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論文名稱: | 石蓴(Ulva lactuca)以Saccharomyces cerevisiae進行饋料批式發酵生產生質乙醇之條件探討
| Study on the Production Conditions of Bioethanol from Ulva lactuca by Fed-Batch Fermentation with Saccharomyces cerevisiae
研究生: | 賴巾玥 Chin-Yueh Lai
指導教授: | 潘崇良 Chorng-Liang Pan
學位類別: | 碩士
學校名稱: | 國立臺灣海洋大學
系所名稱: | 食品科學系
學號: | 10032023
畢業學年度: | 101
語文別: | 中文
關鍵字: | 釀酒酵母 Saccharomyces cerevisiae
| 饋料批式發酵 Fed-Batch
| 生質乙醇 Bioethanol
| 石蓴 Ulva lactuca
全文影像連結: | | (本論文20150728開放校外瀏覽)
| 論文全文(pdf)

論文頁數: | 113
摘要: | 乾燥石蓴粉末含有 75.03% 碳水化合物。以 0.3% 石蓴乾燥粉末做為原料，接種 1% A. salmonicida MAEF108 與 P. vesicularis MA103 菌液誘導至 48 hr，可得知 1% P. vesicularis MA103 得到較高之澱粉酶活性 (1.36 U) 及次高之聚木醣酶活性 (0.98 U) 與接種 1% A. salmonicida MAEF108 得到較高之聚木醣酶活性 (0.78 U) 及次高之澱粉酶活性 (0.66 U)。以 5% 乾燥石蓴粉末先以 4 N HCl 於 121oC 熱萃 20 min，依序以纖維素酶、-澱粉酶與 MA103 與 MAEF108-Ulva 粗酵素液以水解製程 A、B、與 C 製作石蓴寡醣水解液。水解製程 A、B、與 C 之石蓴多醣水解液的總醣量分別為 26.56、13.84、與 7.39 mg/mL，還原醣量分別為 35.78、26.74、與 8.40 mg/mL、及鹽度分別為 78.00、77.50、與 50.00 psu，因此從實驗室規模到先期試驗規模之石蓴寡醣水解液之總醣量。還原醣量、與鹽度分別從 26.56 降低至 7.39 mg/mL、從 35.78 降低至 8.40 mg/mL、及從 78.00 降低至 50.00 psu。由 TLC 之 Rf 值結果得知石蓴寡醣水解液含有鼠李糖與葡萄糖兩種可發酵醣。接種 2-12% S. cerevisiae BCRC 21685、21686、21687、與 21824 於石蓴多醣水解液室溫避光發酵 72 hrs，皆以 10% 總接種量有較高還原醣利用率，分別為 53.52%、52.08%、38.27%、與 42.83%。同時也有較高乙醇濃度與乙醇產率分別為 0.91、0.88、0.56、與 0.90% 及 19.36、18.73、11.92、與 19.15 g/100 g dry weight，其中利用葡萄糖較多者為 10% S. cerevisiae BCRC 21685 並且具有較高之乙醇產率。以 10% S. cerevisiae BCRC 21685 分別接種至石蓴多醣水解液進行批式發酵，於 72 hrs 有較高之乙醇濃度與乙醇產率分別為 0.91% 與 19.34 g/100 g，其還原醣利用率為 53.50%。以 10% S. cerevisiae BCRC 21685 接種至石蓴多醣水解液進行饋料批式發酵，於 120 hrs 有較高之乙醇濃度為 1.02%、乙醇產率為 21.76 g/100 g dry weight (20.14 g/100 g dry weight/day)、與較高還原醣利用率為 58.75%。
| U. lactuca contains 75.03% of carbohydrates. The analyses of enzyme activity on crude enzymes are induced by U. lactuca powder with 1% P. vesicularis MA103 has higher amylase (1.36 U) and xylanase (0.98 U) activity while 1% A. salmonicida MAEF108 has higher xylanase (0.78 U) and amylase (0.66 U) activity. 5% U. lactuca powder sequentially treated with 121oC 0.4 N HCl extraction for 20 min, cellulase hydrolysis, -amylase hydrolysis, and MA103 and MAEF108-Ulva crude enzymes hydrolysis of procedure A, B, and C. The total sugar content of procedure A, B, and C is 26.56, 3.84, and 7.39 mg/mL, the reducing sugar content is 35.78, 26.74, and 8.40 mg/mL, and the salinity is 78.00, 77.50, and 50.00 psu, respectively. U. lactuca polysaccharides hydrolysate at laboratory scale and bench scale resulted in total sugar content, reducing sugar, and salinity from 26.56 decreased to 7.39 mg/mL, from 35.78 decreased to 8.40 mg/mL, and from 78.00 decreased to 50.00 psu, respectively. The thin layer chromatography of the fermentable sugars of the U. lactuca oligosaccharides hydrolysate is glucose and rhamnose. The polysaccharides hydrolysate inoculated 2-12% S. cerevisiae BCRC 21685, 21686, 21687 or 21824 fermentated at room temperature for 72 hrs, resulted in it inoculated 10% yeast has higher reducing sugar utilized are 53.52%, 52.08%, 38.27%, and 42.83%, obtains 0.91%, 0.88%, 0.56%, and 0.90% of the maximum ethanol concentration, and 19.36, 18.73, 11.92, and 19.36, 18.73, 11.92, and 19.15 g/100 g dry weight of the maximum ethanol yield, respectively. Among of them, 10% S. cerevisiae BCRC 21685 has maximum ethanol yield than others. The U. lactuca oligosaccharides hydrolysate with batch fermentated by 10% S. cerevisiae BCRC 21685, obtains 0.91% of the maximum ethanol concentration, 19.34 g/100 g dry weight of the maximum ethanol yield, and 50% of reducing sugar utilized for 72 hrs. The U. lactuca oligosaccharides hydrolysate with fed-batch fermentated by 10% S. cerevisiae BCRC 21685, obtains 1.02% of the maximum ethanol concentration, 21.76 g/100 g dry weight (20.14 g/100 g dry weight/day) of the maximum ethanol yield, and 50% of the maximum reducing sugar utilized for 120 hrs.
