深圳河及沿岸污水处理厂典型雌激素微生物转化研究
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摘要
本论文选取雌激素酮（E1）、雌二醇（E2）、雌三醇（E3）、17α-雌二醇（17α-E2）和雌炔醇（EE2）等5种雌激素，采用气相色谱质谱联用（GC-MS）方法，调查了其在深圳河湾水体及相关污水处理厂中的时空分布和通量变化规律。研究污水厂不同类型活性污泥对雌激素的吸附降解，并采用聚合酶链式反应-变性梯度凝胶电泳（PCR-DGGE）方法剖析雌激素胁迫下微生物群落结构的变化，识别功能微生物种群。模拟研究深圳河水体中雌激素物质在不同溶解氧条件下的衰减转化以及对微生物群落的影响。采用Gaussian03量子化学软件计算和分析雌激素生物转化过程的中间产物和代谢途径。
深圳河湾水体中5种雌激素均有检出，EE2浓度最高，为15.6~443.7ng·L~(-1)，其他4种雌激素浓度低于80ng·L~(-1)。深圳河中游雌激素污染较严重。在沿岸3个污水厂中，EE2在污水厂出水中浓度最高达853.0ng·L~(-1)。二级生物处理工艺对E2、E3和EE2的平均去除率分别约为19.1%、71.7%和81.2%，对E1和17α-E2去除率为负值。雨季，通过上游和中游两座污水厂排入深圳河的雌激素总通量分别是17.7g·d~(-1)和17.8g·d~(-1)，对深圳河相应断面的贡献率分别为25%和12%；旱季，两座污水厂出水雌激素通量分别为43.5g·d~(-1)和69.7g·d~(-1)，相应贡献率上升为39%和52%。活性污泥对雌激素的吸附在30min~1h之内达到平衡，遵循Langmuir和Freundlich模型。活性污泥对天然雌激素E1、E2、E3和17α-E2的生物降解速率分
别约为347μg·L~(-1)·d~(-1)、252μg·L~(-1)·d~(-1)、405μg·L~(-1)·d~(-1)和268μg·L~(-1)·d~(-1)。EE2降解最慢，速率约为14μg·L~(-1)·d~(-1)。在单一雌激素胁迫条件下，E1和E3导致群落多样性增加，而E2、EE2和17α-E2导致群落多样性降低。耐受雌激素长期胁迫的菌群多为γ-变形菌。硝化细菌在EE2和E2转化过程中可能起到重要作用。
深圳河水中溶解氧浓度对雌激素EE2和E2的转化有较大影响。好氧、缺氧和厌氧状态下EE2的平均半衰期分别约为12天、3.5天和20天。E2的半衰期分别为<1天、<1天和3天。缺氧条件下EE2和E2的转化最快。E1和E3是EE2和
E2的转化产物。河水中的主要功能菌群包括常见的污染物降解菌、贫营养环境指示菌以及多种致病菌。
量子化学计算表明，5种雌激素分子中，E1和E3能隙相对较低，反应活性相对较强；E3首先氧化C16位所连羟基，再进一步开环转化；E1分子中C17-C13键级最低，可能在此通过环氧化发生环开裂，生成小分子羧酸等易降解物质。
This thesis focused on the transformation of typical estrogens in Shenzhen River and related wastewater treatment plants (WWTPs). Five steroid estrogens including estrone (E1), estradiol (E2), estriol (E3),17a-estradiol (17a-E2) and ethinylestradiol (EE2) were selected as target chemicals. The distribution and flux shift of estrogens in Shenzhen River and three related sewage treatment plants (STPs) were investigated using gas chromatography-mass spectrometry (GC-MS). Adsorption and biodegradation of estrogens were studied using activated sludge at designed conditions. The microbial community structure under the estrogen stress was analyzed with the method of16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Transformation of estrogens in river water under different dissolved oxygen conditions was explored. The dominant microbial populations were identified in detail. A quantum chemistry software of Gaussian03was applied to identify the intermediates and metabolic pathways of estrogens.
It was found that five selected estrogens including E1, E2, E3,17a-E2and EE2were detected in Shenzhen River water. EE2was measured with concentrations as15.6～443.7ng·L-1, while other four estrogens with concentrations lower than80ng·L-It was showed that midstream was more polluted than upstream and downstream. In the effluents of three wastewater treatment plants (WWTPs), EE2was the predominant estrogen with high concentration reached853.0ng·L-1. Biological treatment processes in WWTPs were effective in removing E2, E3and EE2with mean removals of19.1%,71.7%and81.2%, respectively. However, E1and17α-E2were removed with negative removal ratios.
