论文总字数：19943字

摘 要

众多研究表明，生物油脂有着广阔的应用前景。但如何提高菌株的油脂积累仍是较大的，依旧在被研究的课题。作为普遍公认的安全的生物体，解脂耶式酵母（Yarrowia lipolytica）是研究真菌基础构型的良好模型和各种生物大宗化学品的良好平台。本论文以实验室培育的Y. lipolytica菌株，以菌体生物量和油脂含量为指标，在摇瓶水平上发酵研究培养基营养成分、底物组成和多种培养条件对Y. lipolytica的产油脂的合成影响，以实现油脂在菌内的大量积累。本论文研究如下：（1）以葡萄糖为碳源，确定了无机氮源与有机氮源中的牛肉膏、大豆蛋白胨和硫酸铵是适合用于产油的培养基。确定了以摩尔比为1:2的大豆蛋白胨和硫酸铵的双氮源作为培养基的氮源组成。（2）研究了在不同C/N比的情况下Y. lipolytica产油脂的影响。C/N比为90时Y. lipolytica的油脂平均值最高为1.64 g/l，生物量平均值为4.88 g/l。比作为对照组的C/N比为100的都高。（3）研究在初始pH对Y. lipolytica的产油积累的影响。初始pH值为6.3，以KOH和CH₃COOH为缓冲液调节pH。结论得出在pH为5时，油脂积累最高为1.71g/l，但生物量稍微低点。（4）研究温度对Y. lipolytica产油脂的积累的影响，发现分阶段温控对油脂积累影响较大。高温利于菌体增殖，低温利于油脂积累。发现在实行28℃发酵1天，20℃发酵剩余6天的情况下，油脂积累和生物量最大，分别为1.78 g/l和6.54 g/l。

关键词：解脂耶式酵母 油脂 氮源 C/N比 初始pH值 温度

Optimization of fermentation conditions of Yarrowia lipolytica

for lipid production

ABSTRACT

Numerous studies have shown that lipid has a broad application prospects. However, how to increase the lipid accumulation in the strains is still relatively large and remains the subject of study. As a generally recognized and safe organism, Yarrowia lipolytica is a good model for studying the basic properties of fungi and a platform for various biochemical products. In this paper, a laboratory-grown Y. lipolytica strain was used as an indicator of bacterial biomass and lipid content. To study the effect of fermentative substrate, nutrient medium and culture conditions on the shake flask level on lipid synthesis of Y. lipolytica, in order to achieve large accumulation of lipid in bacteria. This thesis is studied as follows: (1) Using glucose as a carbon source, it was determined that the beef extract, soybean peptone, and ammonium sulfate in the inorganic nitrogen source and the organic nitrogen source are suitable for the production of lipid. A nitrogen source consisting of soy peptone and ammonium sulfate in a molar ratio of 1:2 was determined as the nitrogen source of the medium. (2) The effects of Y. lipolytica lipid production under different C/N ratios was studied. At a C/N ratio of 90, the average lipid content of Y. lipolytica was 1.64 g/l, and the average biomass was 4.88 g/l. It is higher than the control group which has a C/N ratio of 100. (3) Study the effect of initial pH on lipid accumulation of Y. lipolytica. The initial pH is 6.3 and the pH is adjusted with KOH and CH₃COOH buffers. The conclusion is that when the pH is 5, the highest accumulation of lipid is 1.71g/l, but the biomass is slightly lower. (4) To study the effect of temperature on lipid accumulation in Y. lipolytica, it was found that the phased temperature control had a greater effect on the accumulation of lipid. High temperature is conducive to cell proliferation, low temperature conducive to lipid accumulation. It was found that when the fermentation was performed at 28℃. for 1 day and at 20℃, for the remaining 6 days, the lipid accumulation and biomass were the largest, 1.78 g/l and 6.54 g/l, respectively.

Key Words: Yarrowia lipolytica; Temperature; Lipid; Nitrogen source; C/N ratio; pH;

目 录

摘要 I

ABSTRACT i

第一章 文章综述 3

1.1 油脂功能 3

1.2 产油脂微生物 3

1.2.1 微生物产油脂的研究 3

1.2.2 产油微生物的必备条件 4

1.2.3 微生物产油的优势 4

1.2.4 产油微生物的种类 5

1.3 解脂耶式酵母简介 6

1.3.1 解脂耶式酵母简介 6

1.3.2 解脂耶式酵母的产油合成途径 8

1.4 影响酵母产油的因素 9

1.4.1 氮源种类 9

1.4.2 C/N比 9

1.4.3 温度 9

1.4.4 初始pH值 9

1.5 本论文的内容及意义 10

1.5.1 本论文的研究意义 10

1.5.2 本论文的研究内容 10

第二章 实验材料与方法 11

2.1 材料 11

2.1.1 实验试剂 11

2.1.2 实验仪器 11

2.1.3 菌种 12

2.1.4 培养基 12

2.2 培养方法与培养条件 12

2.2.1 不同氮源对酵母产油的影响 13

2.2.2 C/N比对酵母产油的影响 13

2.2.3初始pH值对酵母产油的影响 14

2.2.4 温度对酵母产油的影响 14

2.3 分析方法 14

2.3.1 发酵液残糖测定 14

2.3.2 细胞干重测定 14

2.3.3 油脂提取 15

第三章 结果与讨论 16

3.1氮源对解脂耶式酵母产油发酵的影响 16

3.2 C/N比对酵母产油的影响 18

3.3 初始pH值对酵母产油的影响 19

3.4 温度对酵母产油的影响 20

第四章 展望 22

参考文献 23

致 谢 26

第一章 文章综述

1.1 油脂功能

油脂是一种特殊的酯，是烃的衍生物，是油和脂肪的统称。从化学组成上来讲油脂都是甘油与高级脂肪酸形成的酯。自然界中的油脂大多是多种物质的混合物，其主要成分是一分子甘油与三分子高级脂肪酸脱水形成的酯，称为甘油三酯。动植物是油脂的常规来源。植物油脂大多数是用来食用的，也广泛应用于制造润滑油、油漆、甘油、肥皂和硬化油等。植物油脂含不饱和脂肪酸，溶点较低，常温下呈液态，消化吸收率高。植物油脂肪含量在99%以上，此外含有丰富的维生素E，少量的钾、钠、钙和微量元素。动物油脂可分为陆生温血动物、水生动物和禽类的油脂，例如猪油、鱼油、鸭油等，一般是固体的。主要供食用，如猪脂、牛脂、羊脂等。也广泛应用于制造硬化油、肥皂、甘油、润滑油和制革工业及药品。鱼油是制备涂料的原料，用熬制法取得。

1.2 产油脂微生物

1.2.1 微生物产油脂的研究

产油微生物是在一定的条件下生长，并将碳氢化合物、碳水化合物和一般的油脂作为碳源通过代谢途径转化为体内大量积累油脂的微生物。油脂积累量超过细胞干重20%的才能称为产油微生物，这是在1989年Ratledge^[25]对产油微生物给予的更明确的定义。

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