生长素吲哚乙酸对铜绿微囊藻生理生化及产毒特性的影响

为了探究生长素吲哚乙酸(IAA)对产毒铜绿微囊藻(Microcystis aeruginosa)的影响, 从生长、光合色素含量、叶绿素光诱导荧光特征、脂质氧化和微囊藻毒素合成特性等方面, 研究了IAA对M. aeruginosa CHAB6301生理生化及产毒的影响。结果表明, 在低浓度IAA(0.04和0.2 mg/L)条件下, 铜绿微囊藻生长、叶绿素含量、光合系统(PSⅡ)电子传递效率及藻毒素含量均无明显变化, 藻蓝蛋白、别藻蓝蛋白和丙二醛(MDA)含量均低于对照。高浓度IAA(1和5 mg/L)能够促进细胞生长, 提高叶绿素含量, 但是抑制藻蓝蛋白和别藻蓝蛋白含量, 降低膜脂过氧化程度和细胞内藻毒素合成。综合各指标测定结果, 低浓度IAA对M. aeruginosa CHAB6301生长和光合作用影响不明显, 而高浓度IAA可促进藻细胞生长和光合作用, 增加微囊藻水华形成几率。     

铜绿微囊藻在不同供磷水平下对砷胁迫的响应

采用磷饥饿培养后的微囊藻细胞进行不同供磷水平的五价砷(As(V))暴露实验,考察单一胞外磷变化的情况下As(V)对滇池分离出的铜绿微囊藻FACHB905生长及产毒的影响.结果表明胞外磷浓度变化不会影响铜绿微囊藻对As(V)的耐受阈值(-10-7 mol/L).少磷条件下的半数抑制浓度(IC50)为10-2.79 mol/L,比无磷条件下的IC50(10-5.81 mol/L)高3个数量级,并且少磷条件下As(V)与细胞活性位点的结合常数要远低于无磷条件,因此胞外磷在As(V)对微囊藻的毒性效应上具有关键的作用.As(V)对微囊藻单细胞的叶绿素含量没有显著影响,但是对毒素产量具有剂量效应.在少磷条件下,As(V)浓度大于10-7 mol/L可促进微囊藻FACHB905的胞内产毒量;而在无磷条件下,所有As(V)处理组的胞内产毒量均上升78%左右.由上可知,微囊藻在产毒方面与As(V)具有协同效应,这对于全面了解滇池水华暴发期间毒素的变化规律具有一定的参考价值.     

磷浓度对铜绿微囊藻、大型溞和金鱼藻三者相互作用的影响

为了解磷浓度对生物操纵和水生植被恢复效果的影响,以铜绿微囊藻、大型溞和金鱼藻分别作为浮游植物、浮游动物和大型沉水植物的代表,在25℃、2000—3000lx光强和11mg/L氮浓度条件下,研究两者和三者共培养时4种磷浓度(0.2、0.5、1.0、1.5 mg/L)下各自的增长率和培养液中氮磷去除率的变化。结果表明:两者共培养时,磷浓度不大于0.2mg/L时,有利于大型溞的繁殖和金鱼藻的生长;磷浓度介于0.5—1.5mg/L时,铜绿微囊藻呈正增长趋势,而金鱼藻的生长则明显受抑制。三者共培养时,所有磷浓度下的大型溞数量及金鱼藻生物量均不同程度的升高,且铜绿微囊藻的生长得到了有效抑制,以磷浓度为0.2—0.5mg/L时效果最佳;N/P比值对藻、溞、草间的相互作用有重要影响,在藻-溞系统中,大型沉水植物的加入可以大大提高抑藻效果,减小N/P比值波动带来的不利影响。磷浓度为0.5mg/L时的水体氮磷去除效果好于其他磷浓度梯度。     

微囊藻对柔细束丝藻生长及土臭素合成与释放的影响

文章选择两种特征不同的微囊藻——产毒的铜绿微囊藻(Microcystis aeruginosa)和无毒的惠氏微囊藻(Microcystis wesenbergii),分别以不同的接种比例(1﹕2、1﹕1和2﹕1)与产土臭素(Geosmin)的柔细束丝藻(Aphanizomenon gracile)混合培养,以探索种间相互作用对藻类生长和束丝藻土臭素合成与释放的影响。结果表明,在共培养条件下,两种微囊藻均抑制了柔细束丝藻的生长,而柔细束丝藻却促进了两种微囊藻的生长。惠氏微囊藻促进了柔细束丝藻土臭素的释放(接种比例为1﹕1时,束丝藻胞外土臭素的细胞份额达到269.43 fg/cell),仅在生长早期与生长受到抑制阶段促进了土臭素的合成;铜绿微囊藻在共培养早期促进了束丝藻土臭素的合成,但共培养却抑制了土臭素的释放,而且在共培养的中后期已检测不到土臭素。研究结果表明,在自然水体中束丝藻与微囊藻的季节演替过程中,微囊藻在与束丝藻的竞争中处于优势,且微囊藻对束丝藻的竞争压力促使后者合成异味物质,随着束丝藻的消亡可能伴随大量异味物质的释放,增加异味事件发生风险。     

