USST_Arts_112451339 压力辅助臭氧法提取小球藻油脂研究

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摘要

面对目前全球日益严重的能源短缺和环境污染问题，开发利用无污染的生物

质能源新能源势在必行。与传统制备生物柴油原料的动植物体相比，微藻具有相

对较高的生长速率及油脂密度，可以提供较高的油脂产率。微藻生物柴油由于其

可再生、易生物降解、不污染环境等优点，可作为一种重要的能源取代石化柴油，

受到各国的广泛关注。

本文以小球藻为研究对象，采取压力辅助臭氧法对小球藻细胞进行破壁处理

提取生物油脂。破碎细胞一是利用臭氧的氧化性，二是借助臭氧加压形成的臭氧

微气泡，增大与藻类细胞的接触面积对细胞进行破碎处理，旨在寻求全新的细胞

破壁方法对油脂提取，进而为制备生物柴油生产提供依据。本文主要研究了小球

藻的生长变化趋势，探索了细胞密度与吸光度之间的关系。实验主要考察了油脂

提取过程中的多种影响因素，主要包括压力、循环次数，确定油脂提取的最佳工

艺参数。对破碎后的处理液在电子显微镜以及扫描电镜下观察，初步探究压力辅

助臭氧法破碎藻类细胞的机理。通过与不加压直接通臭氧、超声波破碎处理的对

比进一步阐述论证实验结果，最后通过 GC-MS 分析该方法下的小球藻油脂成分。

超声波破碎处理中主要探索了超声时间以及超声功率对油脂产率的影响，确定油

脂提取的最佳工艺条件，最后结果与压力辅助臭氧法对比分析不同。

结果表明，实验条件下培养的小球藻细胞生长较为缓慢，在 80 天左右达到稳

定期，并且小球藻细胞密度与吸光值之间存在良好的线性关系。压力辅助臭氧法

可以成功破碎小球藻细胞，破碎后体系中氨氮、总氮、有机氮含量明显上升，硝

酸盐含量上升，亚硝酸盐下降，溶解性磷和总磷的含量也有明显增加。压力和循

环数对小球藻破碎有着明显的影响。随着压力的增加，油脂提取率先升高后降低，

而随着循环次数的增加，油脂提取率进一步增加，通过对实验单因素分析得到臭

氧加压法提取小球藻油最佳工艺条件为：压力 0.6MPa，80 个循环，在此条件下小

球藻油最大萃取率为 26.7%。

不同实验条件破碎细胞的机理也不一样，直接通臭氧处理的小球藻细胞表面

有很多隆起，并且在细胞的外围存在一层光圈，而压力辅助臭氧法处理得到的小

球藻细胞颗粒表面的致密结构完全被破坏，细胞基本都存在膨起及内凹情况，一

些细胞的中间出现空洞。超声波处理的细胞表面有类似“撞痕”的存在，细胞颗

粒表面的致密结构遭到破坏。超声波处理中功率对油脂提取率的影响不大，随着

超声时间的延长油脂提取率进一步增加。相对超声处理法，一定条件下臭氧加压

法具有高效、高提取率等优点，应用前景广阔。

另外 GC-MS 检测的结果表明，小球藻油脂中包含多种脂肪酸，其主要成分为

C16:0 与C18:0，相对比例高达 40.4%及48.6%。

关键词：压力辅助臭氧超声小球藻细胞破碎生物柴油

ABSTRACT

Faced with the problem of global energy shortage and environmental pollutions,

we need to develop and utilize biomass energy source imperatively. Compared with

traditional biodiesel raw material animals and plants, the algae growth rate having a

relatively high density and oil, grease can provide higher yields. Microalgae biodiesel

because of its renewable, readily biodegradable, do not pollute the environment, etc.,

can be used as an important source of energy to replace fossil diesel, cause widespread

concern around the world.

