环境中微囊藻毒素的检测、提纯及其紫外光助催化降解的研究
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摘要
随着水体富营养化的日益加剧,蓝藻水华爆发带来的微囊藻毒素污染成为一个全球关注的环境问题。微囊藻毒素MCs是由蓝藻所产生的一种具有强烈致癌作用的肝毒素,其分子结构复杂、种类繁多,以痕量形式稳定地存在于各类富营养化的天然水体中,传统的处理工艺难以有效祛除,这对人类健康构成了极大的潜在威胁。目前迫切要求对MCs进行快速定性、定量分析的同时,也需要大量MCs纯品作为试验材料进行深入研究,以便寻找一种稳定高效的方法降解祛除饮用源水中的MCs。本论文围绕着MCs的分离检测、纯化制备以及UVA光助高级氧化方法(AOP)降解这几个主题,以太湖梅梁湾采集的样品为对象开展了一系列研究工作。具体包括以下内容:
优化建立了基于固相萃取(SPE)-高效液相色谱(HPLC)和商品化的ELISA试剂盒的两种途径检测水样中痕量MCs的方法。通过对样品预处理中小柱、淋洗、洗脱、浓缩定容等关键环节的单因素考察,确立的SPE方法能够高效稳定地将水样中MCs浓缩富集1000倍;洗脱液采用配有DAD检测器的HPLC仪器,以含有三氟乙酸(TFA)的甲醇溶液等度洗脱方式进行MCs检测,SPE–HPLC方法不但可以定性不同种类的MCs,而且可以进行准确定量检测。结果同ELISA方法进行了比较验证。采用这两种方法对夏季太湖梅梁湾的水样进行了检测,结果表明水中主要含有的微囊藻毒素为MC-RR和MC-LR,其中MC-RR含量更高,在峰值9月份两种MCs浓度都已经超过了有关标准,需要采取措施控制。
在室温条件下采用60%的甲醇结合藻细胞超声粉碎方法从水华藻浆中提取了MCs,毒素浓度较高而杂质含量较低,调节pH值使残余色素蛋白变性析出得以分离。在SPE-HPLC仪器上进行了提取MCs分离条件的优化,并运用LC-ESI/MS设备进一步确认鉴定毒素类型,结果表明MC-RR、MC-YR和MC-LR这几种藻毒素是太湖水华藻样中主要的MCs类型,还包括一种去甲基化的MC-RR和一种未见报道的MCs;同样以此提取液为对象,在Flash色谱和制备型HPLC仪器上进行了MCs放大制备试验,在流动相比例为甲醇/水=55/45,流速选定为10mL/min,进样量为400μL的最佳条件下,同时分离、纯化制得约0.1mg纯净MC-RR和MC-LR单体。
不同光照条件下考察了Fenton试剂和Fenton体系对高低不同浓度的提取MC-LR的降解情况,并采用甲基紫褪色光度法对过程中产生的羟自由基进行间接测量,探讨了这些过程中藻毒素降解的机理。结果表明Fenton体系可以部分降解MC-LR,降解速率和程度与Fenton试剂用量成正比,基本遵循羟自由基氧化进攻的机理;在低强度辐照UVA光照下,Fe~(3+)和Fe~(2+)能够有效降解MC-LR,前者更为显著,但过程中的羟自由基产量和毒素降解率的正相关性降低,Fe(OH)~(2+)络合物产生光生羟自由基是导致毒素降解主要的引发剂;UVA-Fenton体系中二者的协同效应加速了体系中Fe~(2+) /Fe~(3+)的循环,极大提高了羟自由基产量,使MC-LR降解速率大于Fenton和Fe~(3+)(Fe~(2+))/UVA体系中降解速率之和;广谱的太阳光参与的Fe~(2+)(Fe~(3+))体系和Fenton体系降解MC-LR比UVA更为高效;在采用太阳光照射和模拟天然水中的浓度和
The occurrence of toxic water bloom of cyanobacteria algae has been a serious pollution problem in recent years. Microcysins (MCs) is a group of extremely hepatotoxic toxin produced by the species of cyanobacteria. Due to complicated structure and diverse congener, the MCs pollution is characteristic of trace-level, recalcitrant and complex in eutrophic freshwater. It has been proven difficult to be removed by conventional water treatments and constitute potential hazard to public health, some necessary measures of monitoring and determination on MCs are urgently needed. Furthermore, purified MCs are also needed to utilize as reagent material in relevant research field, such as degradation of MCs in drinking water with high efficiently. Focused on determination and purification of MCs, as well as degradation by photo-Advanced Oxidation Process, this dissertation selected the water and bloom sample from Meiliang Bay, TaiHu Lake as objective to conduct a series of investigation.
To optimize and develop two methods of trace level MCs determination based on solid phase extraction (SPE)-high performance liquid chromatography (HPLC) and ELISA, single factor comparisons were conducted in such key SPE processes as cartridge, rinse, elution and concentration. MCs in water sample could be condensed 1000 times by this method. When methanol solution with trifluoroacetic acid (TFA) was applied to mobile phase, HPLC assay offered a good potential for a sensitive and selective determination of MCs. The results were compared and validated between SPE-HPLC with ELISA method. According to the results for water samples from TaiHu lake during warm months, MC-RR and MC-LR were main MCs component, and the content of former was usually greater than the later. The maximum contents of both MCs which appeared in September have exceeded the guideline and some measures must be taken to make MCs pollution under control.