金藻吞噬微囊藻产生的无毒变异株与产毒原始株的比较

分别从喂食三株原始产毒铜绿微囊藻Microcystis aeruginosa(AC、DS和PCC 7820)的金藻Poterioochromonassp.培养物中获得三株藻,以Nest PCR方法(引物对CC/CG和CH/CI)确定此三株藻均为微囊藻属藻株.HPLC测试结果显示这三株藻均不产生微囊藻毒素.显示Poterioochromonas sp.具有将产毒微囊藻转化为尤毒微囊藻的能力.比较产毒原始株与无毒变异株的生理特性发现,变异株的类胡萝卜素/叶绿素比值高于原始株;而光反应曲线结果表明,变异株的PSⅡ的量子产率和光合作用活力高于原始产毒株,并且变异株在较低光强下就可达到最大的光合作用活力.显示喂食后产生的变异株比原始株有较高的光合作用效率.变异株的藻蓝蛋白/叶绿素比值则低于原始株,光合作用最适光强低于变异株,并且显示产毒原始株通过增加藻蓝蛋白的相对含量来提高对光照的吸收.变异株具有较高的光合作用效率和藻蓝蛋白含量可能是其能够在微囊藻和金藻混合培养的群体中占优势的原因之一.     

藻类连续培养体系的构建与优化及其在产毒和无毒微囊藻竞争中的应用

利用发酵罐加装外置环形光源构建藻类连续培养系统, 以产毒微囊藻PCC 7806及其无毒突变株PCC 7806 mcyB–为培养材料, 通过对补料时间、接种密度和稀释率参数的优化, 获得最优培养条件, 并应用于产毒与无毒微囊藻的竞争实验中。通过优化得到连续培养的最优培养条件: 补料时间为第4天, 起始接种密度为4×106 cells/mL, 稀释率为0.15/d。在连续培养下, 光照为35 μmol/(m2·s)时, 以1﹕1的起始比例接种产毒与无毒微囊藻, 二者间的竞争会达到平衡, 并以无毒微囊藻占据优势, 且两者以不同的优势度长时间维持不变。在此基础上, 开展了不同光强对产毒与无毒微囊藻竞争影响的实验, 结果表明, 光强为35和80 μmol/(m2·s)时, 无毒株在连续培养中占据优势; 而光强为5和15 μmol/(m2·s)时, 无毒和产毒微囊藻维持起始接种比例不变。研究通过优化连续培养条件为室内藻类竞争实验提供了更为适宜的培养模式。     

不同氮、磷浓度对铜绿微囊藻生长、光合及产毒的影响

对一株从野外分离得到的铜绿微囊藻产毒株进行分批培养,在不同的氮磷条件下研究其生长、光合荧光及毒素含量的变化。结果表明:正磷酸盐浓度不变时,铵氮浓度的改变对铜绿微囊藻的生长有明显影响。叶绿素a(Chl.a)含量在铵氮浓度为1.83-18.3mg/L时明显较大;微囊藻毒素(包括MC-LR和MC-RR)的含量在铵氮浓度为1.83mg/L时达到最大;当铵氮浓度为0-1.83mg/L时,随着铵氮浓度升高,可变荧光FV和MC的产量均增大,同时MC异构体的种类增多;铵氮浓度过大对M.aeruginosa的生长、生理和产毒均有抑制作用。在另一组实验中,即铵氮浓度不变而正磷酸盐浓度增大时,Chl.a含量呈总体下降的趋势,并且与FV/Fm呈显著正相关关系(P<0.01,r=0.97),MC(MC-LR和MC-RR)的含量在正磷酸盐浓度小于0.56mg/L时明显升高,MC-LR与FV/Fm呈显著正相关关系(P<0.01,r=0.967)。       

