鼠李糖脂作用下17α-炔雌醇在水/底泥中的迁移转化规律及生物有效性
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摘要
近年来,药品及个人护理品(PPCPs)作为一类新型污染物,在水体环境中广泛检出。PPCPs中多数物质具有半疏水特性,易吸附沉积于底泥、土壤等固相介质上而长期滞留于环境中。针对疏水毒害有机污染物,生物表面活性剂代替化学合成表面活性剂应用于环境污染物的受控迁移转化和生物强化降解,是现代生物修复技术的重要部分。
鼠李糖脂是研究最为广泛的生物表面活性剂之一,是由微生物分泌的多种同系物组成的混合物。基于鼠李糖脂的分子结构多样性和可选择培养性,目前罕见有关于鼠李糖脂同系物在环境修复各环节中作用效应及差异的研究报道。此外,针对半疏水性PPCPs物质与PAHs等完全疏水物质分子结构特征及环境行为的差异,有关鼠李糖脂对半疏水性物质作用特性及规律的研究亦尚未见到。为此,本研究选择环境雌激素效力最强的人工合成17α-炔雌醇(EE2)为特征PPCPs污染物,在鼠李糖脂同系物组分分离提纯及分子结构、性质表征的基础上,以不同环境介质条件为实验体系,深入系统地探索了鼠李糖脂同系物组分对EE2的胶束增溶、吸附/解吸、及对其生物有效性的影响等作用特性及机制。最后通过易混合置换实验,探讨了鼠李糖脂作用下EE2在模拟饱和底泥柱中的迁移转化规律,为推动基于鼠李糖脂生物表面活性剂的特征PPCPs受控定向迁移和生物强化修复技术的应用奠定了理论基础,丰富了表面活性剂生物修复技术的研究内容。得到的主要研究结果如下:
(1)对诱变菌Pseudomonas aeruginosa MIG-N146的鼠李糖脂发酵产物进行粗提和分离纯化,得到了单鼠李糖脂和双鼠李糖脂同系物组分,分别命名为RL-F1和RL-F2。RL-F1和RL-F2在水溶液中的CMC分别为0.11和0.07 mM。DLS和TEM测试表明,RL-F1聚集胶束粒径的尺寸较小,呈单分散模式。而RL-F2胶束粒径相对较大,分布较广,呈多分散模式,且RL-F2聚集胶束较易随其本身浓度的增加发生形变。
(2)增溶平衡和增溶动力学实验研究表明,与RL-F1相比,RL-F2对EE2有着较大的胶束增溶能力,但EE2向RL-F2准胶束相传质的速率相对较低。溶质增溶后,RL-F2胶束/EE2复合结构的颗粒粒径尺寸及可分散性增加的幅度较RL-F1时大。半疏水性溶质EE2与鼠李糖脂聚集胶束的结合不是单一的作用模式,EE2同时占据在鼠李糖脂胶束中具有中等极性微环境的核-壳内界面处和疏水碳链内核部位。而RL-F2胶束与EE2分子之间的作用更趋于疏水相互作用结合。根据聚集胶束核-壳结构/增溶溶质的复合理论模型分析证明,鼠李糖脂同系物的聚集体类型及其微观形态是影响鼠李糖脂增溶性能的重要因素。
(3)对新鲜配制EE2/底泥的吸附实验体系,RL-F2的存在能有效抑制EE2在底泥相中的吸附。与之相反,RL-F1在较宽的浓度范围内,对EE2的吸附均表现出一定的促进作用。对陈化EE2/底泥的脱附实验体系,两种鼠李糖脂同系物对EE2均有一定的脱附效能。而整体上,RL-F2对EE2的脱附效率均大于RL-F1,这与RL-F2胶束对EE2较高的增溶分配能力是一致的。采用兼顾吸附态表面活性剂和水相胶束竞争作用的HOC分配模型模拟证明,有机污染物-表面活性剂-底泥的复杂体系,鼠李糖脂同系物类型是影响HOC分配的最关键因素。
(4)摇瓶生物降解实验研究表明,鼠李糖脂的投加不会改变水/底泥体系微生物对EE2的生物利用转化过程。RL-F1对EE2生物降解的强化程度远远大于RL-F2。将实际测试的降解速率增效因子与理论值进行对比,可以推断,粗糖脂混合物对有机物生物有效性的作用效应并非简单的同系物之间作用贡献的加和,混合产物体系各组分的作用特性受到抑制。鼠李糖脂同系物组分对EE2的生物强化降解效应与其增溶脱附效应不呈正比例关系。鼠李糖脂作用下微生物摄取有机物的途径有多种,而有机物增溶后鼠李糖脂胶束的生物有效性对有机物的微生物降解起主要作用。
(5)模拟底泥柱的易混合置换实验表明,高孔隙水流速条件下,RL-F2作用体系EE2的淋溶穿透率较高,约达83.3%(此时生物转化部分仅占13.6%),这使得EE2在底泥柱中的最终残留显著降低。双鼠李糖脂对EE2在模拟底泥柱中的传输有较好的增溶洗脱作用。而低孔隙水流速条件下,RL-F1的存在大大促进了EE2在底泥柱中吸附滞留,此时大部分滞留溶质可以通过生物强化降解的途径而被有效去除,EE2的生物降解转化率大大增加(75.3%)。
综上所述,不同环境介质条件下,对不同的作用环节,鼠李糖脂同系物对有机物的作用效应及贡献表现出一定的特异性和非一致性。建议在实际应用中,应针对修复目的、鼠李糖脂产物组成及其在不同环节的作用特性等进行区别、综合的考虑。单鼠李糖脂表现出对EE2较强的吸附固定作用,且能较大程度上提高有机物的生物有效性,这为复杂环境体系有机污染物的修复提供了一种基于单鼠李糖脂的有机物固定-生物强化降解相结合的技术手段。
Recently, as emerging contaminants, pharmaceuticals and personal care products (PPCPs) were widely detected in the aqueous environment. Most of PPCPs have moderate hydrophobic properties. They are tended to adsorb and deposit onto sediments and soils. It thus leads them long-term persistent. Biosurfactants, taking place of chemical synthetic surfactants, are usually applied to change migration and transformation of the hydrophobic toxic contaminants and/or enhance their biodegradation of them, which are important parts of the modern bioremediation technology.