At the room temperature, the 60% methanol solution with ultrasonic method was applied to extract MCs from bloom sample. The massive of phycocyanin protein was removed by adjusting the pH of solvents to the isoelectric point. SPE-HPLC has been employed to isolate MCs and the structures of the obtained MCs were further identified with HPLC, spectrophotometer, and electrospray ionization mass spectrometry (ESI-MS). The result showed that MC-RR, MC-LR and MC-YR were main MCs component in bloom sample, with a small quantity of Dimethyl MC-RR and a kind of unknown MCs. Methods were also established to isolate and purify MCs with Flash chromatography and preparative chromatography. Under the condition of mobile phase with 55% methanol solution, flow rate 10ml/min and injection volume 400μL, about 0.1mg pure MC-RR and MC-LR were obtained.
优化建立了基于固相萃取(SPE)-高效液相色谱(HPLC)和商品化的ELISA试剂盒的两种途径检测水样中痕量MCs的方法。通过对样品预处理中小柱、淋洗、洗脱、浓缩定容等关键环节的单因素考察,确立的SPE方法能够高效稳定地将水样中MCs浓缩富集1000倍;洗脱液采用配有DAD检测器的HPLC仪器,以含有三氟乙酸(TFA)的甲醇溶液等度洗脱方式进行MCs检测,SPE–HPLC方法不但可以定性不同种类的MCs,而且可以进行准确定量检测。结果同ELISA方法进行了比较验证。采用这两种方法对夏季太湖梅梁湾的水样进行了检测,结果表明水中主要含有的微囊藻毒素为MC-RR和MC-LR,其中MC-RR含量更高,在峰值9月份两种MCs浓度都已经超过了有关标准,需要采取措施控制。
在室温条件下采用60%的甲醇结合藻细胞超声粉碎方法从水华藻浆中提取了MCs,毒素浓度较高而杂质含量较低,调节pH值使残余色素蛋白变性析出得以分离。在SPE-HPLC仪器上进行了提取MCs分离条件的优化,并运用LC-ESI/MS设备进一步确认鉴定毒素类型,结果表明MC-RR、MC-YR和MC-LR这几种藻毒素是太湖水华藻样中主要的MCs类型,还包括一种去甲基化的MC-RR和一种未见报道的MCs;同样以此提取液为对象,在Flash色谱和制备型HPLC仪器上进行了MCs放大制备试验,在流动相比例为甲醇/水=55/45,流速选定为10mL/min,进样量为400μL的最佳条件下,同时分离、纯化制得约0.1mg纯净MC-RR和MC-LR单体。
不同光照条件下考察了Fenton试剂和Fenton体系对高低不同浓度的提取MC-LR的降解情况,并采用甲基紫褪色光度法对过程中产生的羟自由基进行间接测量,探讨了这些过程中藻毒素降解的机理。结果表明Fenton体系可以部分降解MC-LR,降解速率和程度与Fenton试剂用量成正比,基本遵循羟自由基氧化进攻的机理;在低强度辐照UVA光照下,Fe~(3+)和Fe~(2+)能够有效降解MC-LR,前者更为显著,但过程中的羟自由基产量和毒素降解率的正相关性降低,Fe(OH)~(2+)络合物产生光生羟自由基是导致毒素降解主要的引发剂;UVA-Fenton体系中二者的协同效应加速了体系中Fe~(2+) /Fe~(3+)的循环,极大提高了羟自由基产量,使MC-LR降解速率大于Fenton和Fe~(3+)(Fe~(2+))/UVA体系中降解速率之和;广谱的太阳光参与的Fe~(2+)(Fe~(3+))体系和Fenton体系降解MC-LR比UVA更为高效;在采用太阳光照射和模拟天然水中的浓度和
The occurrence of toxic water bloom of cyanobacteria algae has been a serious pollution problem in recent years. Microcysins (MCs) is a group of extremely hepatotoxic toxin produced by the species of cyanobacteria. Due to complicated structure and diverse congener, the MCs pollution is characteristic of trace-level, recalcitrant and complex in eutrophic freshwater. It has been proven difficult to be removed by conventional water treatments and constitute potential hazard to public health, some necessary measures of monitoring and determination on MCs are urgently needed. Furthermore, purified MCs are also needed to utilize as reagent material in relevant research field, such as degradation of MCs in drinking water with high efficiently. Focused on determination and purification of MCs, as well as degradation by photo-Advanced Oxidation Process, this dissertation selected the water and bloom sample from Meiliang Bay, TaiHu Lake as objective to conduct a series of investigation.
To optimize and develop two methods of trace level MCs determination based on solid phase extraction (SPE)-high performance liquid chromatography (HPLC) and ELISA, single factor comparisons were conducted in such key SPE processes as cartridge, rinse, elution and concentration. MCs in water sample could be condensed 1000 times by this method. When methanol solution with trifluoroacetic acid (TFA) was applied to mobile phase, HPLC assay offered a good potential for a sensitive and selective determination of MCs. The results were compared and validated between SPE-HPLC with ELISA method. According to the results for water samples from TaiHu lake during warm months, MC-RR and MC-LR were main MCs component, and the content of former was usually greater than the later. The maximum contents of both MCs which appeared in September have exceeded the guideline and some measures must be taken to make MCs pollution under control.
At the room temperature, the 60% methanol solution with ultrasonic method was applied to extract MCs from bloom sample. The massive of phycocyanin protein was removed by adjusting the pH of solvents to the isoelectric point. SPE-HPLC has been employed to isolate MCs and the structures of the obtained MCs were further identified with HPLC, spectrophotometer, and electrospray ionization mass spectrometry (ESI-MS). The result showed that MC-RR, MC-LR and MC-YR were main MCs component in bloom sample, with a small quantity of Dimethyl MC-RR and a kind of unknown MCs. Methods were also established to isolate and purify MCs with Flash chromatography and preparative chromatography. Under the condition of mobile phase with 55% methanol solution, flow rate 10ml/min and injection volume 400μL, about 0.1mg pure MC-RR and MC-LR were obtained.
引文
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