臺灣博碩士論文加值系統

本研究從台灣高雄海岸採集的海水樣本中分離出具有生產類胡蘿蔔素葉黃素特性的微細藻Chlorella sorokiniana Kh12。此研究目的在評估和優化C. sorokiniana Kh12培養條件、葉黃素累積階段、提高葉黃素和脂質的萃取量。使用2X-(HT)-9k 模式的混合營養培養C. sorokiniana Kh12得到3.46 g/L的生物量和13.69 mg/g的葉黃素產量，這是目前相關報告中的最高產量之一。C. sorokiniana Kh12使用珠磨和有機溶劑甲醇震盪 7 分鐘可獲得最大葉黃素萃取率（7.56 mg/g），以上結果顯示C. sorokiniana Kh12具有生產脂質和葉黃素的潛力。 透過生物製程條件優化培養C. sorokiniana Kh12的結果顯示大量營養素和微量營養素的 3x: 3x 比例顯著提升生物量至4.6 g/L 和葉黃素含量至14.02 mg/g; 連續培養在32℃溫度下使C. sorokiniana Kh12的葉黃素含量提升到17.3 mg/g; 在10k lux光照強度下培養C. sorokiniana Kh12得到最高的葉黃素含量14.42 mg/g; C. sorokiniana Kh12培養在鹽度25%至75%之間有利於提升脂質含量為20.5%至26%; 使用兩階段培養C. sorokiniana Kh12策略部分，結果顯示有效提升C. sorokiniana Kh12生物量、葉黃素和脂質產量分別為4.1 g/L、16.01mg/g和246 mg/g，使用 3x:3x 大量微量營養素比例可提高葉黃素生產力和產量（4.05 mg/L/d，56.74 mg/L）。C. sorokiniana Kh12培養在管式生物反應器比普通生物反應器有較高的二氧化碳的利用率而提高生物量產量; 在探討管式生物反應器培養條件對C. sorokiniana Kh12生物量和葉黃素產量的影響方面，實施多種培養策略包含改變光照、切換到管式生物反應器、光/暗、部分圖案遮光、培養六天後改變光照、溫度、營養物添加和組合的兩階段方法; 管式反應器中的 8k 光強度顯示出較好的生物量（6.75 g/L）以及葉黃素生產率和產量（4.43 mg/L/d，62.1mg/L）; 光/暗週期(18:6) 循環以及部分圖案遮光顯示出更好的葉黃素產量（65.48 mg/L 和69.14 mg/L）以及生產力（4.67 mg/L/d 和4.93 mg/L/d ）; 尿素作為碳/氮源，透過基因上調將產量和生產力提高到 82.1 mg/L 和 5.86 mg/L/d，與其他最高產量相比，在有限的養分供給條件下達到最大產量。此外，在兩個階段中觀察到生物量和葉黃素的增加，其中在第二階段添加（BS，HT），並且在第8天後增加2k的光強度產生8.31 g/L的生物量和86.42 mg/L的葉黃素產量和 6.17 mg/L/d 的生產力，這是混合營養分批培養報告中獲得的最高生產力之一。
此外，反應器有限空間造成對細胞的干擾，研究結果雖然獲得較高生物量但卻無法有效提升葉黃素含量。 C. sorokiniana Kh12 是目前研究報告中葉黃素產量最高的微細藻之一，顯示出其作為商業葉黃素生產的潛力藻種。本研究成功分離並優化了微細藻 C. sorokiniana Kh12 的葉黃素生產策略。這些發現為葉黃素生產的生物製程優化提供了重要的研究資訊，並凸顯出這個微細藻種在工業規模上生產高價值葉黃素的潛力。

A study was performed to identify and characterize novel strains capable of producing the carotenoid lutein from seawater samples collected along the coast of Kaohsiung, Taiwan. The microalgae strain, Chlorella sorokiniana Kh12 was found to be a viable option for the synthesis of lutein. The research aimed to optimize the cultivation conditions, lutein accumulation stage, and extraction methods for maximizing lutein and lipid recovery. For Chlorella sorokiniana Kh12, the study focused on evaluating and optimizing various parameters. Under mixotrophic cultivation using a 2X-(HT)-9k mode, Kh12 exhibited high biomass production (3.46 g/L) and lutein yield (13.69 mg/g), making it one of the highest reported yields. MeOH was determined as the most effective solvent for lutein extraction, and bead milling for seven minutes yielded the maximum lutein extraction yield (7.56 mg/g). These results demonstrated the potential for lipid co-production and commercial lutein synthesis. In the case of Chlorella sorokiniana Kh12, the bioprocess conditions for lutein production were optimized by investigating various parameters. A 3x:3x ratio of macro- and micronutrients significantly influenced biomass yield (4.6 g/L) and lutein content (14.02 mg/g). Continuous cultivation at 32 °C increased lutein content to 17.3 mg/g, while an illumination intensity of 10k lux resulted in the highest lutein content (14.42 mg/g). Manipulating salinity levels from 25% to 75% facilitated lipid accumulation, with an optimized lipid content ranging from 20.5% to 26%, and implementing a two-stage strategy further enhanced biomass, lutein, and lipid yields, reaching 4.1 g/L, 16.01, and 246 mg/g, respectively. The use of a 3x:3x macro-micronutrient ratio led to the increase in lutein productivity and yield (4.05 mg/L/d, 56.74 mg/L). Furthermore, the tubular bioreactor gives higher biomass than the normal bioreactor because of better mass transfer utilization of CO2. To increase the biomass and lutein, several tactics such as varying light, switching to a tubular bioreactor, light/dark, patterned light and a two-stage approach involving changing the light, temperature, nutrient feeding, and combination after six days are being implemented.8k light intensity in a tubular reactor showed better biomass which was 6.75 g/L, and lutein productivity and yield (4.43 mg/L/d, 62.1mg/L). Furthermore Light/dark (18:6) cycles as well as pattern light have shown better lutein yields of 65.48 mg/L and 69.14 mg/L and productivity’s 4.67 mg/L/d and 4.93 mg/L/d. respectively. Urea, as carbon/nitrogen source, boosted yield and productivity to 82.1 mg/L and 5.86 mg/L/d by gene upregulation which is maximum with a limited amount of nutrients compared to other maximum yields. Moreover, an increase in biomass as well as lutein was observed in two stages where in the second stage addition of (BS, HT), and 2k increase of light intensity after day 8 yielded 8.31 g/L biomass and 86.42 mg/L lutein yield and 6.17 mg/L/d productivity which is one of the maximum reported in mixotrophic batch cultivation.
Moreover, results with high biomass also slightly decrease lutein due to the interference of cells in the limited space of the reactor. Chlorella sorokiniana Kh12 showed itself as a potential source for commercial lutein production by producing one of the highest lutein yields. This research study successfully isolated and optimized lutein production strategies for microalgae strains, Chlorella sorokiniana Kh12. The findings contribute valuable insights into the bioprocess optimization of lutein production and highlight the potential of these strains for high-value lutein production on an industrial scale.

