臺灣博碩士論文加值系統

由於人類長期的濫捕，近年來全球漁獲量逐漸降低，嚴重影響魚類營養保健食品(如多元不飽和脂肪酸)之來源，而從魚類所取得之多元不飽和脂肪酸又存在許多潛在之風險，例如:魚類過度捕撈、素食者不可食用、毒素累積…等。而微藻可行光合作用進行二氧化碳之吸收固定，且所產生之微藻生質體可用來生產高經濟價值之產物(如多元未飽和脂肪酸、色素等)，是十分理想的多元不飽和脂肪酸替代來源。其中，屬於omega-3多元不飽和脂肪酸之EPA，可促進腦部保健、且有效降低罹患心血管疾病與癌症的機率，是人類重要的營養補充品。
本研究嘗試以自台灣南部海域自行篩選之擬球藻(Nannochloropsis oceanica CY2)與巴夫藻(Pavlova salina CY1)，建構一套可轉換二氧化碳為EPA之微藻生物精鍊系統，並測試目標藻株在不同培養基、氮源濃度(4.41-26.46 mM)、光照強度(100-300 mol m-2 s-1)、光源波長與反應器操作策略條件下，探討其對EPA生產效率之影響。實驗結果發現，N. oceanica CY2相較於P. salina CY1有較佳之藻體產量與EPA產率，故本研究選擇N. oceanica CY2為目標藻株進行後續EPA生產效率提升策略測試。使用BG11培養基，其藻體產量、EPA含量以及EPA產率分別為1.38 g L-1、2.38%與5.49 mg L-1 d-1，明顯優於另兩種培養基(f/2與Basal medium)。為進一步提升EPA生產效率，本研究將氮源濃度由4.4 mM提升至17.6 mM，發現其藻體產量、EPA含量以及EPA產率分別大幅提升至2.45 g L-1、4.31%與 11.49 mg L-1 d-1。觀察藻體生長曲線與EPA含量時發現，在後指數生長期可得最大之藻體EPA含量。而當光照強度由100 mol m-2 s-1提升至200 mol m-2 s-1時，藻體產量亦可提升至3.22 g L-1，但持續將光照強度提高至300 mol m-2 s-1，則會有光抑制的現象發生。此外，不同波長之光源亦對N. oceanica CY2之EPA含量有顯著之影響，以藍色LED作為培養光源，雖對藻體生長有抑制的現象，但可大幅提升藻體EPA含量至5.57%。
發光二極體(LED)具有較高的光電轉換效率、產熱較一般日光燈管低及波長特定等優勢，利用LED作為培養光源，電力消耗產量(electric consumption yield)可降低29.4%至34.9 kw h g-1EPA。由先前之實驗結果發現，相較於波長分布較寬廣的光源(TL5日光燈)，特定波長之單色光源無法提升N. oceanica CY2之藻體生長及EPA產率。故本研究嘗試混合不同色光之LED(白、紅、黃、藍)，探討光源波長分布對藻體生長及EPA產率之影響。實驗結果發現混和色光(紅、黃)可有效提升藻體產率16.7%達0.271 g L-1 d-1，並且利用藍、紅之混合色光可提升EPA產率至12.5 mg L-1 d-1。利用前述之條件進一步進行半批次操作(semi-batch operation)，在培養基取代率為40%之情況下，EPA產率可提升至14.4 mg L-1 d-1。
在大型光生物反應器中，光遮蔽效應(self-shading effect)限制了藻體細胞生長，故本研究嘗試利用新型之浸入式光源生物反應器提升藻體及EPA生產效率，實驗結果顯示，浸入式光源可有效克服光遮蔽效應，並有效提升藻體產率與EPA產率分別達0.235 g L-1 d-1與9.72 mg L-1 d-1；並由於總EPA產量大幅提升，電力消耗產量顯著降低至9.85 kw h g-1EPA。為了進一步降低光源總耗電量，本研究將浸入式光源控制在不同之閃爍頻率(9 Hz、8 Hz、7 Hz)，結果顯示將閃爍頻率控制在9 Hz時，電力消耗產量可進一步降低至8.87 kw h g-1EPA。本研究亦測試不同季節下N. oceanica CY2之戶外培養可行性，結果顯示N. oceanica CY2在秋季與冬季可成功地在戶外培養，最佳藻體產率與EPA產率為0.087 g L-1 d-1與2.5 mg L-1 d-1；但N. oceanica CY2無法在台灣南部夏季的氣溫(〉33oC)下穩定生長。

