土壤酞酸酯污染的微生态效应和真菌—植物联合修复技术研究
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摘要
本文以3种国控酞酸酯为研究对象,研究了酞酸酯复合污染的微生态效应,探讨了真菌、植物联合修复酞酸酯复合污染土壤的可行性。研究方法及结果如下：
(1)在实验室模拟条件下,通过测定土壤基础呼吸、PAEs含量、RAPD-PCR条带数,研究了邻苯二甲酸二甲酯(DMP)、邻苯二甲酸二乙酯(DEP)和邻苯二甲酸二辛酯(DOP)3种PAEs复合污染对农田土壤基础呼吸、微生物多样性的影响。结果表明：PAEs污染初期的土壤基础呼吸被激活,但这种激活作用随着培养时间的延长而减弱,400 mg/kg组土壤基础呼吸最高。土著微生物对PAEs有一定的降解能力,DEP降解最快,其次为DMP,不同浓度的PAEs对微生物的降解能力有影响。不同处理组土壤微生物群落DNA序列Shannon-Weaver指数顺序为0 mg/kg>50 mg/kg>100 mg/kg>200 mg/kg>400 mg/kg。可见,PAEs复合污染提高了土壤基础呼吸,但降低了土壤微生物多样性。
(2)采用3种PAEs复合物梯度驯化,从PAEs污染的农田土壤中筛选出土著降解真菌；通过测定PAEs含量,研究其最佳降解条件和在液体培养基、灭菌土壤中的降解效果。结果表明：筛选出的真菌F2为尖孢镰刀菌(Fusarium oxysporm), F3为棒束梗霉属(Isaria sp.)。正交试验筛选出最佳试验条件为C：N 20：1和pH 7.0,同时,降解菌在pH 5.0-7.0范围内均可较好的存活。F3可在7天内将液体培养基中300 mg/L PAEs降解66.20%；30天内将灭菌土壤中300 mg/kg PAEs降解69.02%。可见,所选菌株对PAEs复合污染物有较好的降解效果。
(3)采用3种PAEs复合污染土壤接种真菌后种植不同根型植物,通过测定植物株高、干重,土壤和植物PAEs含量和土壤DGGE,研究了PAEs对植物生长的影响和土壤、植物中PAEs含量的变化,探讨了建立真菌-植物联合修复模式的可行性。结果表明：PAEs对番茄、大豆的生长均有抑制作用,且对番茄的抑制作用高于大豆,但对香根草生长无影响。添加PAEs、种植植物和接种真菌使土壤基础呼吸和真菌群落发生变化。从DGGE图谱无法判断F3是否存在。土壤PAEs降解是降解菌、土著微生物和植物协同作用的结果。接种F3后可使盆栽土壤中PAEs含量较未接种组平均下降20.67%,植物中PAEs含量平均下降28.86%。可见,接种F3后提高了土壤中复合PAEs的降解效率,同时也降低了PAEs在植物内的积累。最后,综合试验结果提出了三种PAEs污染农田土壤的修复模式。
The impacts of combined phthalate acid ester (PAE) contamination, including dimethyl phthalate (DMP), diethyl phthalate (DEP), and dioctyl phthalate (DOP), on soil microbial diversity were studied. The fungi-plant mode of PAEs bioremediation was also discussed. Research methods and results are as follows:
(1) The impacts of combined PAEs contamination, including DMP, DEP and DOP, on farmland soil basal respiration and microbial diversity were studied by monitoring soil respiration, soil PAEs concentration and random amplified polymorphic DNA (RAPD) marker under laboratory simulation testing. The results showed that the soil basal respiration was activated by PAEs at an early stage, reaching a maximum value at 400 mg/kg PAEs treatment. However, this type of activation effect decreased with prolonged PAEs exposure time. In addition, the concentration of PAEs in the soil decreased, especially the DEP concentration. The Shannon-Weaver index of soil microbial community DNA sequence in all treatments was:0 mg/kg> 50 mg/kg> 100 mg/kg> 200 mg/kg> 400 mg/kg. Overall, the results indicated that the combined PAEs contamination in soil may stimulate soil basal respiration, but decrease soil microbial diversity.
(2) Two PAEs degradation strains F2 and F3 were isolated from PAEs contaminated farmland soils, and DMP, DEP and DOP could be degraded at the same time by those two strains. The strains were identified as Fusarium oxysporm and Isaria sp. respectively. The optimum C:N of those strains was 20:1, and optimum pH was 7.0. The pH 5.0-7.0 was available for their survival. The biodegradation of PAEs in liquid and soil were studied by monitoring PAEs concentration and mycelium weight. The results showed that the concentration of PAEs in liquid culture medium decreased 66.20% in 7days (initial concentrations 300 mg/kg). The concentration of PAEs in the soils decreased 69.02% in 30days (initial concentrations 300 mg/kg). It is clearly that, those two strains were qualified for the biodegrading of combined PAEs contamination.
(3) The degradation of PAEs in farmland soil was tested by monitoring plant biomass and height, PAEs concentration and DGGE. The fungi-plant mode of PAEs bioremediation was also discussed. The results showed that the growth of tomato and soybean was inhibited by PAEs, but no effect on vetiver. The soil basal respiration and fungi community were influence by adding PAEs, plants and inoculate fungi. Bands of F3 were no found in DGGE maps at the end of experiment in F3 inoculate soil. Synergistic effect on PAEs degradation was found between F3, indigenous microorganisms and plants. Compare of non-inoculate treatments, the average concentration of PAEs in pot soil was decreased 20.67% in inoculate treatments, and the average concentration of PAEs in plants was decreased 28.86% in inoculate treatments. It is clearly that, inoculate F3 enhance the biodegradation of PAEs in soil, and decline the accumulation of PAEs in plants. Three types of PAEs bioremediation mode were suggested.
