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系統識別號 U0026-2607201217285800
論文名稱(中文) 利用固定化纖維素分解酵素水解小球藻萃取脂質與葉綠素
論文名稱(英文) Enzymatic Hydrolysis of Chlorella sp. by Immobilized Cellulase for Extraction of Lipid and Chlorophyll
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 100
學期 2
出版年 101
研究生(中文) 尤莉安
研究生(英文) Yuliana Kurniawan
學號 N36997245
學位類別 碩士
語文別 英文
論文頁數 104頁
口試委員 指導教授-吳文騰
共同指導教授-許梅娟
口試委員-張嘉修
中文關鍵字 固定化纖維分解酵  微藻  萃取  脂質  葉綠素 
英文關鍵字 Immobilized cellulase  microalgae  extraction  lipid  chlorophyll 
學科別分類
中文摘要 近年來,利用微藻生產生質燃料被視為具有潛力的替代能源,但由於微藻培養和採集成本居高不下,使之相較於化石燃料顯得較無競爭性,因此在培養微藻生產生質燃料的同時,一併增加其他高附加價值產品(如:葉綠素)的產量,可讓微藻在發展生質能源上具有經濟競爭力。

此研究主要採用溶劑同步自微藻細胞中萃取脂質與葉綠素。首先利用酵素水解微藻細胞壁進行前處理,可促進溶劑萃取時通過細胞壁的滲透率。經聚丙烯腈包覆之氧化鐵磁性奈米粒子,利用amidination reaction活化粒子表面之C≡N官能基,與纖維素分解酵素上之胺基形成共價鍵結。此磁性奈米粒子的優點為反應後能輕易的藉由強力磁石自溶液中分離回收。本實驗將固定化酵素在固定化溫度50℃,pH值為7的條件下應用於水解微藻細胞壁,並藉由產生之還原糖的濃度,決定水解最適化條件,並得蛋白質固定量為88.6 mg/g particle。此固定化纖維分解酵素在重複進行10次批次反應後,仍可維持70.2%之初始活性,存放一星期後則仍有58.2%之初始活性,顯現具有商業化之潛力。

此研究中進一步利用乙醇和正己烷的混合溶劑同步萃取脂質與葉綠素,所探討之實驗參數包括水解時間、混合溶劑比例、溶劑對乾藻重之比例、萃取時間等。萃取後由1g乾藻中可得33.93%脂質 (30.2 mg)與30%葉綠素(10.05 mg),結果顯示此固定化酵素搭配特定溶劑應用於同步萃取脂質與葉綠素具有後續放大可行性。
英文摘要 In recent years, microalgae were considered as potential alternative source for biofuel production. Due to the high cost of cultivation and harvesting, the microalgae biofuel became uncompetitive with fossil fuel. Thus enhancing the interest in offsetting the cost of lipids by co-production of other valuable products (such as: chlorophyll) from microalgae may make microalgae biomass economically competitive for biofuels production.

In this study, lipid and chlorophyll were extracted simultaneously from microalgae by solvent extraction. Enzymatic hydrolysis was applied as a pretreatment to break the cell walls of microalgae in order to facilitate the penetration of the solvent through the microalgae cell wall. The cellulase was immobilized onto PAN coated magnetic nanoparticles by activating the nitrile group in the PAN layer with the amidination reaction. The immobilized cellulase could be removed easily from the reaction system due to the induced moderate magnetic field of magnetic nanoparticles. Under the optimal immobilization conditions, the immobilization yielded a protein loading of 88.6 mg/g particle. The optimal conditions for hydrolysis were also determined by measuring the reducing sugar released at temperature of 50 C and pH 7. The immobilized cellulase retained 70.2% residual activity after ten hydrolysis cycles and 58.2% residual activity after 7 days storage.

