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研究生：	賴彥頴 Lai, Yen-Ying
論文名稱：	本土嗜溫性微藻Desmodesmus sp. 之藻油生產最適化 Optimizing lipid production with an indigenous thermo-tolerant microalga Desmodesmus sp.
指導教授：	張嘉修 Chang, Jo-Shu
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	116
中文關鍵詞：	微藻、鏈帶藻、生質柴油、藻油、自動沉降、光照強度、溫度、氮源、反應曲面法、批次、批次饋料、半連續式、戶外培養
外文關鍵詞：	microalgae, Desmodesmus sp., biodiesel, lipid quality, auto-sedimentation, light intensity, temperature, nitrogen source, response surface methodology, batch, fed-batch, semi-continuous, outdoor cultivation
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工業發展和化石燃料的過度使用在現今已造成了一些嚴重的問題，例如環境污染、氣候變遷、甚至是物種滅絕。因此，使用替代能源(如生質能)被提出來作為解決此問題的方案之一。在眾多生質能的選項中，由於微藻具高生長速率、高油脂含量、可在非耕地培養等優點，近年來利用微藻產生質柴油日益受到重視。本研究首先建立篩選高效能油脂生產藻株之標準方法，針對藻株之油脂生產表現、油脂品質以及自動沉降能力進行評估，基於這些標準由台灣西南方海域分離之四株本土耐熱性微藻(Desmodesmus sp. F2, F32, F44, F51)中挑選出適合生產生質柴油之優勢藻株。由於Desmodesmus sp. F2在相同之培養條件下(光照強度100 μmol m-2 s-1、溫度35 oC)具有較高的油脂含量(52.86 % w/w)、油脂生產速率(113 mg L-1 d-1)、適當的脂肪酸成份分布以及較佳的自動沉降能力(61.97 % hour-1)，因此被選為優勢藻株來進行一系列的微藻油脂生產測試。
本研究探討不同的環境因子(光照強度、溫度、氮源種類、氮源濃度和缺氮時間)對Desmodesmus sp. F2之生長或油脂累積的影響，並結合反應曲面法(Response surface methodology, RSM)，取得以批次程序進行藻油生產之最適化條件。結果顯示，在最適化條件下 (光照強度700 μmol m-2 s-1、溫度35 oC、6.6 mM氮源濃度、缺氮六天) Desmodesmus sp. F2之最大的藻油生產速率約為263 mg L-1 d-1，而油脂含量可達67.57 % w/w。此藻油生產速率是RSM最適化之前的兩倍。
最後，本研究以不同的培養策略(批次饋料及半連續式培養系統)提升藻油生產之表現。在批次饋料(Fed-batch)培養系統中測試不同的進料時間間隔(缺氮2, 4, 6天)，最佳的結果出現在缺氮6天的批次饋料系統中。雖然其油脂含量(49.16 % w/w)和油脂產率(213 mg L-1 d-1)較低，但其藻體濃度可達9.33 g L-1，為批次(Batch)培養系統的三倍以上。此外，本研究亦進行不同藻液和培養基置換比例的在半連續式(Semi-continuous)操作，以半批次培養，在90 %之置換比例下，可達最大的油脂生產速率(302 mg L-1 d-1)，優於批次培養系統之結果，而其油脂含量則為45.57 % w/w。因此，培養系統的選擇必須根據不同的需求作決定。此外，本研究亦進行Desmodesmus sp. F2之50 L戶外批次培養測試。在高溫高光強度下，其藻體產率、油脂含量及油脂生產速率分別為 84 mg L-1 d-1, 33.0 % w/w, and 22 mg L-1 d-1。這些結果顯示利用D. sp. F2進行大規模藻油生產之可行性。

Nowadays, rapid industrial development and the overuse of fossil fuels have caused some severe problems, such as environmental pollutions, climate change, and even the species extinctions. Therefore, using alternative energy (such as biomass energy) has been recognized as one of the strategies to solve these problems. Among the options of biomass energy, microalgae-based biodiesel is consider a promising one since microalgae have high growth rate, high lipid content, can be cultivated on non-arable. In this study, standard criteria (i.e., oil/lipid production performance, lipid quality, and auto-sedimentation ability) were established as a platform technology for screening potential microalgal strains suitable for the use as the oil feedstock for biodiesel production. Based on the aforementioned criteria, the strain with the highest potential as microalgal oil producer was selected from four indigenous thermo-tolerant Desmodesmus sp. isolates (i.e., F2, F32, F44, and F51) obtained from southern coast of Taiwan. Under the same cultivation conditions (light intensity, 100 μmol m-2 s-1; temperature, 35 oC), Desmodesmus sp. F2 appeared to have higher lipid content (52.86 % w/w), higher lipid productivity (113 mg L-1 d-1), proper fatty acid profile, and great auto-sedimentation ability (61.97 % hour-1). Therefore, the F2 strain was chosen as the target strain for further oil production studies.
Next, the effects of environmental factors (i.e., light intensity, temperature, nitrogen source, nitrogen concentration, and duration of nitrogen depletion) on cell growth and lipid accumulation of Desmodesmus sp. F2 were examined. Response surface methodology (i.e., RSM) was used to determine the optimal conditions for batch microalgal oil production. Under the optimal conditions (i.e., light intensity, 700 μmol m-2 s-1; temperature, 35 oC; nitrogen concentration, 6.6 mM; 6-day nitrogen depletion), the maximum lipid productivity and lipid content was about 263 mg L-1 d-1 and 67.57 % w/w, respectively. This lipid productivity is 2 folds higher than that obtained from original conditions before the RSM optimization.
Different cultivation strategies (i.e., fed-batch and semi-continuous cultivation systems) were also applied to improve the microalgal oil production performance. For fed-batch cultivation system, different feeding intervals (i.e., 2, 4, and 6-day nitrogen depletion) were explored. The best results were obtained with 6-day nitrogen depletion feeding, giving a final biomass concentration of 9.33 g L-1, which is 3-fold higher than that obtained in batch cultivation system. Nevertheless, the lipid content (49.16 % w/w) and lipid productivity (213 mg L-1 d-1) were lower than those from batch cultivation. On the other hand, semi-continuous cultivation system was conducted using different replacement ratio of culture broth and fresh medium. Using the semi-batch operation with 90 % replacement ratio, the maximum lipid productivity was 302 mg L-1 d-1, which is higher than using batch cultivation system, whereas the lipid content (45.57%(w/w)) was lower. Thus, the selection of cultivation systems has to depend on different requirements. Moreover, the 50 L outdoor batch cultivation of Desmodesmus sp. F2 was also investigated. The results show that the biomass productivity, lipid content, and lipid productivity was 84 mg L-1 d-1, 33.0 % w/w, and 22 mg L-1 d-1, respectively under the light intensity and high temperature in outdoor environment. The results discussed above suggest that D. sp. F2 seems to be a feasible candidate of oil producer for large-scale biodiesel production.

