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研究生:詹明章
研究生(外文):Ming-ChangChan
論文名稱:本土微藻生產葉黃素之技術開發–微藻培養、萃取方法、保存條件及產量程序最適化
論文名稱(外文):Producing lutein from indigenous microalgae –Optimization of microalgae cultivation, extraction protocols, storage conditions, and mass production processes
指導教授:張嘉修張嘉修引用關係
指導教授(外文):Jo-Shu Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:111
中文關鍵詞:斜生柵藻葉黃素萃取方法光波長光源光強度半批次操作二階段策略
外文關鍵詞:Scenedesmus obliquusluteinextractionproductivitylight qualitylight sourcelight intensitysemi-batch operationtwo-stage process
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葉黃素(lutein)為普遍存在於植物體、藻類與光合生物中的類胡蘿蔔素。除了在植物中扮演光合輔助色素外,文獻中亦指出適量葉黃素可有效延緩老化、調節心血管疾病並預防癌症及其他慢性疾病的發生。近年來,由於微藻具有高生長速率、不受季節採收之限制,相較於金盞花(marigold),微藻也具有較高之葉黃素含量,因此以微藻作為葉黃素生產料源之研究為最近十分熱門的研究主題。
在葉黃素生產過程中,萃取成本為一重要之成本耗費,因此本研究首先建構一高效率之萃取技術,以減低葉黃素生產之萃取成本。此萃取技術,除了能有效地避免提取出葉綠素外,其所提取出之葉黃素濃度亦高於傳統萃取法。而在選擇藻體破壁方法方面,發現以高效率球磨機(bead-beater)在七分鐘的破壁時間下,藻體與破壁媒介的比例大於0.02時,能達到葉黃素之最大萃取效率。在皂化步驟的探討方面,鹼溶液濃度可由原本的60%減低至2.5%,並可省略皂化後的後處理步驟,以有效地節省萃取時間。最後,進行有機溶劑提取步驟的最佳化評估,發現利用乙醚(diethyl ether)作為有機提取溶劑,在適當的S/R(Solvent-to-raffinate ratio)比例下操作,能達到最佳的葉黃素萃取效率。
在葉黃素萃取技術建立之後,本研究亦針對萃取液進行不同保存條件下的葉黃素濃度變化測試。實驗結果發現,在三種不同2,6-Ditert-butyl-4-methylphenol (BHT) 濃度添加下,低溫(4℃及-20℃)均具有較好的葉黃素穩定性。而在0.1% BHT添加及4℃或-20℃保存條件下,葉黃素有最佳之保存效果,在三個月內濃度僅約下降10%。在光照對葉黃素保存性的影響方面,添加適當濃度的抗氧化劑可有效維持葉黃素在光照下的穩定性。
本研究接著將由台灣南方海域所篩選之六株本土柵藻Scenedesmus obliquus AS-6-1, S. obliquus CNW-1, S. obliquus CNW-N, S. obliquus ESP-5, S. obliquus ESP-7與S. obliquus FSP-3進行初步的葉黃素生產能力鑑定。實驗結果發現,S. obliquus FSP-3有最高之葉黃素含量及生產速率(3.63 mg/g及1.39 mg/L/day)。此外,結果顯示在批次實驗的過程中,葉黃素含量會隨著氮源消耗程度不同而有所變化,在氮源完全消耗時,葉黃素含量及產率會達到最大值。再者,本研究針對不同光波長、光源種類及光照強度之策略以探討光照對微藻生產葉黃素的影響。實驗結果顯示在不同光波長照射下,S. obliquus CNW-N與S. obliquus FSP-3均在混合白光有最高的葉黃素生產率(分別約1.09及1.45 mg/L/day)。而在不同混合白光與光強度策略下,發現fluorescent lamp (TL5)在光照強度300 mol/m2/s下時,S. obliquus FSP-3之葉黃素生產速率可達4.08 mg/L/day。利用此光照條件進一步進行半批次操作(semi-batch operation),在培養液取代率為10%的情況下,葉黃素生產速率可提升到5.66 mg/L/day,相較於批次條件,約提升了40%。本研究接著利用二階段策略(two-stage process)提升藻體內的葉黃素含量,當藻體由強光照轉換至低光照強度時,藻體內的葉黃素含量會有顯著或些微的提升,此策略應用在大規模培養時,可有效減低萃取成本。
本研究成功地建立以本土柵藻生產葉黃素之技術平台,針對葉黃素萃取步驟的成本或效能、藻體培養與葉黃素生產效率進行有系統的策略評估,並能有效地提升微藻生產葉黃素的生產速率,此成果應能提供未來進行微藻葉黃素生產商業化之重要參考。

Lutein is a kind of carotenoids widely existing in plants and microorganisms. Literature points out that in addition to acting as photosynthetic pigments, uptake of lutein can also effectively prevent aging, cardiovascular diseases and retinal diseases. Compared to conventional lutein production source (i.e., marigold petals), microalgae have emerged as a promising lutein producer due to its high growth rate, no limitation of seasonal harvesting and most importantly, high lutein content in microalgal biomass. Accordingly, using microalgae as the lutein production sources has become a popular option in recent years.
