【作者】 杨翠云；

【导师】 刘永定；

【作者基本信息】 中国科学院研究生院（水生生物研究所） ， 环境科学， 2007， 博士

【摘要】 蓝藻水华暴发和蓝藻毒素已成为世界性环境问题。蓝藻水华带来的主要危害之一是蓝藻毒素的产生。在已发现的蓝藻毒素中,微囊藻毒素分布最广泛、危害最严重。目前,关于微囊藻毒素的毒性研究大多集中在动物、植物上。微生物(细菌、放线菌、真菌)作为生态系统中的主要分解者和消费者,作为生源元素生物地球化学循环的推动者之一,在生态系统中起着重要的作用;其群落多样性的改变直接影响着水生态系统的结构和功能。本文选择革兰氏阴性和革兰氏阳性代表菌株大肠杆菌和枯草芽孢杆菌为实验材料,研究了微囊藻毒素对其生长及生理学效应。探讨了微囊藻毒素对微生物氮、磷循环中的主要功能类群反硝化细菌和有机磷细菌的生理功能的影响,以期了解微囊藻毒素对微生物在自然界物质循环中是否具有一定的生态学效应,同时也为蓝藻水华污染治理最终目的-生态系统的恢复提供一定的理论基础和依据。1.微囊藻毒素的制备以滇池微囊藻干粉为材料,采用常规的制备方法,大量制备并获得了一定纯度的粗毒素,经HPLC分析,粗毒素主要为MC-RR,纯度为23.58%。MC-RR纯品是粗毒素经制备型HPLC进一步纯化而获得。经HPLC进一步分析,纯度达95%以上,可用于常规的毒理学实验。2. MC-RR对大肠杆菌和枯草芽孢杆菌生长及生化特性的影响低浓度MC-RR处理下,大肠杆菌和枯草芽孢杆菌的生长、细胞活性和对照几乎没有差异,高浓度MC-RR能在较短时间内抑制大肠杆菌和枯草芽孢杆菌的生长和细胞活性,但这种抑制作用仅仅是一种短时效应,随着毒素暴露时间的延长,处理组和对照组的生长曲线几乎呈平行趋势,无明显差异。微生物细胞对毒素胁迫的响应也表现在细胞内可溶性蛋白与可溶性糖的含量改变上。实验结果表明,大肠杆菌和枯草芽孢杆菌在MC-RR处理之初,细胞内蛋白质含量和对照相比有增加的趋势,显著增加量出现的时间不同可能和细菌本身特性有关。但随着时间的延长,处理组细胞内可溶性蛋白的含量有所下降,这可能是因为微生物细胞逐渐适应了这种环境的胁迫,从而使细胞的代谢速率减慢,细胞内可溶性蛋白含量降低。由此可见细菌细胞内含物对环境毒素的胁迫具有一定的应答反应,在一定程度上说明微囊藻毒素对微生物的毒性效应。3. MC-RR对大肠杆菌和枯草芽孢杆菌细胞膜透性的影响研究表明,MC-RR能增加大肠杆菌和枯草芽孢杆菌对溶菌酶的敏感性和渗透性。MC-RR和溶菌酶的协同效应显示了MC-RR对细菌细胞膜渗透性的影响,并且结果显示,这种协同作用具有一定的剂量和时间效应。同时我们的试验还表明,MC-RR能明显促进大肠杆菌和枯草芽孢杆菌细胞内可溶性蛋白和可溶性糖含量的外渗,并且随着毒素浓度的增加,处理组可溶性蛋白和可溶性糖的外渗量和对照相比越明显。结果进一步证明了MC-RR对大肠杆菌和枯草芽孢杆菌细胞膜渗透性的影响,但是这种影响变化非常有限,在毒素处理1 h时,处理组细胞内可溶性蛋白和可溶性糖的含量几乎都达到高峰,随后的处理时间内,变化不明显。4. MC-RR对大肠杆菌的氧化胁迫低浓度MC-RR(0.1,1 mg/L)的处理组ROS含量在整个试验过程中和对照相比均无明显变化,说明不足以引起大肠杆菌细胞的氧化胁迫。而高浓度MC-RR(5,10 mg/L)处理组在1 h时,细胞内ROS含量均显著高于对照,表明细胞受到了氧化胁迫,这种氧化胁迫是由MC-RR引起的,但随着处理时间的延长,处理组ROS含量逐渐下降,恢复到和对照相似的水平,说明细菌通过自身系统的适应和调节逐渐消除了细胞内的活性氧。细胞内TBARS含量变化趋势类似于ROS,低浓度MC-RR不足以引起细胞的膜脂过氧化,整个实验过程中和对照无明显差异,但高浓度MC-RR(5,10 mg/L)处理1 h时,细胞膜脂过氧化程度显著增加,随后由于细胞自身的保护系统,TBARS含量逐渐降低恢复到对照水平。高浓度MC-RR处理下,SOD和CAT酶活性也显著升高,随后逐渐恢复到对照水平。低浓度毒素处理下和对照无明显差异。GSH含量和GR活性变化,均在低浓度MC-RR暴露1 h时低于对照,而在高浓度毒素暴露1 h时明显高于对照,随后逐渐恢复到对照水平。5. MC-RR对枯草芽孢杆菌的氧化胁迫低浓度MC-RR处理枯草芽孢杆菌1 h时细胞内SOD、CAT酶活性以及TBARS、GSH含量和对照相比无明显差异。高浓度MC-RR处理组SOD、CAT、GR酶活性以及TBARS、GSH含量均明显高于对照。10 mg/L MC-RR对枯草芽孢杆菌氧化胁迫的时间效应表明,MC-RR处理1 h时,SOD、CAT、GR酶活性以及TBARS、GSH含量均明显高于对照,但随着处理时间的延长,逐渐恢复到对照水平。6.反硝化细菌及有机磷细菌的分离纯化及鉴定从滇池水样之中分离到了两株反硝化细菌和有机磷细菌,根据形态学和16S rDNA序列分析对其进行了鉴定。结果表明,DN-3属于Bacillus gibsonii,DN-5属于Oceanobacillus iheyensis;P-1和P-2均属于Serratia marcescens.7. MC-RR对反硝化细菌及有机磷细菌生长及生理功能的影响研究结果表明,MC-RR能够抑制反硝化细菌的生长及其反硝化作用,并且随着浓度的增大,这种抑制效果越明显。但随着处理时间的延长,细菌逐渐抵抗了毒素的胁迫,恢复了生长,从而使处理组和对照组生长曲线均成上升趋于平行。MC-RR可能通过抑制反硝化细菌的生长和NR酶活性,从而抑制了培养液中硝态氮含量的减少、亚硝态氮含量的升高,说明MC-RR对反硝化细菌的功能具有一定的影响。高浓度MC-RR同样能够显著抑制有机磷细菌的生长,而低浓度处理组和对照无明显差异,并随时间延长逐渐恢复。MC-RR对ACP和AKP酶活性的影响类似,具有一定的剂量抑制效应。但仅在实验处理的24～48 h内处理组和对照组磷酸酶活性和0 h相比有所变化,随后的处理时间内变化均不明显。整体来看,处理组细胞内ACP和AKP酶活性均低于对照组,说明MC-RR抑制了磷酸酶的活性。高浓度MC-RR能够显著抑制磷细菌培养液中可溶性磷酸盐含量,说明抑制了有机磷细菌的解磷能力,这种解磷能力的降低可能主要归因于对细菌数量以及磷酸酶活性的抑制。更多还原

