- 學位論文
擬球藻饋料培養及基因重組載體建構之研究
The Study Of Fed-Batch Culture And Construction Of Recombinant Plasmid In Nannochloropsis oculata
摘要
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。
並列摘要
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.