Rhamnolipids are among the best known biosurfactants and are usually produced as multicomponent mixtures of homologues. However, the predominance of individual rhamnolipidic homologue in each bioremediation part, and the relationship with their molecular components for such biosurfactant mixtures had not been clearly understood. Because of the moderate hydrophobic property, the environmental chemical behaviors of PPCPs are differentiated from that of the complete hydrophobic hydrocarbons. It thus may lead to different functional performances for rhamnolipids. Accordingly, in this study, molecular structures and micellization properties of the purified rhamnolipidic homologue fractions were characterized. The most estrogenic synthetic 17α-ethinylestradiol (EE2) was chose as typical PPCPs contaminant. Based on these, the rhamnolipidic functioned performances for EE2 including micellar solubilization, sorption and desorption, and enhanced biodegradation were systematically investigated step by step within different environmental medium systems. Finally, EE2 migration and transformation mechanisms in modeled sediment column affected by rhamnolipids were explored by mixable displacement experiment. Research results were as follows:
(1) Two representative rhamnolipidic fractions, RL-F1 and RL-F2, were seperated and purified from the secretions of Pseudomonas aeruginosa mutant strain MIC-N146. They were characterized as mono- and di-rhamnlipids through HPLC-MS and FTIR analyses. The critical micelle concentration of RL-F1 and RL-F2 were 0.11 and 0.07 mM, respectively. DLS and TEM measurements indicated that, the micelle size of RL-F2 was relatively bigger than RL-F1. Size distribution for RL-F2 micelles showed multi-dispersity, while monodisperse mode was observed for RL-F1. It also found that aggregate shape transition was tended to accompany with increase of the RL-F2 bulk concentration.
(2) Solubilization equilibrium and solubilization kenitics experiments were carried out as batch tests. RL-F2 had a higher maximum solubilizaiton capability of EE2, but observed smaller mass transfer rate than RL-F1. Organic solubilization brought about size increase of the micelles. It was found that the increase of micelle size and size dispersity for RL-F2 aggregates were relatively larger after EE2 solubilzation. Combination of the moderate hydrophobic EE2 and rhamnolipidic micelle were not caused by a single interaction mode. Once solubilization, EE2 molecules located in the core-shell interface having moderate polar microevironment, as well as the hydrophobic core part. According to model analysis of the micelle/solubilizate composite structure, the type of rhamnolipid and aggregated geometry has a great impact on the incorporation of solubilizate into surfactant aggregates.