In this work, pressure assisted ozone method was used to disrupt Chlorella vulgaris

cells. The principle can be seen as: First, ozone oxidation. Second, microbubbles formed

during the process of compression and decompression increase the contact area between

ozone and cells. We seek to find a new method to disrupt cell walls to provide a new

evidence for biodiesel production. This paper studies the trend growth of Chlorella,

exploring the relationship between cell density and absorbance. Fragmentation

situations were studied to determine the optimum parameters of oil extraction, as well

as efforts of pressure and cycles conditions on the lipid content of Chlorella vulgaris. By

observing fluid before and after treatment in an electron microscope and scanning

electron microscope, we seek to explore the mechanism of cell disruption. To better

explain the method, we also did ozone directly without pressure and disrupt cell walls

with ultrasonic as a comparison, and finally analysis the Chlorella vulgaris fat

component by GC-MS. Sonication process mainly explored the impact of ultrasonic

power and ultrasonic time on the oil yield, to determine the optimum conditions of oil

extraction, finally compare the difference between pressure-assisted method and

sonication process.

The results showed that chlorella cultured cell growth is relatively slow under the

experimental conditions, in about 80 days it goes into stable stage, and there is a good

linear relationship between the chlorella cell density and absorbance values. And the

pressure-assisted ozonation method can break Chlorella vulgaris cells successfully,

ammonia, nitrate, organic nitrogen content increased significantly, as well as dissolved

phosphorus and total phosphorus, nitrite decreased and total nitrogen content

increased. It turns out that pressure and cycles have a significant effect on cells broken.

With increasing pressure and cycles, the oil extraction rate was increased. By

single-factor experimental analysis, the largest oil extraction rate was 26.7% under the

condition of 0.6MPa, 80cycles. Different experimental conditions have different cell

disruption mechanism, cell surface under ozone directly treatment has many bumps, and

in the periphery of the cell layer has transparent rings. While cell surface dense structure

is completely destroyed under pressure-assisted ozonation treatment, cell convex or

concave and even some voids exist in the middle of cells. Something like “hit mark”

exist in the cell surface of the ultrasonic treatment, dense structure of the cell surface of

the particles is destroyed. Ultrasonic processing power has little effect on the rate of oil

extraction, but as ultrasonic time increase, oil extraction rate increase. To some extent,

pressure-assisted ozonation method was better that ultrasonic treatment in efficiency, oil

extraction rate, which had a broad application prospects. And further GC-MS detection

result showed Chlorella contains many fatty oil, the main component is C16:0 and

C18:0, relative proportions can be as high as 40.4% and 48.6% respectively.

Key Word: Pressure-assisted ozonation, Ultrasonic, Chlorella, Cell

disruption, Biodiesel

摘要

ABSTRACT

第一章绪论 ...................................................................................................................1

1.1 能源与环境问题 ................................................................................................... 1

1.1.1 世界能源与环境 ............................................................................................. 1

1.1.2 中国能源与环境现状 ..................................................................................... 1

1.1.3 新兴能源的利用 ............................................................................................. 2

1.2 生物柴油介绍 ....................................................................................................... 2

1.2.1 生物柴油组成 ................................................................................................. 2

1.2.2 生物柴油优点 ................................................................................................. 3

1.2.3 生物柴油原料的研究进展 ............................................................................. 3

1.2.4 生物柴油的制备方法 ..................................................................................... 4

1.3 微藻开发生物柴油的研究进展 ........................................................................... 5

1.3.1 微藻及微藻油脂 ............................................................................................. 5

1.3.2 微藻产油国内外进展 ..................................................................................... 7

1.3.3 微藻产油的优势及影响因素 ......................................................................... 8

1.4 细胞破碎方法 ..................................................................................................... 11

1.4.1 匀浆器破碎 ................................................................................................... 12

1.4.2 玻珠研磨机破碎 ........................................................................................... 12

1.4.3 超声波破碎 ................................................................................................... 13

1.4.4 反复冻融法破碎 ........................................................................................... 14

