| Several bacteria capable of degrading BSM were isolated,identified and characterized.The effects of herbicide bensulfuron-methyl(methyl alpha-(4,6- dimethoxypyrimidin-2-ylcarbamoyl sulfamoyl)-o-toluate,BSM))on soil microbial ecosystem were studied in paddy soil with a short-term treatment of bensulfuron-methyl at different concentrations.The culturable microorganisms(plate counts),soil enzyme activities and changes in community structure (denaturing gradient gel electrophoresis(DGGE)analysis)were included for biological community assessments.The responses of the antioxidant enzymes,superoxide dismutase, catalase,ATP enzyme activities of Escherichia coli(G-),Bacillus subtilis(G+)and BSM-degrading bacterium,Bacillus megaterium D5(G+),following exposure to BSM was also investigated.The results will be valuable to build up alert index systems in BSM-contaminated paddy soil,environmental quality evaluation and bioremediation of BSM-polluted paddy soil.The main results are reported as follows:1.Four BSM-degrading bacteria strains were isolated from the tested soil samples and one of them which had a high efficiency was named as D5.Based on the general physiological and biochemical characteristics,G+C mol%,16S rDNA gene and BIOLOG-GP,strain D5 was identified as Bacillus megaterium.The other three BSM-degrading bacteria had high similarity with Rhodococcus sp.L4,Brevibacillus parabrevis IFO 12334T and Rhodococcus sp.AN-22, with the similarity of 99.7%,100%and 100%,respectively.Strain D5 could utilize bensulfuron-methyl,chlorsulfuron and chlorsulfuron-methyl.Stain D5 was also able to degrade some other aromatic compounds and phosphate pesticide,including benzene,anthrone,triazophos,dichlorvos and dantox;it could also utilize dimethyl benzene, nitrobenzene,paraphenylenediamine,resorcin,brenzcatechin,phenol,metrifonate,durshan and quintiofos.Strain D5 was sensitive to all tested antibiotics,especially to benzylpenicillin sodium, ampicillin,cephalosporinⅤand cephalosporinⅥwith forming of large inhibition zone.The optimal temperature and pH for the growth of strain D5 and BSM degradation is 30℃and 7.5. Supplement of 2 mg L-1yeast could enhance the degradation rate of BSM.The concentration of BSM could affect the growth of strain D5,and the optimal BSM concentration between given rage for its growth was 50 mg L-1.The optimal ventilatory capacity and Mg2+concentration were 1/5 (V/V)and 400 mg L-1,respectively.The optimal carbon source and nitrogen source were yeast extract and glucose,respectively.The effects of strain D5 pure culture and four strains' combination on the whole biomass were different.In liquid medium,97.7%of BSM was degraded at 84 h;in field-simulated paddy soils, 94.3%of BSM was degraded during the 42 d.The result demonstrated the possibility of bioremediation of BSM-polluted paddy soils.Combination of four bacteria had a good degrading efficiency of four herbicides.Whole cell protein SDS-PAGE maps of strain D5 induced by BSM showed special bands,which might be relative with degradation and detoxification of BSM.Using mutagenesis of ultraviolet mutation,a mutant numbered as 6 was obtained and its degradation characteristics could be genetic.2.The influences of BSM on the cultural microorganisms and soil enzymes activities in paddy soil were investigated.The results showed that each kind of culturable microbial population, respiratory intensity,enzyme activity in the soil samples polluted by BSM at different concentrations,was changed with different degree,respectively,during the whole incubation.The growth of cellulose decomposing bacteria and thiobacillus was increased significantly(P＜0.05;P＜0.05)by application of BSM at low concentration;at day 14,the number of actinomycetes and ammonifier in paddy soil with application of 0.067 and 0.335μg/g dry soil BSM was significantly higher compared with the control;at day 21 and 28,the number of ammonifier declined to the control level.3.553μg/g dry soil BSM suppressed the growth of AHB,actinomycetes,aerobic nitrogen-fixing bacteria,ammonifier and cellulose decomposing bacteria.Although there were some influences on the growth of soil culturable microorganisms in BSM treated soils,the effects were not persistent except for AHB and cellulose decomposing bacteria.So,lower concentrations of BSM were safe for soil culturable microorganisms,higher concentrations of BSM were thought to be passive to culturable microorganisms.Intensity of soil respiration was stimulated by BSM at the beginning of BSM application,and then inhibited slightly,the inhibition was lessen gradually and recovered to the control level.The activity of catalase was stimulated by BSM;there were almost no effects on sucrase;the activity of urase increased firstly,then controlled;there's no influences on polyphenoloxidase activity. 3.Denaturing gradient gel electrophoresis(DGGE)was employed to analyse the variation of soil microbial population structure in BSM-polluted paddy soils.The results obtained from DGGE analysis showed that the application of BSM had a certain influences on the variation of soil microbial population structure.Until the 4thweek,the differences of genetic diversity between BSM-treated and no BSM treated(control)were not evident;while the differences of genetic diversity were not conspicuous,almost the same.It could be seen that there's little distinction on the soil microbial genetic diversity between the soils samples treated with different concentration of BSM.So it could be concluded that low concentrations of BSM were safe to soil microorganisms in paddy soils.This result was the same as the conclusion obtained from the former two parts.The addition of BSM-degrading bacterium D5 could release the side-effect of BSM, simulating the growth of other bacteria and elevating the microbial genetic diversity.There are significant differences between microbial populations in different time intervals among different BSM-treated soil samples.This study is a new attempt to apply the Bacillus megaterium D5 to remediate the BSM-polluted environment.The kinetics of BSM degradation by strain D5 and the combination of D5 and BH were investigated.The results showed that different concentrations of BSM could be degraded at a certain degree;the addition of BSM-degrading bacteria could promote the degradation of BSM in paddy soils,the effect of BSM degradation were higher when added with strain D5 alone.4.The activity response of the antioxidant enzymes superoxide dismutase,catalase,ATP enzyme activities of Escherichia coli(G-),Bacillus subtilis(G+)and Bacillus megaterium D5(G+) following exposure to BSM was studied.The bacterial strains were treated with the different concentrations of BSM(0,20,50 and 100 mg L-1.The results suggested that the responses of SOD,CAT and ATPase in these three bacteria at different growth stage to BSM with various concentrations were different.It indicated that the three tested bacteria were induced by BSM at a certain degree.Induction of BSM on BSM-degrading bacteria was weaker than the other two non-BSM-degrading bacteria.This phenomenon might be explained by that BSM-degrading bacteria could degrade BSM and mitigate the side-effect of BSM.The increase of SOD and CAT is evidence of the adaptability of organisms against the oxidation stress,while there are some changes of ATPase activity during the anti-oxidation process.The concentration of BSM applied in this research had significant effects on these three bacteria at the early stage of incubation,but none of them was persistent.Native polyacrylamide gel electrophoresis and activity staining of SOD revealed that the express amount or composition of SOD was affected by BSM.
|
PDF Full Text Request