生質燃料被視為一種可再生性的能源，相對於石化燃料，可減少長期燃燒所造成溫室氣體排放及全球暖化等問題，進而提升環境的友善度，而微藻具有成為生質柴油原料的潛力，因其生長快速、分布範圍廣泛、屬於非糧食作物、某些藻種具有高含油量，其細胞中油脂可經處理成為生質柴油，因此，以不同培養條件誘導微藻細胞提高油脂含量及產量為目前主要研究目標。
本研究利用兩階段培養小球藻 (Chlorella sp.)：第一階段以提高細胞產量為目的，第二階段以反應曲面法針對營養鹽及環境因子篩選出顯著因子並求得對提高油脂含量及產量較適化之培養條件，由實驗結果可知，第一階段小球藻於尿素濃度1.8 g/L下培養第1-10天，最大細胞量可達3.18 g/L；第二階段以溫度：5 ℃、鹽度：0 g/L、尿素濃度：0 g/L、磷酸鹽濃度：0 g/L、總通氣量：2 vvm、CO2濃度：0.03%、光照強度：12000 lux、照光週期：12/12之條件培養第11-14天，以此兩階段培養可得最大油脂含量為43.51%，最大油脂產量為1458.44 mg/L。

目錄

摘要 i
目錄 ii
圖目錄 v
表目錄 vii

第一章 緒論 1
1.1 前言 1
1.2 研究動機 3
第二章 文獻回顧 4
2.1 藻類簡介 4
2.1.1 藻類的分類與特徵 4
2.1.2 小球藻 (Chlorella sp.)簡介 7
2.1.3 小球藻 (Chlorella sp.)的生殖方式 8
2.1.4 藻類的光合作用 8
2.1.5 藻類油脂生成 11
2.2 藻類生長與脂質生成的影響因子 11
2.2.1 營養源 12
2.2.2 鹽度 14
2.2.3 酸鹼度 14
2.2.4 溫度 15
2.2.5 光照強度 15
2.2.6 生長週期與年齡 16
2.3 藻類培養方式 17
2.3.1 培養系統 17
2.3.2 培養策略 19
2.3.3 兩階段培養策略 20
2.4 微藻於生質柴油生產上的潛力 20
2.5 反應曲面法與設計 26
2.5.1 反應曲面法簡介 26
2.5.2 因子篩選及極值點區域的逼近 26
2.5.3 二階模型的建立 28
第三章 實驗材料與方法 31
3.1 藻株培養 31
3.1.1 藻種 31
3.1.2 培養基組成 31
3.1.3 前培養條件 31
3.2 實驗材料與設備 33
3.2.1藥品 33
3.2.2 儀器設備 34
3.3 分析方法 35
3.3.1 細胞濃度分析 35
3.3.2 尿素濃度分析 35
3.3.3 油脂含量分析 36
3.4 實驗步驟 39
3.4.1 基礎培養條件確立 39
3.4.2 二階段培養 40
第四章 結果與討論 44
4.1 基礎培養條件探討 44
4.1.1 不同緩衝溶液對Chlorella sp.生長及油脂生產之影響 44
4.1.2 培養基pH值對Chlorella sp.生長及油脂生產之影響 48
4.2 兩階段培養探討 51
4.2.1 第一階段：不同起始尿素濃度對Chlorella sp.生長之影響 51
4.2.2 第二階段：篩選顯著因子 52
4.2.3 第二階段：中心複合設計 60
第五章 結論 68
參考文獻 70

圖目錄

圖1-1、美國能源供應中再生能源運用比例 2
圖2-1、類囊體的結構 9
圖2-2、光非依賴型反應:卡式循環 10
圖2-3、藻類三酸甘油脂合成途徑 11
圖2-4、不同型態之密閉式光反應器 18
圖2-5、RSM的逐次性質 28
圖2-6、k = 2和k = 3中心複合設計 30
圖2-7、三因子的Box-Behnken設計 30
圖3-1、杯瓶培養 31
圖3-2、細胞濃度之檢量線 35
圖3-3、尿素濃度之檢量線 36
圖3-4、藻細胞總油脂及脂肪酸含量分析流程 38
圖3-5、第一階段至第二階段培養導入方式 41
圖3-6、反應曲面法實驗設計流程 41
圖4-1、Chlorella sp.培養於不同緩衝溶液下之生長曲線 46
圖4-2、Chlorella sp.培養於不同pH值下之生長曲線 49
圖4-3、Chlorella sp.培養於不同尿素濃度下之生長曲線 53
圖4-4、Chlorella sp.培養期始末之尿素濃度消耗情形 54
圖4-5、油脂含量之柏拉圖 57
圖4-6、油脂含量之主效應圖 58
圖4-7、油脂產量之柏拉圖 59
圖4-8、油脂產量之主效應圖 59
圖4-9、油脂含量之反應曲面圖 65
圖4-10、油脂含量之等高線圖 65
圖4-11、油脂含量及油脂產量 (兩階段培養最佳結果vs.單一階段培養) 67

表目錄

表2-1、藻類的分界 4
表2-2、藻類在系統學上的分類 5
表2-3、藻類與植物區別之特徵 6
表2-4、影響藻類生長及脂質生成的因子 12
表2-5、開放式及密閉式系統的主要特徵 18
表2-6、生質柴油原料來源之比較 22
表2-7、不同微藻之含油量 23
表2-8、微藻油脂生產之生質柴油、柴油及ASTM生質柴油規範特性之比較 25
表3-1、韋因培養基組成 32
