臺灣博碩士論文加值系統

由於現在能源危機及溫室效應的影響，微藻的應用被逐漸重視，微藻具有快速生長、高油脂含量與豐富的營養成分，對水質的淨化也有一定的效果。在本實驗中，利用Scenedesmus obliquus的光自營培養所探討的三個研究方向：(1)改變培養環境或培養基成分，探討S. obliquus濃度與葉黃素含量影響。結果顯示加壓條件對S. obliquus藻細胞生長造成生長遲緩的現象發生；紫外光UV-A連續照光會有明顯抑制生長效果；添加酚(Phenol)會抑制藻細胞生長；不同顏色LED光源條件，可獲得在白光條件下培養S. obliquus濃度最高可至2.45 g/L。葉黃素方面，在最後兩天添加紫外光UV-A照射可增加葉黃素含量至0.48% d.w.； 在加壓條件下與添加酚(Phenol)時對S. obliquus內的葉黃素含量並無太大的影響。(2)探討20.0 L生物光反應器培養S. obliquus與連續式培養之影響。結果顯示，在不同進氣量(vvm)條件下，微藻生長無明顯的差異，0.1 vvm進氣量培養S. obliquus濃度可至1.22 g/L。；重覆式批次進料培養，以0.1 vvm進氣量，不同稀釋倍率，培養21天，其產率(Productivity)均約為0.134 g/L/day；利用連續式進料培養，觀察不同水力滯留時間(HRT)影響，而在HRT為4.44 day時，可得產率與重覆式批次相當。(3)本實驗探討超臨界二氧化碳萃取微藻中葉黃素之最適化條件。利用中心混成設計(CCD)以三個因子進行探討: (a)壓力200~400 bar，(b)溫度40~80 ℃，(c)共溶劑(Co-solvent)Ethanol添加量20~50%。實驗結果顯示，利用反應曲面法(Response surface methodology, RSM)設計實驗，其回歸之最大極值，壓力條件為276.8 bar、溫度條件為70.1 ℃及Ethanol共溶劑添加量50%，與傳統甲醇法萃取葉黃素比較，可達葉黃素最高回收率89.8 %。

Due to the depletion of energy and greenhouse effect recently, the applications of microalgae are getting important. Microalgae are organism with high growing rate, rich in fat contents and nutritional components. Also, it has great impact on water purifying. In this research, we investigate the following aspect by cultivating the microalgae: (1) Investigate the impact on the concentration of Scenedesmus obliquus and lutein content by changing the environment or the medium compositions. The result showed us that adding pressure would delay the growing of S. obliquus cells; the growing of algae could be significantly inhibited by continuous irradiating of ultraviolet UV-A; addition of phenol would inhibit the growth of algal cells; with different colors lighting experiment, culturing with white light earned the maximum concentration to S. obliquus to 2.45 g/L. As the study on the effect on lutein, illuminating ultraviolet UV-A in the last two days could increase the lutein content to 0.48%, while adding phenol and exerting pressure had less impact on lutein content. (2) Investigate the effect on the S. obliquus by cultivated by 20.0 L bio-photoreactor and continuous cultivation. The result showed that using the aeration of 0.1 vvm cultivation, the S. obliquus concentration could increase to 1.22 g/L. Apply with different aerations (vvm) do not show obvious effect on the growing of algal cells; with repeated- batch cultivation, at 0.1 vvm, under different dilution ratio for 21 days, the productivities obtain are about 0.134 g/L/day; with continuous cultivation, we study the effect of different hydraulic retention time (HRT), and in the HRT of 4.44 day, we obtain the maximum productivity, which is 0.134 g/L/day. (3) Investigate the optimal condition for extraction of lutein in S. obliquus with supercritical carbon dioxide. Applying of central composite design (CCD) to explore three factors: (a) pressure of 200 to 400 bar, (b) temperature of 40~80 ℃, (c) addition of co-solvent (ethanol) 20-50%. Results implied that, designed the experiment by response surface methodology (RSM), the largest extreme value of regression is obtained. While setting Co-solvent=50%, P=276.8 bar and T=70.1 ℃, we can obtain the lutein yield ratio up to 89.8%, compared with traditional extraction.

