微囊藻毒素-LR对降解菌Pseudomonas putida细胞影响的研究

发布时间：2017-12-31 16:33

本文关键词：微囊藻毒素-LR对降解菌Pseudomonas putida细胞影响的研究　出处：《暨南大学》2015年硕士论文　论文类型：学位论文

【摘要】：微囊藻毒素-LR(MC-LR)是富营养化污染的次生代谢产物,具有很强烈的生物毒性。本文利用前期分离驯化得到的MC-LR降解菌恶臭假单胞菌(Pseudomonas putida)为研究对象,考察了MC-LR对菌体细胞表面特性的影响,并探讨其对细胞活性、菌体自身代谢和调节能力以及菌体形态的作用,对MC-LR的微生物毒性作用进行了初步的探索。P.putida细胞质膜通透性因为MC-LR的浓度而发生改变,相对较高浓度的MC-LR会使菌体细胞质膜通透性持续上升。高浓度(2.5 mg·L-1)MC-LR作用下,P.putida细胞出现破裂死亡情况,导致胞内物质流出,细胞完整性遭到破坏,同时影响离子在细胞膜两侧的分布,发生去极化现象,使细胞膜电位降低。MC-LR能够影响P.putida的生物活性,加速菌体细胞的死亡。Biolog碳源实验表明,MC-LR作用后的P.putida对碳源的整体利用能力明显弱于对照菌体,其毒害作用使得P.putida代谢能力减弱,活性降低。流式细胞实验检测发现,在MC-LR毒性作用下,菌体细胞活性下降并发生凋亡,2.5 mg·L-1 MC-LR作用5 d,其死亡率比未添加MC-LR的对照体系增加了近30%,体系中菌体生物量持续下降。实验结果表明,MC-LR能够影响菌体离子代谢及可溶性蛋白等物质的分泌。当MC-LR质量浓度由0增大到2.5 mg·L-1时,能促进菌体分泌和释放可溶性糖及Na+、Cl-等离子。通过对P.putida SOD酶和ATPase酶活性测定可知,低浓度的MC-LR能够诱导酶活性的增强;高浓度的MC-LR则对菌体抗氧化和新陈代谢能力都有破坏性的影响,并激发产生活性氧自由基,使P.putida遭受氧化胁迫,对SOD酶和ATPase酶有强烈的抑制作用,使得酶活性总体水平不断降低。扫描电镜和原子力显微镜观察表明,在MC-LR作用下,菌体细胞的微观形貌发生了改变,细胞褶皱加深,在2.5 mg·L-1 MC-LR存在下培养5 d后,大部分细胞破裂,细胞内含物流出,细胞结构损坏严重。微观形貌观察实验也验证了MC-LR对菌体细胞的影响,即MC-LR对P.putida具有一定的毒害作用,且MC-LR浓度越大,作用时间越长,细胞损伤越严重。
[Abstract]:Microcystin -LR (MC-LR) is a secondary metabolite of eutrophication, has very strong biological toxicity. MC-LR degrading bacterium Pseudomonas putida by early isolation and domestication of the (Pseudomonas putida) as the research object, the effects of MC-LR on cell surface properties, and its effect on the cell activity. The cell metabolism and regulation ability and cell morphology, microbial toxicity to MC-LR made a preliminary exploration of.P.putida cell membrane permeability for MC-LR concentration change, relatively high concentration of MC-LR can make the cell membrane permeability increased. The high concentration (2.5 mg L-1) MC-LR under the action of rupture of death P.putida cells, resulting in intracellular substance out of cell integrity is destroyed, while the impact of ion distribution in the cell membrane, occurrence of depolarization phenomenon, cell membrane Potential decreased.MC-LR can affect the biological activity of P.putida,.Biolog showed that accelerated the death of carbon source experimental cell, the overall utilization ability of MC-LR after the action of P.putida on carbon source was weaker than that in the control cells, the toxicity of the metabolic capacity of P.putida decreased, the activity reduced. Flow cytometry assay showed that the toxicity of MC-LR. Cell activity decreased and apoptosis, 2.5 mg - L-1 MC-LR 5 d, the mortality rate increased by nearly 30% compared with the control system without the addition of MC-LR, the biomass decreased continuously in the system. The experimental results show that MC-LR can affect cell metabolism and ion secretion of soluble protein substances. When MC-LR concentration increases from 0 2.5 mg - L-1, can promote the secretion and release of soluble sugar and Na+, Cl-. Through the determination of the plasma cell of P.putida SOD enzyme and ATPase enzyme activity, low concentration of MC-LR can induce The enhanced enzyme activity; high concentrations of MC-LR on antioxidant ability of bacteria and The new supersedes the old. with damaging effects, and stimulate the production of oxygen free radicals, P.putida suffered oxidative stress, has a strong inhibitory effect on SOD enzyme and ATPase enzyme, the overall level of enzyme activity decreased rapidly. Scanning electron microscopy and atomic force microscope observation showed that in the presence of MC-LR changed cell morphology, cell fold deepened, in 2.5 mg L-1 the presence of MC-LR after 5 d culture, most cells rupture, cells containing the logistics, cell structure damaged seriously. The microscopic morphology observation experiments verified the effect of MC-LR on the cell. MC-LR has a toxic effect on P.putida and MC-LR, the greater the concentration, the longer the time, the cell damage was more serious.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X172

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