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研究生：	顏世偉 Yen, Shih-Wei
論文名稱：	使用添加生長載體的連續供料批式反應器以本土破囊壺藻Aurantiochytrium sp. CJ6發酵高粱酒粕產製蝦紅素 Fermentative production of astaxanthin from sorghum distillery residue by an indigenous Aurantiochytrium sp. CJ6 strain using a growth carrier loaded continuous-feeding fed-batch process
指導教授：	張嘉修 Chang, Jo-Shu
學位類別：	博士 Doctor
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2023
畢業學年度：	112
語文別：	英文
論文頁數：	144
中文關鍵詞：	高粱酒糟、蝦紅素、破囊壺菌、連續補料批次、光照強度、生長載體、生物炭、焙燒、熱重分析、粒子群優化、循環經濟
外文關鍵詞：	Sorghum distillery residue, astaxanthin, Aurantiochytrium sp., continuous-feeding fed-batch, light intensity, growth carrier, biochar, torrefaction, Thermogravimetric analysis, particle swarm optimization (PSO), circular economy
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蝦紅素具有很強的抗氧化活性，近年來被廣泛用於營養保健品、醫藥領域、化妝品、水產養殖業的天然色素來源等領域。蝦紅素的來源很多，包含人工合成及天然來源。人工合成的蝦紅素佔商業市場的99%，而天然來源只佔1%。人工合成的蝦紅素主要有三個缺點：(1) 製作過程中會產生大量的環境污染物，(2) 含有不具功效的異構物，及(3)消費者食用上的健康疑慮等。天然蝦紅素主要來自藻類，其中雨生紅球藻是最常用的蝦紅素生產藻株，因其細胞的蝦紅素含量高，可高達4%。然而雨生紅球藻養殖不易，且生長速度緩慢，不利於商業化生產，且生產成本偏高。因此，開發替代雨生紅球藻的天然蝦紅素來源實勢在必行。本研究採用一株高潛力蝦紅素生產菌株，即Aurantiochytrium sp. CJ6，進行蝦紅素之生產，並開發新的發酵策略及幫助菌株生長的載體，以提升其商業應用的可行性。
本研究使用的Aurantiochytrium sp. CJ6菌株為一種消耗有機碳源的破囊壺藻 (又稱破囊壺菌），它是從南台灣沿海篩選出來的本土菌株。為因應循環經濟概念，本研究選用高粱酒醩（sorghum distillery residue, SDR）做為培養基的料源，以進行資源回收再利用。SDR是中式白酒生產過程中產生的生物廢棄物，其中仍殘留大量的澱粉和少量的蛋白質。將SDR進行酸水解，可將澱粉轉化為醣，產生高碳/氮比的高粱酒醩水解液(SDR-hydrolysate)，可直接使用為CJ6生長的培養基，不需額外添加氮源，故可大幅降低培養基的成本。
批次實驗的結果顯示，當鹽度為2.5%，通氣率為0.2-0.4 vvm，pH值控制在7.5時，最大細胞濃度為3.3 g/L。此外，適當控制光照時間，在培養第5天至第9天連續提供光源4天的條件下，可獲得最大的蝦紅素含量為69.23 g/g (乾細胞重量，dry cell weight, DCW)。以上條件為CJ6的最佳批次生長條件。
為加速CJ6菌體之生長，本研究嘗試在醱酵槽中加入生長載體，並以SDR來製備生物炭，做為CJ6菌種的生長載體。SDR的主要成分為澱粉，焙燒後產生的高粱酒醩生物炭(SDR-biochar)具有粗糙的表面，具有約20-80微米孔徑的凹洞，非常適合做為CJ6 (細胞大小約為3-5微米)的生長載體。實驗結果證實添加SDR-biochar可以加速CJ6生長。因此，本研究進一步深入探討自製SDR-biochar載體的製備方式及其特性，並以AI的工具及動力學模型的假設，幾近完美地模擬了真實焙燒反應的動力學，並發現在275oC恆溫焙燒60分鐘可以得到最佳的SDR-biochar 載體。將此SDR-biochar275 (275°C恆溫焙燒產生的SDR-biochar) 加入發酵槽中可以加速CJ6細胞生長，因而大幅提升蝦紅素的生產速率 (productivity)。
最後，我們是以新的醱酵策略CF-FB結合SDR-ciochar275的方式來增加蝦紅素的生產速率。本研究提出的所謂的連續補料批次(Continuous-feeding fed-batch, CF-FB)的新的操作方式，是使用蠕動泵緩慢注入SDR-hydrolysate，以保持醱酵狀態長時間維持在一個「營養飢餓的不利環境」之下。在此操作條件下，CJ6菌體為了要自我保護而加速蝦紅素的合成與累積。具體而言，添加SDR-生物炭可將蝦紅素生產率從 0.026 mg/L/D提高到0.070 mg/L/D，顯著提高169%。因此，SDR不僅適合做為CJ6生長的培養基，而且還可以通過焙燒轉化為生物炭顆粒作為生長載體以促進CJ6細胞的生長。使用SDR-biochar顆粒作為生長載體有助於解決CJ6培養時間過長的問題。SDR-biochar為細胞生長提供有利的環境，生物炭顆粒的使用縮短了培養時間並提高了培養的效率。實驗結果顯示，以SDR做為循環經濟平台，有利於應用SDR養藻產製蝦紅素的高價值再利用，並提升以CJ6菌種產製蝦紅素之商業化生產的可行性。

