探討溫度暨萃取發酵系統對於Saccharomyces cerevisiae連續生產苯乙醇之影響

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周筱軒(Siao-Syuan, Jhou) 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

探討溫度暨萃取發酵系統對於Saccharomyces cerevisiae連續生產苯乙醇之影響
(Enhanced biotransformation of L-phenylalanine into 2-phenylethanol via cultivation temperature control strategy and extractive fermentation)

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摘要(中)

苯乙醇因具有特殊的玫瑰香氣而被大量的應用在香水、化妝品及食品香精中，年產量在全球香料市場中佔有一席之地。利用微生物發酵法，依循Ehrlich pathway的生成途徑，可以透過Saccharomyces cerevisiae將苯丙胺酸轉化成苯乙醇，但在發酵過程中，面臨最大的限制在於產物的抑制效應，會對細胞產生毒性的影響。而且在微生物發酵中，不同培養溫度也會影響微生物的生理狀態和發酵行為，因此本研究主要的目的是在於探討「溫度」環境因子對於S. cerevisiae生產苯乙醇之影響，探討不同培養溫度對於菌體的生長及產物的變化，並結合萃取技術來改善產物抑制的問題。
研究結果發現在溫度為25℃下最適合S. cerevisiae的生長，但在溫度為35℃下則比較有助於單位菌重轉化成產物的能力，於是設計一種兩階段溫度轉換的策略同時提升菌體及產物的產量，在此操作模式下，苯乙醇的產量可以高達2.53 g/L，與在單一發酵溫度25℃跟35℃時相比下分別提升了15.48 %及22.38 %，最後再結合連續萃取發酵裝置，以Hytrel®作為吸附劑的角色，透過 in situ product removal 技術，減輕產物對於菌體生長的限制，最終苯乙醇的累積濃度可達到4.506 g/L。

摘要(英)

2-Phenylethanol (PEA) is one of the important flavor and fragrance compound in the world with a rose-like aroma. In addition, it is widely used in the cosmetics, perfume, and food industries.

PEA can be produced from L-phenylalanine (L-Phe) through Ehrlich pathway using yeasts. In the fermentation process, the growth of yeast will be limited by product inhibition, cultivation temperature will also has a strong connection with microbial growth and fermentation behavior.

In this work, we have explored the effects of temperature on bioconversion of L-Phe into PEA using Saccharomyces cerevisiae BCRC21812 as well as develop strategies to enhance PEA production. Experimental results showed that at 25°C, the optimum cells growth was achieved and favored bioconversion yield. But at 35°C was more favorable for product yield based on biomass. Therefore, two-stage batch fermentation by temperature control was conducted. PEA concentration using two-stage fermentation was 2.53 g/L which corresponds to 15.48 % and 22.38 % higher than single-stage fermentation at 25°C and 35°C, respectively.

To increase the PEA production, the extractive fermentation will be conducted using Hytrel®. In the preliminary test, it showed that Hytrel® can adsorb PEA to reduce the inhibitory effect and non-toxic effect on yeast growth. After the successive extraction and feeding fermentation, the final concentration of PEA was achieved 4.506 g/L.

關鍵字(中)

★ 酵母菌
★ 苯乙醇
★ 溫度效應

關鍵字(英)

★ Saccharomyces cerevisiae
★ 2-Phenylethanol
★ temperature

論文目次

目錄
摘要 ii
Abstract iii
誌謝 v
目錄 vi
表目錄 x
圖目錄 xi
第一章緒論 1
1-1研究動機 1
1-2研究目的 2
第二章文獻回顧 4
2-1 香料香精化學品市場趨勢及發展 4
2-2 芳香化合物-苯乙醇 7
2-2-1 苯乙醇基本性質介紹 7
2-2-2 苯乙醇特性及應用 7
2-2-3 苯乙醇之生產方式 9
2-3 釀酒酵母(Saccharomyces cerevisiae) 14
2-3-1 酵母菌基本介紹 14
2-3-2 酵母菌生殖方式 15
2-3-3 酵母菌代謝途徑 16
2-3-4 酵母菌生長條件 18
2-4 影響發酵之因素 19
2-4-1 培養基組成 19
2-4-2 pH值 20
2-4-3 通氣速率 21
2-4-4 光 21
2-4-5 溫度 21
2-4-6 產物抑制效應 22
2-5 微生物發酵製程改善-ISPR技術 22
2-6 Hytrel®性質及應用 27
第三章材料與方法 29
3-1 實驗規劃 29
3-2 實驗材料與設備 30
3-2-1 實驗菌株 30
3-2-2 實驗藥品 31
3-2-3 實驗儀器與設備 32
3-2-4 數據呈現之參數符號設定 35
3-2-5 實驗裝置 35
3-3 實驗方法 39
3-3-1 菌種保存 39
3-3-2培養基組成 39
3-4 分析方法 43
3-4-1 菌重濃度測定 43
3-4-2 葡萄糖殘糖分析 43
3-4-3 乙醇濃度分析 45
3-4-4 苯丙胺酸和苯乙醇濃度分析 47
第四章實驗結果與討論 50
4-1 發酵培養基最適化 50
4-1-1 碳源濃度對於Saccharomyces cerevisiae發酵動力曲線之影響 50
4-1-2 氮源濃度對於Saccharomyces cerevisiae發酵動力曲線之影響 53
4-2 培養溫度對於發酵實驗之探討 57
4-2-1 Saccharomyces cerevisiae溫度耐受性測試 57
4-2-2 好氧發酵之不同通氣速率比較 59
4-2-3 兩階段溫度控制之轉換時機點 63
4-2-4 兩階段溫度轉換策略之可行性 66
4-3 Hytrel®吸附萃取能力測試 68
4-3-1 Hytrel®基本吸附能力 68
4-3-2 Hytrel®添加量之吸附影響 70
4-3-3 Hytrel®不同脫附劑測試 72
4-3-4 Hytrel®細胞毒性測試 74
4-3-5 Hytrel®萃取次數之穩定度 76
4-4 兩階段溫度轉換發酵模式結合萃取系統應用 79
4-4-1 兩階段溫度轉換饋料測試 79
4-4-2 兩階段溫度轉換發酵結合連續式萃取及饋料操作 81
第五章結論及建議 84
5-1 結論 84
5-2 建議 85
參考文獻 86

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指導教授

徐敬衡(Chin-Hang Shu)

審核日期

2019-8-8

推文