Extraction cost constitutes a large proportion of production cost of lutein. Therefore, this work first made efforts on establishing high-efficiency extraction techniques to lower the cost of lutein extraction. This specific method, a modified version of the method proposed by Miron et al. (2002), can effectively separate carotenoids from chlorophyll and recover lutein with a higher concentration compared with conventional methods. For the optimization of cell-disrupting step, bead-beater exhibited the highest extracting efficiency under the conditions of 7 mins treatment time and a algae-to-bead ratio of 0.02. In addition, without affecting the extraction efficiency, the saponifying concentration could be reduced from 60% to 2.5% with the post-treatment step omitted. Finally, the performance of using different organic solvents on lutein extraction was also evaluated. The results show that diethyl ether appeared to be the most suitable solvent, while different S/R ratios (Solvent-to-Raffinate ratio) were operated under different production purpose (such as the counterbalance of the solvent and time cost).
After the establishment of extraction protocols, the storage methods of lutein extracts under different conditions were investigated. The results show that under the storage addition of 0.01% BHT and low temperature (4oC and -20oC), lutein extracts showed the best stability. As for light exposure effects, the stability of lutein extracts could be greatly maintained under light irradiation if a suitable amount of antioxidant was added.
This work was also undertaken to optimize the microalgae cultivation strategies for achieving better lutein production performance. First, six indigenous microalgal strains isolated from aquatic environments in Southern Taiwan (namely Scenedesmus obliquus AS-6-1, S. obliquus CNW-1, S. obliquus CNW-N, S. obliquus ESP-5, S. obliquus ESP-7 and S. obliquus FSP-3) were examined for their capabilities to accumulate lutein. Among them, S. obliquus FSP-3 showed the highest lutein content and lutein productivity, which were 3.63 mg/g and 1.39 mg/L/day, respectively. The result also showed that under microalgal growth, the maximal lutein content and productivity were obtained in the beginning of nitrogen starvation. For light-related strategies, both of the two strains (i.e., CNW-N and FSP-3) exhibited higher lutein production under the illumination of mixing white light (1.09 and 1.45 mg/L/day, respectively). Moreover, the lutein productivity of S. obliquus FSP-3 could be improved by adjusting the light source and light intensity. When using fluorescent lamp (TL5) at an intensity of 300 mol/m2/s, the lutein productivity of strain FSP-3 could reach 4.08 mg/L/day. Semi-batch operations were conducted using the obtained optimal conditions to further enhance the lutein productivity to 5.66 mg/L/day, which is about 40% increase compared to that obtained from batch tests. Moreover, lutein content could also be improved via a two-stage process, in which the light intensity was set at a higher level to facilitate cell growth to a certain amount but was tuned lower to stimulate lutein accumulation. The results show that a significant or slight increase in lutein content was observed when using the two-stage operation, which could be applied to the large-scale lutein production process to save the lutein extraction cost.