(3) EE2 distribution within sediment-water sorption and desorption systems effected by rhamnolipids were investigated and compared. When freshly-treated sorbate was used, in general, RL-F2 was observed to inhibit EE2 desorption from sedment. In contrast, the coexistence of RL-F1 produced considerable net enhancement for EE2 sorption. When aged sorbate was used, both rhamnolipids could facilitate mobilization of EE2. But the desorption efficiency functioned by RL-F2 was generally larger than that of RL-F1. It was consistent with its higher solubilization capacity for EE2. Experimental data was further estimated by the conceptual model that considered the sorbed rhamnolipids and aqueous micelles for organic partitioning. It demonstrated strongly rhamnolipid type being a most important influencing factor towards HOC distribution.
(4) The effect of rhamnolipids on the bioavailability of EE2 in sediment/water system was tested. Addition of rhamnolipids was observed to not change the process of microbial transformation of EE2. The enhancement extend of EE2 biodegradation functioned by RL-F1 was much larger than that by RL-F2. After comparing the actual degradation-rate enhancing coefficients with the theoretical ones, it could be inferred that the crude rhamnolipids functioned effect for HOC’s bioavailability is not a simple addition action process played by each homologue fraction. The functional effect of individual component will be suppressed within a mixed system. The contribution of rhamnolipid homologue to EE2 biodegradation enhancement was not positively proportional to its corresponding solubilization and mobilization efficacy. When in the present of rhamnolipid, there were diverse paths for HOC uptake by microorganisms. The total bioavailability of the rhamnolipid micelle after HOC solubilizing played a major role for biodegradation.
(5) When with higher pore water velocity, RL-F2 functioned EE2 breakthrough rate was about 83.3%, while biotransformation part covered only 13.6%. It led to a marked decrease in the final residual rate. When the pore water velocity was lower, EE2 was largely retained within the sediment column due to enhanced sorption. Most of the sorbed EE2 went through biodegradation process. The biodegradation rate was enhanced as high as 75.3%. It also led to an efficient removal of HOC.
Taken together, rhamnolipid-homologue functioned performance present high specificity and inconsistency when within different environmental medium and effect process. In actual applications, one should carry out an overall consideration in terms of the remediation target, biosurfactant homologue composition and each homologue performance. Monorhamnolipid showed good sorption enhancement of EE2. It also could increase the EE2 bioavailability in a large extend. This provides an important technique for HOC removal within complex environment solid system, i.e. a combined technique based on monorhamnolipid functioned HOC immobilization and enhanced biodegradation. The results of this study provide useful information for controlled transport of PPCPs and bioremediation enhancement technology. It also enriches the research content for bioremediation technology.
