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姓名	尼瓦娜(Wa Ode Cakra Nirwana)  查詢紙本館藏	畢業系所	化學工程與材料工程學系
論文名稱	探討以Amycolatopsis thermoflava轉化阿魏酸生產香草醛之研究 (Bioconversion of Ferulic Acid to Vanillin by Amycolatopsis thermoflava)
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摘要(中)	通過阿魏酸 (FA) 的微生物轉化生產香草醛最近引起了更多關注，因為從醃製的香草莢生產天然香草醛是一個昂貴的過程。然而，FA和香草醛的毒性、FA和香草醛作為碳源和能源的利用以及香草醛的高度降解是獲得高滴度香草醛的主要限制。 這項工作旨在通過工藝優化最大限度地利用 Amycolatopsis thermoflava 將 FA 生物轉化為香草醛的效率。展示了兩種方法：（第 1 部分）使用商業 FA 作為前體的生物轉化，（第 2 部分）使用來自玉米芯鹼性水解物的 FA 進行生物轉化，以利用農工業廢料作為廉價原材料。 使用商業 FA 生產香草醛（第 1 部分）表明，補充 FA 的時間強烈影響香草醛的形成。在穩定期早期加入FA有利於獲得高細胞密度和高產量。對培養溫度影響的研究表明，45 oC 培養有利於細胞生長，而 30 oC 則有利於高產，因為它可以降低香草醛的降解速度。因此，進行了兩階段分批發酵。這種策略允許使用更高濃度的 FA。為了提高香草醛的產量，通過控制FA的溫度和補料時間實施了兩階段補料分批發酵。與 30 oC 的單階段分批發酵相比，該策略顯著提高了香蘭素的產量和生產力，分別在第 0 小時將 FA 添加到肉湯中。對於分批補料操作中FA的補料方式，延長FA補充時間可以降低其對細胞的毒性作用，從而提高細胞生長速度，縮短生物轉化時間。但是，它也加速了香草醛的降解。 對於第二部分，工作開始於優化超聲輔助提取條件，以使用鹼處理從玉米芯中釋放 FA。最佳水解條件是在 0.5 M NaOH 濃度和 10% 固體負載下提取 30 分鐘，導致相對較高濃度的 FA 715 mg/L 和對香豆酸 1025 mg/L。為了最大限度地從 FA 中形成香草醛，研究了五個參數，包括滅菌方法、營養限制、初始生物量濃度、培養溫度和還原糖控制的影響。結果表明，使用高壓滅菌的水解產物進行生物轉化對細胞生長和香草醛形成都產生了較差的結果。巴氏殺菌更有利，然而，污染物的存在限制了整個過程。因此，制定了營養限制策略。對初始生物量濃度和培養溫度影響的觀察表明，最大香草醛形成分別在 1.5 g/L 和 45 oC 時獲得。為了優化香草醛的生產，開發了一種還原糖控制策略。與使用高壓滅菌水解物的生物轉化相比，香草醛的產量和生產力分別顯著增加了 29.5 倍和 585 倍。
摘要(英)	Vanillin production through a microbial transformation of ferulic acid (FA) has recently attracted more attention since producing natural vanillin from cured vanilla pods is a costly process. However, the toxicity of FA and vanillin, utilization of FA and vanillin as carbon and energy sources, and high degradation of vanillin are the main limitations to obtaining a high titer of vanillin. This work aims to maximize the efficiency of bioconversion of FA to vanillin using Amycolatopsis thermoflava by process optimization. Two approaches were demonstrated: (Part 1) bioconversion using commercial FA as a precursor, (Part 2) bioconversion using FA from alkaline hydrolysate of corn cobs to utilize agro-industrial waste as a cheap raw material. Vanillin production using commercial FA (Part 1) showed that the timing of FA supplementation strongly affects vanillin formation. The addition of FA in the early stationary phase was favorable to obtaining high cell density and high yield. Studies on the effect of culture temperature showed that cultivation at 45 oC favored cell growth, while 30 oC favored high yields since it could reduce the rate of vanillin degradation. Therefore, a two-stage batch fermentation was conducted. This strategy allowed the use of higher concentrations of FA. To increase vanillin productivity, a two-stage fed-batch fermentation by controlling the temperature and feeding time of FA was implemented. This strategy significantly increased the yield and productivity of vanillin by 1.8 and 12.0 folds, respectively, compared to single-stage batch fermentation at 30 oC where FA was initially added to the broth at the 0th h. Concerning the feeding method of FA at fed-batch operation, expanding the time of FA supplementation could lower its toxic effect on cells, thereby increasing cell growth rate and shortening bioconversion time. However, it also accelerated the degradation of vanillin. For part two, the work was started by optimizing the ultrasound-assisted extraction conditions to release FA from corn cobs using alkaline treatment. Optimal hydrolysis conditions were obtained at 0.5 M NaOH concentration and 10% solid loading for 30 min of extraction, resulting in relatively high concentrations of FA 715 mg/L and p-Coumaric acid 1025 mg/L. To maximize vanillin formation from FA, five parameters were investigated including the effects of sterilization method, nutrient limitation, initial biomass concentration, culture temperature, and reducing sugar control. The results showed that bioconversion using autoclaved hydrolysate gave poor results both for cell growth and vanillin formation. Pasteurization was more favorable however, the presence of contaminants limited the entire process. Therefore, a nutrient limitation strategy was developed. Observations on the effects of initial biomass concentration and culture temperature showed that the maximum vanillin formation was obtained at 1.5 g/L and 45 oC, respectively. To optimize vanillin production, a reducing sugar control strategy was developed. The yield and productivity of vanillin significantly increased by 29.5 and 585 folds, respectively, compared to bioconversion using autoclaved hydrolysate.