This study successfully established the platform technology on lutein production from the indigenous Scenedesmus obliquus strains. Systematic evaluation was done on the approaches for lutein extraction, microalgae growth and lutein production, leading to a marked improvement on lutein productivity. The results obtained from this research could be valuable references for future development of commercially viable microalgae-based lutein production processes.

摘要 I
Abstract III
Acknowledgement VI
List of Tables XII
List of Figures XIV
Chapter 1 Introduction 1
1-1 Background 1
1-2 Motivation and purpose 1
Chapter 2 Literature review 4
2-1 Introduction to photosynthesis and photosynthetic pigments 4
2-1-1 Photosynthesis 4
2-1-2 Chlorophyll 7
2-1-3 Carotenoids 8
2-1-4 Lutein 11
2-2 Introduction to microalgae 12
2-3 Microalgae as a lutein feedstock 15
2-3-1 Lutein production from microalgae and other sources 15
2-3-2 Distribution of lutein and other carotenoids in microalgae 18
2-3-3 Lutein biosynthesis: carotenogenesis pathways 21
2-3-4 Function of lutein from microalgae 24
2-3-5 Influence of environmental factors on microalgal lutein
production 25
2-4 Lutein recovery and storage from microalgal biomass 28
2-4-1 Introduction to microalgal pigment extraction 28
2-4-2 Organic solvent-mediated extraction 29
2-4-3 Effect of storage conditions on lutein extracts 30
Chapter 3 Material and methods 32
3-1 Chemicals and materials 32
3-2 Equipments 33
3-3 Experimental methods 35
3-3-1 Microalgae and their culture medium 35
3-3-2 Operation of photo-bioreactor 39
3-3-3 Comparison of conventional and modified methods 39
3-3-4 Effect of cell disruption methods on lutein extraction 40
3-3-5 Effect of saponification process on lutein content 41
3-3-6 Effect of organic solvents on lutein content 41
3-3-7 Effect of preservation conditions on lutein extracts 42
3-4 Data analysis 42
3-4-1 Determination of microalgal biomass concentration and growth kinetic parameters 42
3-4-2 Determination of nitrate concentration 43
3-4-3 Measurement of light intensity 44
3-4-4 Determination of lutein content in the microalgal biomass using high-performance liquid chromatography 45
Chapter 4 Results and discussion 47
4-1 Optimization of extraction protocols 47
4-1-1 Comparison of conventional method and modified method for carotenoids 47
4-1-2 Effect of cell-disrupting methods 50
4-1-3 Effect of cell-disrupting time under different ratio of microalgal biomass to beads 51
4-1-4 Effect of concentration of saponifying reagent and post-treatment time 52
4-1-5 Effect of different organic solvent 55
4-1-6 Effect of multiple extractions 56
4-1-7 Effect of S/R ratio 58
4-1-8 Summary 59
4-2 Purification of lutein extracts 61
4-3 Evaluation of storage condition on preservation efficiency of lutein extracts 63
4-3-1 Effect of heat and BHT addition on lutein stability 63
4-3-2 Effect of light on lutein stability 64
4-3-3 Summary 67
4-4 Cultivation of microalgae 68
4-4-1 Identification of microalgal strains 68
4-4-2 Selection of a suitable candidate for lutein production 71
4-4-3 Evolution of lutein content during microalgae cultivation 74
4-4-4 Effect of light wavelength on biomass production and lutein content 76
4-4-5 Effect of light source on biomass production and lutein content 79
4-4-6 Effect of light intensity on biomass concentration and lutein content 85
4-4-7 Improvement of biomass productivity: semi-batch operation 89
4-4-8 Improvement of lutein content: two-stage operation 92
4-4-9 Summary 95
Chapter 5 Conclusions 96
References 100
Appendix 111
Appendix curriculum vitae 112

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