表3-2、培養基配置 32
表3-3、實驗用藥品及用途 33
表3-4、篩選實驗因子與水準設定 42
表3-5、25-1因子實驗配置 42
表3-6、中心複合設計因子與水準設定 (α=1) 43
表3-7、中心複合設計之實驗配置 43
表4-1、不同緩衝溶液對Chlorella sp.生長及油脂生產之影響 47
表4-2、Chlorella sp.培養於不同緩衝溶液下之脂肪酸組成 47
表4-3、不同pH值對Chlorella sp.生長及油脂生產之影響 50
表4-4、Chlorella sp.培養於不同pH值條件下之脂肪酸組成 50
表4-5、不同尿素濃度對Chlorella sp.生長及油脂生產之影響 54
表4-6、25-1因子實驗設計結果 56
表4-7、25-1因子實驗設計—油脂含量之迴歸分析 57
表4-8、25-1因子實驗設計—油脂產量之迴歸分析 58
表4-9、中心複合設計實驗結果 63
表4-10、中心複合設計—油脂含量之迴歸分析 (未簡化) 64
表4-11、中心複合設計—油脂含量之迴歸分析 (簡化後) 64
表4-12、中心複合設計—油脂含量之變異數分析表 (簡化後) 64
表4-13、培養條件 (兩階段培養最佳結果 vs. 單一階段培養) 66

江永棉、王瑋龍、黃淑芳，1990，台灣海藻簡介，國立台灣博物館。
沈明來，1999，試驗設計學，九州圖書文物有限公司。
林李旺，2009，快速精通實驗設計—邁向Six Sigma的關鍵方法，全華圖書股份有限公司。
洪哲穎、陳國誠，1992，回應曲面實驗設計法在微生物酵素生產上之應用，化工 39 (2):3-18。
徐明光，1999，台灣的淡水浮游藻(I)-通論及綠藻(1)，國立台灣博物館。
梁象秋、方紀祖、楊和荃，1998，水生生物學(型態與分類)，水產出版社。
葉俊良，2006，在光生化反應器中以二階段策略培養微藻生產油脂之研究，國立成功大學化學工程學系碩士論文。
葉修鋒，2009，探討微藻Chlorella sp.油脂生產較適培養條件，國立清華大學化學工程學系碩士論文。
黎正中、陳源樹，2006，實驗設計與分析，高立圖書有限公司。
Alonso, D. L., E. H. Belarbi, J. M. Fernandez-Sevilla, J. Rodriguez-Ruiz, and E. M. Grima. 2000. Acyl lipid composition variation related to culture age and nitrogen concentration in continuous culture of the microalga Phaeodactylum tricornutum. Phytochemistry 54 (5):461-471.
Becker, E. W. 1994. Microalgae: biotechnology and microbiology: University of Cambridge press, New York.
Ben-Amotz, A., Tornabene, T. G. 1985. Chemical profile of selected species of microalgae with emphasis on lipids. J. Phycol. 21:72-81.
Bertoldi, F. C., E. Sant'Anna, M. V. D. Braga, and J. L. B. Oliveira. 2006. Lipids, fatty acids composition and carotenoids of Chlorella vulgaris cultivated in hydroponic wastewater. Grasas Aceites 57 (3):270-274.
Bigogno, C., I. Khozin-Goldberg, S. Boussiba, A. Vonshak, and Z. Cohen. 2002. Lipid and fatty acid composition of the green oleaginous alga Parietochloris incisa, the richest plant source of arachidonic acid. Phytochemistry 60 (5):497-503.
Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Physiol. Pharmacol. 37 (8):911-917.
Bold, H. C., and M. J. Wynne. 1985. Introduction to the Algae: structure and reproduction. 2nd ed: Prentice Hall, Englewood Cliffs, New Jersey.
