土壤中毒死蜱降解质粒pDOC的转移及其促成的生物强化作用
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摘要
毒死蜱作为一种广谱、低毒、低残留、低抗药性和高效的有机磷杀虫剂,在我国被广泛应用,同时其在环境中的污染不容忽视。生物修复是治理毒死蜱污染的一种切实有效的方法。本实验室分离到一株以毒死蜱为唯一碳源和能源生长的细菌DSP,通过形态特征、生理生化特性及16S rDNA序列分析将其鉴定为芽孢杆菌(Bacillus latersporus),并确定了其降解功能位于质粒pDOC。本文通过投加质粒pDOC到土壤进行生物修复,探索质粒pDOC在土壤中的转移机制及其在毒死蜱降解中的作用,以建立一种以质粒转移为基础的农药微生物降解生物强化新途径,从本质上提高土壤微生物对农药的降解能力,克服生物强化中面临的接种菌存活问题。主要研究结果如下：
(1)利用菌株Bacillus latersporus DSP、E. coli DH5a (pZP201-gfp)及E. coli HB101(pRK2013)的抗性交叉差异行为进行三亲杂交构建荧光毒死蜱降解菌Bacillus latersporus DSP (pDOC-gfp)。通过荧光、降解功能及降解性质粒pDOC-gfp检测证明Bacillus latersporus DSP (pDOC-gfp)构建成功。
(2)以杭州华家池桑树田土壤为材料,采用标记质粒pDOC-gfp投加到毒死蜱污染土壤进行生物修复,并从修复30d后的土壤中分离得到4个发荧光且对毒死蜱有降解能力的单菌,分别命名为ZQ1、ZQ2、ZQ3和ZQ4。通过形态特征、生理生化特性及16S rDNA序列证明这4个菌株属于不同种的细菌,由此说明质粒pDOC-gfp在土壤中向土著微生物发生转移。
(3)利用标记菌株Bacillus latersporus DSP (pDOC-gfp)研究了质粒pDOC强化十壤中毒死蜱降解作用的持久性及其对土壤微生物的影响。结果表明,随着施药频率的增加,降解菌对毒死蜱的生物强化效果长期有效；接种降解菌对土壤微生物群落功能多样性没有显著影响,不具破坏性。
(4)利用标记菌株Bacillus latersporus DSP (pDOC-gfp)采用土柱淋滤的方法研究质粒pDOC在饱和流/非饱和流毒死蜱污染土壤中垂直迁移情况及其对毒死蜱降解。研究发现,质粒pDOC在土壤中可以向下发生迁移；同时质粒供体菌的迁移与质粒转移进入土著菌可以强化土壤中毒死蜱的降解作用。
(5)研究质粒pDOC强化毒死蜱微生物降解的影响因子,结果表明：接种质粒供体菌明显促进了毒死蜱生物降解率,E. coli JM109(pDOC-gfp)是本实验条件中长期修复毒死蜱污染土壤的最佳选择；不同条件下E. coli JM109(pDOC-gfp)对毒死蜱的降解作用顺序为：10mg kg-1≈1mg kg-1>50mg kg-1>100mg kg-130℃>40℃>20℃、60%WHC (田间最大持水量)>40%WHC>80%HZ soil(杭州土壤)>JX soil (嘉兴土壤)>XS soil (萧山土壤)>JH soil(金华土壤)。
Chlorpyrifos as a broad spectrum organophosphorus insecticide is widely used in world due to its high effectiveness, low toxicity to non-target organisms and easy biodegradability and caused great pollution in the environment. The use of chlorpyrifos-degrading bacteria for bioremediation of chlorpyrifos-contaminated sites has been proved to be the most potential clean-up method. In our earlier experiments, a bacterial strain capable of degrading chlorpyrifos, Bacillus laterosporus DSP, was isolated from the soil for the biodegradation and/or detoxification of chlorpyrifos, and the pDOC plasmid in the B. laterosporus DSP strain was found to be responsible for the degradation of chlorpyrifos. In the present study, the development of chlorpyrifos degradation capacity in soil into which a plasmid had been introduced was monitored using the degradation rate and the MPN of the chlorpyrifos degraders as the endpoints of the transfer. The transfer event was monitored using a reporter gene (gfp) combined with microscopic observations and the imaging of GFP expressed in the recipients. The main goal of this study was to develop a plasmid-mediated bioaugmentation method that could result in the persistent capacity for the degradation of chlorpyrifos in the soil. The results were summarized as follows:
(1) The insertion of gfp into the plasmid pDOC was performed by triparental mating in which the helper strain E. coli HB101(pRK2013) was used to mobilize gfp segment from the donor strain E. coli DH5a (pZP201-gfp) into the recipient Bacillus laterosporus DSP. The B. laterosporus DSP strain (pDOC-gfp) was selected based on its resistance to antibiotics, its ability to utilize chlorpyrifos as sole carbon and energy source, and the observation of GFP expression by microscopy.