鼠李糖脂是研究最为广泛的生物表面活性剂之一,是由微生物分泌的多种同系物组成的混合物。基于鼠李糖脂的分子结构多样性和可选择培养性,目前罕见有关于鼠李糖脂同系物在环境修复各环节中作用效应及差异的研究报道。此外,针对半疏水性PPCPs物质与PAHs等完全疏水物质分子结构特征及环境行为的差异,有关鼠李糖脂对半疏水性物质作用特性及规律的研究亦尚未见到。为此,本研究选择环境雌激素效力最强的人工合成17α-炔雌醇(EE2)为特征PPCPs污染物,在鼠李糖脂同系物组分分离提纯及分子结构、性质表征的基础上,以不同环境介质条件为实验体系,深入系统地探索了鼠李糖脂同系物组分对EE2的胶束增溶、吸附/解吸、及对其生物有效性的影响等作用特性及机制。最后通过易混合置换实验,探讨了鼠李糖脂作用下EE2在模拟饱和底泥柱中的迁移转化规律,为推动基于鼠李糖脂生物表面活性剂的特征PPCPs受控定向迁移和生物强化修复技术的应用奠定了理论基础,丰富了表面活性剂生物修复技术的研究内容。得到的主要研究结果如下:
(1)对诱变菌Pseudomonas aeruginosa MIG-N146的鼠李糖脂发酵产物进行粗提和分离纯化,得到了单鼠李糖脂和双鼠李糖脂同系物组分,分别命名为RL-F1和RL-F2。RL-F1和RL-F2在水溶液中的CMC分别为0.11和0.07 mM。DLS和TEM测试表明,RL-F1聚集胶束粒径的尺寸较小,呈单分散模式。而RL-F2胶束粒径相对较大,分布较广,呈多分散模式,且RL-F2聚集胶束较易随其本身浓度的增加发生形变。
(2)增溶平衡和增溶动力学实验研究表明,与RL-F1相比,RL-F2对EE2有着较大的胶束增溶能力,但EE2向RL-F2准胶束相传质的速率相对较低。溶质增溶后,RL-F2胶束/EE2复合结构的颗粒粒径尺寸及可分散性增加的幅度较RL-F1时大。半疏水性溶质EE2与鼠李糖脂聚集胶束的结合不是单一的作用模式,EE2同时占据在鼠李糖脂胶束中具有中等极性微环境的核-壳内界面处和疏水碳链内核部位。而RL-F2胶束与EE2分子之间的作用更趋于疏水相互作用结合。根据聚集胶束核-壳结构/增溶溶质的复合理论模型分析证明,鼠李糖脂同系物的聚集体类型及其微观形态是影响鼠李糖脂增溶性能的重要因素。
(3)对新鲜配制EE2/底泥的吸附实验体系,RL-F2的存在能有效抑制EE2在底泥相中的吸附。与之相反,RL-F1在较宽的浓度范围内,对EE2的吸附均表现出一定的促进作用。对陈化EE2/底泥的脱附实验体系,两种鼠李糖脂同系物对EE2均有一定的脱附效能。而整体上,RL-F2对EE2的脱附效率均大于RL-F1,这与RL-F2胶束对EE2较高的增溶分配能力是一致的。采用兼顾吸附态表面活性剂和水相胶束竞争作用的HOC分配模型模拟证明,有机污染物-表面活性剂-底泥的复杂体系,鼠李糖脂同系物类型是影响HOC分配的最关键因素。
(4)摇瓶生物降解实验研究表明,鼠李糖脂的投加不会改变水/底泥体系微生物对EE2的生物利用转化过程。RL-F1对EE2生物降解的强化程度远远大于RL-F2。将实际测试的降解速率增效因子与理论值进行对比,可以推断,粗糖脂混合物对有机物生物有效性的作用效应并非简单的同系物之间作用贡献的加和,混合产物体系各组分的作用特性受到抑制。鼠李糖脂同系物组分对EE2的生物强化降解效应与其增溶脱附效应不呈正比例关系。鼠李糖脂作用下微生物摄取有机物的途径有多种,而有机物增溶后鼠李糖脂胶束的生物有效性对有机物的微生物降解起主要作用。
(5)模拟底泥柱的易混合置换实验表明,高孔隙水流速条件下,RL-F2作用体系EE2的淋溶穿透率较高,约达83.3%(此时生物转化部分仅占13.6%),这使得EE2在底泥柱中的最终残留显著降低。双鼠李糖脂对EE2在模拟底泥柱中的传输有较好的增溶洗脱作用。而低孔隙水流速条件下,RL-F1的存在大大促进了EE2在底泥柱中吸附滞留,此时大部分滞留溶质可以通过生物强化降解的途径而被有效去除,EE2的生物降解转化率大大增加(75.3%)。
综上所述,不同环境介质条件下,对不同的作用环节,鼠李糖脂同系物对有机物的作用效应及贡献表现出一定的特异性和非一致性。建议在实际应用中,应针对修复目的、鼠李糖脂产物组成及其在不同环节的作用特性等进行区别、综合的考虑。单鼠李糖脂表现出对EE2较强的吸附固定作用,且能较大程度上提高有机物的生物有效性,这为复杂环境体系有机污染物的修复提供了一种基于单鼠李糖脂的有机物固定-生物强化降解相结合的技术手段。
Recently, as emerging contaminants, pharmaceuticals and personal care products (PPCPs) were widely detected in the aqueous environment. Most of PPCPs have moderate hydrophobic properties. They are tended to adsorb and deposit onto sediments and soils. It thus leads them long-term persistent. Biosurfactants, taking place of chemical synthetic surfactants, are usually applied to change migration and transformation of the hydrophobic toxic contaminants and/or enhance their biodegradation of them, which are important parts of the modern bioremediation technology.
