产生物表面活性剂菌株的筛选、鉴定及其产物的初步研究-生物化学与分子生物学专业论文.docxVIP

曲阜师 曲阜师范大学 2009 级硕士学位论文 I I 摘 要 本研究以大庆油田地下 3000 米的原油为研究材料，通过血平板以及排油圈实验，筛 选得到了两株能产生物表面活性剂的菌株，分别命名为 DQ-1 和 DQ-2。通过 16S rRNA 亲 缘关系 鉴定 以及 系统 进化树 的构 建， 初步 认定 DQ-1 为铜 绿假 单胞 菌（ Pseudomonas aeruginosa），DQ-2 菌株属于芽孢杆菌属（Bacillus）。以 DQ-1 为研究对象，对菌株的生理 学特性进行分析：通过在不同温度下的生长实验，发现 DQ-1 菌株的最适生长温度 40-45℃， 其中在 42℃生长最快；但是当培养温度达到 50℃时，菌株的生长能力显著降低。结果表 明，该菌株具备一定的耐高温生长能力。pH 梯度实验结果表明，DQ-1 的最适生长 pH 范 围为 7-10，在 pH=11 的条件下仍然能够生长，表明 DQ-1 具备一定的耐碱生长能力，耐碱 范围较广，推测与其生长环境相关。 为了鉴定DQ-1所产生的生物表面活性剂的类型，在本研究中，以廉价的甘油作为碳源， 以发酵法制备了DQ-1所产的生物表面活性剂；通过离心、酸化、萃取、旋蒸、TLC层析等 工艺提取纯化了发酵过程中产生的生物表面活性剂；最后，通过液相色谱和液质联用 （HPLC-MS）鉴定出DQ-1产生的生物表面活性剂就是鼠李糖脂，这与目前文献报道的铜 绿假单胞菌所产生的生物表面活性剂类型一致。 关键词：生物表面活性剂；铜绿假单胞菌；芽孢杆菌；鼠李糖脂 II II Abstract In this study, several strains were isolated from crude oil of Da Qing oil field. By using blood-plate together with oil displacement method, two strains which could synthesize bio-surfactant were obtained and designated DQ-1 and DQ-2, respectively. Results of 16S rRNA alignment and constructed phylogenic tree revealed that DQ-1 was Pseudomonas aeruginosa, while DQ-2 belonged to Bacillus genus. DQ-1 was taken as research object in the following experiment for characteristic assay. When cultured in different temperatures, DQ-1 grew well from 40℃ to 45℃, and exhibited maximal growth under 42℃, which means DQ-1 was somewhat temperature-tolerant. In order to study whether DQ-1 could grow in extreme acid or alkali environments, growth rate of DQ-1 under different pH conditions was measured. Results showed that the optimal pH of DQ-1 fluctuated from 7 to 10, which means DQ-1 was of alkali-resistance. We concluded that all of these characteristics enabled DQ-1 to adapt to its habitat environments effectually. Bio-surfactant synthesized by DQ-1 was identified in the following research. By utilizing glycerol as carbon resource, DQ-1 could produce plenty of bio-surfactant during fermentation. Bio-surfactant was collected by centrifugation, precipitated via acidification, extracted through ethyl acetate