文獻指出微藻可用來降低二氧化碳排放且在特定培養條件下有生產油脂的能力，因此被譽為第三代生質燃料之料源。本研究針對自南台灣篩選出之本土小球藻(Chlorella sp. ESP-31)進行其生產油脂並應用於合成生質柴油之潛力分析。由於光源的提供對微藻培養十分重要，因此，本研究首先探討不同人工光源與光強度對微藻生長的影響。結果顯示，新型日光燈管(TL5)適用於微藻之室內培養，而使用200 mg/L碳酸氫納為碳源培養時，以光照度9 W/m2 (50 umol/m2•s)時有較佳的藻體生產力約0.029 g/L/d。
接著，本研究探討Chlorella sp. ESP-31的最佳培養基與培養條件，乃利用三種培養基(Basal 、Modified Bristol’s和MBL培養基)與四種培養條件(光自營，異營，光異營，混營)進行Chlorella sp. ESP-31之培養。結果發現在光自營(二氧化碳)、光異營(葡萄糖)與混營(二氧化碳加葡萄糖)的條件下，使用氮源充足的培養基(Basal和Modified Bristol's培養基)可得到較好的藻體濃度(2-5 g/L)，而使用氮源缺乏的培養基(MBL培養基)則可得到較高的油脂含量(20-53%)，但其生長較緩慢。無論使用何種培養基，Chlorella sp. ESP-31在混營的情況下可獲得最高的油脂含量(40-53%)與油脂產量(67-144 mg/L/d)。分析此藻株所累積的油脂成分發現，超過60-68%的脂肪酸皆為飽和的棕梠酸和硬脂酸與單一不飽和的油酸，其成分相當適合進行轉酯化以合成生質柴油。
此外，本研究亦建立一套以二氧化碳為碳源並將其轉化為油脂之培養程序，並以氮源缺乏策略及光生物反應器設計以提高Chlorella sp. ESP-31在光自營下之油脂生產與二氧化碳固定之效率。結果顯示，使用降低起始氮源濃度的單一階段氮源缺乏策略(Basal培養基；起始氮源濃度為0.313 g/L KNO3)可有效的增進微藻油脂產量並得到78 mg/L/d的油脂生產力與55.9%的油脂含量。接著，使用高表面對體積比(S/V=109.3 m2/m3)的直立型管狀反應器更可進一步的提高油脂產率至132.4 mg/L/d。實驗中亦發現當氮源缺乏，而油脂含量上升時，其飽和的棕梠酸和硬脂酸與單一不飽和的油酸占總油脂之比例從26%上升至65%，且生產油脂的同時還可在十天內固定6.36克的二氧化碳，其二氧化碳固定效率可達430 mg/L/d。
本研究也探討在光異營下使用不同有機碳源(葡萄糖、果糖、蔗糖、甘油、醋酸鈉與醋酸)對Chlorella sp. ESP-31生長與油脂含量之影響。當未控制pH值時，使用葡萄糖培養Chlorella sp. ESP-31可得最高的藻體產量(3.5 g/L)和油脂含量(26%)。若將pH值控制在8.5時，使用果糖與醋酸鈉也可得到3.2-3.6 g/L的藻體產量與24-25%的油脂含量。此外，使用醋酸進料控制pH值的饋料批次(pH-stat)操作也被用來增進藻體生長與油脂產量，當使用醋酸控制pH在7.0-7.5之間可得到最高的藻體轉換效率(0.68 g/g CH3COOH)、油脂含量(50%)與油脂產率(78 mg/L/d)。然而，實驗結果顯示，該微藻之油脂成分(即脂肪酸成分分佈)並不隨碳源之不同而改變。
最後，本研究將實驗室等級的直立式管狀反應器規模放大至50公升光管反應器，並進行Chlorella sp. ESP-31之室外培養。在最佳光自營(二氧化碳)及光異營(醋酸)的條件下，可得到30-31 mg/L/d的油脂產率且油脂可累積至35-44%。為了增進戶外培養的藻體生長與油脂累積，本研究嘗試使用不同起始藻種接種量進行陪養，結果發現使用0.70 g/L的起始接種量可得到較高的油脂產率(47.6 mg/L/d)，且利用兩階段油脂累積策略可舒緩夏天養藻所面臨嚴重的汙染問題，並提升油脂產率。

Microalgae have the ability of mitigating carbon dioxide emissions and producing oil simultaneously under specific growth conditions, thereby having the potential as the feedstock to produce the third-generation biofuels. Light supply is one of the most important factors affecting autotrophic growth of microalgae. Therefore, this study started with the investigation on the effects of the type and light intensity of artificial light sources on the cell growth of an indigenous microalga Chlorella sp. ESP-31 isolated from southern Taiwan. The results show that a new fluorescent lamps (TL5) was effective in indoor cultivation of microalgae. A higher overall productivity of 0.029 g/L/d was obtained when using TL5 lamps as the light source with a light intensity of 9 W/m2 (or 50 umol/m2•s) using 200 mg/L NaHCO3 as the carbon source.