The omega-3 fatty acids, especially EPA and DHA, are important elements for human health. Nowadays, marine cold water fish are the major source of omega-3 fatty acids. However, the global fish harvest has been nearly saturated. There is an urgent need in developing an alternative source of omega-3 fatty acids. Among the potential alternatives, microalgae have emerged as a new resource of omega-3 fatty acids since microalgae have the ability to synthesize and accumulate large amounts of omega-3 fatty acids in their cells. Besides, producing omega-3 fatty acids from microalgae could avoid many problems arising from fish-based omega-3 fatty acids production, such as limitation of fish harvest, biomagnification of toxin, odd smell, and not vegetarians available).
This work made effort on establishing a suitable cultivation system and strategies for improving the microalgal EPA production. In the beginning, two indigenous marine microalgae strains isolated from coastal water located in southern Taiwan (namely, Pavlova salina CY1 and Nannochloropsis oceanica CY2) were examined for their capabilities of EPA production. Among them, N. oceanica CY2 showed higher potential for microalgal EPA production, giving an EPA content and EPA productivity of 2.34 % and 3 mg L-1 d-1, respectively. Specific engineering strategies were employed to enhance EPA accumulation in the microalgal cells. The results show that BG-11 was the most effective medium to grow N. oceanica CY2, giving an EPA content and biomass concentration of 2.38 % and 1.53 g L-1, respectively. Furthermore, the maximal EPA productivity could further improve to 5.49 mg L-1 d-1. The EPA content nearly doubled (4.31 %) and EPA productivity was significantly improved to 11.49 mg L-1 d-1 when increasing the initial nitrate concentration (NaNO3) from 4.41 mM to 17.64 mM. The results also show that during the microalgal growth, the maximal EPA content occurred between exponential growth phase and stationary phase; in the meantime, the maximal biomass productivity was also achieved in this phase.
The illumination system also markedly affected the EPA content of the photoautotrophic microalga. When using fluorescent lamp (TL5) as the light source, the maximal EPA content decreased with increasing light intensity (100-300 mol m-2 s-1); however, the cell growth improved as light intensity was increased from 100 to 200 mol m-2 s-1. The highest EPA productivity (11.49 mg L-1 d-1) was obtained with a light intensity of 150 mol m-2 s-1. Moreover, using light emitting diodes (LEDs) as light source have the advantages of being durable, possessing high light efficiency, and unique emission wavelength band. Therefore, LEDs were applied for microalgae cultivation. The maximal EPA content could be improved to 5.57 % and 5.41 % by illuminating with LED-Blue and LED-Red, respectively. The electric consumption yield of EPA could reduce by 29.4% to 34.90 kw h g EPA-1. Next, the effect of multiple light wavelengths on cell growth and EPA production was also examined. Two of the four different colors of LEDs (i.e., white, blue, yellow and red) were combined to verify whether the multi-wavelengths could enhance the microalgal cell growth and EPA production of N. oceanica CY2. Compared with single-wavelength (i.e., white-LED), the maximal biomass productivity could be improved 16.7% by combining LED-Red and LED-yellow. Moreover, the maximal microalgal EPA productivity could reach 12.9 mg L-1 d-1 by cultivation with LED-Blue and LED-Red. In addition, the electric consumption yield of EPA could further reduce to 32.94 kw h g EPA-1. Using semi-batch operations with the obtained optimal conditions, the EPA productivity could be enhanced to 14.4 mg L-1 d-1 by operating at 40% replacement ratio.
The self-shading effect of microalgal culture is a critical problem of large-scale microalgae cultivation. In this work, a novel photobioreactor with immersed light source was designed to improve microalgal biomass and EPA production. Compared with control experiments (i.e., external light source only), the immersed light source could enhance maximal overall biomass productivity to 0.235 g L-1 d-1. With the increase of total EPA production, the electric consumption yield significantly decreased to 9.85 kw h g-1EPA. Moreover, the immersed light sources were operated with different flashing-frequencies (9 Hz, 8 Hz and 7 Hz). The results show that the total EPA production slightly decreased by 6.7%, but the energy consumption yield of EPA could decrease to 8.87 kw h g-1EPA. Therefore, the optimal flashing-frequency (9 Hz) was chosen for further studies.
The feasibility of outdoor cultivation of Nannochloropsis oceanica CY2 in different seasons (fall, winter and summer) were also tested. The EPA content of outdoor cultivation was similar to that from indoor cultivation. The maximal biomass productivity and EPA productivity could reach 0.087 g L-1 d-1 and 2.5 mg L-1 d-1, respectively. However, N. oceanic CY2 cannot tolerate the high temperature (ambient temperature up to 33 oC) in the summer time.

摘要 I
Abstract III
Acknowledgment VI
Contents VIII
List of Tables XI
List of Figures XIII
Chapter 1 Introduction 1
1-1 Background 1
1-2 Motivation and purpose 2
Chapter 2 Literature review 4
2-1 Introduction to microalgae 4
2-2 Photosynthesis 8
2-3 Microalgae as EPA feedstock 11
2-3-1 EPA production from microalgae and other sources 11
2-3-2 EPA biosynthesis 14
2-3-3 Influence of environmental factors on microalgal EPA production 18
Chapter 3 Materials and methods 27
3-1 Chemicals and materials 27
3-2 Equipment 28
3-3 The analytical method 29
3-3-1 Measurement of biomass concentration and growth kinetic parameters 29
3-3-2 Determination of the nitrate concentration in culturing medium 30
3-3-3 Measurement of light intensity and light quality 32
3-3-4 Determination of EPA content in the microalgal biomass by direct transesterification method 33
3-4 Experimental methods 34
3-4-1 Microalgal strain and culturing medium 34
3-4-2 Operation of photobioreactor and cultivation media 37
3-4-3 The adjustment of nitrate concentration in culturing medium 37
3-4-4 Selection of suitable light intensity to enhance the EPA production rate 38
3-4-5 Selection of suitable light quality (wavelength) for the EPA production rate 38
3-4-6 Effect of photobioreactor strategies on EPA production rate 39
3-4-7 Large scale photobioreactor with immersed light source 40
Chapter 4 Results and discussion 42
4-1 The selection of microalgal strain 42
4-2 The effect of medium on EPA production 44
4-3 The effect of nitrate concentration on EPA production 47
4-4 Evolution of fatty acid profiles of N. oceanica CY2 at different growth phases 50
4-5 The effect of light intensity on EPA production 53
4-6 The effect of light quality (wavelength) on EPA production 56
4-7 The operation strategy for improving EPA production rate 67
4-8 The novel photobioreactor with immersed light source 73
4-9 The Outdoor cultivation test 81
Chapter 5 Conclusion 84
References 88
Appendix 101

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