Chlorella sp. 藻壁含有纖維素 (cellulose)，可以藉由纖維素分解酵素 (cellulase) 水解即可形成還原性葡萄糖 (glucose)。研究目的最佳 Chlorella sp. 培養的參數以利其藻壁之纖維素水解生產還原性葡萄糖。
實驗探討分成綠球藻培養及藻壁切解兩階段分別進行。在培養微藻部份，針對不同操作因子如光源、進料含氮濃度及二氧化碳進流速率以探討對 Chlorella sp. 生長速率及消耗營養源狀況之影響，也進行不同之進料策略以提高綠球藻產率，以尋求對Chlorella sp. 之較佳培養條件。實驗結果顯示，半連續式培養策略可使培養環境較長期地保持在較佳的狀態，與批次培養、饋料批次培養比較，生長速率較快，藻體產率較高，使每四天與每三天收獲一次之在半連續式培養下之微藻的藻體產率平均可達到0.295 g L-1 d-1 與0.341 g L-1 d-1，其藻體對應尿素生成率分別為 12.01 與 10.68。而在酵素水解形成還原糖的部份，主要是針對不同的反應條件進行實驗，取得較佳的條件以提昇還原糖之轉化率。結果顯示，在溫度 60℃ 與 pH=6 的環境下，反應 24 hr的效果最好，可生成還原單糖濃度達到 1.254 ± 0.019 g/L/(45 mg‧15 U)，其轉化率為 8.36%。
由此可知，以纖維素分解酵素水解綠球藻藻壁取得還原醣已確認可行，更進一步地，也將以這些收集的濃縮藻糖液當作進料，提供其他菌株如嗜鹽菌 (Halobacteria sp.) 作為培養之營養源。

The cell wall of Chlorella sp. contains cellulose, which can be hydrolyzed by cellulose to form reducing sugar. The purpose of this study was to investigate the optimal parameters for the cultivation of Chlorella sp. to produce reducing sugar by hydrolysis of cellulose from the cell wall of Chlorella sp..
The microalgae, Chlorella sp., was cultivated in different culture conditions to improve biomass productivity. Light intensity, nitrogen feeding concentration, and carbon dioxide (CO2) flow rate were used as the affecting factors to observe the influence of culture conditions for the growth rate of Chlorella sp.. To increase the biomass productivity of Chlorella sp., different feeding strategies were also designed and executed. The experimental results that the average biomass productivity of 0.295 g L-1 d-1 and 0.341 g L-1 d-1 and the biomass yield w.r.t. urea of 12.01 and 10.68 during a 4-day and 3-day refleshment in the semi-continuous cultivation, could be achieved. The results were comparably superior to that obtained from batch and fed-batch cultivations. The optimal reaction condition to achieve maximum productivity of reducing sugar by the cellulose hydrolysis of Chlorella sp. were investigated. The results demonstrated that glucose concentrations of 1.254 ± 0.019 g/L/(45 mg‧15 U) can be obtained. The reducing sugar conversion yield was about 8.36 % under the optimal condition of 60 ℃, pH 6.0.
This research work confirmed that reducing sugar can be formed by the cellulase hydrolysis of cellulose from the cell wall of Chlorella sp. In the future, the hydrolyzed reducing sugar collected would be further supplied to used other strains such as halophilic bacteria as their nutrients for growth.

1. Courties C, Vaquer A, Trousselier M, Lautier J, Chrétiennot-Dinet M-J,Neveux J, Machado C, Claustre H, Smallest eukaryotic organism, Nature, 370, 255, 1994.
2. Matsuzaki M, Misumi O, Shin IT, Maruyama S, Takahara M, MiyagishimaSY, Mori T, Nishida K, Yagisawa F, Nishida K, Yoshida Y, Nishimura Y,Nakao S, Kobayashi T, Momoyama Y, Higashiyama T, Minoda A, SanoM, Nomoto H, Oishi K, Hayashi H, Ohta F, Nishizaka S, Haga S, Miura S,Morishita T, Kabeya Y, Terasawa K, Suzuki Y, Ishii Y, Asakawa S, TakanoH, Ohta N, Kuroiwa H, Tanaka K, Shimizu N, Sugano S, Sato N, NozakiH, Ogasawara N, Kohara Y, Kuroiwa T, Genome sequence ofthe ultrasmall unicellular red alga Cyanidioschyzon merolae, 10D. Nature, 428, 653-657, 2004.