【Abstract】 Toxic cyanobacterial blooms in fresh water bodies have been becoming a kind of ecological disaster worldwide. One of the most harmful effects of cyanobacterial blooms is typically from the toxins produced by the algae,which represent a major threat to human, livestock and wildlife health. Of all the algal toxins, microcystins are among the most abundant and diverse. Most investigations into the toxicity of the microcystins are focused on animals and higher plants. In this paper the ecophysiological effects of microcystins on bacteria and its possible mechanism were studied. The main results are as follows:1. Microcystins from Microcystis bloom in Lake Dianchi were extracted and purified with high performance liquid chromatography (HPLC). MC-RRwith a purity over 95% was finally obtained by preparative HPLC.2. The growth and biochemical response of Escherichia coli (E.coli) and Bacillus subtilis (B. subtilis) to MC-RR were studied. The results showed that the growth and cell viability of bacteria were inhibited for a short period compared with that of the control when exposed to MC-RR. But the dose of MC-RR did not have a lethal effect. E.coli and B. subtilis only showed growth inhibition at the initial growth phase when cells were treated with MC-RR. Indeed the normal rate of growth was gradually re-established and the growth curves of the toxin-treated and untreated bacteria became parallel. The cell viability of E.coli and B. subtilis were also inhibited for a short period compared with that of the control which was similar to the growth of bacteria. The contents of protein and soluble sugar in cells increased compared with that of the control at the beginning of MC-RR exposure and then gradually decreased. The results showed the stress of MC-RR on E.coli and B. subtlis. The change of protein and soluble sugar make cells suit for the stress of MC-RR gradually.3. The permeabilising ability of MC-RR under different concentrations to the cell outer membrane of E. coli and B. subtilis was demonstrated by a rapid and sustained reduction in the OD675 values of lysozyme-treated cells. And the decrease of the absorbance values significantly showed a time- and dose-effect. The extravasations of protein and soluble sugar increased with the increment of the treated-concentration of MC-RR and the prolonged of the treated-time. The results showed that MC-RR could increase the permeability of cell outer membrane of E.coli and B.subtilis. Though the permeability effect of microcystins on cell outer membranes is still controversial and inconclusive our results once again showed that MC-RR could increase the permeability of cell outer membrane of E.coli and B.subtilis. And the synergistic effects of MC-RR and lysozyme on bacteria might indicate that MC-RR might play the ecological role on bacteria by combining with other substance in some aquatic environments.4. In