論文目次: | 目錄    i
| 表目錄    v
| 圖目錄    vi
| 附錄目錄    viii
| 中文摘要    ix
| 英文摘要 (Abstract)    x
| 壹、前言    1
| 貳、文獻整理    3
| 一、生質能源    3
| 1-1. 生質能源之定義    3
| 1-2. 生質能源之基本原理    3
| 1-3. 生質乙醇    3
| 1-3-1. 生質乙醇之特性    3
| 1-3-2. 生質乙醇之產量    4
| 1-3-3. 生質乙醇之能源作物    4
| 1-3-3-1. 第一代生質乙醇之料源    4
| 1-3-3-2. 第二代生質乙醇之料源    5
| 1-3-3-3. 第三代生質乙醇之料源：大型海藻    6
| 1-3-3-3-1. 大型海藻簡介    6
| 1-3-3-3-2. 大型海藻生質乙醇    6
| 二、石蓴 (Ulva lactuca)    7
| 2-1. 石蓴之簡介    7
| 2-2. 石蓴之營養成分    8
| 三、海藻多醣    8
| 3-1. 海藻多醣之定義    8
| 3-2. 海藻多醣之萃取方法    9
| 3-3. 石蓴多醣之組成    9
| 3-3-1. 石蓴硫酸多醣 (Ulvan)    9
| 3-3-2. 纖維素 (Cellulose)    10
| 3-3-3. 木糖葡聚醣 (Xyloglucan)    10
| 3-3-4. 葡萄糖酸聚醣 (Glucuronan)    10
| 四、海藻寡醣    10
| 五、石蓴粉末誘導 Pseudomonas vesicularis MA103 與 Aeromonas
| salmonicida MAEF108 之酵素    11
| 5-1. 洋菜酶 (Agarase)    11
| 5-5-1. 洋菜酶之種類    11
| 5-1-2. 洋菜酶之分子量    12
| 5-1-3. 洋菜酶之應用    13
| 5-2. 聚木醣酶（Xylanase）    13
| 5-2-1. 聚木醣酶之種類    13
| 5-2-2. 聚木醣酶之分子量    15
| 5-2-3. 聚木醣酶之應用    16
| 5-3. 澱粉酶 (Amylase)    15
| 5-3-1. 澱粉酶之種類    16
| 5-3-2. 澱粉酶之分子量    18
| 5-3-3. 澱粉酶之應用    18
| 5-4. 鹿角菜膠酶 (Carrageenase)    18
| 5-4-1. 鹿角菜膠酶之種類    19
| 5-4-2. 鹿角菜膠酶之分子量    19
| 5-4-3. 鹿角菜膠酶之應用    19
| 5-5. 纖維素酶 (Cellulase)    20
| 5-5-1. 纖維素酶之種類    20
| 5-5-2. 纖維素酶之分子量    20
| 5-4-3. 纖維素酶之應用    20
| 六、酵母菌發酵生產生質乙醇    21
| 6-1. 酵母菌之形態    21
| 6-2. 酵母菌之應用    21
| 6-3. 釀酒酵母 (Saccharomyces cerevisiae)    22
| 6-4. 酵母菌之乙醇發酵理論    22
| 6-5. 發酵之方式    22
| 6-5-1. 批式培養 (Batch culture)    22
| 6-5-2. 饋料批式培養 (Fed-batch culture)    23
| 6-5-3. 連續式培養 (Continuous culture)    24
| 參、實驗設計    25
| 肆、實驗材料與方法    26
| 一、實驗材料    26
| 1-1. 原料    26
| 1-2. 實驗菌株    26
| 1-2-1. 細菌菌株    26
| 1-2-2. 酵母菌菌株    26
| 1-3. 試驗藥品    26
| 1-3-1. 藥品    26
| 1-3-2. 酵素    27
| 1-3-3. 培養基組成    27
| 1-3-4. 還原醣量反應試劑    29
| 1-3-5. 薄層色層分析矽膠片    29
| 1-3-6. 酵素反應基質    29
| 1-3-6-1. 洋菜酶反應基質    29
| 1-3-6-2. 聚木醣酶反應基質    29
| 1-3-6-3. 澱粉酶反應基質    29
