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题名: 铜绿微囊藻对不同形态砷胁迫的响应
作者: 龚艳
答辩日期: 2008-06-15
导师: 宋立荣 ; 刘剑彤
专业: 环境科学
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 铜绿微囊藻 ; 砷 ; 磷 ; 微囊藻毒素 ; 毒素释放 ; 氧化胁迫 ; 细胞超微结构 ; 修复能力 ; 协同效应
其他题名: Responses of Microcystis aeruginosa to Different Arsenic Species
摘要: 日益严重的水体富营养化导致有害藻华频发已成为全球性的生态环境问题之一。微囊藻水华和微囊藻毒素的研究是目前国内外学者广泛关注的研究热点之一,滇池就是其中的典型代表。1989年国家“七五”科技攻关项目-滇池污染物环境容量研究报告显示,滇池草海上层水和沉积物中的砷化物含量分别为0.139 mg/L和331.85 mg/L。随后973项目“湖泊富营养化过程与蓝藻水华暴发机理研究”2006年年度总结报告中指出:可溶态砷是滇池全湖中含量最高的微量元素。磷是藻类生长所必需的营养元素之一,且砷与磷同处于元素周期表VA族,具有相似性,由此,砷对微囊藻生长及产毒能力是否存在影响不得不引发我们的思考。本文研究了三种不同产地铜绿微囊藻对砷胁迫的生理响应和磷营养在微囊藻响应砷胁迫的辅助作用,试图解释不同产地铜绿微囊藻是否对砷具有不同的生理响应,探讨铜绿微囊藻的产毒能力是否受到不同形态砷的影响,揭示磷对于微囊藻响应砷胁迫的作用。具体研究内容和结果如下: (1) 当水样中同时存在砷和磷时,砷的含量会影响总磷的测量。本实验采用单一影响因素研究和正交因素水平试验相结合的方法消除钼锑抗法测磷过程中砷的干扰,结果表明不同形态砷的干扰强度依此是,五价砷As(V) > 三价砷As(III) >> 二甲基胂DMAA。硫代硫酸钠过少,不能完全消除砷的干扰,过多则会成为新的干扰因素。As(V)的浓度从0.1 mg/L-5.0 mg/L,考虑到实际操作,以及实际水样中的As(V)的含量等因素,选择0.5 mL为最佳量, 15 min为最佳显色时间。 (2) 比较了铜绿微囊藻FACHB905对As(V),As(III)和DMAA三种形态砷胁迫的生理响应。在BG-11培养基中,藻细胞对As(V)具有高耐受性,耐受值达到10-3 M。As(III)对M. aeruginosa FACHB905的毒性阈值介于10-5-10-4 M间;有趣的是,微囊藻细胞在10-4 M As(III)处理下,第六天出现生长和产毒同时恢复的现象。10-8-10-4 M DMAA对藻细胞生长有微弱的抑制作用。产毒方面的响应是:As(V)可促进MC-LR的产毒量,从而As(V)可显著提高微囊藻的胞内毒性;微囊藻总产毒量与As(III)的处理浓度具有剂量关系,并形成典型的hormesis现象,峰值出现在10–7 M As(III);浓度小于10-4 M的DMAA对藻细胞的产毒能力没有任何影响。 (3) 探讨铜绿微囊藻FACHB905是否对As(V)和P具有分辨能力,可从侧面解答该藻株为何对As(V)具有高耐受性。结果表明,在本研究设定的浓度范围内 (5-130 μg/L),As(V)不影响磷酸盐动力学参数,从侧面反映出微囊藻对砷和磷具有一定的选择性。 (4) 选取三株不同产地铜绿微囊藻:分离自滇池的M. aeruginosa FACHB905,分离自荷兰的藻株M. aeruginosa PCC7806,分离自台湾桃园的M. aeruginosa TY-1。这三株铜绿微囊藻虽然分离自不同的生态地区,但是它们在亲缘与生理活性上具有高度相似性。三株铜绿微囊藻处于不同磷营养下,对As(V)胁迫的响应却有所不同。磷饥饿预处理14天后的微囊藻,即胞内不含多聚磷酸体的藻细胞进行As(V)处理时,可以看出:铜绿微囊藻FACHB905的生长受到明显的抑制,并且分析出As(V)进入胞内后取代P与活性酶结合,从而抑制藻细胞生长;而相同磷营养条件下,M. aeruginosa PCC7806的生长未受到影响。没有进行磷饥饿处理的三株微囊藻,即胞内含有多聚磷酸体的藻细胞对As(V)均表现出高耐受性。 (5) As(V)可促进这三株不同产地铜绿微囊藻的总产毒量,但是这种促进效应与胞内外磷均有关联。胞内无磷的M. aeruginosa FACHB905进行As(V)处理时,由于As(V)进入胞内与活性位点结合后,抑制藻细胞生长,因此其胞内毒素产量的升高主要源自生长率的下降。但是胞内含磷的这三株铜绿微囊藻进行As(V)处理时,As(V)会促进胞内MC-LR的产量及总毒素产量,并且与生长率没有关联。M. aeruginosa FACHB905,M. aeruginosa PCC7806和M. aeruginosa TY-1在As(V)处理下,胞内单细胞产毒量最大增幅分别为对照的1.4倍,3.8倍和1.4倍。对比以往研究结果可知,在单细胞水平上,As(V)对铜绿微囊藻的胞内毒素产量的影响水平,高于常规的环境因子。 (6) 目前还没有一个详尽的标准来衡量胞外微囊藻毒素主要来自于活细胞的主动释放还是衰亡细胞的被动扩散。因此,藻细胞的毒素释放还是个颇具争议的话题。胞内含有多聚磷酸体的这三株铜绿微囊藻进行As(V)处理后,发现M. aeruginosa TY-1本身具有毒素释放的能力,则As(V)促进毒素合成的同时也提高其毒素释放能力;M. aeruginosa PCC7806和FACHB905不具备毒素释放的能力,As(V)促进其毒素合成,但是绝大部分毒素都分布于胞内。另外发现,当磷饥饿处理后的M. aeruginosa PCC7806培养于1.0 μM 的培养液中时,As(V)可帮助微囊藻细胞主动释放毒素。研究铜绿微囊藻是否主动释放毒素,以及释放毒素的能力是否发生改变,As(V)处理似乎给这方面的研究提供新的思路。 (7) 考察铜绿微囊藻FACHB905和PCC7806在As(III)毒性阈值附近 (25,50和75 μM)的生理响应,发现M. aeruginosa FACHB905的毒性阈值介于25和50 μM间,并且具有自我修复能力; 而M. aeruginosa PCC7806的毒性阈值小于25 μM,不具备此修复的能力。As(III)对于两株藻的毒害机理主要在于氧化胁迫,SOD和CAT活性都明显升高;但是SOD活性只在第一天升高,而CAT活性在前四天都有升高。M. aeruginosa FACHB905在As(III)处理十天后的胞内产毒量与对照的产毒量相当,但是M. aeruginosa PCC7806的单细胞毒素含量随着As(III)处理浓度的增大呈下降趋势。另外发现M. aeruginosa FACHB905在50和75 μM As(III)胁迫下的超微结构出现异常:细胞分裂为类似四分分裂的形式,细胞类囊体和其它一些质粒的空间分布发生改变;而铜绿微囊藻PCC7806在高浓度As(III)胁迫下仅出现细胞衰亡的现象。基于本研究的现象,M. aeruginosa FACHB905超微结构的改变可能跟该藻株具有自我修复能力有关,另外M. aeruginosa FACHB905有可能成为研究原核细胞的胞质分裂的一个新的实验材料。由上述研究结果可知,富营养水体中砷磷并存时,铜绿微囊藻细胞可正常生长,并且铜绿微囊藻在产毒方面与As(V)具有协同效应,这对于全面了解砷污染水体,如滇池,在水华爆发期间毒素的变化规律具有一定的参考价值。
