臺灣博碩士論文加值系統

Yarrowia lipolytica酵母菌是一種油脂微生物，能夠從其中萃取出許多油脂，常被視為是一種有潛力的生質柴油來源。本實驗探討利用不同的碳源 (葡萄糖、甘油、木糖)及其碳氮源莫爾數比(2、5、10和20) ，測試對於 Y. lipolytica Po1g的產油率。並額外加入廢食用油做為誘發劑促使產油。此實驗的最佳結果為，使用葡萄糖在碳氮莫爾數比為20下，可以產出生物量X=12.4 g/L，油脂含量YL/S = 25.7%，油脂產率L= 1.95 g/L 於第三天收成。以甘油為主要碳的此實驗中，生物量X = 3.6-10.4 g/L及油脂含量YL/X = 8-16%。而木糖於此實驗中，無法當作單一碳源為增長生物量及油脂。有趣的是Y. lipolytica可藉由同化程序將木糖轉換成木糖醇。利用廢食用油為誘發劑，加入YPD基質中，其結果並無有效地提升產油。

Yarrowia lipolytica may be a promising microorganism for lipid accumulation, which could be utilized for biodiesel production. This study was aimed to investigate the ability of Y. lipolytica to accumulate lipid using glucose, glycerol, xylose as the carbon source at different C/N 2, 5, 10, 20, and the addition of waste cooking oil as an inducer to accumulate lipid. In this study the best results were obtained (lipid content YL/X = 25.70% (g/g) and lipid yield L= 1.95 g/L for C/N = 20) with glucose as the carbon source after fermentation for 3 days. While the use of glycerol resulted in a biomass (X = 3.6-10.4 g/L) and lipid content (YL/X = 8-16%), xylose on the other hand was found not capable of promoting biomass growth and lipid accumulation when used as the sole carbon source. Nevertheless it is interesting that Y. lipolytica assimilates xylose and in the process produces xylitol. As for the addition of waste cooking oil, biomass growth and lipid content were not significantly improved.

CONTENTS

中文摘要 I
Abstract II
Acknowledgement III
CONTENTS IV
LIST OF FIGURES VII
LIST OF TABLES IX
CHAPER ONE
Introduction 1
1.1. Background study 1
1.2 Objective of this study 2
CHAPTER TWO 3
Literature review 3
2.1 Oleaginous microorganisms 3
2.1.1 Introduction 3
2.2 Yarrowia lipolytica 4
2.2.1 Yarrowia lipolytica Strain: Po1g 4
2.2.2 Metabolic traits 5
2.2.3 Overview of the main metabolic pathways for Y. lipolytica 5
2.3 Nutrient for oleaginous microorganisms 7
2.3.1 Glucose as a carbon source 7
2.3.2 Glycerol as a carbon source 8
2.2.3 Xylose as a carbon source 9
2.3.4 Waste cooking oil as a carbon source 11
2.4 Soxhlet extraction 12
CHAPTER THREE 15
Methodology 15
3.1 Experimental materials 15
3.1.1 Microorganism 15
3.2 Apparatus and equipment 16
3.3 Flow char 17
3.4 Fermentation 17
3.4.1 Microorganism maintaining 17
3.4.2 Inoculum 18
3.4.3 Microbial fermentation 18
3.4.4 Biomass 18
3.4.4 .1 Small scale 18
3.4.4.2 Large scale 18
3.5 Soxhlet extraction 19
3.6 Sugar analysis 19
3.6.1Materials 19
3.6.2 Procedure 19
3.7. Glycerol and xylose analyses 20
3.8 Effect of C/N ratio on yeast growth and lipid accumulation 20
3.8.1 Glucose as carbon source 20
3.8.2 Glycerol as carbon source 21
3.8.3 Xylose as carbon source 22
3.8.4 Waste cooking oil as a growth inducer 22
3.8.5 Two-stage culture 23
3.8.6 Xylose with other carbon source 23
CHAPTER FOUR 25
Results and Discussion 25
4.1 Y. lipolytica grew on YPD medium 25
4.2 Effect of C/N ratio on yeast growth and lipid accumulation 26
4.2.1 Glucose as sole carbon source 26
4.2.2 Glycerol as sole carbon source 26
4.2.3 Xylose as sole carbon source 27
4.3.1. Effect of waste cooking oil 32
4.3.2 Effect of carbon source with co-substrate glycerol 35
4.4. Two-stage culture 38
4.4.1 Glucose as initial carbon source 38
4.4.2 Glycerol as initial carbon source 40
4.4.3 Xylose metabolize with other carbon source 43
CHAPTER FIVE 47
CONCLUSION 47
REFERENCES 48
APPENDIX 53

