 50mg L-1 MSM within 7 days.A little alkaline condition benefit the biodegradation of MSM.The optimum temperature for their biodegradability was all 30?,and the isolates XJ-412 and XJ-412-1 could still sustain a broad range of temperature.The feasible salt concentration for the MSM degradation was 20?25g.L-1 the influence of aeration on the biodegradability of the isolates was trivial.Adding yeast extract and glucose could accelerate the biodegradation of MSM,while the soil extract could not accelerate just because of the poor nutriment in the soils.The ion except Hg2+ and Ni2+ had no definite inhibition on the degradation of MSM by the isolates.Strain XJ-412-1 was capable of degrading a wide range of sulfonylurea herbicides,including thifensulfuron-methyl,chlorsulfuron,bensulfuron-methyl and nicosulfuron.Thifensulfuron-methyl was the most preferred substrate,nicosulfuron was degraded slower than chlorsulfuron.Bensulfuron-methyl was the most persistent.The initial degradation products of metsulfuronmethyl,thifensulfuron-methyl,and bensulfuron-methyl by XJ-412-1 were identified as corresponding deesterified derivatives by liquid chromatography-mass spectrometry,which indicated a primary pathway of the deesterification of these three sulfonylurea herbicides.The carboxyesterase activity of the stain was also assayed.Studies also found that the MSM-degrading enzyme was a constitutive and not inductive enzyme.To extract the enzyme,culture XJ-412-1 in LB liquid medium for 72?84 hours,then break it up with lysozyme,ultrasonic and French pressure cell press,and the French pressure cell press was the best method.The best reaction system as following:incubation 20?L crude enzyme in pH7.0 PBS for 10?20min at 30?.The enzyme was jarless among pH 7.0~8.0,and temperature 4~45 ?,the ion Hg2+?Ni2+ and Zn2+ could inhibit the carboxyesterase activity,adding Triton-100 would inhibit the enzyme activity slightly,but Tween 80 and SDS would inhibit enzyme activity seriously when the adding concentration was 10mM.It showed the highest activity with p-nitrophenyl acetate,and the activities decreased with the increase of the aliphatic chain length,indicating that the hydrolase was an esterase and not a lipase.The carboxyesterase was completely inhibited by phenylmethylsulfonyl fluoride,indicating that the enzyme seems a serine esterase.The carboxyesterase was partially purified from strain XJ-412-1 by ammonium sulfate precipitation and DEAE Sepharose Fast Flow.The influences of MSM 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 MSM.The concentration of MSM applied is an important factor affecting populations of various microorganisms,except those characteristics of MSM itself.When the concentration of MSM was higher than 10?g.kg-1 dry soil(d.w.),the total number of bacteria in the soil samples polluted by MSM 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.The fungi growth in the treatment soils was stimulated by the MSM at the concentration higher than 20?g kg-1 dry soil(d.w.)within the first week,and then recovered to the same level as the control group.The actinomycete growth in the treatment soils was stimulated by the MSM at low concentration,but was inhibited at high concentration.Aerobic nitrogen-fixing bacteria in the soil samples polluted by MSM were inhibited evidently during incubation.The soil respiration was inhibited by the MSM at the concentration higher than lO?g kg-1 dry soil(d.w.)in the first two weeks,and the higher the applied concentration was,the stronger the inhibition was observed.The results of enzymological studies indicated that the MSM had a great effect on urease activity in the soil samples planted with vegetable.In the first week,the catalase activity was inhibited by the MSM at all of the applied concentrations,and the higher the applied concentration was,the stronger the inhibition was observed.In the first week,the urease activity was stimulated by MSM at the concentration of 5,10 and 20?g.kg-1 dry soil,and resumed to the normal level after 28d.In addition,dehydrogenase and sucrase were inhibited after the first four weeks of MSM treatment,and then resumed to the normal level subsequently.The bioremediation of metsulfuron-methyl contaminated soil by inoculating XJ-412-1 was studied under laboratory conditions.After addition of 106 cells g-1 dry soil into soil,81.3%of metsulfuron-methyl at concentration of 10 mg kg-1 dry soil was degraded at 30d.Only 43.2%of metsulfuron-methyl was degraded when the concentration of metsulfuron-methyl was 50 mg kg-1 dry soil.The optimal temperature for metsulfuron-methyl degradation by XJ-412-1 in soil was 30?.The addition of glucose could accelerate the degradation of metsulfuron-methyl.Pour root with XJ-412-1 could protect maize from the phytotoxicity of metsulfuron-methyl of 40,80?g.kg-1 in varying degrees.When the concentration of metsulfuron-methyl increased to 120?g.kg-1,the effects were not distinct.It suggested that the metsulfuron-methyl in soil could be degraded effectively by inoculating XJ-412-1. |
PDF Full Text Request