荧光原位杂交技术和流式细胞仪用于环境样品中产毒及非产毒微囊藻的定量监测

全球范围内,高频次、大范围暴发的蓝藻水华对淡水水体环境造成严重影响.微囊藻因其在生长特别是衰亡过程中向水体释放微囊藻毒素而威胁人类健康.因此,分析其产毒株及非产毒株在环境样品中的组成,建立产毒蓝藻的预报及评价体系显得极为重要.本文采用荧光原位杂交技术结合流式细胞技术实现对环境样品中产毒藻株的鉴别与定量.针对目标基因mcyA设计的、以地高辛标记的双链DNA探针可有效应用于产毒微囊藻FACHB905和PCC7806的鉴别.分别对来自滇池、太湖和关桥的11个样品进行分析显示,该方法与传统的形态学鉴定及PCR方法有较好的匹配.荧光原位杂交技术与流式细胞相结合可有效鉴别产毒与非产毒微囊藻,尤其可以对野外样品中产毒与非产毒藻株进行简便、可视化地鉴别,从而达到对产毒微囊藻水华早期预警的目的.     

微囊藻毒素缓解过氧化氢胁迫下铜绿微囊藻损伤的初步研究

过氧化氢可抑制藻类生长, 同时会导致微囊藻毒素(Microcystins, MCs)的释放, 实验设置4个处理组探讨了外源微囊藻毒素MC-LR对H2O2胁迫下铜绿微囊藻生理生化变化的影响。结果表明: 在H2O2胁迫下, 微囊藻的生长和光合活性受到显著抑制, 藻细胞存活率降低, ROS含量明显增加, SOD活性上升。与单独H2O2胁迫相比, 加入MC-LR能增加微囊藻细胞的存活率。250 mol/L H2O2处理24h和48h后, 在培养基中加入200 ng/mL MC-LR可以缓解H2O2对铜绿微囊藻光合系统PSII活性的抑制作用。当微囊藻暴露于250 mol/L H2O2环境中时, 添加了MC-LR处理组藻细胞中的ROS含量明显减少(P0.05)。在相同浓度H2O2且加入了外源MC-LR后藻细胞SOD活性下降(P0.05)。因此, 微囊藻毒素MC-LR可缓解250 mol/L H2O2引起的氧化损伤并增强微囊藻自身的生存能力。研究结果有利于阐明H2O2胁迫影响产毒蓝藻生长代谢的途径及MCs生物学意义。       

利用3对引物鉴定产毒和非产毒微囊藻

根据微囊藻毒素合成酶基因簇序列 ,合成了 3对引物epF/mb1R ,mcF/teR ,mcF/umR ,通过全细胞PCR的方法检测了 19种不同来源微囊藻产毒的情况。 3种引物对 15株产毒微囊藻中均可扩增到预期大小的片段 ,测序结果证明这些片段是微囊藻毒素合成酶基因片段。PCR反应结果与HPLC分析所得到的结果有良好的对应性。在此基础上 ,初步确定了 3对引物检测产毒微囊藻对细胞浓度要求的下限。与其它引物相比 ,3对引物的特异性强 ,扩增条带大小适中 ,便于观察     

东湖蓝藻水华毒性的研究 Ⅱ.季节变化及微囊藻的毒性

1984年至1986年间武汉东湖(包括湖边池塘)的水华有7种,即铜绿微囊藻、大型铜绿微囊藻、边缘微囊藻、水华鱼腥藻、卷曲鱼腥藻、美丽颤藻和束丝藻。经生物测定,除束丝藻未测、卷曲鱼腥藻和美丽颤藻未测出毒性外,其余4种皆为有毒水华。东湖的水华随着季节变化而有不同类型的更迭,其出现格局为:卷曲鱼腥藻、微囊藻、颤藻、微囊藻。微囊藻水华的毒性在不同季节也有较大差异,毒性最低在8—9月份,最高在12月份(LD_(50)=24mg/kg鼠重),随着温度的降低而提高。讨论了某些环境参数与微囊藻水华形成及其毒性变化的关系。此外,还用脑内注射和腹腔注射方法,研究了微囊藻毒素的毒性表现。     

Toxicity of Microcystis species isolated from natural blooms and purification of the toxin.

