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改性粘土对短凯伦藻产毒及其毒素代谢产物的影响
申慧慧
Subtype博士
Thesis Advisor宋秀贤
2021-11-25
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Keyword改性粘土 短凯伦藻 短裸甲藻毒素代谢物 吸附作用
Abstract

短凯伦藻(Karenia brevis)是一种能产生短裸甲藻毒素代谢物(BrevetoxinsBTXs)的微藻,可形成赤潮,破坏生态系统,造成巨大的经济损失,甚至威胁人类健康。K. brevis主要分布在美洲中部的墨西哥湾,在亚洲的中国以及澳洲的新西兰等国家沿海海域也有检出。目前,我国已发现了K. brevis藻株,还尚无中国株K. brevis产毒的相关报道。改性粘土技术是一种目前国内外普遍认可的赤潮应急处置方法。本研究以中国株K. brevis 165为研究对象,揭示了不同生长期K. brevis 165产生BTXs毒素代谢物的特征和变化规律;在此基础上,开展了改性粘土对主要毒素BTX2的吸附作用及吸附特征研究;基于K. brevis 165的产毒特征和改性粘土对BTXs的吸附作用,通过室内模拟实验,进一步开展了改性粘土对K. brevis 165产生的BTXs毒素代谢物的影响研究,揭示了改性粘土对K. brevis 165产毒影响的作用机制。本文的主要研究成果如下:

1. 基于液-质联用技术,研究短凯伦藻产生的藻毒素代谢物  以中国株K. brevis 165为研究对象,基于液相色谱-高分辨率质谱(LC-HR-MS)和液相色谱-多级质谱(LC-MS-MS)技术,优化了富集藻培养液中BTXs毒素代谢物的样品前处理方法,建立了筛查、鉴定藻胞内外BTXs毒素代谢物的方法,并应用此方法对不同生长阶段K. brevis 165的胞内外BTXs毒素代谢物轮廓进行分析研究,首次报道了中国境内藻株K. brevis 165能够产生BTXs毒素代谢物。实验结果表明,利用亲水亲油平衡萃取柱(HLB萃取柱)富集海水体系中BTXs毒素的回收率高于以往的C18固相萃取柱萃取法;在K. brevis 165胞内外分别发现了5种和8BTXs毒素代谢物,并在培养液中首次发现BTXs毒素代谢物的拮抗剂(Brevenal),充分证明了建立筛查鉴定BTXs毒素代谢物方法的必要性。

对不同生长阶段K. brevis 165产生BTXs毒素代谢物的分析结果表明,胞内单细胞总BTXs毒素代谢物含量为6.78 pg/cell~21.53 pg/cell,培养液中总毒素代谢物浓度为10.27 μg/L~449.11 μg/L,而且不同生长阶段的BTXs毒素代谢物种类和含量存在明显差异。因此,当赤潮暴发时,准确测定了解BTXs毒素代谢物的含量分布,有助于评估生态灾害风险,从而规避造成的危害,达到灾害预警和防灾减灾的目的。

2. 改性粘土对藻毒素BTX2的吸附作用研究  K. brevis培养液中BTXs毒素代谢物的含量不容忽略,因此,有必要深入探究改性粘土控制赤潮的同时对培养液中BTXs毒素代谢物的影响作用。因此,本章研究了改性粘土(聚合氯化铝(PAC)改性粘土,称为MCI)对海水中BTX2的吸附作用及特征。基于前人的研究,通过X射线衍射(XRD)、傅里叶红外光谱(FTIR)和比表面积(BET)等分析技术,对MCI进行了表征,探讨了MCI吸附BTX2的吸附动力学、吸附等温线和热力学特性。研究结果表明,高岭土原土的主要组成是高岭石和石英,该原土经过PAC改性后其比表面积变化不明显,但表面电性由负变正,这有利于MCI对电负性物质的吸附。进一步分析MCI吸附BTX2的特征时发现,高岭土原土和MCI对海水中BTX2的吸附过程符合准二级吸附动力学模型,吸附等温线符合Freundlich等温吸附模型,属于自发的吸热过程。进一步对影响吸附的环境因素进行分析发现,在一定温度下,当海水中BTX2的初始浓度逐渐增大时,高岭土原土和MCⅠ对海水中BTX2的吸附量均呈现增加的趋势;随着温度升高,两种材料的吸附容量也呈现升高的规律。但是,MCⅠ吸附K. brevis培养液中BTX2时,会降低对BTX2的吸附量。在改性粘土控制产毒赤潮的现场应用中,该结果能够为评估改性粘土作用下毒素造成的生态危害研究提供理论支持。