(1)在实验室模拟条件下,通过测定土壤基础呼吸、PAEs含量、RAPD-PCR条带数,研究了邻苯二甲酸二甲酯(DMP)、邻苯二甲酸二乙酯(DEP)和邻苯二甲酸二辛酯(DOP)3种PAEs复合污染对农田土壤基础呼吸、微生物多样性的影响。结果表明：PAEs污染初期的土壤基础呼吸被激活,但这种激活作用随着培养时间的延长而减弱,400 mg/kg组土壤基础呼吸最高。土著微生物对PAEs有一定的降解能力,DEP降解最快,其次为DMP,不同浓度的PAEs对微生物的降解能力有影响。不同处理组土壤微生物群落DNA序列Shannon-Weaver指数顺序为0 mg/kg>50 mg/kg>100 mg/kg>200 mg/kg>400 mg/kg。可见,PAEs复合污染提高了土壤基础呼吸,但降低了土壤微生物多样性。
(2)采用3种PAEs复合物梯度驯化,从PAEs污染的农田土壤中筛选出土著降解真菌；通过测定PAEs含量,研究其最佳降解条件和在液体培养基、灭菌土壤中的降解效果。结果表明：筛选出的真菌F2为尖孢镰刀菌(Fusarium oxysporm), F3为棒束梗霉属(Isaria sp.)。正交试验筛选出最佳试验条件为C：N 20：1和pH 7.0,同时,降解菌在pH 5.0-7.0范围内均可较好的存活。F3可在7天内将液体培养基中300 mg/L PAEs降解66.20%；30天内将灭菌土壤中300 mg/kg PAEs降解69.02%。可见,所选菌株对PAEs复合污染物有较好的降解效果。
(3)采用3种PAEs复合污染土壤接种真菌后种植不同根型植物,通过测定植物株高、干重,土壤和植物PAEs含量和土壤DGGE,研究了PAEs对植物生长的影响和土壤、植物中PAEs含量的变化,探讨了建立真菌-植物联合修复模式的可行性。结果表明：PAEs对番茄、大豆的生长均有抑制作用,且对番茄的抑制作用高于大豆,但对香根草生长无影响。添加PAEs、种植植物和接种真菌使土壤基础呼吸和真菌群落发生变化。从DGGE图谱无法判断F3是否存在。土壤PAEs降解是降解菌、土著微生物和植物协同作用的结果。接种F3后可使盆栽土壤中PAEs含量较未接种组平均下降20.67%,植物中PAEs含量平均下降28.86%。可见,接种F3后提高了土壤中复合PAEs的降解效率,同时也降低了PAEs在植物内的积累。最后,综合试验结果提出了三种PAEs污染农田土壤的修复模式。
The impacts of combined phthalate acid ester (PAE) contamination, including dimethyl phthalate (DMP), diethyl phthalate (DEP), and dioctyl phthalate (DOP), on soil microbial diversity were studied. The fungi-plant mode of PAEs bioremediation was also discussed. Research methods and results are as follows:
(1) The impacts of combined PAEs contamination, including DMP, DEP and DOP, on farmland soil basal respiration and microbial diversity were studied by monitoring soil respiration, soil PAEs concentration and random amplified polymorphic DNA (RAPD) marker under laboratory simulation testing. The results showed that the soil basal respiration was activated by PAEs at an early stage, reaching a maximum value at 400 mg/kg PAEs treatment. However, this type of activation effect decreased with prolonged PAEs exposure time. In addition, the concentration of PAEs in the soil decreased, especially the DEP concentration. The Shannon-Weaver index of soil microbial community DNA sequence in all treatments was:0 mg/kg> 50 mg/kg> 100 mg/kg> 200 mg/kg> 400 mg/kg. Overall, the results indicated that the combined PAEs contamination in soil may stimulate soil basal respiration, but decrease soil microbial diversity.
(2) Two PAEs degradation strains F2 and F3 were isolated from PAEs contaminated farmland soils, and DMP, DEP and DOP could be degraded at the same time by those two strains. The strains were identified as Fusarium oxysporm and Isaria sp. respectively. The optimum C:N of those strains was 20:1, and optimum pH was 7.0. The pH 5.0-7.0 was available for their survival. The biodegradation of PAEs in liquid and soil were studied by monitoring PAEs concentration and mycelium weight. The results showed that the concentration of PAEs in liquid culture medium decreased 66.20% in 7days (initial concentrations 300 mg/kg). The concentration of PAEs in the soils decreased 69.02% in 30days (initial concentrations 300 mg/kg). It is clearly that, those two strains were qualified for the biodegrading of combined PAEs contamination.
(3) The degradation of PAEs in farmland soil was tested by monitoring plant biomass and height, PAEs concentration and DGGE. The fungi-plant mode of PAEs bioremediation was also discussed. The results showed that the growth of tomato and soybean was inhibited by PAEs, but no effect on vetiver. The soil basal respiration and fungi community were influence by adding PAEs, plants and inoculate fungi. Bands of F3 were no found in DGGE maps at the end of experiment in F3 inoculate soil. Synergistic effect on PAEs degradation was found between F3, indigenous microorganisms and plants. Compare of non-inoculate treatments, the average concentration of PAEs in pot soil was decreased 20.67% in inoculate treatments, and the average concentration of PAEs in plants was decreased 28.86% in inoculate treatments. It is clearly that, inoculate F3 enhance the biodegradation of PAEs in soil, and decline the accumulation of PAEs in plants. Three types of PAEs bioremediation mode were suggested.
引文
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