the present paper, E.coli was undertaken to determine the effect of microcystin-RR on microbes. For this purpose, six biochemical parameters including reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione (GSH) were investigated in E.coli when exposed to 0.1, 1, 5, 10 mg/L MC-RR. With high concentrations MC-RR exposure, the results showed that the ROS level and TBARS contents of E.coli were obviously increased after 1 h exposure to MC-RR. At the same time, the activities of antioxidant enzymes including SOD and CAT as well as GR activity and GSH contents in bacterial cells were also increased when exposed to MC-RR for 1 h, subsequently decreased. These results suggested that MC-RR could cause the accumulation of ROS in E.coli and induce the oxidant stress for a short period. The antioxidant system protects E.coli from oxidative damage.5. B. subtilis was undertaken to determine the dose- and time- effect of MC-RR, and the results showed that the activity of antioxidant enzymes including SOD and CAT was significantly increased than that of the control when exposed to 5 or 10 mg/L MC-RR for 1 h. The contents of TBARS and GSH as well as GR activity were obviously increased only when exposed to 10 mg/L MC-RR. For the time-effect of MC-RR on B. subtilis, the activities of antioxidant enzymes including SOD and CAT as well as GR activity and TBARS, GSH content in B. subtilis were first significantly increased and than subsequently decreased. These results suggested that MC-RR could induce the oxidative stress of B. subtilis for a short period. The antioxidant system protects B. subtilis from oxidative damage.6. Two strains denitrifying bacteria and Organic phosphorus bacteria were isolated to further study the effect of MC-RR on the population of functional bacteria. From the morphological characteristics and results of 16S rDNA sequence analysis, the denitrifying bacteria were identified as Bacillus gibsonii and Oceanobacillus iheyensis, the organic phosphorus bacteria were identified as Serratia marcescens.7. The growth and denitrification function of denitrifying bacteria were prolonged by MC-RR. It showed a time- and dose-effect. The decrease of Nitrate and the increase of nitrite were both inhibited as a result of the growth and NR activity inhibition. Indeed with the toxin-treated time increased, the normal rate of growth was gradually re-established and the growth curves of the toxin-treated and untreated bacteria became parallel. The effect of MC-RR on organic phosphorus bacteria was similar to that on denitrifying bacteria. During all the experiment the enzyme activity of ACP and ALP were lower than that of the control and finally the content of phosphate in liquid medium decreased with the exposure of MC-RR.更多还原