【作者】 吴庆；

【导师】 马润宇； 丛威；

【作者基本信息】 北京化工大学 ， 生物化工， 2004， 硕士

【摘要】 随着海洋资源的利用越来越受到重视，海藻作为海洋资源的重要来源之一，对海藻的人工养殖和培养，以及从其中提取各种生物活性物质，特别是多不饱和脂肪酸被证明有多方面的生理功能，这方面的研究成为世界各国的热点。本文初步研究了微藻的生物反应器培养和从微藻中提纯富集多不饱和脂肪酸的工艺和方法。从一种新型的黄绿色单细胞超微型藻Nannochloropsis sp.藻种出发，研究了海藻从在摇瓶培养到20L光生物反应器中培养。并且采用尿素包合法对海藻多不饱和脂肪酸进行富集。以期为海藻多不饱和脂肪酸的利用提供新思路和途径。（1）Nannochloropsis sp. 藻细胞的培养，确定了培养条件为接种浓度63±3 mg/L（干重），通气流量为400 ml min-1，光强为70.8 μmol photon m-2 s-1，培养温度23±0.2℃。研究了碳源对藻细胞生长的影响，特别是加入葡萄糖作为碳源对生物量有大幅提高。经研究发现Nannochloropsis sp.藻细胞在3L气升式内环流光生物反应器中混养条件培养时，培养8天后细胞浓度可达857.1 mg DW l-1，为摇瓶培养的2倍（432.9 mg DW l-1）。在扩大培养研究中，Nannochloropsis sp.藻细胞在20L光生物反应器混养条件下培养获得生物量为633.1 mg l－1。但其延滞期较3L气升式内环流光生物反应器培养更长。由此可见，Nannochloropsis sp.的扩大培养存在一定的适应期，其大规模培养存在一定的困难。对细胞生长进行模型模拟，所得模型可以较好的反应细胞的生长情况。（2）从海藻细胞中提纯富集多不饱和脂肪酸的研究，选定了研磨法破碎藻细胞、石油醚－乙醚混合溶剂萃取海藻粗油、以甲醇－KOH作为皂化试剂的实验工艺。所得细胞破碎率可达65％；对溶剂萃取海藻粗油的各影响因子作正交实验，得出实验最佳提取工艺条件为乙醚和石油醚的体积比为1：2、提取温度20℃、提取时间5h时，并分析了各因素对提纯影响的差异；对皂化后所得海藻油<WP=5>脂作气相色谱分析，色谱峰分离效果好，无峰拖尾现象发生。尿素包合法提纯多不饱和脂肪酸，可以基本除去海藻粗油中的饱和脂肪酸和低不饱和脂肪酸。着重对尿素浓度这一关键因素对提纯效果的影响。实验研究得出，在尿素－甲醇质量分数为20％时，提纯富集的总多不饱和脂肪酸占总脂的约90％。更多还原

【Abstract】 With the use of ocean resources being more and more paid attentions, the research of algae’ cultivation and purifying it’s biological active substance, especially the found of the PUFAs’ physiological function being proved by the scientists, has become the hotspot in the world widely, as the algae is one of the most important resources in the ocean.The article primarily explores the cultivation of a microalgae which is a new style Kelly single microalgae Nannochloropsis sp., in flask 、3l and 20L photobioreactor and the extraction and enrichment of Polyunsaturated fatty acids (PUFAS), which were 16:2、16:3、18:2 and 20:5 fatty acids(e.g. 16:2 means there were 16 carbons and 2 double bounds in the carbon chain of the fatty acid) by urea complexion, in order to provide new ways for the use of algae PUFAs.(1) In the cultivation of the Nannochloropsis sp., cultivation condition is inoculation concentration 63±3 mg/L(dry biomass weight), ventilation flux 400 ml min-1, and luminous intensity 70.8 μmol photon m-2 s-1. It has been found that the carbon resources influence the biomass in the cultivation, and adding glucose can enhance the biomass obviously. Through the research , the biomass in the 3L airlift circumfluence photobioreactor can reach 857.1 mg DW l-1 after 8d by mix-cultivate. In the scale cultivation in 20L airlift photobioreactor, the biomass is 633.1 mg l－1 in the 10d by mix-cultivate. So scale cultivation of the Nannochloropsis sp. still has limits. And for the biomass in the flask、<WP=7>3L photobioreactor and 20L photobioreactor, the models for the cell growth has been built. The values according to the models are answered for the values measured in the experiments. (2) In the research of PUFAs’ extraction and enrichment from the algae Nannochloropsis sp.. in the experiment we choose grinding for cytocrosis, petroleum benzin-ether for extraction and methanol-KOH for saponification. The ratio value of the cytocrosis can reach 65%, and in the extractive experiment, the best technological conditions we select are V(petroleum benzin)/V(ether) 2/1, temperature 20℃ and extraction for 5h. There is good result of gas chromatograph of the lipid, without tailed peak and time-lag in the chromatograph peak.In use of the method of urea complexion to purify the PUFAs, the saturated and low-unsaturated lipids can be removed well. We put the emphasis in the concentration of the urea-methanol, and when the mass concentration is 20%, The mass of the PUFAs purified is about 90% of the total lipid extracted.更多还原