Microcystis strains (2 toxic and 18 nontoxic to mice) were isolated from toxic waterblooms that had been collected from Lake Kasumigaura, Ibaraki Prefecture, Japan, in August 1985. Thirteen of the strains (2 toxic and 11 nontoxic) were Microcystis aeruginosa, 2 (nontoxic) were Microcystis wesenbergii, and the other 5 were difficult to identify. Six (1 toxic and 4 nontoxic M. aeruginosa and 1 M. wesenbergii) of these 20 strains were established as axenic cultures. A toxic and axenic strain of M. aeruginosa, K-139, was used to study the relationship between growth conditions and toxicity. Cells in early-to-mid-log phase showed the highest toxicity (50% lethal dose, 7.5 mg of cells per kg of mouse), and maximum toxicity was not affected by growth temperatures between 22 and 30 degrees C. Purification and characterization of the toxins from K-139 cells were also conducted, and at least two toxins were detected. One of the toxins (molecular mass, 980 daltons) has not been reported previously. The main target of the toxin in mice was the liver. Marked congestion and necrosis in the parenchymal cells around the central veins of the liver were observed microscopically in specimens that had been prepared from the mice with acute toxicity after injection with the toxin.     

微囊藻碳酸酐酶活性在不同环境因素下的调节与适应

测定了3种微囊藻水华中的优势种类,即铜锈微囊藻（Microcystis aetlzginosa Kutz．）,绿色微囊藻（Microcystis viridis（A．Br．）Lemm）,惠氏微囊藻（Microcystis wesenbergii（Kom．）Kom．）,以及微囊藻573（Microcystis sp．573）的碳酸酐酶活性;研究了无机碳、pH、温度、光强、NIP比等环境因素和外源葡萄糖对铜锈微囊藻碳酸酐酶活性的影响,发现微囊藻碳酸酐酶活性受环境中碳酸氢根浓度的调节,故推断碳酸氢根是铜锈微囊藻利用的主要无机碳形式;相比添加葡萄糖进行混合营养培养的细胞,无外源葡萄糖和暗饥饿培养的微囊藻细胞会产生高约6倍的碳酸酐酶活性;光强的改变也会影响碳酸酐酶的活性。     

The role of microcystins in maintaining colonies of bloom-forming Microcystis spp

Microcystis is a cosmopolitan genus of cyanobacteria and occurs in many different forms. Large surface blooms of the cyanobacterium are well known in eutrophic lakes throughout the globe. We evaluated the role of microcystins (MCs) in promoting and maintaining bloom-forming cell aggregates at environmentally relevant MC concentrations (0.25-10 μg l(-1)). MCs significantly enhanced Microcystis colony sizes. Colonial diameters in microcystin-RR (MC-RR)-treated cultures (at 1 μg l(-1)) were significantly larger than control colonies, by factors of 1.5, 2.6 and 2.7 in Microcystis wesenbergii DC-M1, M. ichthyoblabe TH-M1 and Microcystis sp. FACHB1027 respectively. Depletion of extracellular MC concentrations caused Microcystis colony size to decrease, suggesting that released MCs are intimately involved in the maintenance of Microcystis colonial size. MC-RR exposure did not influence Microcystis growth rate, but did significantly increase the production of extracellular polysaccharides (EPS). In addition, MC-RR exposure appeared to trigger upregulation of certain parts of four polysaccharide biosynthesis-related genes: capD, csaB, tagH and epsL. These results strongly indicate that induction of polysaccharides by MC-RR was the major mechanism through which MCs enhanced colony formation in Microcystis spp. Cellular release of MCs, therefore, may play a key role in the persistence of algal colonies and the dominance of Microcystis.     

Toxic cyanobacteria strains isolated from blooms in the Guadiana river (southwestern Spain)

This paper describes the occurrence of toxic cyanobacteria along the Guadiana River over its course between Mérida and Badajoz (Extremadura, Spain). Water sampling for phytoplankton quantification and toxin analysis was carried out regularly between 1999 and 2001 in six different locations, including two shallow, slow-flowing river sites, two streamed river sites and two drinking water reservoirs. The cyanobacterial community differed significantly between these locations, especially during the summer. The predominant genera were Microcystis, Oscillatoria, Aphanizomenon and Anabaena. Using an ELISA assay the total microcystin contents of natural water samples from the most eutrophic locations ranged from 0.10 - 21.86 microg mcyst-LR equivalent x L(-1) in Valdelacalzada and 0.10-11.3 microg mcyst-LR equivalent x L(-1) in Vitonogales, and a seasonal variation of toxin content was observed. The amount of microcystins produced by each strain was determined by ELISA assay and the detection and identification of microcystin variants of three toxic strains of Microcystis aeruginosa was performed by high performance liquid chromatography (HPLC). The analysis of microcystins of the cultured strains revealed that toxin production was variable among different strains of M. aeruginosa isolated either from different blooms or from the same bloom.     