(2) The soil used in this study was collected from Hangzhou, Zhejiang province, China, and contained no detectable amount of residual chlorpyrifos. To explore the development of the chlorpyrifos degradation capacity in the soil by plasmid transfer, the gfp-tagged plasmid was applied to study the bioremediation in soil. Four of GFP-positive strains, ZQ1, ZQ2, ZQ3, and ZQ4, were isolated from the pDOC-gfp-treated soil30days after treatment, which were capable to chlorpyrifos degradation. The isolates were identified as different species due to their morphological characteristics, physiological and biochemical characteristics and16S rDNA sequences. Moreover, by comparing the relative mobilities of the plasmids, all were found to be identical to that in the initial strain, Bacillus laterosporus DSP, suggesting that new chlorpyrifos degraders were formed by the transfer of pDOC.
(3) To investigate the persistence of bioremediation in chlorpyrifos-polluted soil by plasmid donor strains B. latersporus DSP (pDOC-gfp) and its impact on soil microbial functional diversity, the BIOLOG technique and soil respiration were used to understand microbial community structure and function. The results indicated that the degradation efficiency of chlorpyrifos in soil was always greatly enhanced by inoculation with strain B. latersporus DSP (pDOC-gfp) after repeated application. Soil microbial functional activity and its activity in inoculated soil was not affected and damaged.
(4) By the infiltration test of soil column, the dispersal of plasmid donor strain B. latersporus DSP (pDOC-gfp) in unsaturated and saturated chlorpyrifos contaminated soil was studied. It has been found that chlorpyrifos degraders were throughout the entire length of the column under unsaturated or saturated flow conditions in chlorpyrifos contaminated soil. The enhanced the degradation of chlorpyrifos in soil column may due to the transport of donor strain and its degradative plasmid transfer.
(5) The effect of the plasmid donor, temperature, moisture, and soil type on plasmid transfer was studied. The results indicated that the degradation efficiency of chlorpyrifos in soil was greatly enhanced by inoculation with donor strains. The degradation of chlorpyrifos in the E. coli JM109(pDOC-gfp)-treated soils appeared to be more rapid than the degradation in other donor strain inoculated soils. Therefore, the E. coli JM109(pDOC-gfp) inoculated appeared to be more suitable for bioaugmentation. Strain E. coli JM109(pDOC-gfp) was used to study the effect of various factors, such as temperatures, humidity and soil types, which effect the bioremediation of chlorpyrifos in soil. The results showed that the chlorpyrifos degradation efficiency under different conditions were as follows:10mg kg-1≈1mg kg-1>50mg kg-1>100mg kg-1,30℃>40℃>20℃,60%WHC (water-holding capacity)>40%WHC>80%WHC, and HZ soil (Hangzhou soil)> JX soil (Jiaxing soil)> XS soil (Xiaoshan soil)> JH soil (Jinhua soil).
(1)利用菌株Bacillus latersporus DSP、E. coli DH5a (pZP201-gfp)及E. coli HB101(pRK2013)的抗性交叉差异行为进行三亲杂交构建荧光毒死蜱降解菌Bacillus latersporus DSP (pDOC-gfp)。通过荧光、降解功能及降解性质粒pDOC-gfp检测证明Bacillus latersporus DSP (pDOC-gfp)构建成功。
(2)以杭州华家池桑树田土壤为材料,采用标记质粒pDOC-gfp投加到毒死蜱污染土壤进行生物修复,并从修复30d后的土壤中分离得到4个发荧光且对毒死蜱有降解能力的单菌,分别命名为ZQ1、ZQ2、ZQ3和ZQ4。通过形态特征、生理生化特性及16S rDNA序列证明这4个菌株属于不同种的细菌,由此说明质粒pDOC-gfp在土壤中向土著微生物发生转移。
(3)利用标记菌株Bacillus latersporus DSP (pDOC-gfp)研究了质粒pDOC强化十壤中毒死蜱降解作用的持久性及其对土壤微生物的影响。结果表明,随着施药频率的增加,降解菌对毒死蜱的生物强化效果长期有效；接种降解菌对土壤微生物群落功能多样性没有显著影响,不具破坏性。
(4)利用标记菌株Bacillus latersporus DSP (pDOC-gfp)采用土柱淋滤的方法研究质粒pDOC在饱和流/非饱和流毒死蜱污染土壤中垂直迁移情况及其对毒死蜱降解。研究发现,质粒pDOC在土壤中可以向下发生迁移；同时质粒供体菌的迁移与质粒转移进入土著菌可以强化土壤中毒死蜱的降解作用。
(5)研究质粒pDOC强化毒死蜱微生物降解的影响因子,结果表明：接种质粒供体菌明显促进了毒死蜱生物降解率,E. coli JM109(pDOC-gfp)是本实验条件中长期修复毒死蜱污染土壤的最佳选择；不同条件下E. coli JM109(pDOC-gfp)对毒死蜱的降解作用顺序为：10mg kg-1≈1mg kg-1>50mg kg-1>100mg kg-130℃>40℃>20℃、60%WHC (田间最大持水量)>40%WHC>80%HZ soil(杭州土壤)>JX soil (嘉兴土壤)>XS soil (萧山土壤)>JH soil(金华土壤)。