誌謝 I
摘要 II
ABSTRACT III
目錄 V
圖目錄 IX
表目錄 XII
第一章　緒論 1
第二章 文獻回顧 3
2.1 微藻簡介 3
2.1.1 微藻介紹 3
2.1.2 影響微藻生長之環境因子 7
2.1.3 微藻培養方式 11
2.1.4 微藻培養介紹 11
2.1.5 氣舉式生物反應器介紹 14
2.2 類胡蘿蔔素簡介 15
2.2.1 葉黃素(Lutein)介紹 16
2.2.2 葉黃素的生理活性 17
2.2.3 葉黃素的生產 19
2.2.4 影響葉黃素合成的營養及環境因子 22
2.3 超臨界流體簡介 27
2.3.1 超臨界流體歷史發展 27
2.3.2 超臨界流體定義 28
2.3.3 超臨界流體性質 29
2.3.4 影響超臨界流體萃取之因素 32
2.3.5 超臨界流體萃取的應用 36
2.4 反應曲面法介紹 37
第三章　實驗材料與方法 40
3.1　實驗材料 40
3.1.1　藻種 40
3.1.2　實驗藥品 41
3.2　實驗儀器 42
3.3　分析方法 43
3.3.1　光照強度測量方法 43
3.3.2　藻體濃度測量方法(吸光值測量&利用紅外線水分蒸發儀測量) 43
3.3.3　葉黃素(Lutein)濃度測量方法 44
3.4　實驗方法 45
3.4.1　藻種保存 45
3.4.2　藻種活化 45
3.4.3　培養基組成 45
3.5　實驗架構 46
3.6　實驗培養條件 47
3.6.1　改變環境因素(abiotic stress)實驗探討 47
3.6.2　20.0 L氣舉式光反應器連續培養實驗探討 49
3.6.3　超臨界流體萃取最佳化條件實驗探討 51
3.7　實驗設備圖 52
3.7.1　500 ml反應器設備圖 52
3.7.2　20.0 L 氣舉式反應器設備圖 53
3.7.3　超臨界二氧化碳萃取設備圖 54
第四章 結果與討論 55
4.1 藉由改變環境因素，探討藻細胞和葉黃素含量之影響 55
4.1.1 不同加壓比例對Scenedesmus obliquus生長之影響 55
4.1.2 紫外光(UV-A)對Scenedesmus obliquus生長和葉黃素含量之影響 58
4.1.3 酚(Phenol)的添加對Scenedesmus obliquus生長和葉黃素含量之影響 60
4.1.4 不同LED色光對Scenedesmus obliquus生長和葉黃素含量之影響 62
4.1.5 改變環境因素(abiotic stress)之綜合探討 64
4.1.6 改變環境因素(abiotic stress)產生之葉黃素與文獻進行比較 65
4.2 20.0 L光反應器實驗探討 66
4.2.1 不同通氣量對Scenedesmus obliquus生長之影響 66
4.2.2 探討利用重覆批次(repeated-batch)方式培養Scenedesmus obliquus其生 長情況之影響 68
4.2.3 探討利用連續式(continuous)方式培養Scenedesmus obliquus其生長情況之影響 70
4.3 超臨界二氧化碳萃取葉黃素之最佳化探討 72
4.3.1 有機溶劑萃取葉黃素 72
4.3.2 探討添加填充顆粒(packing particles)對超臨界萃取Scenedesmus obliquus之葉黃素的影響 72
4.3.3 反應曲面法進行超臨界萃取中的Scenedesmus obliquus葉黃素最佳探討 74
4.3.4 反應曲面模式適切性之統計檢驗 77
4.3.5 反應曲面圖 78
4.3.5.1 Temperature 和 Co-solvent 對葉黃素回收率之反應曲面圖 78
4.3.5.2 Co-solvent 和 Pressure 對葉黃素回收率之反應曲面圖 79
4.3.5.3 Temperature 和 Pressure 對葉黃素回收率之反應曲面圖 80
第五章 結論與未來展望 81
5.1 結論 81
5.2 未來展望 82
參考文獻 83
附錄 88

蔣安國、蔣安仁、丁敬哲、鍾健平(2005)。以模擬技術和統計方法建構專案排程之可靠度。第一屆管理與決策2005年學術研討會特刊，119-130。
王明輝、陳韻研、何炎明(2008)。紫外線照射對綠藻Scenedesmus dimorphus生長繁殖和葉綠素含量的影響。安徽農業科學，第36卷，第23期，9863-9864。
鄭俊明、劉清雲(2007)。微藻產業。科學發展。415期，34-40。
陳 峰、姜 悅(1999)。微藻生物技術。中國輕工業出版社，第一版。
鄭建新、鄒登峰(2006)。生物轉換法從金盞菊提取物中製備葉黃素的研究。中國藥理學通報，第22卷，第12期，1528-1531。
謝誌鴻、吳文騰(2009)。綠色生質能源。科學發展，433期，36-41。
陳曉薇、陳茂景(2008)。「微」小世界的無限潛能-台電公司在微藻減碳技術的發展。能源報導，12月，8-10。
李秀霞、張傑明(1997)。土壤復育技術：超臨界流體萃取實驗之研究。化工期刊，第44卷，第3期，67-78。
鄒樹平(2007)。微藻的綜合開發利用。水產科學，第26卷，第3期，179-181。
侯嘉龍(2010)。綠色寶藏-綠藻的開發與應用。化工，第57卷，第2期，53-64。
劉龍軍(2006)。利用微藻生產特種天然類胡蘿蔔素的研究發展。研究綜述，第30卷，第9期，63-68。
郭志有(2010)。葉黃素的功能及應用。河北農業科學，第14卷，第2期，52-53。
雷劍芬(2009)。氣升式反應器在發酵工業中的應用研究進展。化工時刊，第23卷，第6期，67-69。
劉靜瑀(2008)。微藻減碳，捕捉二氧化碳量較植樹高兩倍。中央廣播電視台，8月11日。
張建芬(2006)。葉黃素的微藻生物法生產技術。食品科技，第12期，106-109。
潘忠政(2001)。整合鹼液吸收及光合作用以固定二氧化碳。大葉大學環境工程研究所碩士論文。
洪志瑞(2007)。油脂性微藻培養於新型光生化反應器之研究。成功大學化學工程學系研究所碩士論文。
鄭玟芩(2008)。海洋微藻在氮源限制下固定CO2與生質潛能組成之研究。成功大學環境工程學系研究所碩士論文。
