氯嘧磺隆降解及其对土壤微生态影响的研究
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摘要
磺酰脲类除草剂氯嘧磺隆(Chlorimuron-ethyl)残留药害对农业产生巨大的影响,本研究针对这一问题做了系统的研究。从多年施用氯嘧磺隆的土壤中分离出具有较高降解效果的菌株,研究其生长特性和降解特性,通过模拟土壤环境研究其实际应用效果,还从土壤酶和土壤微生物类群方面研究了氯嘧磺隆对土壤微生态的影响。
1、采用多年施用氯嘧磺隆的大豆田土壤作为研究材料,经过多次驯化培养后,最终得到菌株24株,其中细菌13株、真菌7株、放线菌4株,该菌种资源为氯嘧磺隆降解机制研究和商业化菌剂开发奠定了基础。在此基础上,采用高效液相色谱分析进一步测定其对氯嘧磺隆的降解率,获得了具有较高降解效率的菌株4个,降解率均达50%以上；并对其进行了菌种鉴定,D3为枯草芽孢杆菌(Bacillus subtilis)、L1为蜡样芽孢杆菌(Bacillus cereus)、L3巨大芽孢杆菌(Bacillus megaterium)和C4a为长枝木霉(Trichoderma longibrachiatum)。结合生物学方法,进一步分析了不同环境因子对生长和降解效率的影响,并且实验室模拟环境测定菌株L3对氯嘧磺隆污染改良的效果。
2、以氯嘧磺隆降解菌株巨大芽孢杆菌L3为试材,研究显示该菌株氯嘧磺隆降解酶为胞内酶,酶促反应最适温度为35℃,最适pH值为7.5。在pH6-10之间均有较高的降解率。随着Cu2+、Ba2+和Ca2+浓度增大而降解率降低；K+和Mn2+对酶促反应影响较小；当Zn2+、Fe2+和Fe3+添加浓度为5mM时,其降解率突然下降。
3、氯嘧磺隆对6种土壤酶活性均有一定的影响,过氧化氢酶、多酚氧化酶、蔗糖酶和脱氢酶菌表现出激活现象。过氧化氢酶与多酚氧化酶类似,都是实验的中期其激活作用最大,最后刺激作用消失。而蔗糖酶前期的激活作用达到顶峰,随后逐渐下降。蛋白酶和脲酶则表现出被氯嘧磺隆抑制的现象,都是前期具有较大的抑制效果,后期逐渐消失。总体来说,低浓度的氯嘧磺隆对各种酶的影响相对较小,试验后期都能恢复到与对照值一致,而高浓度处理并不是所有的处理都恢复到对照值的水平。
4、变性梯度凝胶电泳结果显示,氯嘧磺隆的施加对土壤微生物种群结构有一定的影响。低浓度的氯嘧磺隆对土壤中微生物种群的影响较小,高浓度的氯嘧磺隆对土壤微生物群落影响比较显著；氯嘧磺隆对土壤中细菌菌群组成的影响是双向的,氯嘧磺隆可以抑制某些细菌的生长,也可以促进某类细菌的生长,或者对其生长没有影响。加入氯嘧磺隆后仍可快速生长的类群可能是能够降解氯嘧磺隆的种类,进一步分析可能获得更多的高效降解氯嘧磺隆的微生物类群。
Chlorimuron-ethyl, as one of the most versatile class of herbicides, was widely used in agricultural production. However, during the using process, the residual of the herbicide had badly effect on the soil, and it might cause serious problems in agricultural production process. The purpose of this study was to solve this problem by using a series of research. In our study, we isolated chlorimuron-ethyl degrading bacteria from herbicide-contaminating soil, the growth characteristics of the strains and the degradation characteristics of chlorimuron-ethyl were investigated and effects of various factors on the degradation were studied. We investigated the application effects of the strains by adding them into the herbicide-contaminating soil environment. Moreover, we studied the enzymes and microbial structures in the contaminated soil. The main results were as follows:
(1)We used the soybean field soil, which was using chlorimuron-ethyl as herbicide, as research material. We gradually increased the chlorimuron-ethyl concentration in the soil to let the microbes adapt the contaminated substrate. After several domesticated culture,24strains were finally isolated, including3bacteria,7fungi and4actinomycetes. Those strains laid the groundwork for the further work. The degradation rate of chlorimuron-ethyl was mesured by using the HPLC analysis, and four strains, which had the most degradation efficiency, were further used in our study. All of the four strains could degrade more than50%of chlorimuron-ethyl. We used several methods to identify the strains, and the results showed that strain D3belong to Bacillus subtilis, strain L1belong to Bacillus cereus, strain L1belong to Bacillus megaterium, strain C4a belong to Bacillus megaterium, Trichoderma longibrachiatum. We investigated the degrading characteristics of the strains and studied the effects of various environmental factors on the degradation. Moreover, we detected the efficiency by using L3to degrade chlorimuron-ethyl in the improved conditions.
