【作者】 汤晓；

【导师】 孟琴；

【作者基本信息】 浙江大学 ， 生物化工， 2006， 硕士

【摘要】 铜绿假单胞菌被广泛用于原油等有机物的生物降解,其分泌的鼠李糖脂是一种用途广泛的生物表面活性剂,尤其在治理原油污染方面占有独特地位。目前鼠李糖脂的发酵多用各类植物油作为碳源,发酵周期一般需要一周左右,产量普遍只有10～30g/L。正是由于鼠李糖脂产量较低造成生产成本较高,使得它与化学表面活性剂相比,还无法实现大规模的工业化应用。本实验的目的在于寻找一株优良的铜绿假单胞菌,希望它具有较强的分泌鼠李糖脂以及生物降解原油的潜力,初步探索其在工业规模水平生产鼠李糖脂的可行性。 本文分六个部分。第一部分,综述了鼠李糖脂的生产和应用的现状,以及铜绿假单胞菌用于生物降解的发展,提出了本文的研究思路:筛选一株铜绿假单胞菌,考察其产鼠李糖脂及降解原油的双重特性,并提高其鼠李糖脂产量,缩短发酵周期,初步探索其在工业规模水平生产鼠李糖脂的可行性,同时观察该优化菌对原油的降解能力。 第二部分,研究筛选得的铜绿假单胞菌鼠李糖脂分泌与原油降解之间的相互关系。保存在原油培养基内的菌株被转接至甘油培养基进行若干次培养时,其产鼠李糖脂能力在最初几次培养时逐渐提高,并可维持若干次数,直至第七次培养起突然下降;而菌株从甘油培养基转接至原油培养基培养时,原油的乳化和降解能力也在最初几次培养时得到提高,直至第七次培养起显著下降。菌株利用甘油发酵生产鼠李糖脂时,产量可达12. 88g/L;同时能在较短的时间内降解胜利油田原油的许多组分,主要为中等碳链的烷烃(C₁₀-C₂₄)。 第三部分,通过紫外诱变提高鼠李糖脂产量。通过两次紫外诱变,并结合自然分离进行复筛,最终获得的高产变株经过一次甘油培养,产鼠李糖脂能力比出发菌株提高88. 4%,而且鼠李糖脂短时间内积累量较多。该变株在甘油培养基内连续培养三次,鼠李糖脂产量可获得较大提高。 第四部分,对培养条件进行了优化。经比较,以含量为3%（w/v）的菜油发酵较为合适。培养基经优化后,发酵120小时,鼠李糖脂产量可达48. 43g/L。利用菜油进行摇瓶发酵,到达发酵终点时,鼠李糖脂产量达56. 1g/L,菜油利用率为92. 3%。 第五部分,利用50L搅拌通气式反应器发酵鼠李糖脂。采取间歇补料的培更多还原

【Abstract】 Rhamnolipid secreted by Pseudomonas aeruginosa is a kind of biosurfactant with extensive use, and Pseudomonas aeruginosa is also widely used in the biodegradation of many organic pollutants. At present, various vegetable oils have been used in rhamnolipid biosynthesis. Generally, the production is 10~30 g/L and the fermentation period is around one week. Although rhamnolipid is very effective in the treatment of environmental pollution and will not be harmful to the environment, it cannot be widely used on industrial scale presently while comparing to chemical surfactants, for the consideration of economic cost. Therefore, high production of rhamnolipid with low material cost should be considered in order to apply this biosurfactant to industrial level.This thesis was divided into six sections. Section 1 reviewed rhamnolipid production and application, 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, as well as to improve its productivity of rhamnolipid and shorten its fermentation period.In Section 2, we discussed the relationship between rhamnolipid production and crude oil biodegradation after preservation in crude oil. While the strain preserved in crude oil-containing medium was transferred to glycerol medium for several passages, it expressed increased rhamnolipid productivity, which sustained for six passages and sharply decreased after the seventh passage. The rhamnolipid productivity was closely related to its biodegradability in crude oil-containing medium, which could be correspondingly sustained for six subcultures and then decreased sharply after the seventh passage. This strain could degrade many compositions in crude oil from Shengli Oil Field within a short period. The main compositions degraded were alkanes with intermediate chains and the rhamnolipidproduction in glycerol medium could reach 12.88 g/L.In Section 3, U.V. mutation was conducted to improve rhamnolipid production. After two times of U.V. mutation and the natural selection, a strain with high productivity was selected. The rhamnolipid production in glycerol medium was increased by 88.4% for the first passage and rhamnolipid accumulation was rapid. While this strain was subcultured in glycerol medium for three passages, its rhamnolipid productivity could be increased.In Section 4, the culture medium was optimized. Rapeseed oil was selected as the carbon source. After optimization, rhamnolipid production could reach 48.43 g/L within 120h. It reached to 56.1 g/L at the end of fermentation and 92.3% of rapeseed oil was used.In Section 5, rhamnolipid was produced in a bioreactor. The fed-batch fermentation was conducted and a foam recycling system was set. The strain had a rapid growth and a high cell concentration. Rhamnolipid was accumulated rapidly and the production could reach 59.28 g/L. The recovery ratio of the product was 72.9% with a purity of 76.89%.Finally, this paper was summarized in Section 5. And the genetic research of improving the rhamolipid productivity and crude oil biodegradability by preservation in crude oil, as well as rhamnolipid production by using frying oil was prospected in the end.更多还原