跳到主要內容

臺灣博碩士論文加值系統

(18.204.48.64) 您好!臺灣時間:2021/08/03 13:16
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:朱書賢
研究生(外文):Shu-Hsien Chu
論文名稱:擬球藻饋料培養及基因重組載體建構之研究
論文名稱(外文):The Study Of Fed-Batch Culture And Construction Of Recombinant Plasmid In Nannochloropsis oculata
指導教授:李文乾
指導教授(外文):Wen-Chien Lee
口試委員:李文乾吳昭燕黃光策
口試委員(外文):Wen-Chien LeeJau-Yann WuKuang-tse Huang
口試日期:2012-07-02
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:142
中文關鍵詞:全球暖化擬球藻高密度培養基因重組
外文關鍵詞:global warmingNannochloropsus oculatahigh cell density cultureDNA recombination of microalgae
相關次數:
  • 被引用被引用:1
  • 點閱點閱:394
  • 評分評分:
  • 下載下載:6
  • 收藏至我的研究室書目清單書目收藏:0
由於全球經濟快速發展,人類大量使用煤、石油和天然氣等石化燃料作為動力,造成二氧化碳及溫室氣體大幅增加,使全球碳循環遭破壞,造成全球暖化現象。可能解決的方案之一,是利用微藻的光合作用來減少大氣中二氧化碳含量。利用微藻生長速度快速,且吸收固化二氧化碳等特性,來減少工業所排放二氧化碳的廢氣,加上其富含油脂,可經萃取、轉化後,再生成為生質燃料。因此微藻固定二氧化碳具有相當之經濟潛力,與減緩暖化效應之能力。 
本研究利用光生物反應器培養方式,探討饋料策略、光照強度及CO2濃度對擬球藻生長之影響。在研究中,發現相同條件下,改良式Walne’s medium比起原來Walne’s medium微藻生長乾重(g/L)高出66.2%。在整體相同培養基含量下,也發現擬球藻之饋料培養策略比批次培養的生長乾重高了25%。在饋料策略培養上,起始培養基利用改良式Walne’s medium比起Walne’s medium培養的擬球藻,生長乾重高了32%。擬球藻在饋料培養情況下,光照強度為影響擬球藻生長之重要因素。在光照強度提升2.5倍,隨著不同起始藻濃度,擬球藻生長乾重增加4.1至29.4%不等。同時,在前一相同培養條件下,改變二氧化碳濃度,發現2%二氧化碳所培養出的擬球藻生長乾重為最高。
另外,本研究亦嘗試建構一可用於在藻類表現外源基因之重組質體,此重組質體包含可用於微藻表現之啟動子-RBCs1 promoter、RBCs1 terminator以及Zeocinr基因。首先在擬球藻之Zeocin抗藥性研究方面,發現擬球藻對Zeocin濃度之CC50在0.01~0.1 μg/ml間。在未來重組擬球藻細胞之篩選上,可利用0.1 μg/ml Zeocin針對野生與重組擬球藻進行初步篩選,以取得合適的重組微藻。



Due to rapidly global economic development, human heavy use of fossil fuels such as coal, oil and gas resulted in a substantial increase in carbon dioxide and greenhouse gases emissions, causing the destruction of the global carbon cycle and the global warming. One of possible solution to the problem of global warming is the use of microalgal photosynthesis for atmospheric carbon dioxide mitigation. Microalgaes can grow quickly and fix carbon dioxide to reduce carbon dioxide emission by manufacture factories; and because of its oil-rich they can be used for the production of renewable biofuel by extraction and transesterification. Thus the microalgal biofixation of carbon dioxide has considerable economic potential, and the ability to slow down the effects of global warming.
This study uses the photobioreactor culture to investigate the feeding effects on the light intensity and CO2 concentration of Nannochloropsus oculata. In the study, we found that microalgal biomass yield increased in 66.2% if the Walne’s medium were replased by modified Walne’s medium. Using the modified medium the biomass yield of N. oculata in fed-batch culture was 1.25 times higher than that in batch culture. Also, in the fed-batch culture the microalgal growth rate in modified Walne’s medium was 1.32 times higher than that in Walne’s medium. Light intensity was an important factor of microalgal growth in the high cell density culture. As the light intensity increased up to 2.5 times, microalgal growth increased from 4.1 to 29.4%, depending on the initial algal concentration. Under the conditions for high cell density culture, the highest microalgal biomass could be achieved in 2% CO2.
In this study, we also tried to construct a recombinant plasmid that could express the target gene encoding foreign protein in microalgae. The recombinant plasmid includes RBCs1 promoter, RBCs1 terminator and zeocin-resistance gene. In the study of N. oculata resistance to zeocin, we found that CC50 with N. oculata to zeocin concentration was 0.01-0.1 μg/ml. In the future, we can use 0.1 μg/ml zeocin for the screening of transgenic N. oculata based on these preliminary results.