(2) We used the most effective degradation strain-L3to investigate the degrading details of chlorimuron-ethyl. The results showed that the enzyme, which was responsible for chlorimuron-ethyl degrading, was intracellular enzyme. With chlorimuron-ethyl as substrate, the optimal temperature and pH for the enzyme were35℃and7.5, respectively. The enzyme was stable and high activity was maintained from pH6to10. The activity of the enzyme was inhibited by Cu2+, Ba2+and Ca2+, when the concentration of these three ions increased, the enzyme activity decreased. The enzyme activity appeared to be almost unaffected by K+and Mn2+. When the concentration of Zn2+, Fe2+and Fe3+was increased to5mM, the degrading rate decreased suddenly.
(3) Chlorimuron-ethyl had great effect on all the six kinds of enzyme from the soil, and it could activate catalase, polyphenol oxidase, saccharase, dehydrogenase activity. Both catalase and polyphenol oxidase showed the highest activity at the medium term of the test, and then the stimulation effect disappeared. Saccharase got the highest activity when it was first stimulated by chlorimuron-ethyl, and then the stimulation effect decreased. Both protease and urease were inhibited by chlorimuron-ethyl. The two kinds of enzyme could be inhibited greatly at the earlier stage of the test. However, the inhibition effect disappeared at the later stage of the test. In conclusion, low concentration of chlorimuron-ethyl had little effect on the enzyme in the soil, most of the enzyme activity could be recovery at the end of the test. However, when the system contained high concentration of chlorimuron-ethyl, the enzyme activities were not easy to recover.
(4) PCR-DGGE analyses indicated that the microbial community had changed significantly with adding chlorimuron-ethyl to the soil. Comparatively speaking, the low concentration of chlorimuron-ethyl had small effect on microbial community, while high concentration of chlorimuron-ethyl had great influence on microbial community. With adding chlorimuron-ethyl to the soil, the changes of relative quantities of various microbes were detected in the profiles. The quantities of some microbes increased, whereas others decreased. Some microbes maintained their populations. The consortia which could grow quickly might be responsible for chlorimuron-ethyl degradation, and more degrading strains could be found after the consortia be further analysed.
1、采用多年施用氯嘧磺隆的大豆田土壤作为研究材料,经过多次驯化培养后,最终得到菌株24株,其中细菌13株、真菌7株、放线菌4株,该菌种资源为氯嘧磺隆降解机制研究和商业化菌剂开发奠定了基础。在此基础上,采用高效液相色谱分析进一步测定其对氯嘧磺隆的降解率,获得了具有较高降解效率的菌株4个,降解率均达50%以上；并对其进行了菌种鉴定,D3为枯草芽孢杆菌(Bacillus subtilis)、L1为蜡样芽孢杆菌(Bacillus cereus)、L3巨大芽孢杆菌(Bacillus megaterium)和C4a为长枝木霉(Trichoderma longibrachiatum)。结合生物学方法,进一步分析了不同环境因子对生长和降解效率的影响,并且实验室模拟环境测定菌株L3对氯嘧磺隆污染改良的效果。
2、以氯嘧磺隆降解菌株巨大芽孢杆菌L3为试材,研究显示该菌株氯嘧磺隆降解酶为胞内酶,酶促反应最适温度为35℃,最适pH值为7.5。在pH6-10之间均有较高的降解率。随着Cu2+、Ba2+和Ca2+浓度增大而降解率降低；K+和Mn2+对酶促反应影响较小；当Zn2+、Fe2+和Fe3+添加浓度为5mM时,其降解率突然下降。
3、氯嘧磺隆对6种土壤酶活性均有一定的影响,过氧化氢酶、多酚氧化酶、蔗糖酶和脱氢酶菌表现出激活现象。过氧化氢酶与多酚氧化酶类似,都是实验的中期其激活作用最大,最后刺激作用消失。而蔗糖酶前期的激活作用达到顶峰,随后逐渐下降。蛋白酶和脲酶则表现出被氯嘧磺隆抑制的现象,都是前期具有较大的抑制效果,后期逐渐消失。总体来说,低浓度的氯嘧磺隆对各种酶的影响相对较小,试验后期都能恢复到与对照值一致,而高浓度处理并不是所有的处理都恢复到对照值的水平。
4、变性梯度凝胶电泳结果显示,氯嘧磺隆的施加对土壤微生物种群结构有一定的影响。低浓度的氯嘧磺隆对土壤中微生物种群的影响较小,高浓度的氯嘧磺隆对土壤微生物群落影响比较显著；氯嘧磺隆对土壤中细菌菌群组成的影响是双向的,氯嘧磺隆可以抑制某些细菌的生长,也可以促进某类细菌的生长,或者对其生长没有影响。加入氯嘧磺隆后仍可快速生长的类群可能是能够降解氯嘧磺隆的种类,进一步分析可能获得更多的高效降解氯嘧磺隆的微生物类群。
Chlorimuron-ethyl, as one of the most versatile class of herbicides, was widely used in agricultural production. However, during the using process, the residual of the herbicide had badly effect on the soil, and it might cause serious problems in agricultural production process. The purpose of this study was to solve this problem by using a series of research. In our study, we isolated chlorimuron-ethyl degrading bacteria from herbicide-contaminating soil, the growth characteristics of the strains and the degradation characteristics of chlorimuron-ethyl were investigated and effects of various factors on the degradation were studied. We investigated the application effects of the strains by adding them into the herbicide-contaminating soil environment. Moreover, we studied the enzymes and microbial structures in the contaminated soil. The main results were as follows:
