大肠杆菌对有机溶剂的耐受性研究

发布时间：2018-03-15 18:24

本文选题：大肠杆菌　切入点：有机溶剂　出处：《天津科技大学》2008年硕士论文　论文类型：学位论文

【摘要】： 在有机溶剂／水两相体系生物转化技术应用中,有机溶剂对细胞的毒害是其中的重要障碍。研究有机溶剂对细胞的毒害及微生物对有机溶剂的耐受机制,对改造、选育耐有机溶剂的菌株,增强在生物转化中的应用有着重大意义。本文以大肠杆菌(Escherichia coli) K12为模式菌株,研究了在有机溶剂／水两相系统中,有机溶剂甲苯、四氯化碳、正己烷、邻苯二甲酸二乙酯对E.coli K12生长和E.coli K12细胞膜的影响,初步探讨了E.coli K12对有机溶剂的耐受机制。通过研究甲苯、四氯化碳、正己烷、邻苯二甲酸二乙酯对E.coli K12生长的影响可以得到以下结果： ①E.coli K12对四种logP值不同的有机溶剂的耐性不同：对正己烷耐性较强,依次是邻苯二甲酸二乙酯、四氯化碳,对甲苯的耐受性最弱。 ②大肠杆菌对不同浓度的有机溶剂耐受性不同：在1.5%、1.0%、0.3%(v／v)的甲苯、四氯化碳、正己烷、邻苯二甲酸二乙酯存在下,可以看到明显的OD值变化的差异。 ③在0.3%的甲苯、四氯化碳等溶剂存在下,大肠杆菌存活率随作用时间延长而降低,甲苯尤其明显。在添加0.3%的甲苯30mmin后,大肠杆菌的存活率仅为24.8%。采用罗丹明123(Rh123)、二乙酸荧光素(FDA)荧光法测定在0.3%的甲苯、四氯化碳等有机溶剂存在下,大肠杆菌细胞膜电位、细胞膜通透性的变化。结果发现在添加0.3%的四氯化碳30mmin后,Rh123和FDA荧光强度下降了16.3%、34.5%,即相当于膜电位下降了16.3%,膜渗透性增加了34.5%。当培养液中添加0.3%的甲苯时,FDA荧光强度下降65.9%；表明细胞膜完整性受到严重破坏。用气相色谱法研究了大肠杆菌细胞膜脂肪酸组成的变化,发现在邻苯二甲酸二乙酯、四氯化碳存在的情况下,细胞膜脂肪酸组成中饱和脂肪酸的含量增加。尽管甲苯对大肠杆菌的抑制作用最强,但甲苯对膜脂肪酸组成影响却不明显。脂肪酸饱和度的增加改变了细胞膜的流动性,增强了对有机溶剂的耐受性。
[Abstract]:In the application of organic solvent / water biphasic biotransformation technology, the toxicity of organic solvent to cells is an important obstacle. It is of great significance to select strains resistant to organic solvents and enhance their application in biotransformation. In this paper, the organic solvents toluene, carbon tetrachloride, n-hexane in organic solvent / water two-phase system were studied using Escherichia coli K12 as the model strain. The effects of diethyl phthalate on the growth of E. coli K12 and the cell membrane of E. coli K12 were studied. The tolerance mechanism of E. coli K12 to organic solvents was studied. By studying the effects of toluene, carbon tetrachloride, hexane and diethyl phthalate on the growth of E. coli K12, the following results were obtained:. The tolerance of E. coli K12 to four organic solvents with different logP values was different: the tolerance to n-hexane was stronger, followed by diethyl phthalate, carbon tetrachloride and the weakest tolerance to toluene. (2) the tolerance of Escherichia coli to different concentrations of organic solvents was different: in the presence of toluene, carbon tetrachloride, hexane and diethyl phthalate, significant differences in OD values could be seen in the presence of 1.5 ~ 1.0 and 0.3% v / v) of toluene, carbon tetrachloride, hexane and diethyl phthalate. 3 in the presence of 0.3% toluene, carbon tetrachloride and other solvents, the survival rate of Escherichia coli decreased with the prolongation of time, especially toluene. After adding 0.3% toluene for 30 minutes, the survival rate of Escherichia coli was only 24.8%. The membrane potential of Escherichia coli in the presence of 0.3%% toluene, carbon tetrachloride and other organic solvents was determined by rhodamine 123H Rh 123H and fluorescein diacetate fluorimetry. The change of membrane permeability. The results showed that the fluorescence intensity of Rh123 and FDA decreased by 16.3% and 34.5mm after adding 0.3% carbon tetrachloride for 30mmin, that is, the membrane potential decreased 16.3% and membrane permeability increased 34.50.When 0.3% toluene was added to the culture medium, the fluorescence of FDA was increased. The intensity decreased by 65.9, indicating that the integrity of the cell membrane was seriously damaged. The change of fatty acid composition of E. coli cell membrane was studied by gas chromatography. It was found that in the presence of diethyl phthalate and carbon tetrachloride, Although toluene had the strongest inhibitory effect on Escherichia coli, toluene had no obvious effect on membrane fatty acid composition. The increase of fatty acid saturation changed the fluidity of cell membrane. Enhanced tolerance to organic solvents.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378

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