Due to its potent antioxidant activity, astaxanthin is extensively utilized in nutraceutical supplements, the pharmaceutical industry, cosmetics, and as a natural pigment source in aquaculture. While it can be artificially synthesized or naturally sourced, the former accounts for 99% of the market. Artificial synthesis, however, poses environmental, efficacy, and health concerns. Among natural sources, microalgae like Haematococcus pluvialis, containing up to 4% astaxanthin in its cells, are prominent. This study aimed to explore new natural astaxanthin sources beyond H. pluvialis, focusing on the astaxanthin-rich strain Aurantiochytrium sp. CJ6, and its commercial viability.
The CJ6 strain, a high organic carbon-consuming thraustochytrid, was isolated from Taiwan's southern coast. The study used sorghum distillery residue (SDR), a byproduct of Chinese liquor production, as the feedstock for the culture medium. Acid hydrolysis of SDR converts its abundant starch into sugars, resulting in a high carbon-to-nitrogen ratio in the hydrolysate. This makes it an ideal culture medium for CJ6 growth without the need of additional nitrogen sources.
Batch experiment results indicated optimal growth conditions for CJ6 as 2.5% salinity, an aeration rate of 0.2-0.4 vvm, and a pH of 7.5. With these conditions and proper light exposure control, the maximum astaxanthin content obtained was 69.23 μg/g (dry cell weight, DCW).
To promote growth, a growth carrier was introduced into the fermenter. The SDR-biochar, produced after torrefaction of SDR, provided a suitable habitat for CJ6 cells due to its rough surface with large cavities. Experimental results confirmed accelerated CJ6 growth with the addition of SDR-biochar, optimized through AI and kinetic modeling to be produced by torrefying at 275°C for 60 minutes.
SDR-biochar was created through isothermal torrefaction for use as a growth carrier. The two-step parallel reaction (TPR) kinetic model was used to investigate the torrefaction kinetics of SDR, optimized with the Particle Swarm Optimization (PSO) algorithm. The resulting SDR-biochar275 was added to the culture medium, promoting CJ6 growth.
Finally, the study aimed to increase astaxanthin productivity by combining the CF-FB strategy with SDR-biochar275. The slow infusion of SDR-hydrolysate maintained an unfavorable environment, triggering astaxanthin synthesis in cells under high light stress. This approach significantly improved astaxanthin productivity by 169%. This study demonstrated that SDR is not only suitable as a culture medium for CJ6 cultivation but can also be converted into a growth-promoting biochar. This approach shortens cultivation time, improves efficiency, and offers a potential solution for biowaste disposal. The study also suggests that cultivating CJ6 for astaxanthin production in this manner greatly enhances commercial-scale production feasibility.