The extraction of lipid and chlorophyll by using a solvent mixture of ethanol and hexane was examined in this study. The influences of the operating parameters including the hydrolysis time, the ratio between ethanol to hexane, the ratio of mixed solvents to algal biomass (dry weight), and the extraction time were investigated. After hydrolysis, the recovery of lipid and chlorophyll yields of 33.93% (30.2 mg of lipid) and 30% (10.05 mg of chlorophyll) could be extracted from one gram of microalgae. These results represent the feasibility of the proposed applications of immobilized cellulase for extraction of lipid and chlorophyll from microalgae.
論文目次 ABSTRACT i
摘要 ii
ACKNOWLEDGEMENT iii
TABLE OF CONTENTS iv
LIST OF FIGURES viii
LIST OF TABLES xi
CHAPTER 1 1
1.1 Research Background and Motivation 1
1.2 Objectives of The Study 3
1.3 Outline of the thesis 4
CHAPTER 2 6
2.1 Chlorella sp. 6
2.1.1 The potential of microalgae as bio-energy feedstock 6
2.1.2 Biology of Chlorella sp. and Its Applications 7
2.2 Lipids 8
2.2.1 Lipid Classification 8
2.2.2 Fatty Acids 10
2.2.3 Lipid Extraction by Solvent Extraction 11
2.2.4 Transesterification 16
2.3 Chlorophyll 18
2.3.1 Chlorophyll Classification 18
2.3.2 Uses of Chlorophyll 19
2.3.3 Chlorophyll Extraction by Organic Solvent Extraction 20
2.4 Cellulase enzyme 22
2.4.1 Brief Introduction of Cellulase 22
2.4.2 Cellullose Hydrolysis Mechanism 24
2.5 Enzyme Immobilization 26
2.5.1 Brief Introduction of Enzyme Immobilization 26
2.5.2 Methods of enzyme immobilization 28
2.6 Magnetic Nanoparticles 34
2.6.1 The Synthesis and Magnetism of Magnetic Nanoparticles 34
2.6.2 Magnetic Nanoparticles in Enzyme Immobilization 37
CHAPTER 3 39
3.1 Chemicals and Materials 39
3.2 Experimental Instruments 40
3.3 Experimental Methods 44
3.3.1 Preparation of Magnetic Nanoparticles 44
3.3.2 Preparation of Polyacrylonitrile (PAN) - Coated Magnetic Nanoparticles 45
3.3.3 Cellulase Immobilization 45
3.3.4 Preparation of Substrate (microalgae cells) 46
3.3.5 Preparation of Acetate Buffer Solution 47
3.3.6 Hydrolysis of Microalgae Cell Walls with Immobilized Cellulase 47
3.3.7 Extraction of Lipid and Chlorophyll 48
3.3.8 Reusability 49
3.3.9 Storage Stability 49
3.4 Experimental Analysis 49
3.4.1 Transmission Electron Microscopy (TEM) 49
3.4.2 Fourier Transform Infra Red (FT-IR) 50
3.4.3 Magnetization Measurements 50
3.4.4 Protein Loading Amount 50
3.4.5 Cellulase Activity Assays 52
3.4.6 Measurement of Lipid Content 54
3.4.7 Measurement of Chlorophyll 54
CHAPTER 4 56
4.1 Characterization of Magnetic Nanoparticles and Immobilized Cellulase 56
4.1.1 Particles Morphology and Size 56
4.1.2 Chemical Bonding (Functional Groups) 58
4.1.3 Magnetic Property 60
4.2 Optimization of Immobilizing Cellulase onto PAN-Coated Magnetic Nanoparticles 63
4.2.1 The Effect of pH Value on Cellulase Immobilization 63
4.2.2 The Effect of Temperature on Cellulase Immobilization 65
4.2.3 The Effect of Enzyme Concentration on Cellulase Immobilization 66
4.2.4 The Effect of Immobilization Time on Cellulase Immobilization 69
4.3 Enzymatic Activity and Stability of Immobilized Cellulase 70
4.3.1 The Effect of pH on Activity of Immobilized Cellulase 70
4.3.2 The Effect of Temperature on Activity of Immobilized Cellulase 71
4.3.3 The Effect of Substrate Concentration on Activity of Immobilized Cellulase 73
4.3.4 Reusability of Immobilized Cellulase 74
4.3.5 Storage Stability of Immobilized Cellulase 76
4.4 The Extraction of Lipid and Chlorophyll 76
4.4.1 The Effect of Solvent Mixtures on Extraction of Lipid and Chlorophyll 77
4.4.2 The Effect of Pretreatment on Lipid and Chlorophyll Extraction 78
4.4.3 The Effect of Hydrolysis Time on Lipid and Chlorophyll Extraction 80
4.4.4 The Effect of Solvent Ratio on Lipid and Chlorophyll Extraction 81
4.4.5 The Effect of Solvent Volume on Lipid and Chlorophyll Extraction 83
4.4.6 The Effect of Extraction Time on Lipid and Chlorophyll Extraction 84
CHAPTER 5 86
5.1 Conclusions 86
5.2 Suggestions 87
REFERENCES 89
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