利用蔗渣水解液培養Yarrowia lipolytica PO1g之生質在以生產脂肪酸甲基酯

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研究生：	Yeshitila Yeshitila - Asteraye Tsigie
論文名稱：	利用蔗渣水解液培養Yarrowia lipolytica PO1g之生質在以生產脂肪酸甲基酯 Synthesis of Fatty Acid Methyl Esters From Yarrowia lipolytica PO1g Biomass Grown In Sugarcane Bagasse Hydrolysate
指導教授：	朱義旭 Yi-Hsu Ju
口試委員:	Suryadi Ismadji Suryadi Ismadji Setiyo Gunawan Setiyo Gunawan Warmadewanthi Warmadewanthi Ahmed Fazary Ahmed Fazary Truong Chi Thanh Truong Chi Thanh 劉志成 Jhy-Chern Liu 王孟菊 Wang Meng Jiy
學位類別：	博士 Doctor
系所名稱：	工程學院 - 化學工程系 Department of Chemical Engineering
論文出版年：	2012
畢業學年度：	101
語文別：	英文
論文頁數：	112
中文關鍵詞：	生質柴油、脂肪酸甲基酯、蔗渣水解液、次臨界水、中性脂質、次臨界甲醇
外文關鍵詞：	Sugarcane bagasse hydrolysate, Sub-critical water, Neutral lipid
相關次數：	點閱：232 下載：5
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本研究探討以去毒素後之蔗渣水解液(DSCBH)作為介質以培養Yarrowia lipolytica Po1g生產油脂及生質柴油之可行性。以2.5%鹽酸水解蔗渣可得到最大之總醣含量(21.38 g/L)，其中13.59 g/L為木糖、3.98 g/L is為葡萄糖、2.78 g/L為阿拉伯糖。利用中和可將5-羟甲基糠醛及糠醛之濃度分別減少21.31%及24.84%。以蛋白胨為氮源在DSCBH中培養Y. lipolytica Po1g可得到最大之生質濃度為11.42 g/L，若改以為氮源則最大生質濃度僅有6.49 g/L。以蛋白胨為氮源時，利用DSCBH為碳源所得生質濃度(11.42 g/L)比用D-葡萄糖(10.19 g/L)、D-木糖(9.89 g/L)及未去毒素之蔗渣水解液(5.88 g/L)為碳源所得生質濃度都要好。Y. lipolytica Po1g生長在以蛋白胨為氮源之DSCBH介質中時最大之脂質含量、脂質產量及脂質生產率分別為58.5%、6.68 g/L及1.76 g/L-day。
將經次臨界水(SCW)處理及未經次臨界水處理後之Y. lipolytica Po1g以溶劑萃取出中性脂質決定其含量及其脂肪酸組成，並探討處理溫度(125, 150 or 175 oC)、含水量(20, 30 or 40 mL/g biomass)及時間(10, 20 or 30 min)對脂質產量之影響。結果顯示在175 oC、20 mL水及20 min條件下可得到最大中性脂質量(42.69 %, w/w)。未經SCW處理時最大中性脂質量只有23.21%。經及未經SCW處理所得脂質脂肪酸組成沒有顯著差異，但是在經SCW處理所得脂質中發現有長鍊脂肪酸。經SCW處理後所得中性脂質量可增加兩倍。
Y. lipolytica Po1g之生質在沒有酸或鹼存在下直接與甲醇及水在次臨界狀態下(175 oC, 23 bar)反應。本研究所提出之方法可於合理的反應時間內得到高生質柴油產量。此方法應用於其他含大量脂肪酸及水之生質之可能性也一併加以討論。