摘要	I
Abstract	III
Acknowledgments	V
Contents	VI
List of Tables	X
List of Figures	XII
Chapter 1  Introduction	1
1-1 Background	1
1-2 Motivation and purpose	2
1-3 Research scheme	3
Chapter 2  Literature review	7
2-1 Introduction of astaxanthin	7
2-2 The potential of thraustochytrids in astaxanthin production	13
2-3 Acid hydrolysis and reuse of SDR in fermentation and animal feed	17
2-4 Cultivation conditions for astaxanthin-producing strains	21
2-5 Biochar and torrefaction	26
2-6 Torrefaction kinetics simulation with an artificial intelligence tool, PSO algorithm, for optimization of the self-made growth carrier (SDR-biochar) production	29
2-7 The utilization of SDR-biochar as a growth carrier and its underlying mechanisms	31
2-8 Culture strategies and CF-FB	33
Chapter 3  Materials and methods	34
3-1 Chemicals and materials	34
3-2 Equipment	35
3-3 The producing strain: Aurantiochytrium sp. CJ6	38
3-4 Source and proximate analysis of SDR	40
3-5 Preparation of culture medium	41
3-6 Various parameters for optimizing CJ6 cultivation	42
3-7 Self-made growth carrier: SDR-biochar	43
3-7-1 Thermogravimetric analysis	44 
3-7-2 Isothermal torrefaction kinetics	45
3-7-3 Two-step reaction mechanism	47
3-7-4 Particle Swarm Optimization (PSO)	48
3-7-5 Preparation of growth carrier	52
3-8 Fed-batch cultivation with SDR-biochar for improvement of CJ6 growth	53
3-9 Determination of inhibitors and metal ions for mechanism stud	55
3-10 Analytical methods	55
3-10-1 Determination of total carbohydrates	55
3-10-2 Determination of the total nitrogen concentration	56
3-10-3 Determination of the cell mass concentration	57
3-10-4 Determination for the content, concentration and productivity of astaxanthin	58
Chapter 4  Results and Discussion for the optimization of culture conditions	61
4-1 Chemical composition of sorghum distillery residue (SDR)	61
4-2 Hydrolysis with different inorganic acids	62
4-3 The C/N ratio	64
4-4 Effect of salinity on cell growth	65
4-5 Effect of aeration on cell growth	67
4-6 Effect of pH value in cultivation on cell growth	69
4-7 Effects of luminance on cell growth and astaxanthin content	71
4-8 Fermentation strategies: PF-FB vs. CF-FB	75
Chapter 5  Results and discussion for the self-made growth carrier	79
5-1 Basic properties and thermogravimetric analysis	79
5-2 Isothermal torrefaction	83
5-3 Torrefaction kinetics from the traditional Model-Free approach	89
5-4 Torrefaction kinetics from PSO approach	94
Chapter 6  Results and discussion for the culture strategy	101
6-1 Effect of the adding amount of SDR-biochar on cell mass production of Aurantiochytrium sp. CJ6	101
6-2 Effect of light intensity on astaxanthin accumulation in Aurantiochytrium sp. CJ6	107
6-3 Fed-batch fermentation with growth carrier for the enhancement of astaxanthin production	110
Chapter 7  Conclusions	118
7-1 The optimization of the culture and fermentation conditions for Aurantiochytrium sp. CJ6	118
7-1-1 Summary of the findings	118
7-1-2 Research highlights	119
7-1-3 Novelty and significance of the results	119
7-2 Optimization of production conditions for the self-made SDR-biochar used as growth carrier for CJ6	121
7-3 Cultivation of Aurantiochytrium sp. CJ6 in a CF-FB fermenter using SDR-biochar as the growth carrier	123
7-3-1 Summary of findings	123
7-3-2 Research highlights of the results	124
7-3-3 Novelty and significance of the results	126
7-4 Circular economy for SDR	127
7-5 Future perspectives	129
Appendix	131
A-1 The calibration curve of total carbohydrates and absorbance values	131
A-2 The calibration curve of astaxanthin	132
A-3 The HPLC Chromatograms of astaxanthin	133
A-4 The calibration curve of acetic acid	134
A-5 The calibration curve of furfural	135
A-6 The calibration curve of 5-HMF	136
References	137
                                    

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