List of Content
ACKNOWLEDGEMENT………………………………………………………….… iii
摘要 ………………………………………………………………………………….. iv
ABSTRACT…………………………………………………………………………… vi
ABBREVIATIONS……………………………………………………………………. viii
LIST OF FIGURES………………………………………………………………..…. xii
CHAPTER 1- INTRODUCTION…………...……..………………………………… 1
1.1 Research background…………………………………………………………….. 1
1.2 Research objectives………………….……………………………………..……. 5
CHAPTER 2 – LITERATURE REVIEW……………………...…...………………. 6
2.1 Overview of microalgae lutein…………………………………………...……… 6
2.1.1 Microalgae isolation and selection…………………………………………. 6
2.1.2 Effect of macro and micronutrients………………………………………… 7
2.1.3 Effect of light intensity…………………………………………………….. 7
2.1.4 Effect of temperature………………………………………………………. 8
2.1.5 Effect of salinity……………………………………………………………. 8
2.1.6 Enhancement strategies: upstream and downstream processing…………… 9
2.1.7 Two-stage cultivation strategies………….………………………………… 9
2.1.8 Challenges and future directions……………………………………………. 10
CHAPTER 3 - MATERIALS AND METHOD……………………...……………… 12
3.1. Chemicals and materials....................................................................................... 12
3.2 Field collection of microalgae samples and enrichment in media......................... 12
3.3 Initial evaluation of algal culture for enhanced biomass production……………. 12
3.4 Screening identification and cultivation parameters of the microalgae strain....... 13
3.5 Lutein extraction.................................................................................................... 16
3.5.1 Impact of solvent on the extraction of lutein................................................ 16
3.5.2 The impact of the cell disruption technique.................................................. 16
3.6 Optimizing parameters for the production of lutein............................................... 18
3.6.1 Micro- and macronutrient effects.................................................................. 18
3.6.2 Influence of temperature, light, and salinity................................................. 18
3.6.3 Analysis of ROS in different conditions……………………..…………..... 19
3.6.4 Effect of combination of salinity and temperature………………………… 19
3.6.5 Impact of different light intensities on tubular bioreactor…………..……. 19
3.6.6 Impact of pattern and light/dark cycles in a tubular bioreactor……………. 20
3.6.7 Effect of nitrogen sources and two-stage strategies……….…..…………... 21
3.7 Analytical methods................................................................................................. 22
3.7.1 Biomass, lutein, and lipid estimation............................................................ 23
3.7.2 Chromatography............................................................................................ 23
3.7.3 Statistical analysis......................................................................................... 23
CHAPTER 4 - RESULTS AND DISCUSSIONS......................................................... 25
4.1 Identification and characterization of promising microalgal strains.................. 25
4.2 Lutein, biomass, and lipid yield under autotrophic, mixotrophic, and
nutrient-stressed conditions.................................................................................. 26
4.3 Impact of solvent on the extraction of lutein....................................................... 32
4.4 Enhancing lutein extraction from Chlorella sorokiniana Kh12 through optimized cell disruption……………………………………………………... 35
4.5 Influence of variation of micro and macronutrients............................................. 36
4.6 Influence of different illumination intensities on Kh12....................................... 40
4.7 Influence of temperatures ................................................................................... 43
4.8 Influence of salinity ............................................................................................ 45
4.9 Effect of short-term stress of temperature and salinity on cells ROS generation. 47
4.10 Influence of different light intensity in tubular bioreactor……………………... 49
4.11 Effect of light/dark condition…………………………………………………... 51
4.12 Effect of pattern bioreactor…………………………………………………….. 53
4.13 Effect of nitrogen sources ……………………………………….…………….. 55
4.14 Two-stage cultivation strategies ……………………………………………….. 57
CHAPTER 5 – SUMMARY AND FUTURE DIRECTIONS………………............. 61
5.1 Summary ............................................................................................................... 61
5.2 Future perspectives ............................................................................................... 63
REFERENCES .............................................................................................................. 65

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