英文摘要: Eutrophication of lakes or reservoirs is occurred more frequently all over the world. In China, contamination by toxic blooms and toxins is becoming more and more serious in many freshwater bodies, such as Dianchi Lake, the 6th largest freshwater lakes in China. In Dianchi Lake, cyanobacterial blooms are mainly Microcystis. It can produce microcystins, which are the most common cyanotoxins and cause liver damage. A survey of Chaohai region in this lake showed that the inorganic arsenic concentration (arsenate plus arsenite) was 139 μg L−1 in epilimnetic water and 331.85 mg kg−1 in sediment in 1989 (state "seven-five" science and technology support program). Recently, the report of 973 Program in 2006 told that the concentration of dissolved arsenic was the highest in all trace elements in Dianchi Lake. It is still unclear to verify whether occurrence of Microcystis blooms in the lake is linked with arsenic abundance, and whether arsenic affects the toxin production of Microcystis species if the linkage exists. In this study, responses of three Microcystis aeruginosa strains to different arsenic species were systematically studied and discussed. The main contents and the results are as follows: 1. When arsenic co-exists in water samples, it can interfere with Mo-Sb-Vc-method determination of phosphate in water samples. In this study, we applied methods of the single affecting factor and the orthogonal test to determine the best addition amount of sodium thiosulfate and the best displaying time. The present study showed that the interference effect of arsenic species followed this trend: arsenate > arsenite >> dimethylarsinic acid. The response of adsorption values to the addition amount of sodium thiosulfate was a U-shape curve, and the lowest adsorption in the curve represented the best addition amount to defilade arsenic. When the arsenate concentration was in the range of 0.1-5.0 mg/L, the best amount of sodium thiosulfate (40 mg/mL) was 0.5 mL and the best displaying time was 15 min when considering the practical operation. 2. Responses of Microcystis aeruginosa FACHB905 to As(V), As(III) and DMAA were performed. In BG-11 media, M. aeruginosa FACHB905 was shown to be tolerant to inorganic arsenic, and no inhibitory effect on its growth was found below 10–5 M As(III) and 10–3 M As(V); while 10-8-10-4 M DMAA slightly inhibited its growth. It is interesting that the recovery growth and microcystin production of M. aeruginosa FACHB905 occurred after 6 days’ exposure to 10-4 M As(III). Exposure to As(V) enhanced the toxicity of M. aeruginosa FACHB905, since its microcystin-LR content per cell increased. Total microcystin production was stimulated in the presence of 10–7 M As(III), and the response of this M. aeruginosa strain to As(III) seemed to be a typical inverted U-shaped hormesis. However, the concentration of DMAA below 10–4 M was no effect on microcystin production. 