摘要 I
Abstract III
Acknowledgment VI
Contents VIII
Lists of tables XII
Lists of figures XIV
Chapter 1	Introduction	1
1-1 Background	1
1-2 Motivation and purpose	2
1-3 Research scheme of the thesis	4
Chapter 2	Literature review	6
2-1 Biodiesel	6
2-1-1 Introduction of biodiesel	6
2-1-2 Biodiesel production	7
2-2 Algae	9
2-2-1 Macroalgae	9
2-2-2 Microalgae	10
2-3 Introductions of cell growth and lipid formation of microalgae	11
2-3-1 Mechanism of photosynthesis	11
2-3-2 Mechanism of lipid formation	13
2-3-3 Lipid production from microalgae	17
2-4 Factors affecting the cell growth or lipid accumulation of microalgae	18
2-4-1 Light intensity	18
2-4-2 Temperature	19
2-4-3 Medium composition	20
2-4-4 Stress conditions	23
2-4-5 Cultivation systems	24
Chapter 3	Materials and methods	27
3-1 Chemicals and materials	27
3-1-1 Chemicals used for Desmodesmus sp. strains cultivation	27
3-1-2 Chemicals used for lipid analysis	28
3-1-3 Others	28
3-2 Equipments	28
3-3 Isolation and identification of Desmodesmus sp. strains	30
3-4 Composition of D. sp. culture medium	32
3-5 Operation of cultivation system	33
3-5-1 Batch operation with indoor tubular-typed photobioreactor	33
3-5-2 Fed-batch operation with indoor tubular-typed photobioreactor	34
3-5-3 Semi-continuous operation with indoor tubular-typed photobioreactor	35
3-5-4 Batch operation with outdoor tubular-typed photobioreactor	36
3-6 Test tube auto-sedimentation experiment	37
3-7 Statistical experimental design methodology	38
3-7-1 Experimental design using light intensity and temperature as design factors and biomass productivity as response	38
3-7-2 Experimental design using nitrogen concentration and duration of nitrogen depletion as design factors and lipid productivity as response	39
3-8 The analytical methods	41
3-8-1 Measurement of biomass concentration	41
3-8-2 Measurement of residual nitrate concentration	42
3-8-3 Determination of lipid content and fatty acid profile	43
3-8-4 Determination of carbohydrate content	45
3-8-5 Determination of protein content	46
3-9 Data analysis	47
3-9-1 Definition of specific growth rate	47
3-9-2 Definition of overall biomass and lipid productivity	47
3-9-3 Calculation of microalgal settling efficiency and maximum auto-sedimentation rate	47
Chapter 4	Results and discussions	49
4-1 Screening of promising oil producer from four indigenous microalga Desmodesmus sp. isolates	49
4-1-1 Identification of biomass production, lipid content, and lipid productivity of four Desmodesmus sp. isolates	50
4-1-2 Effect of duration of nitrogen depletion on lipid profile of the four D. sp. isolates	53
4-1-3 Performances of auto-sedimentation of four D. sp. isolates	57
4-1-4 Summary	59
4-2 Improving lipid production performance for the target strain of Desmodesmus sp. F2	60
4-2-1 Effects of culture conditions on cell growth and lipid accumulation	60
4-2-2 RSM of light intensity and temperature on maximum biomass productivity	74
4-2-3 RSM of nitrate concentration and nitrogen depletion time on overall lipid productivity	76
4-2-4 Summary	80
4-3 The effect of photobioreactor operation strategy on oil production of Desmodesmus sp. F2 and outdoor cultivation tests	81
4-3-1 Fed-batch cultivation system	82
4-3-2 Semi-continuous cultivation system	87
4-3-3 50 liter outdoor batch cultivation system	89
4-3-4 Summary	93
Chapter 5	Conclusions	94
Reference 97
Appendix 113
Appendix A. European Biodiesel Standard-EN 14214	113
Appendix B. American Biodiesel Standard-ASTM D6751	114
Appendix C. Taiwan Biodiesel Standard-CNS 15072	115
Appendix D. Properties of different kinds of fatty acids (Knothe, 2009)	116

                                    

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