Box, G. E., and K. B. Wilson. 1951. On the experiment attainment optimum conditions. J. Roy. Statist. Soc. B13:1-45.
Butler, W. R., J. Calaman and S. Bean. 1996. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J. Anim. Sci. 74: 858-865.
Carvalho, A. P., L. A. Meireles, and F. X. Malcata. 2006. Microalgal reactors: A review of enclosed system designs and performances. Biotechnol. Prog. 22 (6):1490-1506.
Chaumont, D. 1993. Biotechnology of algal biomass production-a review of systems for outdoor mass-culture. J. Appl. Phycol. 5 (6):593-604.
Chi, Z. Y., Y. Liu, C. Frear, and S. L. Chen. 2009. Study of a two-stage growth of DHA-producing marine algae Schizochytrium limacinum SR21 with shifting dissolved oxygen level. Appl. Microbiol. Biotechnol. 81 (6):1141-1148.
Chisti, Y. 2007. Biodiesel from microalgae. Biotechnol. Adv. 25 (3):294-306.
Chisti, Y. 2008. Biodiesel from microalgae beats bioethanol. Trends Biotechnol. 26 (3):126-131.
Cifuentes, A. S., M. A. Gonzalez, I. Inostroza, and A. Aguilera. 2001. Reappraisal of physiological attributes of nine strains of Dunaliella (Chlorophyceae): Growth and pigment content across a salinity gradient. J. Phycol. 37 (2):334-344.
Cohen, Z., A. Vonshak, and A. Richmond. 1988. Effect of environmental conditions on fatty acid composition of the red alga Porphyridium cruentum correlation to growth-rate. J. Phycol. 24 (3):328-332.
Converti, A., A. A. Casazza, E. Y. Ortiz, P. Perego, and M. D. Borghi. 2009. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis oculata and Chlorella vulgaris for biodiesel production. Chem. Eng. Process. 48 (6):1146-1151.
Endo, T., U.　Schreiber, and　K. Asada. 1995. Suppression of quantum yield of photosystem-II by hyperosmotic stress in Chlamydomonas reinhardtii. Plant Cell Physiol. 36 (7):1253-1258.
Energy Information Administration, Monthly Energy Review. 2009. From http://www.eia.doe.gov
Fabregas, J., A. Maseda, A. Dominguez, and A. Otero. 2004. The cell composition of Nannochloropsis sp. changes under different irradiances in semicontinuous culture. World J. Microbiol. Biotechnol. 20 (1):31-35.
Fabregas, J., A. Otero, A. Maseda, and A. Dominguez. 2001. Two-stage cultures for the production of astaxanthin from Haematococcus pluvialis. J. Biotechnol. 89 (1):65-71.
Fabregas, J., V. Vazquez, B. Cabezas, and A. Otero. 1993. Tris not only controls the pH in microalgal cultures, but also feeds bacteria. J. Appl. Phycol. 5 (5):543-545.
Floreto, E. A. T., and S. Teshima. 1998. The fatty acid composition of seaweeds exposed to different levels of light intensity and salinity. Bot. Marina 41 (5):467-481.
Gehl, K. A., and B. Colman. 1985. Effect of external pH on the internal pH of Chlorella saccharophila. Plant Physiol. 77 (4):917-921.
Guckert, J. B., and K. E. Cooksey. 1990. Triglyceride accumulation and fatty-acid profile changes in Chlorella (chlorophyta) during high pH-induced cell-cycle inhibition. J. Phycol. 26 (1):72-79.
Guschina, I. A., and J. L. Harwood. 2006. Lipids and lipid metabolism in eukaryotic algae. Prog. Lipid Res. 45 (2):160-186.
Hervas, M., J. A. Navarro, and M. A. De La Rosa. 2003. Electron transfer between membrane complexes and soluble proteins in photosynthesis. Accounts Chem. Res. 36 (10):798-805.
Higashiyama, K., K. Murakami, H. Tsujimura, N. Matsumoto, and S. Fujikawa. 1999. Effects of dissolved oxygen on the morphology of an arachidonic acid production by Mortierella alpina 1S-4. Biotechnol. Bioeng. 63 (4):442-448.
Hsieh, C. H., and W. T. Wu. 2009. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour. Technol. 100 (17):3921-3926.
Hu, Q. 2004. Handbook of Microalgal Culture : biotechnology and applied phycology. Edited by A. Richmond: Oxford: Blackwell Science.
Hu, Q., M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, and A. Darzins. 2008. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J. 54 (4):621-639.