Abreu, F.R., Alves, M.B., Macedo, C.C.S., Zarab, L.F., Suareza, P.A.Z. 2005. New multi-phase catalytic systems based on tin compounds active for vegetable oil transesterification reaction. Journal of Molecular Catalysis A, 227, 263-267.
Ageitos, J.M., Vallejo, J.A., Patricia V-C., Villa T.G. 2011. Oily yeasts as oleaginous cell factories. Applied Microbiology and Biotechnology. 90(4), 1219–1227.
Agarwal, A. K. 2007. Biofuels (alcohols and biodiesel) applications as fuels for internal combustion engines. Progress in Energy and Combustion Science, 33(3), 233-271.
Aggelis, G., Sourdis, J. 1997. Prediction of lipid accumulation degradation in oleaginous microorganisms growing on vegetable oils. Antonie Leeuwenhoek 72(2), 159–165.
Alexander, M.A, Chapman, T.W., Jeffries, T.W. 1988. Xylose metabolism by Candida Shehatae in continuous culture. Applied Microbiology and Biotechnology, 28(4-5), 478-486.
Angerbauer, C., Siebenhofer, M., Mittelbach, M., Guebitz G.M. 2008. Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production. Bioresource Technology, 99(8), 3051–3056.
Makri, A., Fakas, S., Aggelis G. 2010. Metabolic activities of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures. Bioresource Technology, 101(7), 2351-2358.
Barth, G., &; Gaillardin, C. 1997. Physiology and genetics of the dimorphic fungus
Yarrowia lipolytica. FEMS Microbiol Reviews, 19(4), 219-237.

Beopoulos, A., Cescut, J., Haddouche, R., Uribelarrea, J-L., Molina-Jouve, C., Nicaud, J.M. 2009. Yarrowia lipolytica as a model for bio-oil production. Progress in Lipid Research, 48(6), 375–387.
Cheirsilp, B., Louhasakul, Y. 2013. Industrial wastes as a promising renewable source for production of microbial lipid and direct transesterification of the lipid into biodiesel. Bioresource Technology, 142, 329–337.
Fontanille, P., Kumar, V., Christophe, G., Nouaille, R., Larroche , C. 2012. Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Yarrowia lipolytica. Bioresource Technology, 114, 443-449.
Gao, D., Zeng, J., Zheng, Y., Yu, X., Chen, S. 2013. Microbial lipid production from xylose by Mortierella isabellina. Bioresource Technology, 133, 315–321.
Gong Z., Wang Q., Shen H., Hu C., Jin G, Zhao Z.K.2012. Co-fermentation of cellobiose and xylose by Lipomyces starkeyi for lipid production. Bioresource Technology, 117, 20–24.
Heredia, L., Ratledge, C. 1988. Simultaneous utilization of glucose and xylose by Candida curvata D in continuous culture. Biotechnology Letters, 10(1), 25–30.
Jacob, Z. 1992. Linear growth and lipid synthesis in the oleaginous yeast Rhodotorula gracilis. Folia Microbiologica, 37(2), 117–121.
Kapturowska, A.U., Stolarzewicz, I.A., Krzyczkowska, J., Białecka-Florjan’czyk E. 2012. Studies on the lipolytic activity of sonicated enzymes from Yarrowia lipolytica. Ultrasonics Sonochemistry, 19(1), 186–191.
Luque de Castro M.D., Priegeo-Capote F. 2010. Soxhlet extraction: Past and present panacea. Journal of chromatography A, 1217(16), 2383-2389.