Phylogenetic Inference of Colony Isolates Comprising Seasonal Microcystis Blooms in Lake Taihu, China

Blooms of the toxin-producing cyanobacterium, Microcystis spp., are an increasingly prevalent water quality problem and health hazard worldwide. China's third largest lake, Lake Taihu, has been experiencing progressively more severe Microcystis blooms over the past three decades. In 2009 and 2010, individual Microcystis colonies, consisting of four different morphospecies, were isolated and genotyped using a whole-cell multiplex PCR assay. The 16S-23S rDNA-ITS sequences were aligned based on Bayesian inference and indicated that one morphospecies was genetically unique (Microcystis wesenbergii) and three were indistinguishable (Microcystis aeruginosa, Microcystis flos-aquae, and Microcystis ichthyoblabe). Microcystin (mcyB) genes were detected intermittently in two of the morphospecies while the other two morphospecies lacked the mcyB gene in all samples. Water temperature was found to influence bloom formation and morphotype prevalence, and chlorophyll a and temperature were positively and significantly correlated with microcystin concentration. Cooler water temperatures promoted toxigenic strains of Microcystis. Wind appeared to influence the distribution of morphotypes across the lake, with M. aeruginosa and M. ichthyoblabe being more susceptible to wind stress than M. wesenbergii and M. flos-aquae. The results of this study indicated that the blooms were composed of a variety of Microcystis morphospecies, with more genotypes observed than can be attributed to individual morphotypes. We conclude that morphology is not a reliable indicator of toxigenicity in Lake Taihu, and caution should be exercised when the M. aeruginosa morphotype is present because it is capable of producing MC-LR, the most toxic microcystin isoform.     

七株微囊藻系统进化关系的RAPD-PCR分析

应用RAPD-PCR的方法,选用24个随机引物,分析来自不同地区的7株微囊藻的基因组多态性。结果显示,Microcystis.viridis及M.wesenbergii明显与M.aeruginosa区分开。M.aeruginosa分为两个可视为不同种的异源分类单位。作为对照的Anabaena sp.7120与其他微囊藻株表现出完全不同的基因型及更远的遗传距离。 此项研究表明,以基因型而不是表现型为基础,分析蓝藻种内及种间区别是可能的。因此,为解决蓝藻分类问题,特别是在种和属的水平上,提供了重要的线索。结合正在进行的用特异性及准确性强的引物区分微囊藻产毒及非产毒株的方法,RAPD-PCR可望将微囊藻产毒及非产毒株进化关系澄清。     

Toxicity and toxins of natural blooms and isolated strains of Microcystis spp. (Cyanobacteria) and improved procedure for purification of cultures.

All samples of cyanobacterial blooms collected from 1986 to 1989 from Lake Kasumigaura, Ibaraki Prefecture, Japan, were hepatotoxic. The 50% lethal doses (LD50s) of the blooms to mice ranged from 76 to 556 mg/kg of body weight. Sixty-eight Microcystis cell clones (67 Microcystis aeruginosa and 1 M. viridis) were isolated from the blooms. Twenty-three strains (including the M. viridis strain) were toxic. However, the ratio of toxic to nontoxic strains among the blooms varied (6 to 86%). Microcystins were examined in six toxic strains. Five toxic strains produced microcystin-RR, -YR, and -LR, with RR being the dominant toxin in these strains. Another strain produced 7-desmethylmicrocystin-LR and an unknown microcystin. This strain showed the highest toxicity. Establishment of axenic strains from the Microcystis cells exhibiting extracellularly mucilaginous materials was successful by using a combination of the agar plate technique and two-step centrifugation.     

Toxicity and toxins of natural blooms and isolated strains of Microcystis spp. (Cyanobacteria) and improved procedure for purification of cultures

All samples of cyanobacterial blooms collected from 1986 to 1989 from Lake Kasumigaura, Ibaraki Prefecture, Japan, were hepatotoxic. The 50% lethal doses (LD50s) of the blooms to mice ranged from 76 to 556 mg/kg of body weight. Sixty-eight Microcystis cell clones (67 Microcystis aeruginosa and 1 M. viridis) were isolated from the blooms. Twenty-three strains (including the M. viridis strain) were toxic. However, the ratio of toxic to nontoxic strains among the blooms varied (6 to 86%). Microcystins were examined in six toxic strains. Five toxic strains produced microcystin-RR, -YR, and -LR, with RR being the dominant toxin in these strains. Another strain produced 7-desmethylmicrocystin-LR and an unknown microcystin. This strain showed the highest toxicity. Establishment of axenic strains from the Microcystis cells exhibiting extracellularly mucilaginous materials was successful by using a combination of the agar plate technique and two-step centrifugation.