3. Barbier G, Oesterhelt C, Larson MD, Halgren RG, Wilkerson C, GaravitoRM, Benning C, Weber AP, Comparative genomics of two closelyrelated unicellular thermo-acidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular basis of the metabolicflexibility of Galdieria sulphuraria and significant differences in carbohydratemetabolism of both algae, Plant Physiology, 137, 460-474, 2005.
4. 張碧芬, 袁紹英, 游呈祥, 「微生物學的世界」，原生生物、藻類、真菌, 天下遠見出版股份有限公司, 142-143, 2004。
5. Armin Hallmann, Algal Transgenics and Biotechnology, Transgenic Plant Journal, 81-98, 2007.
6. Grima E. M., J. A. S. Perez, F. G. Camacho, et al. , Effect of Growth-Rate on the Eicosapentaenoic Acid and Docosahexaenoic Acid Content of Isochrysis-Galbana in Chemostat Culture, Applied Microbiologyand Biotechnology 41(1): 23-27,1994.
7. Becker W, Microalgae in human and animal nutrition. In: Richmond A (Ed) Handbook of Microalgal Culture, Blackwell, Oxford, pp 312-351, 2004.
8. Pulz O, Gross W, Valuable products from biotechnology of microalgae, Applied Microbiology and Biotechnology 65, 635-648, 2004.
9. 劉翠玲, 許嘉伊, 藻類應用商機無限, 全球水產藻類發展現況與趨勢, 全球綠色商機與農業發長趨勢, 35, 43-49, 2011。
66
10. 劉翠玲, 全球保健食品市場現況, 農業生技產業季刊, 23, 1-8, 2010。
11. Spolaore P, Joannis-Cassan C, Duran E, Isambert A, Commercial applicationsof microalgae, Journal of Bioscience and Bioengineering 101, 87-96 , 2006.
12. Chien-Ya Kao, Sheng-Yi Chiu, Tzu-Ting Huang, Le Dai, Guan-Hua Wang, Ching-Ping Tseng, Chiun-Hsun Chen, Chih-Sheng Lin, A mutant strain of microalga Chlorella sp. for the carbon dioxide capture from biogas, biomass and bioenergy, 36, 132-14 0,2012.
13. Sheng-Yi Chiu, Chien-Ya Kao, Tzu-Ting Huang, Chia-Jung Lin, Seow-Chin Ong, Chun-Da Chen,Jo-Shu Chang, Chih-Sheng Lin, Microalgal biomass production and on-site bioremediation of carbon dioxide,nitrogen oxide and sulfur dioxide from flue gas using Chlorella sp. cultures, Bioresource Technology, 102, 9135–9142, 2011.
14. Pulz O, Gross W, Valuable products from biotechnology of microalgae, Applied Microbiology and Biotechnology 65, 635-648, 2004.
15. http://zh-yue.wikipedia.org/wiki/Photosynthesis
16. Jian-Ming Lv, Li-Hua Cheng, Xin-Hua Xu, Lin Zhang, Huan-Lin Chen, Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions, Bioresource Technology, 101, 6797–6804, 2010.
17. Kuei-Ling Yeh, Jo-Shu Chang, Wen-ming chen, Effect of light supply and carbon source on cell growth and cellular composition of a newly isolated microalga Chlorella vulgaris ESP-31, Engineering in Life Sciences, 10, 201-208, 2010.
18. James C. Ogbonna, Hideo Tanaka, Light requirement and photosynthetic cell cultivation – Development of processes for efficient light utilization in photobioreactors, Journal of Applied Phycology, 12, 207-218, 2000.
19. T. Mazzuca Sobczuk, F. Garcı´a Camacho, F. Camacho Rubio, F. G. Acie´n Ferna´ ndez, E. Molina Grima, Carbon Dioxide Uptake Efficiency by Outdoor Microalgal Cultures in Tubular Airlift Photobioreactors, BIOTECHNOLOGY AND BIOENGINEERING, 465–475, 2000.
20. Sheng-Yi Chiu, Chien-Ya Kao, Ming-Ta Tsai, Seow-Chin Ong, Chiun-Hsun Chen, Chih-Sheng Lin, Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration, Bioresource Technology, 100, 833–838, 2009.
21. Kuei-Ling Yeh, Jo-Shu Chang, Wen-ming chen, Effect of light supply and carbon source on cell growth and cellular composition of a newly isolated microalga Chlorella vulgaris ESP-31, Eng. Life Sci., 10, 201–208, 2010
22. Zhi-Yuan Liu, Guang-Ce Wang , Bai-Cheng Zhou, Effect of iron on growth and lipid accumulation in Chlorella vulgaris, Bioresource Technology, 99, 4717–4722, 2009.