Madzak C., Treton B., Blanchin-Roland, S. 2000. Strong hybrid promoters and integrative expression/ secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. Journal of Molecular Microbiology and Biotechnology, 2(2), 207-16.
Neidig, H.A. 1977. Separation acid and neutral compounds by solvent extraction. Chemical Education Resources.
Papanikolaou, S., Aggelis, G., 2011. Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production. European Journal of Lipid Science and Technology, 113(8), 1031–1051.
Papanikolaou, S., Galiotou-Panayotou, M., Chevalot, I., Komaitis, M., Marc, I.,Aggelis, G. 2006. Influence of Glucose and Saturated Free-Fatty Acid Mixtures on Citric Acid and Lipid Production by Yarrowia lipolytica .Current Microbiology, 52(2), 134–142.
Papanikolaou1, S. , Galiotou-Panayotou1, M. , Fakas1, S., Komaitis1, M., Aggelis, G.
2007. Lipid production by oleaginous Mucorales cultivated on renewable carbon sources. European Journal of Lipid Science and Technology, 109( 11), 1060–1070.
Ratledge, C., Wynn, J.P. 2002. The biochemistry and molecular biology of lipid accumulation in oleaginous microorgaisms. Advances in Applied Microbiology, 51, 1-51.
Ratledge, C. 2004. Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie, 86(11), 807–815.
Rywinska, A., Juszczyk, P.,Wojtatowicz, M., Robak, M., Lazar, Z., Tomaszewska, L., Rymowicz, W. 2013. Glycerol as a promising substrate for Yarrowia lipolytica biotechnological applications. Biomass and Bioenergy, 48, 148-177.
Smedes, F. and Askland, T.K. 1999. Revisiting the development of the Bligh and Dyer total lipid determination method. Marine Pollution Bulletin, 38(3), 193- 201.
Sampaioa, F.C., Chaves-Alvesa, V. M., Convertib, A., Passosa,. F. M. L., Coelhoa J. L. C. 2008. Influence of cultivation conditions on xylose-to-xylitol bioconversion by a new isolate of Debaryomyces hansenii. Bioresource Technology. 99 (3), 502–508.
Turcotte G, Kosaric N. 1989. Lipid biosynthesis in oleaginous yeasts. Advances in Biochemical Engineering/Biotechnology , 40, 73–92.
Van Maris, A.J., Winkler, A.A., Kuyper, M., de Laat, W.T., van Dijken, J.P., Pronk , J.T. 2007. Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component. Advances in Biochemical Engineering/ Biotechnology, 108, 179–204.
Vicente, G., Martinez, M., Aracil, J. 2004. Integrated biodiesel production: a comparison of different homogeneous catalysts systems. Bioresource Technology, 92(3), 297-305.
Wang, Z., Zhunge, J., Fang, H., Prior, B.A. 2001. Glycerol production by microbialfermentation: a review. Biotechnology Advances, 19(3), 201-223.
Winkelhausen, E., Kuzmanova, S. 1998. Microbial conversion of d-xylose to xylitol. Journalof Fermentation and Bioengineering, 86(1), 1-14.
Zhao, X., Kong, X., Hua, Y., Feng, B., Zhao, Z.K. 2008. Medium optimization for lipid production through co-fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi. European Journal of Lipid Science and Technology, 110(5), 405–412.

Zhang, G., French, W.T., Hernandez, R., Alley E., Paraschivescu, M. 2011. Effects of furfural and acetic acid on growth and lipid production from glucose and xylose by Rhodotorula glutinis. Biomass and Bioenergy, 35(1), 734 -740.