Illman, A. M., A. H. Scragg, and S. W. Shales. 2000. Increase in Chlorella strains calorific values when grown in low nitrogen medium. Enzyme Microb. Technol. 27 (8):631-635.
Jiang, Y., and F. Chen. 1999. Effects of salinity on cell growth and docosahexaenoic acid content of the heterotrophic marine microalga Crypthecodinium cohnii. J. Ind. Microbiol. Biotechnol. 23 (6):508-513.
Johnson, E. T., and C. Schmidt-Dannert. 2008. Light-energy conversion in engineered microorganisms. Trends Biotechnol. 26 (12):682-689.
Khozin-Goldberg, I., and Z. Cohen. 2006. The effect of phosphate starvation on the lipid and fatty acid composition of the fresh water eustigmatophyte Monodus subterraneus. Phytochemistry 67 (7):696-701.
Laing, I. 1991. Cultivation of marine, unicellular algae. Lab. Leaflet. MAFF Direct. Fish. Res., Lowestoft 67:1-31.
Li, Q., W. Du, and D. Liu. 2008a. Perspectives of microbial oils for biodiesel production. Appl. Microbiol. Biotechnol. 80 (5):749-756.
Li, Y., M. Horsman, B. Wang, N. Wu, and C. Q. Lan. 2008b. Effects of nitrogen sources on cell growth and lipid accumulation of green alga Neochloris oleoabundans. Appl. Microbiol. Biotechnol. 81 (4):629-636.
Liang, Y., J. Beardall, and P. Heraud. 2006. Changes in growth, chlorophyll fluorescence and fatty acid composition with culture age in batch cultures of Phaeodactylum tricornutum and Chaetoceros muelleri (Bacillariophyceae). Bot. Marina 49 (2):165-173.
Lynn, S. G., S. S. Kilham, D. A. Kreeger, and S. J. Interlandi. 2000. Effect of nutrient availability on the biochemical and elemental stoichiometry in the freshwater diatom Stephanodiscus minutulus (Bacillariophyceae). J. Phycol. 36 (3):510-522.
Mader, S. S. 2000. Inquiry into Life. 10th ed: McGraw-Hill Companies.
Metting, F. B. 1996. Biodiversity and application of microalgae. J. Ind. Microbiol. 17:477-489.
Miao, X., and Q. Wu. 2006. Biodiesel production from heterotrophic microalgal oil. Bioresour. Technol. 97:841-846.
Murata, N. 1989. Low-temperature effects on cyanobacterial membranes. J. Bioenerg. Biomembr. 21 (1):61-75.
Najafpour, G. 2007. Biochemical Engineering and Biotechnology: Elsevier Science.
Nishida, I., and N. Murata. 1996. Chilling sensitivity in plants and cyanobacteria: The crucial contribution of membrane lipids. Annu. Rev. Plant Physiol., Plant Mol. Biol. 47:541-568.
Nuutila, A. M., A. M. Aura, M. Kiesvaara, and V. Kauppinen. 1997. The effect of salinity, nitrate concentration, pH and temperature on eicosapentaenoic acid (EPA) production by the red unicellular alga Porphyridium purpureum. J. Biotechnol. 55 (1):55-63.
Orcutt, D. M., and G. W. Patterson. 1974. Effect of light-intensity upon lipid-composition of Nitzschia closterium (Cylindrotheca fusiformis). Lipids 9 (12):1000-1003.
Otsuka, H. 1961. Changes of lipid and carbohydrate contents in Chlorella cells during sulfur starvation, as studied by technique of synchronous culture. J. Gen. Appl. Microbiol. 7 (1):72-77.
Perrin, D. D., and B. Dempsey. 1974. Buffers for pH and Metal Ion Control. 1st ed: Chapman and Hall, London; Wiley, New York.
Philips, S. 1997. A Biology of the Algae. 2nd ed: University of Cambridge press, London.
Pulz, O. 2001. Photobioreactors: production systems for phototrophic microorganisms. Appl. Microbiol. Biotechnol. 57 (3):287-293.
Reitan, K. I., J. R. Rainuzzo, and Y. Olsen. 1994. Effect of nutrient limitation on fatty-acid and lipid-content of marine microalgae. J. Phycol. 30 (6):972-979.
Renaud, S. M., H. C. Zhou, D. L. Parry, L. V. Thinh, and K. C. Woo. 1995. Effect of temperature on the growth, total lipid content and fatty acid composition of recently isolated tropical microalgae Isochrysis sp, Nitzschia closterium, Nitzschia paleacea, and commercial species Isochrysis sp (clone T ISO). J. Appl. Phycol. 7 (6):595-602.