中文摘要 i
Abstract iii
目錄 v
圖目錄 x
表目錄 xiv
第一章 緒論 1
1.1 前言 1
1.2 研究目的 3
第二章 文獻回顧 4
2.1 全球暖化 4
2.1.1 溫室效應 5
2.1.2 生態系統的碳循環 6
2.1.3 二氧化碳之封存方式 8
2.1.4 生物固定 10
2.2 藻類簡介 11
2.3 藻類生理特性之介紹 15
2.3.1 光合作用 15
2.3.2 呼吸作用 17
2.3.3 影響藻類生長之環境因子 19
2.3.4 藻類之生長曲線 23
2.4 藻類基因重組之相關研究 24
2.4.1 基因轉殖方法 26
2.4.1.1 轉形作用 27
2.4.1.1 轉導作用 28
第三章 實驗材料、設備與方法 31
3.1 實驗材料 31
3.1.1 實驗藻種 31
3.1.2 藻類培養 32
3.1.3 大腸桿菌培養與保存 33
3.1.4 基因重組 34
3.1.5 DNA瓊脂凝膠電泳分析 35
3.2 實驗設備 36
3.3 實驗方法 38
3.3.1 藻體培養之最適化 38
3.3.1.1 光生化反應器裝置 38
3.3.1.2 培養基組成配置 39
3.3.1.3 藻種保存之固定化技術 41
3.3.1.4 藻種活化 42
3.3.1.5 藻種繼代培養 42
3.3.1.6 藻種滲透壓之測試 43
3.3.1.7 吸光值對乾重之檢量線 43
3.3.1.8 比生長速率之計算 43
3.3.1.9 批次培養 44
3.3.1.10 饋料培養 44
3.3.1.11 光照度對饋料培養擬球藻之生長影響 45
3.3.1.12 CO2濃度對饋料培養藻類之生長影響 45
3.3.2 基因重組載體pCR®2.1-RBCs1PT-FZF之建構 46
3.3.2.1 Zeocin抗藥性測試 46
3.3.2.2 基因來源 46
3.3.2.3 寡核酸引子設計 49
3.3.2.4 質體pCR®2.1-RBCs1PT-FZF建構之流程 50
3.3.2.5 重組質體抽取 54
3.3.2.6 聚合酶連鎖反應(Polymerase Chain Reaction 54
3.3.2.7 DNA 瓊脂凝膠電泳分析 56
3.3.2.8 重組質體的建構 57
3.3.2.9 TA cloning 59
3.3.2.10 重組菌篩選 60
第四章 結果與討論 61
4.1 擬球藻最適化之培養 61
4.1.1 擬球藻滲透壓測試 61
4.1.2 吸光值對藻體乾重之檢量線 62
4.1.3 培養基對擬球藻生長之影響 63
4.1.4 不同培養策略對擬球藻生長之影響 64
4.1.5 不同培養基饋料對擬球藻生長之影響 66
4.1.6 光照強度對擬球藻生長之影響 68
4.1.7 CO2濃度對擬球藻生長之影響 72
4.2 微藻基因重組載體之建構 77
4.2.1 擬球藻Zeocin抗藥性之測試 77
4.2.2 pCR®2.1-RBCs1 PT-FZF載體之建構 80
4.2.2.1 pCR®2.1-RBCs1 terminator 之建構 80
4.2.2.2 pCR®2.1-RBCs1 promotor 之建構 85
第五章 結論與建議 91
第六章 參考文獻 93
附錄A 102
附錄B 108
附錄C 116

方清吉(1989) ,“二氧化碳與地球氣溫暖化問題”,船舶科技,第23期

朱鴻鈞、劉翠玲、楊玉婷(2011),“全球水產植物藻類研發現況與趨勢”,水產生技 26: 6-12.

