【作者】 姚斌；

【导师】 孟琴；

【作者基本信息】 浙江大学 ， 生物化工， 2007， 硕士

【摘要】 随着经济的快速发展和对能源需求的增加，海上或沿海一带原油污染事件频频发生，它已经成为环境污染的一种重要来源。由于原油难以被微生物摄取，再加上微生物易受高盐度海水的抑制，使得原油在海上的降解速率很慢，因此我们需要选育出能耐高盐环境并产表面活性剂的微生物来促进原油降解和环境修复。铜绿假单胞菌（命名为P．aeruginosa ZJU）是一种既能分泌鼠李糖脂又能降解原油的优良菌种，目前对它的研究主要集中在低盐度环境下铜绿假单胞菌用于原油废水和污泥的生物降解，而对高盐度环境下的原油降解研究比较少。因此，本实验旨在于寻找一株优良的铜绿假单胞菌，希望其能适应高盐度的环境，同时又具有较强的分泌鼠李糖脂以及降解原油的能力，并初步探索其在高盐度环境下降解原油的可行性。本文包括以下几个内容：首先，综述了原油污染的现状，铜绿假单胞菌用于生物修复的优势和特点，提出了本文的研究思路：通过驯化筛选出一株铜绿假单胞菌，评价其在高盐度下产鼠李糖脂及降解原油的双重特性，然后分析影响原油降解的几个主要因素，初步探索该菌在高盐度环境下降解原油的应用可行性。其次，利用S35合成海水作为驯化培养基，使铜绿假单胞菌在高盐度环境下长期驯化，分离得到驯化后的铜绿假单胞菌（命名为P．aeruginosa ZJU-X），考察驯化前后铜绿假单胞菌在生长以及鼠李糖脂产量方面的变化。结果发现P．aeruginosa ZJU-X能够很好地适应高盐度环境。而海水中高浓度的钙盐和镁盐在一定程度上抑制了铜绿假单胞菌的生长和鼠李糖脂的分泌。以菜油为碳源，驯化后的铜绿假单胞菌在S25、S35和S40合成海水培养基中的鼠李糖脂产量分别达到了14.6g／L、11.8g／L和10.8g／L。然后考察了鼠李糖脂发酵液的生物表面活性。鼠李糖脂发酵液的临界胶束浓度（CMC）只有24.4mg／L，且鼠李糖脂主要由Rha-C₁₀C₁₀和Rha-Rha-C₁₀C₁₀这两种主要成分构成。通过比较原油的最短乳化时间，可以看到，相同发酵液（含鼠李糖脂）用量所产生的对原油的乳化效果要明显好于化学表面活性剂十二烷基硫酸钠（SDS）和石油磺酸盐。最后，分析了盐度和初始原油浓度对P．aeruginosa ZJU-X降解原油的影响。原油降解率随盐度增加而降低，同样，对高浓度原油的降解率也比较低。通过气相色谱分析，发现当原油浓度低于0.5％（w／v）时，P．aeruginosa ZJU-X能在7天内明显降解原油，而当原油浓度高于1.0％（w／v）时，P．aeruginosa ZJU-X降解原油的效果欠佳。通过高盐度驯化后，菌株P．aeruginosa ZJU-X明显改善了对原油的降解效果，例如，在原油浓度为0.5％（w／v）时，其降解率达到了61.8％。总之，本论文进行了高盐环境下原油降解的探索性研究，而实际应用还有待于今后的进一步开发研究。更多还原

【Abstract】 With the rapid demands and transportations of crude oil, oil spills have frequently occurred and put enormous pollution into the marine environment. The spilled oil are slowly degraded due to the low bioavailability as well as the harsh saline environment for the microorganism. In these situations, the most important requirement for bioremediation is the presence of hydrocarbon-degrading microorganisms, which could survive in the high saline environment and produce biosurfactant.Pseudomonas aeruginosa ZJU is such a typical hydrocarbon-degrading microorganism used for biodegradation of crude oil in the wastewater or contaminated soil. Therefore, P. aeruginosa with high ability of crude oil degradation and rhamnolipid production and surviving in high salinity condition should be preferred for practical application in seashore or in the sea.This thesis can be divided into the following sections:First, we reviewed the treatments of the spilled oil and the advantages of Pseudomonas aeruginosa for biodegradation, as well as the use of Pseudomonas aeruginosa in biodegradation. At the end of this section, the research outline was illustrated for selecting a strain of Pseudomonas aeruginosa to study its characteristics of rhamnolipid production and crude oil biodegradation under high salinity environment, as well as the factors determining the biodegadation process.Secondly, P. aeruginosa ZJU was acclimated by using the S35 synthetic seawater, then the acclimated P. aeruginosa ZJU （named P. aeruginosa ZJU-X） was separated. By comparing the cell growth and rhamnolipid excretion between P. aeruginosa ZJU-X and P. aeruginosa ZJU, we concluded P. aeruginosa ZJU-X could survive in the high salinity condition. However, the high concentration of Ca²⁺ and Mg²⁺ showed negative effect on the cell growth and rhamnolipid production. P. aeruginosa ZJU-X could obtain a maximal productivity of rhamnolipids at 14.6g/L、11.8g/L and 10.8g/L in vegetable oil-culture medium varied with high salinity of 25‰、35‰ and 40‰, respectively.Then, the characteristics of the rhamnolipids were analyzed. The rhamnolipidsproduced by P. aeruginosa ZJU-X were identified with two major components, Rha-Q（10）C（10） and Rha-Rha-C（10）C（10）. The Critical Micelle Concentration （CMC） of the cell-free culture 24.4mg/L. The crude oil emulsification ability of rhamnolipid curlture broth was much better than the chemical surfactant sodium dodecyl sulfate （SDS）.Last, the effect of salinity and the concentration of crude oil on the biodegradation were investigated. The extent of biodegradation of crude oil declined with the increase of salinity and the concentration of crude oil. When the concentration of crude oil was below 0.5%（w/v）, P. aeruginosa ZJU-X could degrade a majority components of the crude oil. However, the degradation of crude oil was largely inhibited with crude oil concentration over 1.0%（w/v）. By analysis of the residual crude oil, we concluded that P. aeruginosa ZJU-X degraded crude oil more thoroughly than P. aeruginosa ZJU at high saline environment.更多还原