纳米银对念珠藻的毒性效应及其机理研究

发布时间：2018-03-10 18:32

  本文选题：纳米银　切入点：银离子　出处：《华南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：纳米银(AgNPs)是目前国内外生产和使用量最大的纳米材料之一,由于AgNPs具有多种优越的性能,被广泛应用于工业生产、医疗卫生和环境保护等领域。AgNPs可能在产品生产、使用和回收等环节中通过多种途径进入到水体环境中,对水生生态系统中的非靶标生物造成潜在威胁。关于AgNPs对一些微藻和微型水生动物的毒性已经有很多的报道,但是关于AgNPs对具有重要生态功能的丝状固氮蓝藻影响的研究鲜有报道。同时,关于AgNPs对微型藻类的毒性效应机理存在争议。因此,本论文选用念珠藻(Nostoc sp.)为实验材料,研究用柠檬酸钠还原法制备的AgNPs和银离子(Ag+)对念珠藻的急性和亚急性毒性效应,试图从藻形态结构、生理、生化改变等角度揭示AgNPs对丝状固氮蓝藻-念珠藻的致毒机理。主要研究结果如下:急性毒性实验(4d)中,在0~48h内,AgNPs和Ag+处理后,培养液中溶解态银浓度均逐渐下降并趋于平衡。AgNPs处理的小颗粒银浓度在0~6h内急剧下降,但在24h后趋于稳定。Ag+处理的小颗粒银浓度在0~12h内几乎没有变化,但到12h后显著升高到24h达到最高。显微观察发现高浓度的AgNPs和Ag+均可引起细胞收缩、质壁分离、胞内物质紊乱和细胞壁裂解等现象,AgNPs处理后疑似有银颗粒进入了细胞内部。所有浓度AgNPs和Ag+处理4d后的念珠藻干重、Chla含量、藻体TC含量、藻体碳/氮比和固氮酶活性均显著低于CK,而藻体总氮含量(TN)均显著高于对照。AgNPs处理后,培养液中碳/氮比均显著高于CK,而0.04 mg·L-1 Ag+处理组碳氮比(C/N)低于CK,但0.06 mg·L-1 Ag+处理则与CK比无显著差异。Ag+处理4d后,藻叶绿素荧光参数值Fm、Fv/Fm、Ψ0、ΦE0和PIABS均显著低于CK,而ΦD0、ABS/RC、TR0/RC和DI0/RC均显著高于CK,特别是ABS/RC和DI0/RC增加了97%和126%。但0.6mg·L-1AgNPs处理仅F0、Fm和ΦD0显著降低,而Fv/Fm、Ψ0、ΦE0和PIABS显著升高。同时,0.4 mg·L-1AgNPs处理所有参数均与CK无显著差异。说明短期作用后,Ag+和AgNPs均对藻产生胁迫作用,但AgNPs对藻光合系统II的影响比Ag+小。亚急性毒性实验(9d)中,仅高浓度AgNPs和Ag+处理使念珠藻干重和Chla含量显著低于CK。Ag+处理均使藻体C/N显著降低,但AgNPs处理无影响。0.6mg·L-1AgNPs处理使培养液中C/N、藻体可溶性蛋白和藻胆蛋白含量显著高于CK,其余处理组无显著变化。Ag+显著降低念珠藻固氮酶活性,AgNPs则对固氮酶活性无显著影响。处理9d后藻细胞蛋白二维电泳记录到超过200个差异蛋白点,选择与CK表达差异超过2.5倍以上的132个点进行了蛋白定性,只匹配到51蛋白质,扣除12个重复的蛋白,总共定性差异蛋白39个。当0.06 mg·L-1 Ag+处理后,藻细胞中26个蛋白显著上调,但6种蛋白的表达下调,而0.6 mg·L-1 AgNPs处理时,22个蛋白显著上调,3个显著下调。对两种形态银处理,上调蛋白中有相同蛋白10个,下调蛋白中有1个相同,但唯有一种在光合作用的卡尔文循环中起关键作用蛋白-转酮醇酶在AgNPs处理时显著上调,Ag+处理时显著下调。同时,Ag+处理抑制固氮酶表达,但AgNPs处理没有变化,这与前述乙炔还原法测定到固氮酶活性结果一致。AgNPs处理时别藻蓝蛋白上调,但Ag+处理未发现变化,这与光度法测得结果一致。另外,Ag+使微藻抗氧化的重要酶类-超氧化物歧化酶上调,但是AgNPs处理没有这种变化。Ag+处理9d后,藻叶绿素荧光参数值F0、Fm、Fv/Fm、Ψ0、ΦE0、PIABS和ET0/RC均显著高于对照,但ΦD0、VJ、ABS/RC、TR0/RC和DI0/RC值均显著低于CK,特别是ABS/RC和DI0/RC两个参数值分别降低超过70%。但对于AgNPs,仅0.4 mg·L-1和0.6 mg·L-1处理F0值和0.6 mg·L-1处理Fm值显著高于CK以外,其他参数均与CK无显著差异。这与急性实验的参数值变化趋势几乎完全相反。上述结果说明AgNPs和Ag+对念珠藻细胞光合作用、固氮作用、蛋白合成的影响存在明显差异。另外,实验结果表明随AgNPs和Ag+浓度的增加,藻体银含量增加,0.6 mg·L-1 AgNPs处理时藻到富集量最大,且同种处理方式下藻体银含量在第9d的含量均低于第4d。结合上述生长和生理生化数据,说明念珠藻在短期受到高浓度AgNPs和Ag+的毒害比较严重,但从处理9d后的生理生化指标变化的结果来看,念珠藻对Ag+和AgNPs均产生了相当程度的适应,但对两种形态银的适应策略存在明显差异。
[Abstract]:Silver nanoparticles (AgNPs) is one of the largest domestic production and consumption of nano materials, because AgNPs has many excellent properties, is widely used in industrial production, health and environmental protection in areas such as.AgNPs may in the production, use and recycling and other aspects through a variety of ways to enter the aquatic environment, causing potential the threat to the aquatic ecosystem in non target organisms. On the toxicity of AgNPs to some microalgae and micro aquatic animal that there have been many reports, but about AgNPs of filamentous cyanobacteria affect important ecological function has not been studied. At the same time, a toxic effect mechanism of AgNPs on micro algae is controversial. Therefore, this thesis selection of Nostoc (Nostoc sp.) as experimental materials, research using sodium citrate reduction method to prepare AgNPs and silver ion (Ag+) on acute and subacute toxicity effect of Nostoc Should try to from the algae morphology, physiological, biochemical changes of filamentous cyanobacteria reveal AgNPs - Nostoc toxicity mechanism. The main results are as follows: acute toxicity test (4D), in 0~48h, AgNPs and Ag+ after treatment, the concentration of dissolved silver culture was gradually decreased and tended to be small particles of silver concentration decreased sharply in balance with.AgNPs 0~6h, but in 24h tends to small Ag concentration and stable.Ag+ treatment in 0~12h almost no change, but 12h increased significantly to the highest 24h. Microscopic observation results showed that high concentration of AgNPs and Ag+ could induce cell contraction, plasmolysis, cell substances in disorder and cell wall cracking phenomenon, after AgNPs treatment of suspected silver particles into the interior of the cell. All the concentrations of AgNPs and Ag+ 4D after the treatment of Nostoc dry weight, Chla content, TC content of algae, algae carbon / nitrogen ratio and nitrogenase activity were significantly lower 浜嶤K,鑰岃椈浣撴，

本文编号：1594546