艾爾‧高爾著;張瓊懿,欒欣譯(2007),“不願面對的真相”,商周出版社

呂權蓁(2009),“利用農桿菌方式轉殖單細胞綠藻”,國立中興大學生物科技學研究所碩士論文

周明顯、鄭文熙、張仁瑞、朱信(2007),“以藻類及植物光合作用回收再利用二氧化碳技術研發”,國立中山大學環境工程研究所國科會計畫編號NSC96-EPA-Z110-001

林安秋(1984),“作物之光合作用”,台灣商銀印書館

林殿順(2010),“台灣二氧化碳地質封存潛能及安全性”,經濟前瞻. 2-5

林靜宜、詹富智(2005),“無篩選標示基因 (marker-free) 轉基因植物之構築及其最新發展”,植物病理學會刊 14(3): 159-176.

洪志瑞(2007),“油質性微藻培養於新型光生化反應器之研究”,國立成功大學化學工程研究所碩士論文

張泉湧(2011),“全球氣候變遷”,五南圖書

張惟閔(2005),“微藻培養於新型光生化反應器之系統開發”,國立清華大學化學工程研究所碩士論文

許晃雄(1998),“人為的全球暖化與氣候變遷”,「民間能源會議-因應溫室效應的民間觀點」研討會

郭耀綸、何婉清、夏良宙、陳國、葉慶龍、葉信平、鄭秋雄、戴永禔、彭仁君、孫元勳(2001),“生態學”,睿煜出版社.

陳建初(1983),“水質管理”,九大圖書公司.

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研究所碩士論文



劉翠玲、許嘉伊(2011),“藻類應用商機無限全球水產藻類發展現況與趨勢”,臺灣經濟研究月刊 34(3): 43-49.

闕壯群(2009 ),“微藻類固碳工程”,科學發展 433期. 6-11

蘇惠美(1999),“餌料生物之培養與利用”,台灣省水產試驗所東港分所

蘇惠美、雷淇祥、廖一久(1990),“溫度、光照度及鹽度對骨藻生長速率之影響”,臺灣水產學會刊 17(3): 213-222.

Adams E, Caldeira K. 2008. Ocean storage of CO2. Elements 4: 319-324

Apt KE, Kroth-Pancic P, Grossman A. 1996. Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Molecular and General Genetics MGG 252: 572-579.

Becker EW. 1994. Microalgae: biotechnology and microbiology: Cambridge Univ Pr.

Belay A, Ota Y, Miyakawa K, Shimamatsu H. 1993. Current knowledge on potential health benefits of Spirulina. Journal of Applied Phycology 5: 235-241.

Cai XH, Brown C, Adhiya J, Traina SJ, Sayre RT. 1999. Growth and heavy metal binding properties of transgenic Chlamydomonas expressing a foreign metallothionein gene. International Journal of Phytoremediation 1: 53-65.

Chen C, Durbin EG. 1994. Effects of pH on the growth and carbon uptake of marine phytoplankton. Marine Ecology-Progress Series 109: 83-83.

Chen YC. 2001. Immobilized microalga Scenedesmus quadricauda (Chlorophyta, Chlorococcales) for long-term storage and for application for water quality control in fish culture. Aquaculture 195: 71-80.

Cheney D, Metz B, Stiller J. 2001. Agrobacterium-mediated genetic transformation in the macroscopic marine red alga Porphyra yezoensis. J. Phycol 37.

Chiu SY, Kao CY, Tsai MT, Ong SC, Chen CH, Lin CS. 2009. Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration. Bioresource technology 100: 833-838.