To determine suitable medium and cultivation conditions for the growth of Chlorella sp. ESP-31, different media (Basal, Modified Bristol’s and MBL medium) and cultivation conditions, including phototrophic growth (NaHCO3 or CO2, with light), heterotrophic growth (glucose, without light), photoheterotrophic growth (glucose, with light) and mixotrophic growth (glucose and CO2, with light) were applied. Chlorella sp. ESP-31 preferred to grow under phototrophic (CO2), photoheterotrophic and mixotrophic conditions on nitrogen-rich medium (i.e., Basal medium and Modified Bristol's medium), reaching a biomass concentration of 2-5 g/L. The growth on nitrogen-limiting MBL medium resulted in higher lipid accumulation (20-53%) of macroalgal biomass at the expense of a lower growth rate. Higher lipid content (40-53%) and lipid productivity (67-144 mg/L/d) were obtained under mixotrophic cultivation regardless of the culture medium used. The fatty acid composition of the microalgal lipid comprises over 60-68% of saturated fatty acids (i..e., palmitic acid (C16:0), stearic acid (C18:0)) and monounsaturated acids (i.e., oleic acid (C18:1)). This lipid composition is appropriate for biodiesel production.
To develop the cultivation technology able to achieve both CO2 mitigation and biofuel production from the microalgal strain, the nitrogen starvation strategies and photobioreactor design were applied to examine their effects on the performance of lipid production and of CO2 fixation of Chlorella sp. ESP-31 under phototrophic condition. Comparison of single-stage and two-stage nitrogen starvation strategies shows that using single-stage cultivation on Basal medium with lower initial nitrogen source concentration (i.e., 0.313 g/L KNO3) was the most effective approach to enhance microalgal lipid production, attaining a lipid productivity of 78 mg/L/d and a lipid content of 55.9%. The lipid productivity of Chlorella sp. ESP-31 was further upgraded to 132.4 mg/L/d when it was grown in a vertical tubular PBR with a high surface to volume ratio of 109.3 m2/m3. The proportion of saturated (i.e., palmitic acid (C16:0) and stearic acid (C18:0)) and monounsaturated (i.e., oleic acid (C18:1)) fatty acids increased from 26% to 65% of the total microalgal lipid during nitrogen starvation. The high lipid productivity was also accompanied by fixation of 6.36 g of CO2 during the 10-day phototrophic growth with a CO2 fixation rate of 430 mg/L/d.
The growth and lipid production of Chlorella sp. ESP-31 were also investigated under photoheterotrophic cultivation using different carbon sources (namely, glucose, fructose, sucrose, glycerol, sodium acetate and acetic acid). In the absence of pH control, growing Chlorella sp. ESP-31 on glucose obtained the highest biomass concentration (3.5 g/L) and lipid content (26%). By controlling pH at 8.5, the growth on fructose and sodium acetate was improved, obtaining a biomass concentration of 3.2-3.6 g/L and a lipid content of 24-25%. Moreover, an economical fed-batch operation with pH-stat feeding of acetic acid was employed to enhance biomass and lipid production. When the pH-stat culture was conducted at pH 7.0-7.5 with acetic acid feeding, the best photoheterotrophic growth performance was obtained, resulting in the highest biomass yield, lipid content, and lipid productivity of 0.68 g/g CH3COOH, 50%, and 78 mg/L/d, respectively. The fatty acid profile of the microalgal lipid did not change significantly while using different carbon sources.
The scale of the indoor vertical tubular-type PBR was elevated to 50 liter PBR for outdoor cultivation of Chlorella sp. ESP-31. Under phototrophic (CO2) and photoheterotrophic (acetic acid) conditions, the lipid productivities and lipid contents of 30-31 mg/L/d and 35-44%, respectively, were obtained. The effect of inoculum sizes on the performance of biomass and lipid production under phototrophic cultivation was also investigated. The lipid productivity of Chlorella sp. ESP-31 was further improved to 47.6 mg/L/d when a higher inoculum size of 0.70 g/L was used. Finally, to avoid severe contamnination problem during outdoor microalgae cultivation in the summer time, a two-stage lipid accumulation strategy was applied. It shows that the proposed two-stage process was able to mitigate the bacterial contamination and enhanced the lipid content and lipid productivity of Chlorella sp. ESP-31.

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