3. Researchers recently did much work to know whether algae were capable of discriminating between phosphate and arsenate. Doing similar work with M. aeruginosa FACHB905 could give some indirect evidence to the cyanobacterial ability of tolerance to arsenate. The results showed that arsenate had no effect on the parameters of phosphate kinesics, which give some evidence indirectly that M. aeruginosa FACHB905 had the ability of discriminating between phosphate and arsenate. 4. Three Microcystis aeruginosa strains were selected in this study. They were M. aeruginosa FACHB905 isolated from the epilimnetic water of Dianchi Lake, Yunnan, China; M. aeruginosa PCC7806 isolated from Braakman Reservoir, the Netherlands; M. aeruginosa TY-1 isolated from Taoyuan, Taiwan, China. Though three Microcystis strains were isolated from different ecotypic regions, they were suggested to have a close genetic and physiological homogenicity. However, the three strains had different responses to arsenate under various phosphate conditions. When the cyanobacterial cells were preconditioned to phosphate starvation, the growth of M. aeruginosa FACHB905 was inhibited by arsenate. It was presumed that arsenate, transported into the cells, successfully competed with phosphate for enzymatic sites, then inhibited the growth of M. aeruginosa FACHB905. Yet, the growth of M. aeruginosa PCC7806 preconditioned to phosphate starvation was not affected by arsenate. Three Microcystis strains not preconditioned to phosphate starvation were all highly resistant to arsenate. 5. Arsenate could stimulate total microcystin yields of the three different Microcystis strains. But it is suggested a close association between microcystin stimulation and the intra- and extra-phosphate content. Microcystin yields of M. aeruginosa FACHB905 preconditioned to phosphate starvation was stimulated because of the lower specific growth rates by arsenate. Arsenate could also enhance microcystin production of the three Microcystis strains not preconditioned to phosphate starvation, and the stimulated microcystins had no association with the specific growth rate. In the present study, cellular microcystin content per cell of M. aeruginosa FACHB905, M. aeruginosa PCC7806 and M. aeruginosa TY-1 in relation to arsenate varied by factors of 1.4, 3.8 and 1.4, respectively. Contrasts to reports in recent literature, the normal environmentally-induced changes in cellular microcystin content on a per cell basis were lower than that induced by arsenate. 