Estrogen flux contributed by WWTP effluents were one of the main estrogen sources of Shenzhen River. In wet season, average fluxes of total estrogens discharged from two WWTPs were17.7g·d-1and17.8g·d-1, respectively, which took25%and12%proportions of Shenzhen River fluxes. In dry season, the average WWTPs effluent fluxes increased up to43.5g·d-1and69.7g·d-1with39%and52%contribution to Shenzhen River, respectively.
The sorption equilibrium was attained in30min-lh and the biodegradation took relatively longer time. Under aerobic conditions, the average degrading rates of E1, E2, E3and17α-E2were347μg·L-1·d-1,252μg·L-1·d-1,405μg-L-1·d-1and268μg·L-1·d-1, respectively. However, the degradation rate of EE2was as low as14μg·L-1·d-1.16S rDNA analysis showed that E1and E3were able to increase the microbial diversity, while E2,17α-E2and EE2led to diversity decrease, γ-proteobacteria were identified as the dominant populations which were acclimatized under the estrogen stress in the long run. It was found that nitrifying bacteria was likely the significant functional population in EE2and E2transformation.
Dissolved oxygen was a key factor for estrogen transformation in Shenzhen River. It was demonstrated that the average half-life time of EE2was12days under aerobic conditions and relatively long half-life time of20days under anaerobic conditions. However, the EE2half-life time under anoxic conditions was considerably short as3.5days. The average half-lives of E2under aerobic, anoxic and anaerobic conditions were <1day,<1day and3days, respectively. Estrone (E1) and estriol (E3) were detected as the intermediates in EE2and E2transformation. Bacterial communities in overlying water shifted gradually. Dominant populations included common biodegraders and typical oligotrophic microorganisms. There were also some pathogenic bacteria found in Shenzhen River.
Molecular structural parameters including electron cloud, energy gap, dipole moment and bond order were demonstrated to be closely related to the reactivity and transformation properties of estrogens. Calculation results showed that the energy gaps of the frontier electron orbit were higher in E2,17α-E2and EE2than that in E1and E3, which increased the reactivity of E1and E3. It was demonstrated that hydroxyl oxidation on C16atom of E3occurred initially before further transformation. E1may be attacked at C13-C17bond due to the lowest bond order, which led to further epoxidation and cleavage of D ring on E1molecule.
深圳河湾水体中5种雌激素均有检出，EE2浓度最高，为15.6~443.7ng·L~(-1)，其他4种雌激素浓度低于80ng·L~(-1)。深圳河中游雌激素污染较严重。在沿岸3个污水厂中，EE2在污水厂出水中浓度最高达853.0ng·L~(-1)。二级生物处理工艺对E2、E3和EE2的平均去除率分别约为19.1%、71.7%和81.2%，对E1和17α-E2去除率为负值。雨季，通过上游和中游两座污水厂排入深圳河的雌激素总通量分别是17.7g·d~(-1)和17.8g·d~(-1)，对深圳河相应断面的贡献率分别为25%和12%；旱季，两座污水厂出水雌激素通量分别为43.5g·d~(-1)和69.7g·d~(-1)，相应贡献率上升为39%和52%。活性污泥对雌激素的吸附在30min~1h之内达到平衡，遵循Langmuir和Freundlich模型。活性污泥对天然雌激素E1、E2、E3和17α-E2的生物降解速率分
别约为347μg·L~(-1)·d~(-1)、252μg·L~(-1)·d~(-1)、405μg·L~(-1)·d~(-1)和268μg·L~(-1)·d~(-1)。EE2降解最慢，速率约为14μg·L~(-1)·d~(-1)。在单一雌激素胁迫条件下，E1和E3导致群落多样性增加，而E2、EE2和17α-E2导致群落多样性降低。耐受雌激素长期胁迫的菌群多为γ-变形菌。硝化细菌在EE2和E2转化过程中可能起到重要作用。
深圳河水中溶解氧浓度对雌激素EE2和E2的转化有较大影响。好氧、缺氧和厌氧状态下EE2的平均半衰期分别约为12天、3.5天和20天。E2的半衰期分别为<1天、<1天和3天。缺氧条件下EE2和E2的转化最快。E1和E3是EE2和
E2的转化产物。河水中的主要功能菌群包括常见的污染物降解菌、贫营养环境指示菌以及多种致病菌。
量子化学计算表明，5种雌激素分子中，E1和E3能隙相对较低，反应活性相对较强；E3首先氧化C16位所连羟基，再进一步开环转化；E1分子中C17-C13键级最低，可能在此通过环氧化发生环开裂，生成小分子羧酸等易降解物质。