Rhamnolipids are among the best known biosurfactants and are usually produced as multicomponent mixtures of homologues. However, the predominance of individual rhamnolipidic homologue in each bioremediation part, and the relationship with their molecular components for such biosurfactant mixtures had not been clearly understood. Because of the moderate hydrophobic property, the environmental chemical behaviors of PPCPs are differentiated from that of the complete hydrophobic hydrocarbons. It thus may lead to different functional performances for rhamnolipids. Accordingly, in this study, molecular structures and micellization properties of the purified rhamnolipidic homologue fractions were characterized. The most estrogenic synthetic 17α-ethinylestradiol (EE2) was chose as typical PPCPs contaminant. Based on these, the rhamnolipidic functioned performances for EE2 including micellar solubilization, sorption and desorption, and enhanced biodegradation were systematically investigated step by step within different environmental medium systems. Finally, EE2 migration and transformation mechanisms in modeled sediment column affected by rhamnolipids were explored by mixable displacement experiment. Research results were as follows:
(1) Two representative rhamnolipidic fractions, RL-F1 and RL-F2, were seperated and purified from the secretions of Pseudomonas aeruginosa mutant strain MIC-N146. They were characterized as mono- and di-rhamnlipids through HPLC-MS and FTIR analyses. The critical micelle concentration of RL-F1 and RL-F2 were 0.11 and 0.07 mM, respectively. DLS and TEM measurements indicated that, the micelle size of RL-F2 was relatively bigger than RL-F1. Size distribution for RL-F2 micelles showed multi-dispersity, while monodisperse mode was observed for RL-F1. It also found that aggregate shape transition was tended to accompany with increase of the RL-F2 bulk concentration.
(2) Solubilization equilibrium and solubilization kenitics experiments were carried out as batch tests. RL-F2 had a higher maximum solubilizaiton capability of EE2, but observed smaller mass transfer rate than RL-F1. Organic solubilization brought about size increase of the micelles. It was found that the increase of micelle size and size dispersity for RL-F2 aggregates were relatively larger after EE2 solubilzation. Combination of the moderate hydrophobic EE2 and rhamnolipidic micelle were not caused by a single interaction mode. Once solubilization, EE2 molecules located in the core-shell interface having moderate polar microevironment, as well as the hydrophobic core part. According to model analysis of the micelle/solubilizate composite structure, the type of rhamnolipid and aggregated geometry has a great impact on the incorporation of solubilizate into surfactant aggregates.
(3) EE2 distribution within sediment-water sorption and desorption systems effected by rhamnolipids were investigated and compared. When freshly-treated sorbate was used, in general, RL-F2 was observed to inhibit EE2 desorption from sedment. In contrast, the coexistence of RL-F1 produced considerable net enhancement for EE2 sorption. When aged sorbate was used, both rhamnolipids could facilitate mobilization of EE2. But the desorption efficiency functioned by RL-F2 was generally larger than that of RL-F1. It was consistent with its higher solubilization capacity for EE2. Experimental data was further estimated by the conceptual model that considered the sorbed rhamnolipids and aqueous micelles for organic partitioning. It demonstrated strongly rhamnolipid type being a most important influencing factor towards HOC distribution.
(4) The effect of rhamnolipids on the bioavailability of EE2 in sediment/water system was tested. Addition of rhamnolipids was observed to not change the process of microbial transformation of EE2. The enhancement extend of EE2 biodegradation functioned by RL-F1 was much larger than that by RL-F2. After comparing the actual degradation-rate enhancing coefficients with the theoretical ones, it could be inferred that the crude rhamnolipids functioned effect for HOC’s bioavailability is not a simple addition action process played by each homologue fraction. The functional effect of individual component will be suppressed within a mixed system. The contribution of rhamnolipid homologue to EE2 biodegradation enhancement was not positively proportional to its corresponding solubilization and mobilization efficacy. When in the present of rhamnolipid, there were diverse paths for HOC uptake by microorganisms. The total bioavailability of the rhamnolipid micelle after HOC solubilizing played a major role for biodegradation.
(5) When with higher pore water velocity, RL-F2 functioned EE2 breakthrough rate was about 83.3%, while biotransformation part covered only 13.6%. It led to a marked decrease in the final residual rate. When the pore water velocity was lower, EE2 was largely retained within the sediment column due to enhanced sorption. Most of the sorbed EE2 went through biodegradation process. The biodegradation rate was enhanced as high as 75.3%. It also led to an efficient removal of HOC.
Taken together, rhamnolipid-homologue functioned performance present high specificity and inconsistency when within different environmental medium and effect process. In actual applications, one should carry out an overall consideration in terms of the remediation target, biosurfactant homologue composition and each homologue performance. Monorhamnolipid showed good sorption enhancement of EE2. It also could increase the EE2 bioavailability in a large extend. This provides an important technique for HOC removal within complex environment solid system, i.e. a combined technique based on monorhamnolipid functioned HOC immobilization and enhanced biodegradation. The results of this study provide useful information for controlled transport of PPCPs and bioremediation enhancement technology. It also enriches the research content for bioremediation technology.
引文
[1] Zhou W. Zhu L. Enhanced desorption of phenanthrene from contaminated soil using anionic/ nonionic mixed surfactant. Environmental Pollution. 2007. 147: 350-357
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