林熤庭(2002)。黃斑色素形成機轉之研究。國立陽明大學醫學工程研究所碩士論文。
林育如(2003)。增加年輕人飲食中蔬果的攝取對血漿類胡蘿蔔素濃度的影響。台北醫學大學保健營養學系碩士論文。
孫證雄(2011)。自營環境下培養柵藻生成葉黃素與超臨界流體萃取葉黃素之探討。東海大學化學工程與材料工程學系碩士論文。
李孟洲(2007)。以微藻生產氫氣作為再生能源。海洋大學水產養殖系博士論文。
Arnal, E., M. Miranda, I. Almansa, M. Muriach, J. M. Barcia, F. J. Romero, M. Diaz-Llopis, and F. Bosch-Morell, "Lutein prevents cataract development and progression in diabetic rats," Graefes Arch Clin Exp Ophthalmol, 247, 115 (2009).
Ayhan Demirbas, M. Fatih Demirbas, "Importance of algae oil as a source of biodiesel," , Energy Conversion and Management, 24,1 (2010)
Bendich, A., "Carotenoids and the immune response," Journal of Nutrition., 119, 112 (1989).
Bone, R. A., J. T. Landrum, and A. Cains, "Opticaal density spectra of the macular pigment in vivo and in vitro," Vision Research, 32, 105 (1992).
Box, G. E. & N. R. Draper," Empirical Model Building and Response Surface," John Wiley and Sons, New York (1997).
Brunner, G., "Supercritical fluids: technology and application to food processing, " Journal of Food Engineering, 67, 21 (2005).
Berg, H. V. D., R. Faulks, H. F. Granado, J. Hirschberg, B. Olmedilla, G. Sandmann, S. Southon, and W. Stahl, "The potential for the improvement of carotenoid levels in foods and the likely systemic effects," Journal of the Science of Food and Agriculture, 80, 880 (2000).
Carpentier, S., M. Knaus, and M. Suh, "Associations between lutein, zeaxanthin, and age-related macular degeneration: an overview," Critical Reviews in Food Science and Nutrition, 49, 313 (2009).
Campo, J. A. D., J. Moreno, H. Rodríguez, M. A. Vargas, J. Rivas, and M. G. Guerrero, "Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp.(Chlorophyta)," Journal of Biotechnology, 76, 51 (2000).
Campo, J. A. D., H. Rodríguez , J. Moreno, , M. A. Vargas, J. Rivas, and M. G. Guerrero, "Lutein production by Muriellopsis sp. in an outdoor tubular photobioreactor," Journal of Biotechnology, 85, 289 (2001).
Fang, Y. Z., S. Yang, and G. Wu, "Free radicals, antioxidants, and nutrition," Nutrition, 18, 872 (2002).
Fernández-Sevilla, J. M., F. G. Acién Fernández, and E. M. Grima, "Biotechnological production of lutein and its applications," Applied Microbiol Biotechnol, 86, 27 (2010).
F.I. Dromgoole, " The effects of oxygen on dark respiration and apparent photosynthesis of marine macro-algae," Aquatic Botany, 4, 218(1978).
Goksan, T., Y. A. Durmaz, and E. Gokpinar, "Effects of light path lengths and initial culture density on the cultivation of Chaetoceros muelleri," Aquaculture, 217, 431 (2003).
Heber, D. and L. Qing-Yi, "Overview of mechanisms of action of lycopene," Experimental Biology and Medicine, 227, 920 (2002).