This study investigated the possibility of utilizing detoxified sugarcane bagasse hydrolysate (DSCBH) as the carbon source to culture Yarrowia lipolytica Po1g for microbial oil and biodiesel production. Sugarcane bagasse hydrolysis with 2.5% HCl resulted in maximum total sugar concentration (21.38 g/L) in which 13.59 g/L is xylose, 3.98 g/L is glucose, and 2.78 g/L is arabinose. Detoxification of SCBH by Ca(OH)2 neutralization reduced the concentration of 5-hydroxymethylfurfural and furfural by 21.31% and 24.84%, respectively. Growth of Y. lipolytica Po1g in DSCBH with peptone as the nitrogen source gave maximum biomass concentration (11.42 g/L) compared to NH4NO3 (6.49 g/L). With peptone as the nitrogen source, DSCBH resulted in better biomass concentration than D-glucose (10.19 g/L), D-Xylose (9.89 g/L) and NDSCBH (5.88 g/L). The maximum lipid content, lipid yield and lipid productivity of Yarrowia lipolytica Po1g grown in DSCBH and peptone was 58.5%, 6.68 g/L and 1.76 g/L-day, respectively.
The amount of neutral lipids and fatty acid profiles in neutral lipids extracted from Y. lipolytica Po1g with and without subcritical water (SCW) pre-treatment were investigated. The effects of temperature (125, 150 or 175 oC), amount of water (20, 30 or 40 mL/g biomass) and time (10, 20 or 30 min) on lipid yield were studied. The results show that maximum neutral lipid (42.69 %, w/w) could be achieved at 175 oC using 20 mL water for 20 min. The maximum neutral lipid from unpretreated samples was 23.21%. No difference in fatty acid profiles was observed, but long chain fatty acids were observed in higher amount in SCW pretreated samples. SCW pretreatment increased biodiesel yield by two fold.
Biomass of Y. lipolytica Po1g was reacted with methanol and water under subcritical condition (175 oC, 23 bar) without the presence of acid or base. The proposed method was able to give high biodiesel yield in reasonably short time. The possibility of using this method for biodiesel preparation from wet biomass with high acid and water contents as feedstock was discussed.

TABLE OF CONTENTS

CONTENTS                                                                                                                          PAGE
COVER
APPROVAL LETTER
CHINESE ABSTRACT                                                                                                                   I
ENGLISH ABSTRACT                                                                                                                  II
ACKNOWLEDGEMENTS                                                                                                           IV
TABLE OF CONTENTS                                                                                                                V
LIST OF ABBREVIATIONS                                                                                                         X
LIST OF FIGURES                                                                                                                      XII
LIST OF TABLES                                                                                                                      XIII
CHAPTER ONE                                                                                                                              1
Introduction                                                                                                                                      1
0.	  Background of the study                                                                                                          1
1.	  Objectives of the study                                                                                                             6
CHAPTER TWO                                                                                                                             7
LITERATURE REVIEW                                                                                                                7
1.	 Oleaginous microorganisms                                                                                                   7
1.1.	  Introduction                                                                                                            7
1.2.	  Oil contents of some microorganisms                                                                    7
1.3.	  Lipids from yeasts                                                                                                  9
1.4.	Yarrowia lipolytica                                                                                                  9
1.4.1.	 Introduction                                                                                                    9
1.4.2.	  Media for culturing Yarrowia lipolytica                                                     10
1.4.3.	   Yarrowia lipolytica Strain: Po1g                                                               11
1.5.	Carbon sources for oleaginous microorganisms                                                    12
2.	Sugarcane bagasse                                                                                                                   13
2.1.	Composition of sugarcane bagasse                                                                        13
2.1.1.	   Cellulose                                                                                                 13
2.1.2.	   Hemicellulose                                                                                         14
2.2.	Pretreatment of sugarcane bagasse for hydrolysate production                             15
2.2.1.	Types of pretreatment methods on sugarcane bagasse                              16
2.3.	Detoxification of SCB Hydrolysate                                                                       16