3. 改性粘土对BTXs毒素代谢物的影响  本研究通过室内模拟实验,探究了MCI絮凝K. brevis 165BTXs毒素代谢物的影响及作用机制。具体来说,在MCI絮凝K. brevis 165后,跟踪混合实验体系中BTXs毒素代谢物和环境参数的变化,以期揭示MCⅠK. brevis产生的BTXs代谢物的影响机制。研究结果表明,MCⅠ浓度为0.1 g/L时,对K. brevis 165的去除率为78 ± 2.8%,上层残留藻细胞无法在短时间内增殖达到赤潮密度。与未添加改性粘土的空白对照组相比,MCⅠ可以有效降低实验组中BTX2BTX3的总含量,同时BTXs毒素代谢物(BTX2BTX3Open-ring BTX3Open-ring BTX2BTX-B5Open-ring BTX-B5)总含量也有所降低。MCⅠ还可以促进BTX2BTX-B5Open-ring BTX2Open-ring BTX-B5BTX3Open-ring BTX3代谢转化。改性粘土MCI对实验组中的pH和溶解态硝酸盐含量没有影响,但降低了溶解态磷酸盐的含量,最低含量为5.81±0.84 μM此含量并没有对K. brevis 165的生长造成P限制,推测不会胁迫K. brevis 165产生更多的BTXs毒素代谢物。总体而言,改性粘土在调节水质的同时,可以有效降低K. brevis 165产生的BTXs毒素代谢物含量和毒性,为改性粘土技术现场大规模治理该类产毒藻赤潮提供了理论支持。

Other Abstract

Karenia brevis is a red-tide alga that can produce brevetoxins (BTXs). The K. brevis harmful algal blooms can destroy the marine ecosystem, cause huge economic losses and threaten the human health. K. brevis is mainly distributed in the Gulf of Mexico, and also detected in the coastal waters of China and New Zealand. At present, K. brevis has been found in China, but there are no reports about the BTXs metabolites produced by Chinese K. brevis strain. The modified clay technology has been successfully applied to control harmful algal blooms. In this study, the characteristics of BTXs metabolites produced by Chinese K. brevis165 in China were firstly revealed. On this basis, the adsorption effect and mechanism of modified clay on BTX2 were studied. Furthermore, the effect of modified clay on BTXs metabolites produced by K. brevis 165 was studied through laboratory experiment, and the mechanism of modified clay on the BTXs was revealed. Specific research results are as follows:

1. Profiling of Brevetoxin Metabolites Produced by Karenia brevis 165 Based on Liquid Chromatography-Mass Spectrometry In this study, K. brevis165 was used to research BTXs metabolites. The sample pretreatment method for the enrichment of BTXs metabolites in an algal culture media was improved here. The method for screening and identifying intracellular and extracellular BTXs metabolites was established based on liquid chromatography-time-of-flight mass spectrometry (LC-ToF-MS) and liquid chromatography triple quadrupole tandem mass spectrometry (LC-QqQ-MS/MS). Results showed that the recovery rates for BTX toxins enriched by a hydrophilic-lipophilic-balance (HLB) extraction column were higher than those with a C18 extraction column. This method was used to analyze the profiles of extracellular and intracellular BTXs metabolites at different growth stages of K. brevis 165. This is the first time to report a Chinese strain of K. brevis could produce toxic BTX metabolites. Five and eight kinds of BTXs metabolites were detected in the cell and culture media of K. brevis 165, respectively. Brevenal, a toxic BTX metabolite antagonist, was found especially for the first time in the culture media. The toxic BTX metabolites and brevenal in the K. brevis 165 cell and culture media were found to be fully proven in terms of the necessity of establishing a method for screening and identifying toxic BTX metabolites.