Kohn, P. M. and P.R. Savage, "Supercritical fluids try for CPI application," Chemical Engineering, 86, 41 (1979).
Richmond, A., "Book review," Journal of Appied Phycology, 16, 159 (2004).
Lee, Y. K. and H. S. Tay, "High CO2 partial pressure depresses productivity and bioenergetic growth yield of Chlorella pyrenoidosa culture," Journal of Applied Phycology, 3, 95 (1991).
Larson, K. A. and M.L. King, "Evaluation of Supercritical Fluid Extraction in the Pharmaceutical Industry, "Biotechnology Process, 2, 73 (1986).
Lenucci, M. S., D. Cadinu, M. Taurino, G. Piro, and G. Dalessandro, "Antioxidant composition in cherry and high-pigment tomato cultivars," Journal of Agricultural and Food Chemistry, 54, 2606 (2006).
Macías-Sánchez, M. D., J. M. Fernández-Sevilla, F. G. A. Fernández, M. C. C. García, and E. M. Grima, "Supercritical fluid extraction of carotenoids from Scenedesmus almeriensis," Food Chemistry, 123, 928 (2010).
Marcelo C. Matsudo, R. P. Bezerra, S. Sato, P. Perego, A. Converti and J. C. M. Carvalho, "Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as nitrogen source," Biochemical Engineering Journal, 43, 52 (2008).
Masojidek, J. and H. K. Mangold, "Extraction with supercritical fluids: a progress report from Germany," Journal of the American Oil Chemists Society, 59, 673 (1982).
Myers, R. H. & Montyomery, D. C., "Response Surface Methodology, Process and Product Optimization Using Designed Experiments," John Wiley and Sons, New York (1995).
Piccaglia, R., M. Marotti, and S. Grandi, "Lutein and lutein ester content in different types of Tagetes patula and T. erecta," Industrial Crops and Products, 8, 45 (1998).
Qiang, H., M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, and A. Darzins, "Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances," The Plant Journal, 54, 621 (2008).
Raven, P. H., Johnson, and G. B., “Biology”, McGraw-Hill, sixth edition,(2002).
Richmond, A., "Principles for attaining maximal microalgal productivity in photobioreactor: an overview, Hydrobiologia, 512, 33 (2004).
Renaud, S. M., L. V. Thinh, G. Lambrinidis, and D. L. Parry, "Effect of temperature on growth, chemical composition and fatty acid composition of tropical Australian microalgae grown in batch culture," Aquaculture, 211, 195 (2002).
Roberts, R., J. Green, and B. Lewis, "Lutein and zeaxanthin in eye and skin health," Clinics in Dermatology, 27, 195 (2009).
Sánchez, J. F., J. M. Fernández-Sevilla, F. G. Acién, M. C. Cerón, J. Pérez-Parra, and E. Molina-Grima, "Biomass and lutein productivity of Scenedesmus almeriensis: influence of irradiance, dilution rate and temperature," Applied Microbiology Biotechnology, 79, 719 (2008).
Sánchez, J. F., J. M. Fernández, F. G. Acién, A. Rueda, J. Pérez-Parra, and E. Molina, "Influence of culture conditions on the productivity and lutein content of the new strain Scenedesmus almeriensis," Process Biochemistry, 43, 398 (2008).
Shi, X. M. and F. Chen, "Stability of lutein under various storage conditions," Nahrung, 41, 38 (1997).
Sakaki, H., H. Nochide, S. Komemushi, and W. Miki, "Effect of active oxygen species on the productivity of torularhodin by Rhodotorula glutinis No. 21," Journal of bioscience and bioengineering, 93, 338 (2002).
Schrott, E. L., "Carotenoids in plant photoprotection," Applied Chemistry., 57, 729 (1985).
Suh, I. S. and S. B. Lee, "Cultivation of a cyanobacterium in an internally radiation air-lift photobioreactor," Journal of Applied Phycology, 13, 381 (2001).
Tukaj, Z., K. Matusiak-Mikulin, J. Lewandowska, and J. Szurkowski, "Changes in the pigment patterns and the photosynthetic activity during a light-induced cell cycle of the green alga Scenedesmus armatus," Plant Physiology and Biochemistry, 41, 337 (2003).
V. Klekner and N. Kosaric, " Degradation of phenols by algae, " Environmental Technology,13,493 (1992).
Wu, Z., S. Wu, and X. Shi, "Supercritical fluid extraction and dterminationof lutein in heterotrophically cultivated Chlorella pyrenoidosa," Journal of Food Process Engineering, 30, 174 (2007).
Willis, M. S. and F. H. Wians, "The role of nutrition in preventing prostate cancer: a review of the proposed mechanism of action various dietary substances," Clinica Chimica Acta., 330, 57 (2003).