Chow KC, Tung W. 1999. Electrotransformation of Chlorella vulgaris. Plant Cell Reports 18: 778-780.
Craven G. 2009. What’s The Worst That Could Happen? Commonwealth pulishing CO., Ltd.

Cummings B., 2006. Biology. Pearson Education, Inc. Concept 8.1. pp160-162.

Cummins J, Ho MW, Ryan A. 2000. Hazardous CaMV promoter? Nature Biotechnology 18: 363-364.

Demirbas A. 2010. Use of algae as biofuel sources. Energy Conversion and Management 51: 2738-2749.

Dunahay T. 1993. Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. Biotechniques 15: 452.

Dunahay TG, Jarvis EE, Dais SS, Roessler PG. 1996. Manipulation of microalgal lipid production using genetic engineering. Applied Biochemistry and Biotechnology 57: 223-231.

Feng S, Xue L, Liu H, Lu P. 2009. Improvement of efficiency of genetic transformation for Dunaliella salina by glass beads method. Molecular Biology Reports 36: 1433-1439.

Field CB, Behrenfeld MJ, Randerson JT, Falkowski P. 1998. Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281: 237-240.

Goldman JC. 1973. Carbon dioxide and pH: Effect on species succession of algae. Science (Washington, DC);(United States) 182.

Hirata H, Murakoshi M. 1977. Effects of aeration volume on the growth of marine Chlorella in culture. Mem. Fac. Fish., Kagoshima Univ 26: 15-21.

Holub DJ, Holub BJ. 2004. Omega-3 fatty acids from fish oils and cardiovascular disease. Molecular and Cellular Biochemistry 263: 217-225.

Huijgen W. 2003. Carbon dioxide sequestration by mineral carbonation. Energy: 1-52.

Huerlimann R, De Nys R, Heimann K. 2010. Growth, lipid content, productivity, and fatty acid composition of tropical microalgae for scale‐up production. Biotechnology and Bioengineering 107: 245-257.

Kathiresan S, Chandrashekar A, Ravishankar G, Sarada R. 2009. Agrobacterium-Mediated Transformation in the green alga Haematococcus Pluvialis. . Journal of phycology 45: 642-649.

Kindle KL, Schnell RA, Fernández E, Lefebvre PA. 1989. Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. The Journal of Cell Biology 109: 2589-2601.

Kumar A, Ergas S, Yuan X, Sahu A, Zhang Q, Dewulf J, Malcata FX, Van Langenhove H. 2010. Enhanced CO2 fixation and biofuel production via microalgae: recent developments and future directions. TRENDS in Biotechnology 28: 371-380.

Li SS, Tsai HJ. 2009. Transgenic microalgae as a non-antibiotic bactericide producer to defend against bacterial pathogen infection in the fish digestive tract. Fish & Shellfish Immunology 26: 316-325.

Mallick N. 2002. Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review. Biometals 15: 377-390.

Maroto-Valer MM. 2010. Developments and innovation in carbon dioxide (CO2) capture and storage technology: Volume 2. carbon dioxide (CO2) storage and utilisation: Woodhead Publishing Ltd.

Su MH.,Su SM., Liao IC. 1997. Preliminary results of providing various combinations of live foods to grouper (Epinephelus coioides) larvae. Hydrobiologia 358: 301-304.


Nagao K, Takai Y, Ono M. 1991. Exercises of growing mice, and the effect of the intake of Spirulina platensis upon the hapten-specific immune response. Sci. Phys. Power 40: 187-194.

Ohnuma M, Yokoyama T, Inouye T, Sekine Y, Tanaka K. 2008. Polyethylene glycol (PEG)-mediated transient gene expression in a red alga, Cyanidioschyzon merolae 10D. Plant and Cell Physiology 49: 117-120.

Outchkourov N, Peters J, De Jong J, Rademakers W, Jongsma M. 2003. The promoter-terminator of chrysanthemum rbcS1 directs very high expression levels in plants. Planta 216: 1003-1012.

Phang SM. 2004. Handbook of microalgal culture. Biotechnology and applied phycology. Journal of Applied Phycology 16: 159-160.