The results found by qualitatively and quantitatively analyzing BTX metabolites produced by K. brevis 165 at different growth stages showed that the total toxic BTX metabolite contents in single cells ranged between 6.78-21.53 pg/cell, and the total toxin concentration in culture media ranged between 10.27-449.11 μg/L. There were significant differences for the types and contents of toxic BTX metabolites with varying growth stages. Therefore, when harmful algal blooms occur, the accurate determination of BTX metabolite types and concentrations will be helpful to assess the ecological disaster risk in order to avoid hazards and provide appropriate disaster warnings.

2. Adsorption of BTX2 from seawater by modified clay The content of BTXs metabolites in K. brevis culture media cannot be ignored, so, it is necessary to further explore the effect of modified clay on BTXs metabolites in culture media while controlling harmful red tide. Therefore, the adsorption effect and characteristics of modified clay (PAC-modified clay, known as MCI) on BTX2 in seawater were studied. In this chapter, the MCI was characterized by X-ray diffraction (XRD) technology, Fourier transform infrared spectrometer (FTIR) and BET surface area analyzer technology based on previous studies. Then, the adsorption kinetics, adsorption isotherm and adsorption thermodynamic of MCI on BTX2 were studied. The results showed that the main components of the clay are kaolinite and quartz, and the BET surface area of MCI did not change significantly, but the surface electrical properties changed from negative to positive, which was beneficial to the adsorption of electronegative substances by MCI. The adsorption characteristics of BTX2 by Kaolinite clay and MCI was further analyzed. The results showed that the adsorption process of BTX2 in seawater by Kaolinite clay and MCI conformed to the pseudo-second-order adsorption kinetics model, and the adsorption isotherm conformed to the Freundlich isothermal adsorption model, which was a spontaneous endothermic process. In addition, the factors affecting the adsorption were analyzed. It was found that at a certain temperature, when the initial concentration of BTX2 in seawater gradually increased, the adsorption quantity of BTX2 from seawater increased for Kaolinite clay and MCI. The adsorption capacity of the two materials also increases with the increase of temperature. However, K. brevis culture media can reduce the adsorption capacity of MCI to BTX2. When modified clay is used to control harmful algal blooms in the field, the study can provide theoretical support for evaluating ecological damage caused by marine toxins under the modified clay.

3. Effect of modified clay on BTXs metabolites produced by Karenia brevis In this study, the effect and mechanism of MCI flocculation of K. brevis 165 on BTXs metabolites were investigated by laboratory experiments. Specifically, when K. brevis 165 was flocculated by MCI, the changes of BTXs metabolites and environmental parameters in the whole mixed experimental system were studied to reveal the effect mechanism of MCI on BTXs metabolites produced by K. brevis 165. The results showed that when MCI was 0.1 g/L, the removal rate of K. brevis 165 was 78 ±2.8%, and the residual algae cells could not cause harmful algal blooms again. Compared with the blank control group without modified clay, MCI can reduce the total content of BTX2BTX3 and BTXs metabolites (BTX2, BTX3, Open-Ring BTX3, open-Ring BTX2, BTX-B5 and open-Ring BTX-B5) in the experimental group. MCI can also promote the transformation of BTX-B5 and BTX2 to BTX3, open-ring BTX3, open-ring BTX2 and open-ring BTX-B5. Previous studies have shown that changes in K. brevis culture conditions will affect the production of BTXs metabolites, and the limitation of phosphate will make the algae produce more BTXs metabolites. In our study, the content of dissolved phosphate in MCI group decreased from 9.90 μM to 5.81 μM, but it did not reach the P-restricted concentration for K. brevis 165 growth, and did not stimulate K. brevis 165 to produce more BTXs metabolites. Therefore, the MCI can not only regulate water quality, but also effectively reduce the content of BTXs metabolites in K. brevis 165 culture system, which provides theoretical support for large-scale field application of modified clay.

Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/177041
Collection海洋生态与环境科学重点实验室
Recommended Citation
GB/T 7714
申慧慧. 改性粘土对短凯伦藻产毒及其毒素代谢产物的影响[D]. 中国科学院海洋研究所. 中国科学院大学,2021.
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