3.	  Subcritical water                                                                                                                    16
4.	  Biodiesel                                                                                                                               17
4.1.	 Introduction                                                                                                           17
4.2.	Feedstocks for biodiesel preparation                                                                     18
4.3.	Preparation of biodiesel                                                                                         21
4.3.1.	  Transesterification                                                                                    21
4.3.1.1.	Base catalyzed transesterification                                                  22
4.3.1.2.	Acid catalyzed transesterification                                                  24
4.3.1.3.	Enzyme catalyzed transesterification                                             24
4.3.1.4.	Non-catalyzed transesterification                                                   25
5.	  Experimental Design                                                                                                              26
CHAPTER  THREE                                                                                                                      27
EXPERIMENTAL SECTION                                                                                                       27
0.	   Apparatus and Chemicals                                                                                                     27
1.	Methodology                                                                                                                      27
1.1.	Lipid production from Y. lipolytica PO1g grown in SCBH                                 27
1.1.1.	 SCBH Preparation                                                                                  27
1.1.2.	 Detoxification of SCBH                                                                         29
1.1.3.	Microorganism, Media Preparation, Precultivation and Cultivation                                                                                   29
1.1.4.	  Biomass and Cell Concentration Determination                       30
1.1.5.	  Total Sugar and Inhibitors Concentration Determination         30
1.1.6.	  Lipid Content and Fatty Acids Determination                          31
1.2.	Biodiesel preparation from Y. lipolytica PO1g biomass                                      32
1.2.1.	 Biomass pretreatment and sample preparation                             32
1.2.2.	  Experimental design                                                                     33
1.2.3.	  Lipid content and fatty acids determination                                 34
1.2.4.	  Determination of FAMEs contents                                              34
1.3.	Biodiesel production from wet Y. lipolytica Po1g biomass under subcritical conditions                                                                                                             36
1.3.1.	  Sample preparation from Y. lipolytica Po1g biomass                36
1.3.2.	  Experimental setup                                                                     36
1.3.3.	  Production of biodiesel from biomass                                        37
1.3.4.	  Qualitative analysis using thin layer chromatography (TLC)    39
1.3.5.	Quantitative analysis using gas chromatography (GC)                39
1.3.6.	  Determination of FAMEs contents                                             39
CHAPTER FOUR                                                                                                                          41
RESULTS AND DISCUSSION                                                                                                    41
1.	 Lipid production from Y. lipolytica PO1g grown in SCBH                                                  41
1.1.	Composition of SCBH before And after Neutralization with Ca(OH)2                41
1.2.	Effect of nitrogen source on growth of Y. lipolytica Po1g in DSCBH Medium   44
1.3.	Comparison of growth of Y. lipolytica Po1g in SCBH medium and other carbon sources                                                                                                                                45
1.4.	Lipid content of Y. lipolytica Po1g cells grew in DSCBH medium                      49
1.5.	Comparison of lipid production from Y. lipolytica strains in different media       53
2.	   Biodiesel production from Y. lipolytica Po1g biomass using SCW pretreatment                54
2.1.	Lipid, wax and gum contents of Y. lipolytica Po1g                                               54
2.2.	 Effects of pretreatment parameters on composition of extract                             56
2.3.	Effect of temperature on the neutral lipid profiles of Y. lipolytica Po1g               57
2.4.	 Comparison of fatty acid composition of neutral lipids of Y. lipolytica Po1g with or without SCW pretreatment                                                                                61
2.5.	FAMEs composition of untreated and SCW pretreated samples                           63
3.	 Biodiesel production from Y.  lipolytica Po1g biomass using subcritical methanol             65
3.1.	Effect of amount of water                                                                                      65
3.2.	Effect of methanol amount                                                                                     67
3.3.	Effect of reaction time                                                                                           69
3.4.	Effect of adding sulfuric acid                                                                                 70
3.5.	FAMEs composition                                                                                              72
CHAPTER FIVE                                                                                                                           74
CONCLUSIONS AND FUTURE PROSPECTS                                                                          74
REFERENCES                                                                                                                              76
APPENDIX A                                                                                                                                90
APPENDIX B                                                                                                                                91
CURRICULUM VITAE                                                                                                                92
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