| 1-3-6-4. 鹿角菜膠酶反應基質    29
| 1-3-6-5. 纖維素酶反應基質    30
| 1-4. 儀器設備    30
| 二、實驗方法    31
| 2-1. 海藻之一般成份分析    31
| 2-1-1. 水分含量    31
| 2-1-2. 灰分含量    31
| 2-1-3. 粗蛋白含量    32
| 2-1-4. 粗脂肪含量    32
| 2-2. 石蓴粉末誘導 P. vesicularis MA103 與 A. salmonicida MAEF
| 108 粗酵素液    32
| 2-2-1. 菌株之保存    32
| 2-2-2. 菌株之活化    32
| 2-2-3. 石蓴粉末誘導 P. vesicularis MA103 與 A. salmonicida
| MAEF108 之粗酵素液的製備    33
| 2-2-4. 酵素活性測定方法    33
| 2-2-4-1. 洋菜酶活性測定    33
| 2-2-4-2. 聚木醣酶活性測定    33
| 2-2-4-3. 澱粉酶活性測定    33
| 2-2-4-4. 鹿角菜膠酶活性測定    34
| 2-2-4-5. 纖維素酶活性測定    34
| 2-2-4-6. 酵素活性單位定義    34
| 2-3. 石蓴熱萃多醣水解條件    34
| 2-3-1. 石蓴熱酸萃取多醣水解液之製備    34
| 2-3-2. 石蓴多醣水解液水解成寡單醣水解液之水解製程    34
| 2-4. 酵母菌菌株之最適乙醇發酵條件探討    35
| 2-4-1. 菌株保存    35
| 2-4-2. 菌株活化    35
| 2-4-3. 酵母菌菌株對鼠李糖或葡萄糖利用性之篩選    35
| 2-4-4. 單一酵母菌菌株接種菌量探討    35
| 2-4-5. 石蓴多醣水解液之批式發酵試驗    36
| 2-4-6. 蓴多醣水解液之饋料批式發酵試驗    36
| 2-5. 分析方法    36
| 2-5-1. 總醣量測定    36
| 2-5-2. 還原醣量測定    36
| 2-5-3. 酵母菌量測定    36
| 2-5-4. 氣相層析 (GC) －乙醇濃度測定    37
| 2-5-5. 乙醇產率計算    37
| 2-5-6. 超過濾 (Molecular weight cut off, MWCO)    37
| 2-5-7. 薄層色層分析 (TLC)    37
| 2-5-8. 高壓液相層析 (HPLC)    37
| 三、統計分析    38
| 伍、結果與討論    39
| 一、石蓴之一般成分分析    39
| 二、石蓴粉末誘導 P. vesicularis MA103 與 A. salmonicida MAEF 108
| 粗酵素液    39
| 三、石蓴多醣水解液之水解條件    41
| 1. 石蓴多醣水解液之水解製程    41
| 2. 石蓴多醣水解液中可發酵醣之薄層色層分析    43
| 四、酵母菌菌株之最適乙醇發酵條件探討    44
| 1. 酵母菌菌株對葡萄糖或鼠李糖利用性之篩選    44
| 2. 單一酵母菌菌株接種菌量探討    45
| 3. 單一酵母菌株 (10%) 發酵石蓴多醣水解液後殘醣之薄層色層分析    46
| 4.  S. cerevisiae BCRC 21685 (10%) 接種於石蓴多醣水解液之批式
| 發酵試驗    47
| 5.  S. cerevisiae BCRC 21685 (10%) 接種於石蓴多醣水解液之批式
| 發酵試驗殘醣之薄層色層分析    48
| 6.  S. cerevisiae BCRC 21685 (10%) 接種於石蓴多醣水解液之饋料
| 批式發酵試驗    48
| 7.  S. cerevisiae BCRC 21685 (10%) 接種於石蓴多醣水解液之饋料
| 批式發酵試驗殘醣之薄層色層分析    50
| 8.  石蓴多醣水解液與發酵液之葡萄糖與鼠李糖含量之高壓液相
| 層析    50
| 陸、結論    52
| 柒、參考文獻    53
| 捌、附錄    109
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2013 年 8 月 5 日 23:08