6. Microcystin leakage is a much debated subject since there is no criterion to distinguish whether the dissolved microcystins mainly result from excretion by intact cells or mainly liberation from cell lysis. M. aeruginosa TY-1 was suggested to have the inherent ability of excreting microcystins. Both total microcystin yields and microcystin excretion of M. aeruginosa TY-1 increased with 10-8-10-6 M arsenate. Meanwhile, Microcystis PCC7806 and FACHB905 did not have the ability of microcystin excretion, and then extracellular phosphate did not affect the leakage of microcystins, but could take similar effect on total microcystin yields as the strain TY-1 when they were not preconditioned to phosphate starvation. Besides, it is interesting to found that arsenate could help to actively export microcystins from living cells of M. aeruginosa PCC780 when preconditioned to phosphate starvation and incubated with the medium containing 1.0 μM phosphate. Toxic Microcystis exposed to arsenate may give a new direction to resolve present debated questions whether Microcystis could excrete microcystins from their cells, and whether environmental factors could influence microcystin excretion. 7. Microcystis aeruginosa FACHB905 and PCC7806 exposed to 25, 50 and 75 μM arsenite were explored. The inhibitory threshold dose of arsenite to M. aeruginosa FACHB905 was in the range of 25-50 μM, while that of arsenite to M. aeruginosa PCC7806 was lower than 25 μM. When M. aeruginosa FACHB905 was treated with 50 and 75 μM arsenite, the recovery growth occurred after three days’ exposure. But such phenomenon was not found in M. aeruginosa PCC7806. The toxicity of arsenite to Microcystis cells was mainly oxidative stress. The activities f antioxidative enzyme, such as superoxide dismutase (SOD) and catalase (CAT), were significantly increased in the two strains exposed to arsenite. However, the activities of SOD were just increased on the first day, while those of CAT were increased within the first four days. After 10 days’ exposure to arsenite, microcystin content per cell was not different from that of the control, while cellular microcystin content per cell of M. aeruginosa PCC7806 decreased along with increasing concentrations of arsenite. The ultrastructure of M. aeruginosa FACHB905 treated with 50 and 75 μM arsenite was malformed. It was mainly related to alterations in cell reproductive pattern, which were from binary fission to quartered-fission-like division. And the spatial distribution of thylakoids and some granules in cells was changed. Such phenomenon was also not found in the strain PCC7806. Thus, this phenomenon observed in our study provided further evidence that M. aeruginosa FACHB905 may be a new material to study the prokaryotic cytokinesis and the malformations of cells induced by arsenite may have some association with the ability of recovery. Totally, Microcystis aeruginosa could grow well in eutrophication waters co-existing arsenate and phosphate, and the synergistic effect of arsenate and microcystin production of M. aeruginosa is of definite significance for complete understanding microcystin production in the blooms of lakes contaminated by arsenic, such as Dianchi Lake.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12308
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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铜绿微囊藻对不同形态砷胁迫的响应.龚艳[d].中国科学院水生生物研究所,2008.20-25
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