23. Chih-Hung Hsieh, Wen-Teng Wu, Cultivation of microalgae for oil production with a cultivation strategy of urea limitation, Bioresource Technology, 100, 3921–3926, 2009. 24. Yujie Feng, Chao Li, Dawei Zhang, Lipid production of Chlorella vulgaris cultured in artificial wastewater medium, Bioresource Technology, 102, 101–105, 2011.
25. Ahmad, I., Hellebust, J., Regulation of chloroplast development by nitrogen source and growth conditions in a Chlorella protothecoides strain. Plant Physiology, 94, 944–949, 1990.
26. Sheng-Yi Chiu, Chien-Ya Kao, Chiun-Hsun Chen, Tang-Ching Kuan, Seow-Chin Ong, Chih-Sheng Lin, Reduction of CO2 by a high-density culture of Chlorella sp.in a semicontinuous photobioreactor, Bioresource Technology, 99, 3389–3396, 2008.
27. Pingzhong Feng, Zhongyang Deng, Zhengyu Hu,, Lu Fan, Lipid accumulation and growth of Chlorella zofingiensis in flat plate photobioreactors outdoors, Bioresource Technology, 102, 10577–10584, 2011.
28. Chih-Hung Hsieh, Wen-Teng Wu, A novel photobioreactor with transparent rectangular chambers for cultivation of microalgae, Biochemical Engineering, 46, 300–305, 2009.
29. HU QIANG, DAVID FAIMAN, AND AMOS RICHMOND, Optimal tilt angles ofenclosed reactors for growing photoautotrophic microorganisms outdoors, JOURNAOL F FERMENTATION AND BIOENOINEERING, 85, 230-236, 1998.
30. Octavio Perez-Garcia, Froylan M.E. Escalante, Luz E. de-Bashan, Yoav Bashan, Heterotrophic cultures of microalgae: Metabolism and potential products, water research, 45, 11-36, 2011.
31. Fu-Ying Feng, Wen Yang, Gui-Zhen Jiang, Yi-Nong Xu, Ting-Yun Kuang, Enhancement of fatty acid production of Chlorella sp. (Chlorophyceae) by addition of glucose and sodium thiosulphate to culture medium, Process Biochemistry, 40, 1315–1318, 2005.
32. Pierre B guin Jean-Paul Aubert, The biological degradation of cellulose, FEMS Microbiology Reviews, 13, 25-58, 1994.
33. E. C. Sherrard, F. W. Kressman, Review of processes in the United States prior to World War II. Industrial Engineering Chemistry, 37, 5-8, 1945.
34. Henry R. Bungay, Confessions of a bioenergy advocate, TRENDS in biotechnology, 22 , 67-71, 2004.
35. Bhat, M. K., Bhat, S., Cellulose degrading enzymes and theirpotential industrial applications, Biotechnology advances, 15, 583-620, 1997.
36. Rajai H. Atalla, Akira Isogai, Celluloses, Comprehensive Natu al Products II, 6, 493-539, 2010.
37. Matsudo, M.C., Bezerra, R.P., Sato, S., Perego, P., Converti, A., Carvalho, J.C.M., Repeated fed-batch cultivation of Arthrospira (Spirulina) platensis using urea as, nitrogen source. Biochem. Eng. J. 43, 52–57, 2009.
38. Danesi, E.D.G., Rangel-Yagui, C.O., Carvalho, J.C.M., Sato, S, An investigation of effect of replacing nitrate by urea in the growth and production of chlorophyll by Spirulina platensis. Biomass Bioenerg, 23, 261–269, 2002. 39. Miriam T. Knorst , Reinhard Neubert , Wolfgang Wohlrab , Analytical methods for measuring urea in pharmaceutical formulations , Journal Pharmaceutical and Biomedical Analysis , 15 , 1627-1632 , 1997.
40. HAROLD H. BROWN , Determination of Blood Urea with p-dimethylamino benzaldehyde , ANALYTICAL CHEMISTRY , 31 , 1959.
41. GEORGE W. WATT and JOSEPH D. CHRISP , Spectrophotometric Method for Determination of urea , ANALYTICAL CHEMISTRY, 26, 452-453, 1954.
42. 許憲銘, 蘇卓群, 陳俊明, 尤瓊琦, 微藻自動化生產反應器系統之設計與開發, 農林學報, 58, 289-306, 2009.
43. Chun-Chong Fu , Tien-Chieh Hung , Jing-Yi Chen , Chia-Hung Su , Wen-Teng Wu , Hydrolysis of microalgae cell walls for production of reducing sugar and lipid extraction , Bioresource Technology, 101 , 8750-8754 , 2010.