(1)We used the soybean field soil, which was using chlorimuron-ethyl as herbicide, as research material. We gradually increased the chlorimuron-ethyl concentration in the soil to let the microbes adapt the contaminated substrate. After several domesticated culture,24strains were finally isolated, including3bacteria,7fungi and4actinomycetes. Those strains laid the groundwork for the further work. The degradation rate of chlorimuron-ethyl was mesured by using the HPLC analysis, and four strains, which had the most degradation efficiency, were further used in our study. All of the four strains could degrade more than50%of chlorimuron-ethyl. We used several methods to identify the strains, and the results showed that strain D3belong to Bacillus subtilis, strain L1belong to Bacillus cereus, strain L1belong to Bacillus megaterium, strain C4a belong to Bacillus megaterium, Trichoderma longibrachiatum. We investigated the degrading characteristics of the strains and studied the effects of various environmental factors on the degradation. Moreover, we detected the efficiency by using L3to degrade chlorimuron-ethyl in the improved conditions.
(2) We used the most effective degradation strain-L3to investigate the degrading details of chlorimuron-ethyl. The results showed that the enzyme, which was responsible for chlorimuron-ethyl degrading, was intracellular enzyme. With chlorimuron-ethyl as substrate, the optimal temperature and pH for the enzyme were35℃and7.5, respectively. The enzyme was stable and high activity was maintained from pH6to10. The activity of the enzyme was inhibited by Cu2+, Ba2+and Ca2+, when the concentration of these three ions increased, the enzyme activity decreased. The enzyme activity appeared to be almost unaffected by K+and Mn2+. When the concentration of Zn2+, Fe2+and Fe3+was increased to5mM, the degrading rate decreased suddenly.
(3) Chlorimuron-ethyl had great effect on all the six kinds of enzyme from the soil, and it could activate catalase, polyphenol oxidase, saccharase, dehydrogenase activity. Both catalase and polyphenol oxidase showed the highest activity at the medium term of the test, and then the stimulation effect disappeared. Saccharase got the highest activity when it was first stimulated by chlorimuron-ethyl, and then the stimulation effect decreased. Both protease and urease were inhibited by chlorimuron-ethyl. The two kinds of enzyme could be inhibited greatly at the earlier stage of the test. However, the inhibition effect disappeared at the later stage of the test. In conclusion, low concentration of chlorimuron-ethyl had little effect on the enzyme in the soil, most of the enzyme activity could be recovery at the end of the test. However, when the system contained high concentration of chlorimuron-ethyl, the enzyme activities were not easy to recover.
(4) PCR-DGGE analyses indicated that the microbial community had changed significantly with adding chlorimuron-ethyl to the soil. Comparatively speaking, the low concentration of chlorimuron-ethyl had small effect on microbial community, while high concentration of chlorimuron-ethyl had great influence on microbial community. With adding chlorimuron-ethyl to the soil, the changes of relative quantities of various microbes were detected in the profiles. The quantities of some microbes increased, whereas others decreased. Some microbes maintained their populations. The consortia which could grow quickly might be responsible for chlorimuron-ethyl degradation, and more degrading strains could be found after the consortia be further analysed.
引文
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