Rakoczy-Trojanowska M. 2002. Alternative methods of plant transformation-a short review. Cellular and molecular biology letters 7: 849-858.

Reitan KI, Rainuzzo JR, Řie G, Olsen Y. 1997. A review of the nutritional effects of algae in marine fish larvae. Aquaculture 155: 207-221.

Rocha J, Garcia JEC, Henriques MHF. 2003. Growth aspects of the marine microalga Nannochloropsis gaditana. Biomolecular Engineering 20: 237-242.

Rubio FC, Fernandez F, Perez J, Camacho FG, Grima EM. 1999. Prediction of dissolved oxygen and carbon dioxide concentration profiles in tubular photobioreactors for microalgal culture.
Biotechnology and Bioengineering 62: 71-86.

Schwartz J, Shklar G. 1987. Regression of experimental hamster cancer by beta carotene and algae extracts. Journal of Oral and Maxillofacial surgery 45: 510-515.

Shimogawara K, Fujiwara S, Grossman A, Usuda H. 1998. High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148: 1821-1828.

Singh M. 2005. Essential fatty acids, DHA and human brain. Indian Journal of Pediatrics 72: 239-242.
Siripornadulsil S, Traina S, Verma DPS, Sayre RT. 2002. Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae. The Plant Cell Online 14: 2837-2847.

Sobczuk TM, Camacho FG, Rubio FC, Fernández F, Grima EM. 2000. Carbon dioxide uptake efficiency by outdoor microalgal cultures in tubular airlift photobioreactors. Biotechnology and Bioengineering 67: 465-475.

Solomon S. 2007. Climate change 2007: the physical science basis: contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change: Cambridge Univ Pr.
Takagi M, Yoshida T. 2006. Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. Journal of Bioscience and Bioengineering 101: 223-226.

Tan C, Qin S, Zhang Q, Jiang P, Zhao F. 2005. Establishment of a micro-particle bombardment transformation system for Dunaliella salina. Journal of Microbiology-Seoul- 43: 361.

Te MR, Miller DJ. 1998. Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs. The Plant Journal 13: 427-435.
Teng C, Qin S, Liu J, Yu D, Liang C, Tseng C. 2002. Transient expression of lacZ in bombarded unicellular green alga Haematococcus pluvialis. Journal of Applied Phycology 14: 497-500.

Thakur A, Kumar H. 1999. Nitrate, ammonium, and phosphate uptake by the immobilized cells of Dunaliella salina. Bulletin of Environmental Contamination and Toxicology 62: 70-78.

Tsuchihashi N, Watanabe T, Takai Y. 1987. Effect of Spirulina platensis on caecum content in rats. Bull Chiba Hygiene College 5: 27-30.

Ussery W., 1998. Life on Planet Earth. Chapter 8

Wang B, Li Y, Wu N, Lan CQ. 2008. CO 2 bio-mitigation using microalgae. Applied Microbiology and Biotechnology 79: 707-718.

Watanabe Y, Saiki H. 1997. Development of a photobioreactor incorporating Chlorella sp. for removal of CO2in stack gas. Energy Conversion and Management 38: S499-S503.

White CM, Strazisar BR, Granite EJ, Hoffman JS, Pennline HW. 2003. Separation and capture of CO2 from large stationary sources and sequestration in geological formations—coalbeds and deep saline aquifers. Journal of the Air & Waste Management Association 53.

Yamane Y, Fukino H, Icho T, Kato T, Shimamatsu H. 1988. Effect of Spirulina (Spirulina platensis) on the renal toxicity induced by inorganic mercury and para-aminophenol. Summary of Abstracts 108.
You T, Barnett SM. 2004. Effect of light quality on production of extracellular polysaccharides and growth rate of Porphyridium cruentum. Biochemical Engineering Journal 19: 251-258.

Zaslavskaia L, Lippmeier J, Shih C, Ehrhardt D, Grossman A, Apt K. 2001. Trophic conversion of an obligate photoautotrophic organism through metabolic engineering. Science 292: 2073.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top