【作者】 赵春海；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 微生物学， 2011， 博士

【摘要】 近些年研究表明生物柴油是一种清洁、可降解、可再生生物能源,现在的引擎无需改造就可以直接使用生物油脂作为燃料,并且产生更少的有害气体,环境污染和能源危机使得生物柴油备受关注。目前生物油脂的高额成本是其工业化生产的主要障碍。寻求廉价原料是降低生物柴油成本的关键。我们实验室以前的研究中发现胶红酵母(Rhodotorula mucilaginosa)TJY15是一种产油脂酵母菌。以胶红酵母(R. mucilaginosa)为实验菌种,菊粉水解液为原料,分批培养时该酵母细胞干重和油脂的含量分别为14.8g/L和48.8%;以菊芋根块的水解液为底物分批发酵和分批补料发酵时该酵母细胞干重和油脂的含量分别为14.4 g/L、48.6%和19.47g/L、52.2%。对所产脂肪酸分析表明C16:0, C18:1和C18:2含量较高,三者之和占总脂肪酸的87.6%,说明以菊芋原料经过该酵母转化产生的油脂脂肪酸组成与植物油脂脂肪酸组成类似。为了建立直接转化菊糖生产油脂的工艺,把季也蒙毕赤酵母(Pichia guilliermondii)M-30细胞进行固定化,固定化的突变株M-30细胞与游离的胶红酵母细胞混合培养,研究结果发现适合季也蒙毕赤酵母M-30包埋的最佳条件是:聚乙烯醇7%、海藻酸钠1.4%、pH8.0、氯化钙3%。季也蒙毕赤酵母M-30在固定化培养中酶活达到169.3% U/mL,对照游离细胞酶活只有124.3 U/mL。胶红酵母和季也蒙毕赤酵母M-30联合培养,以菊粉、菊芋提取物为底物,经过48h混合培养,胶红酵母油脂和细胞干重分别为53.16%、12.24g/L;55.43%、12.83 g/L。经过48h发酵罐混合培养,胶红酵母油脂产量达到最高值56.57%,细胞干重达到19.64 g/L,发酵培养的胶红酵母细胞中的C16:0, C18:1 ,C18:2这三种脂肪酸占总脂肪酸的90.0%。为了建立一步发酵菊糖产生油脂的工艺,将来源于马克斯克鲁维酵母(Kluyveromyces marximum)CBS 6556的酶基因INU1在产油脂解脂亚罗威亚酵母中表达:首先将菊粉酶基因INU1与表达质粒pINA1317连接,并在产油脂解脂亚罗威亚酵母(Y. lipolytica) ACA-DC50109中进行了功能表达,得到了重组子Z31。Z31培养72 h经测定粗酶液酶活力为41.7±0.4 U/mL。优化了Z31的产油脂条件,选择5%糖(菊芋)为碳源,氮源选择硫酸铵和酵母膏,且C/N为350,即硫酸铵0.017%和酵母膏0.0355%,发酵78 h油脂达到峰值,油脂和细胞干重分别为46.3%和11.6 g/L。通过2-L发酵罐分别以菊粉和菊芋提取物为底物发酵78h菌体产油量分别为48.3%和50.57%,菌体干重分别达到13.3 g/L和14.63 g/L, C16:0, C18:0 ,C18:1这三种脂肪酸占总脂肪的91.5%。重组油脂酵母产油与单独培养红酵母产油、固定化联合培养产油相比,产油含量和细胞干重相似,从而建立了利用重组油脂酵母菌一步转化菊糖和菊芋根块提取液产生油脂的工艺,大大简化了上述的油脂生产工艺。更多还原

【Abstract】 In the past years, biodiesel, which is biodegradable, can be used without modifying existing engines, and produces less harmful gas emissions such as sulfur oxide has received increasing attention because of the environmental pollution and energy crisis world wide. At present the high production cost of biodiesel is a major barrier to its commercialization. Therefore, using a low cost raw material is crucial in reducing the cost of biodiesel production.In our previous study, it was found that Rhodotorula mucilaginosa TJY15a isolated from the marine fish Synechogobius hasta could accumulate a large amount of oil from glucose and hydrolysate of cassava starch. In this study, we found that R. mucilaginosa TJY15a could accumulate 48.8% (w/w) oil from hydrolysate of inulin and its cell dry weight reached 14.8 g/l during the batch cultivation while it could accumulate 48.6% (w/w) oil and 52.2% (w/w) oil from hydrolysate of extract of Jerusalem artichoke tubers and its cell dry weight reached 14.4 g/l and 19.5 g/l during the batch and fed-batch cultivations, respectively. At the end of the fed-batch cultivation, only 0.04% of reducing sugar and 0.08% of total sugar were left in the fermented medium. Over 87.6% of the fatty acids from the yeast strain TJY15a cultivated in the hydrolysate of extract of Jerusalem artichoke tubers was C16:0, C18:1 and C18:2, especially C18:1 (54.7%). Therefore, the results show that hydrolysates of inulin and extract of Jerusalem artichoke tubers were also the good materials for single cell oil production.In order to directly convert inulin and the extract of Jerusalem artichoke tubers into single cell oil by R.mucilaginosa TJY15a and avoid mixing the cells of R. mucilaginosa, the cells of the mutant M-30 were immobilized. Then the immobilized cells were co-cultured with the free cells of the oleaginous yeast R. mucilaginosa TJY15. It was found that the immobilized inulinase-producing cells of P. guilliermondii M-30 can produce 169.3 U/mL of inulinase activity while the free cells of the same yeast strain only produces 124.3 U/mL of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells are co-cultivated with the free cells of R. mucilaginosa TJY15a, R. mucilaginosa TJY15a can accumulate 53.2% oil from inulin in its cells and cell dry weigh reaches 12.2 g/L. Under the similar conditions, R. mucilaginosa TJY15a can accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reaches 12.8 g/L within 48 h. When the co-cultures are grown in 2-L fermentor, R. mucilaginosa TJY15a can accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weigh reaches 19.6 g/L within 51 h. About 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers is C16:0, C18:1 and C18:2.In order to convert inulin and inulin-containg materias into single cell oil by one step fermentation, the gene of exo-inulinase cloned from Kluyveromyces marxianusCBS6556 was expressed in the oleaginous yeast Y. lipolytica ACA-DC50109. First, the INU1 gene was ligated into the expression plasmid pINA1317 and expressed in the cells of the oleaginous yeast. Then, the inulinase activity of different transformants was determined. The activity of the inulinase by the transformant Z31 of the transformants obtained was found to be 41.7 U/mL after cells growth for 78h. After optimization of the medium and cultivation conditions for single cell oil production, the transformant could accumulate 46.3% (w/w) oil from inulin in its cells and cell dry weight was 11.6 g/Lwithin 78 h at the flask level. During the 2-L fermentation, the transformant could accumulate 48.3% (w/w) oil from inulin in its cells and cell dry weight was 13.3 g/L within 78 h while the transformant could accumulate 50.6% (w/w) oil from extract of Jerusalem artichoke tubers in its cells and cell dry weight was 14.6 g/L within 78 h. At the end of fermentation, most of the added sugar was utilized by the transformant cells. Over 91.5% of the fatty acids from the transformant cultivated in the extract of Jerusalem artichoke tubercles was C16:0, C18:0 and C18:1, especially C18:1 (58.5%).更多还原