This thesis focused on the transformation of typical estrogens in Shenzhen River and related wastewater treatment plants (WWTPs). Five steroid estrogens including estrone (E1), estradiol (E2), estriol (E3),17a-estradiol (17a-E2) and ethinylestradiol (EE2) were selected as target chemicals. The distribution and flux shift of estrogens in Shenzhen River and three related sewage treatment plants (STPs) were investigated using gas chromatography-mass spectrometry (GC-MS). Adsorption and biodegradation of estrogens were studied using activated sludge at designed conditions. The microbial community structure under the estrogen stress was analyzed with the method of16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Transformation of estrogens in river water under different dissolved oxygen conditions was explored. The dominant microbial populations were identified in detail. A quantum chemistry software of Gaussian03was applied to identify the intermediates and metabolic pathways of estrogens.
It was found that five selected estrogens including E1, E2, E3,17a-E2and EE2were detected in Shenzhen River water. EE2was measured with concentrations as15.6～443.7ng·L-1, while other four estrogens with concentrations lower than80ng·L-It was showed that midstream was more polluted than upstream and downstream. In the effluents of three wastewater treatment plants (WWTPs), EE2was the predominant estrogen with high concentration reached853.0ng·L-1. Biological treatment processes in WWTPs were effective in removing E2, E3and EE2with mean removals of19.1%,71.7%and81.2%, respectively. However, E1and17α-E2were removed with negative removal ratios.
Estrogen flux contributed by WWTP effluents were one of the main estrogen sources of Shenzhen River. In wet season, average fluxes of total estrogens discharged from two WWTPs were17.7g·d-1and17.8g·d-1, respectively, which took25%and12%proportions of Shenzhen River fluxes. In dry season, the average WWTPs effluent fluxes increased up to43.5g·d-1and69.7g·d-1with39%and52%contribution to Shenzhen River, respectively.
The sorption equilibrium was attained in30min-lh and the biodegradation took relatively longer time. Under aerobic conditions, the average degrading rates of E1, E2, E3and17α-E2were347μg·L-1·d-1,252μg·L-1·d-1,405μg-L-1·d-1and268μg·L-1·d-1, respectively. However, the degradation rate of EE2was as low as14μg·L-1·d-1.16S rDNA analysis showed that E1and E3were able to increase the microbial diversity, while E2,17α-E2and EE2led to diversity decrease, γ-proteobacteria were identified as the dominant populations which were acclimatized under the estrogen stress in the long run. It was found that nitrifying bacteria was likely the significant functional population in EE2and E2transformation.
Dissolved oxygen was a key factor for estrogen transformation in Shenzhen River. It was demonstrated that the average half-life time of EE2was12days under aerobic conditions and relatively long half-life time of20days under anaerobic conditions. However, the EE2half-life time under anoxic conditions was considerably short as3.5days. The average half-lives of E2under aerobic, anoxic and anaerobic conditions were <1day,<1day and3days, respectively. Estrone (E1) and estriol (E3) were detected as the intermediates in EE2and E2transformation. Bacterial communities in overlying water shifted gradually. Dominant populations included common biodegraders and typical oligotrophic microorganisms. There were also some pathogenic bacteria found in Shenzhen River.
Molecular structural parameters including electron cloud, energy gap, dipole moment and bond order were demonstrated to be closely related to the reactivity and transformation properties of estrogens. Calculation results showed that the energy gaps of the frontier electron orbit were higher in E2,17α-E2and EE2than that in E1and E3, which increased the reactivity of E1and E3. It was demonstrated that hydroxyl oxidation on C16atom of E3occurred initially before further transformation. E1may be attacked at C13-C17bond due to the lowest bond order, which led to further epoxidation and cleavage of D ring on E1molecule.
引文
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