Chlorpyrifos as a broad spectrum organophosphorus insecticide is widely used in world due to its high effectiveness, low toxicity to non-target organisms and easy biodegradability and caused great pollution in the environment. The use of chlorpyrifos-degrading bacteria for bioremediation of chlorpyrifos-contaminated sites has been proved to be the most potential clean-up method. In our earlier experiments, a bacterial strain capable of degrading chlorpyrifos, Bacillus laterosporus DSP, was isolated from the soil for the biodegradation and/or detoxification of chlorpyrifos, and the pDOC plasmid in the B. laterosporus DSP strain was found to be responsible for the degradation of chlorpyrifos. In the present study, the development of chlorpyrifos degradation capacity in soil into which a plasmid had been introduced was monitored using the degradation rate and the MPN of the chlorpyrifos degraders as the endpoints of the transfer. The transfer event was monitored using a reporter gene (gfp) combined with microscopic observations and the imaging of GFP expressed in the recipients. The main goal of this study was to develop a plasmid-mediated bioaugmentation method that could result in the persistent capacity for the degradation of chlorpyrifos in the soil. The results were summarized as follows:
(1) The insertion of gfp into the plasmid pDOC was performed by triparental mating in which the helper strain E. coli HB101(pRK2013) was used to mobilize gfp segment from the donor strain E. coli DH5a (pZP201-gfp) into the recipient Bacillus laterosporus DSP. The B. laterosporus DSP strain (pDOC-gfp) was selected based on its resistance to antibiotics, its ability to utilize chlorpyrifos as sole carbon and energy source, and the observation of GFP expression by microscopy.
(2) The soil used in this study was collected from Hangzhou, Zhejiang province, China, and contained no detectable amount of residual chlorpyrifos. To explore the development of the chlorpyrifos degradation capacity in the soil by plasmid transfer, the gfp-tagged plasmid was applied to study the bioremediation in soil. Four of GFP-positive strains, ZQ1, ZQ2, ZQ3, and ZQ4, were isolated from the pDOC-gfp-treated soil30days after treatment, which were capable to chlorpyrifos degradation. The isolates were identified as different species due to their morphological characteristics, physiological and biochemical characteristics and16S rDNA sequences. Moreover, by comparing the relative mobilities of the plasmids, all were found to be identical to that in the initial strain, Bacillus laterosporus DSP, suggesting that new chlorpyrifos degraders were formed by the transfer of pDOC.
(3) To investigate the persistence of bioremediation in chlorpyrifos-polluted soil by plasmid donor strains B. latersporus DSP (pDOC-gfp) and its impact on soil microbial functional diversity, the BIOLOG technique and soil respiration were used to understand microbial community structure and function. The results indicated that the degradation efficiency of chlorpyrifos in soil was always greatly enhanced by inoculation with strain B. latersporus DSP (pDOC-gfp) after repeated application. Soil microbial functional activity and its activity in inoculated soil was not affected and damaged.
(4) By the infiltration test of soil column, the dispersal of plasmid donor strain B. latersporus DSP (pDOC-gfp) in unsaturated and saturated chlorpyrifos contaminated soil was studied. It has been found that chlorpyrifos degraders were throughout the entire length of the column under unsaturated or saturated flow conditions in chlorpyrifos contaminated soil. The enhanced the degradation of chlorpyrifos in soil column may due to the transport of donor strain and its degradative plasmid transfer.
(5) The effect of the plasmid donor, temperature, moisture, and soil type on plasmid transfer was studied. The results indicated that the degradation efficiency of chlorpyrifos in soil was greatly enhanced by inoculation with donor strains. The degradation of chlorpyrifos in the E. coli JM109(pDOC-gfp)-treated soils appeared to be more rapid than the degradation in other donor strain inoculated soils. Therefore, the E. coli JM109(pDOC-gfp) inoculated appeared to be more suitable for bioaugmentation. Strain E. coli JM109(pDOC-gfp) was used to study the effect of various factors, such as temperatures, humidity and soil types, which effect the bioremediation of chlorpyrifos in soil. The results showed that the chlorpyrifos degradation efficiency under different conditions were as follows:10mg kg-1≈1mg kg-1>50mg kg-1>100mg kg-1,30℃>40℃>20℃,60%WHC (water-holding capacity)>40%WHC>80%WHC, and HZ soil (Hangzhou soil)> JX soil (Jiaxing soil)> XS soil (Xiaoshan soil)> JH soil (Jinhua soil).
引文
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