Renaud, S. M., L. V. Thinh, G. Lambrinidis, and D. L. Parry. 2002. Effect of temperature on growth, chemical composition and fatty acid composition of tropical Australian microalgae grown in batch cultures. Aquaculture 211 (1-4):195-214.
Rex, M. C., D. C. E. Dawson, W. H. Elliott, and K. M. Jones. 1986. Data for Biochemical Research. 3rd ed: Oxford: Clarendon Press.
Roessler, P. G. 1988. Changes in the activities of various lipid and carbohydrate biosynthetic-enzymes in the diatom Cyclotella　cryptica in response to silicon deficiency. Arch. Biochem. Biophys. 267 (2):521-528.
Sanchez-Luna, L. D., R. P. Bezerra, M. C. Matsudo, S. Sato, A. Converti, and J. C. M. de Carvalho. 2007. Influence of pH, temperature, and urea molar flowrate on Arthrospira platensis fed-batch cultivation: A kinetic and thermodynamic approach. Biotechnol. Bioeng. 96 (4):702-711.
Sato, N., M. Hagio, H. Wada, and M. Tsuzuki. 2000. Recent Advances in the Biochemistry of Plant Lipids. Edited by J. L. Harwood and P. J. Quinn: London: Portland Press Ltd.
Schenk, P. M., S. R. Thomas-Hall, E. Stephens, U. C. Marx, J. H. Mussgnug, C. Posten, O. Kruse, and B. Hankamer. 2008. Second generation biofuels: high-efficiency microalgae for biodiesel production. Bioenerg. Res. 1 (1):20-43.
Seto, A., H. L. Wang, and C. W. Hesseltine. 1984. Culture conditions affect eicosapentaenoic acid content of Chlorella minutissima. J. Am. Oil Chem. Soc. 61 (5):892-894.
Spijkerman, E., E. Garcia-Mendoza, H. C. P. Matthijs, E. Van Hunnik, and P. F. M. Coesel. 2004. Negative effects of P-buffering and pH on photosynthetic activity of planktonic desmid species. Photosynthetica 42 (1):49-57.
Spolaore, P., C. Joannis-Cassan, E. Duran, and A. Isambert. 2006. Commercial applications of microalgae. J. Biosci. Bioeng. 101: 87-96.
Spolaore, P., C. Joannis-Cassan, E. Duran, and A. Isambert. 2006. Optimization of Nannochloropsis oculata growth using the response surface method. J. Chem. Technol. Biotechnol. 81 (6):1049-1056.
Starr, C., and R. Taggart. 1989. Biology: the unity and diversity of life. 5th ed: Wadsworth Publishing Company.
Sukenik, A., Y. Yamaguchi, and A. Livne. 1993. Alterations in lipid molecular-species of the marine Eustigmatophyte Nannochloropsis sp. J. Phycol. 29 (5):620-626.
Takagi, M., Karseno, and T. Yoshida. 2006. Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. J. Biosci. Bioeng. 101 (3):223-226.
Tatsuzawa, H., E. Takizawa, M. Wada, and Y. Yamamoto. 1996. Fatty acid and lipid composition of the acidophilic green alga Chlamydomonas sp. J. Phycol. 32 (4):598-601.
Thompson, G. A. 1996. Lipids and membrane function in green algae. Biochim. Biophys. Acta Lipids and Lipid Metabolism 1302 (1):17-45.
Thompson, P. A., M. X. Guo, and P. J. Harrison. 1992. Effects of variation of temperature: I. On the biochemical composition of eight species of marine phytoplankton. J. Phycol. 28 (4):481-488.
Turpin, D. H. 1991. Effects of inorganic N availability on algae photosynthesis and carbon metabolism. J. Phycol. 27 (1):14-20.
Ursi, S., M. Guimaraes, and E. M. Plastino. 2008. Deleterious effect of TRIS buffer on growth rates and pigment content of Gracilaria birdiae Plastino & EC Oliveira (Gracilariales, Rhodophyta). Acta Bot. Bras. 22 (3):891-896.
Walne, P. R. 1974. Culture of Bivalve Molluscs : 50 years' experience at Conwy. 2nd ed: Fishing News Books, Farnham.
Weissman, J. C., R. P. Goebel, and J. R. Benemann. 1988. Photobioreactor design-mixing, carbon utilization, and oxygen accumulation. Biotechnol. Bioeng. 31 (4):336-344.
Zhang, D. H., and Y. K. Lee. 2001. Two-step process for ketocarotenoid production by a green alga, Chlorococcum sp. strain MA-1. Appl. Microbiol. Biotechnol. 55 (5):537-540.