1.4.5 反应釜加温加压破碎 ................................................................................... 14

1.4.6 化学法 ........................................................................................................... 15

1.4.7 酶溶法 ........................................................................................................... 15

1.5 本课题的意义和研究内容 ................................................................................. 16

1.5.1 课题的提出与意义 ....................................................................................... 16

1.5.2 研究内容 ....................................................................................................... 17

第二章实验方法及材料 ...............................................................................................18

2.1 实验材料与试剂 ................................................................................................. 18

2.2 实验仪器与装置 ................................................................................................. 18

2.2.1 实验仪器 ....................................................................................................... 18

2.2.2 实验装置 ....................................................................................................... 19

2.3 藻种的保存与培养 ............................................................................................. 20

2.3.1 藻种保存 ....................................................................................................... 20

2.3.2 小球藻培养 ................................................................................................... 20

2.4 分析方法 ............................................................................................................. 20

2.4.1 叶绿素含量测定 ........................................................................................... 20

2.4.2 COD 值测定 .................................................................................................. 21

2.4.3 臭氧含量的测定 ........................................................................................... 21

2.4.4 破碎指标的测定 ........................................................................................... 21

2.4.5 小球藻藻粉细胞元素含量测定 ................................................................... 21

2.4.6 藻细胞计数 ................................................................................................... 22

2.4.7 臭氧加压氧化破碎细胞 ............................................................................... 22

2.4.8 小球藻油脂提取 ........................................................................................... 22

2.4.9 生物柴油分析 ............................................................................................... 23

2.5 实验设计安排 ..................................................................................................... 24

2.5.1 臭氧不加压破碎藻类提取油脂 ................................................................... 24

2.5.2 压力辅助臭氧法不同循环条件提取小球藻油脂 ....................................... 24

2.5.3 压力辅助臭氧法不同压力条件提取小球藻油脂 ....................................... 24

2.5.4 超声波法不同超声时间提取小球藻油脂 .................................................... 24

2.5.5 超声波法不同超声功率提取小球藻油脂 ................................................... 25

2.5.6 细胞破碎情况分析 ....................................................................................... 25

2.5.7 压力辅助臭氧法与超声波法油脂成分对比分析 ....................................... 25

第三章小球藻生长情况变化 .......................................................................................26

3.1 细胞密度与特定波长下吸光度之间的关系 .................................................. 26

3.2 细胞生长曲线 .................................................................................................. 27

3.3 小球藻细胞基本元素组成 .............................................................................. 28

3.4 本章小结 .......................................................................................................... 29

第四章压力辅助臭氧法破碎小球藻细胞提取生物油脂 ...........................................30

4.1 压力辅助臭氧法破碎效果分析 ......................................................................... 30

4.1.1 不同处理电子显微镜及 SEM 图片分析 ..................................................... 30

4.1.2 不同处理破碎指标分析 ............................................................................... 32

4.2 不同实验条件对油脂产率的影响 ..................................................................... 33

4.2.1 直接通臭氧对油脂产率的影响 ................................................................... 33

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摘要：

摘要面对目前全球日益严重的能源短缺和环境污染问题，开发利用无污染的生物质能源新能源势在必行。与传统制备生物柴油原料的动植物体相比，微藻具有相对较高的生长速率及油脂密度，可以提供较高的油脂产率。微藻生物柴油由于其可再生、易生物降解、不污染环境等优点，可作为一种重要的能源取代石化柴油，受到各国的广泛关注。本文以小球藻为研究对象，采取压力辅助臭氧法对小球藻细胞进行破壁处理提取生物油脂。破碎细胞一是利用臭氧的氧化性，二是借助臭氧加压形成的臭氧微气泡，增大与藻类细胞的接触面积对细胞进行破碎处理，旨在寻求全新的细胞破壁方法对油脂提取，进而为制备生物柴油生产提供依据。本文主要研究了小球藻的生长变化趋势，探索...

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