- 學位論文
擬球藻的最適化饋料培養及其應用於表達海藻糖合成酶基因
Nannochloropsis Oculata In Optimized Fed-Batch Cultivation And Its Application For Expressing Trehalose Synthase Gene
摘要
微藻可行光合作用，利用太陽光能將水和二氧化碳(CO2)轉變為生物能以供生長，其具有營養豐富、環境適應力強、生長速率快、易於培養之特性，在營養充足的培養環境下，光和二氧化碳是影響微藻生長的主要限制因子。本研究利用光生物反應器，以二倍修正Walne培養基進行饋料批次(Fed-batch)培養海洋微藻—擬球藻，探討饋料培養時光照強度及CO2的濃度對於擬球藻生長特性的影響。研究結果顯示，曝氣條件為空氣(約含0.03%CO2)，將光照度設定在40,000 Lux時，其生物質產量0.545 g/L；比生長速率0.192 d-1。結果可以發現，擬球藻利用饋料培養，其耐受光照強度可高達40,000 Lux。 探討擬球藻的CO2濃度耐受性方面，結果顯示，以光照度40,000 Lux培養，在曝氣2 %CO2其生物產量2.240 g/L、比生長速率0.328 d-1為最高。且擬球藻在15% CO2下生長之生物產量、比生長速率分別為1.518 g/L、0.288 d-1，顯示擬球藻在高濃度CO2增殖能力仍然相當高。綜合光照強度與CO2濃度兩生長因子比較，以曝氣2 %CO2，培養在光照強度40,000 Lux的生物產量為10,000 Lux的2.4倍；更高於4000 Lux 3.1倍。所以建議用強光照度40,000 Lux、曝氣2 %CO2濃度進行饋料培養擬球藻，以達高密度養殖之目標。且擬球藻生長耐受CO2濃度高，亦可利用於微藻捕獲工業廢氣中高濃度的CO2，對於生物固碳之應用為一大助益。 另外，本實驗選殖Pseudomonas putida F1之海藻糖合成酶基因，完成重組質體pRBCptH-TS之建構，再使用電脈衝法轉殖擬球藻，並以含有抗生素之固態Walne培養基進行可表現海藻糖合成酶之重組藻株篩選，目前成功篩選四株重組擬球藻，可望進行放大藻液體積之培養。
並列摘要
The characteristics of microalgae are nutrient-rich, strong environmental adaptability, growth rate fast and easy to culture. This microalgae can grow by photosynthetic that is converted water and carbon dioxide into biomass using light as an energy source. In nutrient-adequate cultivate environment, the light is major limiting factor which affecting the growth of microalgae. In the study, Nannochloropsis oculata was cultured in 2X modified Walne’s medium by photobioreactor. The effect of light intensity and tolerance of carbon dioxide was investigated during fed-batch cultivation. In order to studied the effect of light intensity the result demonstrated that the illuminated three different light intensity was separately used to studied as 4000, 10,000 and 40,000 Lux under a photoperiod of 12 hr (light):12 hr (dark) cycle and included air (0.03%CO2). An N. oculata biomass concentration of 0.545 g dry wt/L and specific growth rate of 0.192 d-1 were achieved when the microalgae was illuminated 40,000 Lux. This result showed that the maximum biomass yield of N. oculata was obtained at 40,000 Lux of illuminated the light intensity under fed-batch cultivation condition. Therefore, the fed-batch cultivation using the high light intensity illumination was suitable. For the effect of carbon dioxide, the tolerance capability of carbon dioxide of N. oculata was studied under light intensity of 40,000 Lux. N. oculata cultivation was aerated with different CO2 concentrations including 0.03% (air), 2%, 5%, 7.5%, 10% and 15% CO2. Results showed that the N. oculata cultivation at 2% CO2 could yield the maximum biomass of 2.240 g/L and the maximum specific growth rate of 0.328 d-1. Moreover, the N. oculata could grow at CO2 concentration as high as 15%. The biomass and specific growth rate achieved at 1.518 g dry wt/L and 0.288 d-1, respectively. When aerated with 2% CO2, the use of 40,000 Lux of light intensity promoted the production of biomass in 2.43 and 3.11 times, in comparison with 10,000 Lux and 4000 Lux of light intensity, respectively. In conclusion, the high light intensity could enhance microalgae photosynthetic efficiency. Also, the cultivation at 2% CO2 could enhance growth rate of N. oculata and reduce the mutual shading effect. Therefore, microalgae cells could be cultivated to high cell density at the conditions of 2% CO2 and 40000 Lux of light intensity. Moreover, N. oculata could tolerate high CO2 concentration, suggesting that this microalgae could be used for capturing CO2 from industrial process. Finally, the gene of trehalose synthase from Pseudomonas putida F1 was amplified and cloned into a plasmid vector. The restlant recombinant plasmid pRBCptH-TS was then transformed to N. oculata by electroporation. Four stains of recombinant N. oculata were screend from the plate containing antibiotics in Walne medium.