關鍵字(中)	★ 生物轉化 ★ 香草醛 ★ 阿魏酸 ★ 農業廢棄物 ★ 玉米棒子 ★ Amycolatopsis thermoflava	關鍵字(英)	★ Bioconversion ★ Vanillin ★ Ferulic acid ★ Agrowaste ★ Corn cobs ★ Amycolatopsis thermoflava
論文目次	摘要 i ABSTRACT iii ACKNOWLEDGEMENT v TABLE OF CONTENTS vi LIST OF FIGURES ix LIST OF TABLES xv CHAPTER 1 INTRODUCTION 1 1-1 Background 1 1-2 Aim of the research 3 CHAPTER 2 LITERATURE REVIEW 5 2-1 Vanilla and Vanillin 5 2-2 Vanillin Production 6 2-2-1 Vanillin from vanilla plant 6 2-2-2 Vanillin from chemical synthetics 10 2-2-3 Vanillin from biotechnological routes 11 2-2-3-1 De novo biosynthetic 11 2-2-3-2 Bioconversion or biotransformation 13 2-3 Bioconversion of ferulic acid to vanillin 13 2-4 Agro-industrial wastes for vanillin production 19 2-4-1 Releasing ferulic acid from agro-industrial waste 19 2-4-2 Bioconversion of ferulic acid from agro-industrial waste to vanillin 20 2-4-3 Bioconversion of ferulic acid from corn cobs to vanillin 21 CHAPTER 3 MATERIALS AND METHODS 22 3-1 Scheme of the research 22 3-2 Chemicals 23 3-3 Equipment and Instruments 24 3-4 Microorganism 25 3-5 Medium and culture conditions 26 3-5-1 Bioconversion of commercial ferulic acid to vanillin by Amycolatopsis thermoflava 26 3-5-1-1 Effect of culture temperature, ferulic acid initially added at the 0th h 27 3-5-1-2 Effect of initial feeding time of ferulic acid 27 3-5-1-3 Effect of culture temperature, ferulic acid initially added in the early stationary phase 27 3-5-1-4 Two-stage batch fermentation 28 3-5-1-5 Two-stage batch fermentation with high concentrations of ferulic acid 28 3-5-1-6 Two-stage fed-batch fermentation 28 3-5-2 Bioconversion of ferulic acid from corn cobs to vanillin by Amycolatopsis thermoflava 28 3-5-2-1 Releasing ferulic acid from corn cobs by alkaline treatment 28 3-5-2-2 Effect of sterilization method 29 3-5-2-3 Effect of nutrient limitation 29 3-5-2-4 Effect of initial biomass concentration 30 3-5-2-5 Effect of temperature 30 3-5-2-6 Effect of reducing sugar control 30 3-6 Analysis methods 31 3-6-1 Biomass concentration 31 3-6-2 Reducing sugar 31 3-6-3 Phenolic compounds concentration 32 CHAPTER 4 RESULTS AND DISCUSSION 36 4-1 Bioconversion of commercial ferulic acid to vanillin by Amycolatopsis thermoflava 36 4-1-1 Effect of culture temperature, ferulic acid initially added at the 0th h 36 4-1-2 Effect of initial feeding time of ferulic acid 42 4-1-3 Effect of culture temperature, ferulic acid initially added in the early stationary phase 47 4-1-4 Two-stage batch fermentation 53 4-1-5 Two-stage batch fermentation using high concentrations of ferulic acid 54 4-1-6 Two-stage fed-batch fermentation 58 4-2 Bioconversion of ferulic acid from corn cobs to vanillin by Amycolatopsis thermoflava 62 4-2-1 Releasing ferulic acid from corn cobs by alkaline treatment 62 4-2-2 Effect of sterilization method 64 4-2-3 Effect of nutrient limitation 68 4-2-4 Effect of initial biomass concentration 75 4-2-5 Effect of temperature 79 4-2-6 Effect of reducing sugar control 85 CHAPTER 5 CONCLUSION AND RECOMMENDATIONS 88 5-1 Conclusion 88 5-2 Recommendations 88 REFERENCES 89
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指導教授	徐敬衡(Chin-Hang Shu)	審核日期	2022-11-14
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