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論文名稱: | 馬尾藻以 Clostridium spp. 進行批式發酵生產生質丁醇之條件探討
| Study on the Production Conditions of Biobutanol from Sargassum sp. by Batch Fermentation with Clostridium spp.
研究生: | 陳建志 Chieh-Chih Chen
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: | 10032020
畢業學年度: | 101
語文別: | 中文
關鍵字: | 馬尾藻多醣水解液 (SPSH) Sagassum siliquosum polysaccharide hydrolysates (SPSH)
| 梭狀芽孢桿菌 Clostridium spp.
| 褐藻膠裂解酶 alginate lyase
| 攪拌發酵 agitating fermentation
| 靜置發酵 static fermentation
全文影像連結: | | (本論文20160728開放校外瀏覽)
| 論文全文(pdf)

論文頁數: | 97
摘要: | 本研究之目的在利用馬尾藻經熱酸水解、纖維素酶、澱粉酶、及馬尾藻多醣誘導之 Aeromonas salmonicida MAEF108 與 Pseudomonas vesicularis MA103 粗酵素液依序水解後，以所得馬尾藻多醣水解液 (SPSH) 利用梭狀芽孢桿菌 (Clostridium spp.) 發酵生產丁醇反應條件之探討。不同濃度稀鹽酸與檸檬酸進行熱酸水解之實驗結果顯示使用 0.4 N 稀鹽酸進行熱酸水解所得還原醣產率為最高，達到 12.70%。由 15 g 乾燥馬尾藻經 0.4 N 稀鹽酸於 121oC 下水解 20 min 後，以 13,680 U 纖維素酶、22,000 U 澱粉酶、及 100U (Alginate lyase activity) 馬尾藻多醣誘導之 MA103 與 MAEF108 粗酵素液水解，所得馬尾藻多醣水解液之總醣量與還原醣量分別為 42.30 及 29.86 mg/mL，再以超過濾離心區分出分子量小於 3 kDa 之寡醣，利用 TLC 分析發現水解液中含有單醣及雙醣等簡單醣類，以 HPLC 分析發現隨著水解程序進行，水解液中葡萄糖量逐漸增加，所得馬尾藻多醣水解液之葡萄糖產率為 56.77% (g/g dry biomass)。在耐鹽性試驗中 Clostridium acetobutylicum BCRC10639 與 Clostridium beijerinckii BCRC17950 之生長量 (OD600nm) 皆隨鹽度上升 (0-3%) 而下降，表示 2 株產丁醇菌在鹽度 > 2% 之培養基中生長較緩慢，甚至無法生長。由醣類利用性試驗之實驗結果可知 C. acetobutylicum BCRC10639 可以阿拉伯糖、葡萄糖、以及乳糖做為主要碳源進行發酵，而 C. beijerinckii BCRC17950 無法利用阿拉伯糖及乳糖做為主要碳源。以 60 g/L 葡萄糖發酵液為基質接種 10% C. acetobutylicum BCRC10639 進行攪拌批式發酵，經過 168 hr 後消耗 41.14 g/L 葡萄糖，產生 4.32 g/L 丙酮 (Acetone)、10.95 g/L 丁醇 (Butanol)、及 1.41 g/L 乙醇 (Ethanol)，而 ABE 產率則分別為 10%、27%、與 3% (g/g glucose)。將 10% C. beijerinckii BCRC17950 接種至 60 g/L 葡萄糖發酵液中進行攪拌批式發酵，在 168 hr 時消耗 40.99 g/L 葡萄糖，丙酮、丁醇、與乙醇之產量分別為 4.11、10.38 以及 1.53 g/L，而三者之產率分別為 10%、25% 以及 4% (g/g glucose)。之後以 3 倍稀釋之馬尾藻多醣水解液做為發酵基質，接種 10% C. acetobutylicum BCRC10639 進行攪拌批式發酵，總醣量與還原醣量分別由 14.10 與 9.95 mg/mL 下降至 5.61 與 3.72 mg/mL，而丙酮濃度與丁醇濃度分別為 0.40 與 0.49 g/L，丙酮與丁醇之產率分別為 0.80% 與 0.98% (g/g dry biomass)。利用 3 倍稀釋之馬尾藻多醣水解液進行靜置發酵，接種 10% C. acetobutylicum BCRC10639 發酵 120 hr 後產生 0.01% 丙酮與 0.09% 丁醇，其產率分別為 0.06% 與 0.46% (g/g dry biomass)；接種 10% C. beijerinckii BCRC17950 發酵 96 hr 生成丙酮與丁醇之濃度分別為 0.01% 與 0.04%，二者之產率分別為 0.04% 與 0.21% (g/g dry biomass)。
| The purpose of this study is using Sagassum siliquosum polysaccharide hydrolysates (SPSH) that produced from Sagassum siliquosum dry powder treated with 0.4 N HCl heat-acid extraction, cellulase hydrolysis, amylase hydrolysis, and Sagassum siliquosum polysaccharides induced MA103 and MAEF108 crude enzyme hydrolysis as fermentation substrate for biobutanol production by Clostridium spp. The results of heat-acid extraction with different concentrations of diluted HCl and citric acid indicated that the reducing sugars yield of 0.4 N HCl heat-acid extraction was 12.70%. 15 g Sagassum siliquosum dry powder sequentially treated with 121oC hot water extraction for 20 min with 0.4 N HCl, 13,680 U cellulase hydrolysis, 22,000 U amylase hydrolysis, and 100 U (Alginate lyase) MA103 and MAEF108-Sar crude enzymes hydrolysis, the Sagassum siliquosum polysaccharide hydrolysates (SPSH) content 42.30 mg/mL total sugars and 29.86 mg/mL reducing sugars. Then, distinguished the oligosaccharide (OS) which molecular weight less than 3 kDa by ultrafiltration. The TLC analysis showed that SPSH containing monosaccharides and disaccharides. In HPLC analysis, The monosaccharide content increased gradually with hydrolysis procedures, and the glucose yield of SPSH was 56.77% (g/g dry biomass). In salt tolerance test of Clostridium spp., the growth amount of Clostridium acetobutylicum BCRC10639 and Clostridium beijerinckii BCRC17950 decreased with increasing of salinity. This result indicated that 2 Clostridium spp. grew slowly in medium with salinity > 2%, even can not grow. According to the experimental results of carbohydrate utilization test, C. acetobutylicum BCRC10639 can use arabinose, glucose, and lactose as the main carbon source for fermentation, and C. beijerinckii BCRC17950 can not use arabinose and lactose as the main carbon source. Using 60 g/L glucose fermentation solution as substrate inoculated C. acetobutylicum BCRC10639 to stirring batch fermentation, after 168 hr fermentation the glucose consumption was 41.14 g/L and produced 4.32 g/L acetone, 10.95 g/L butanol, and 1.41 g/L ethanol. The ABE yield of C. acetobutylicum BCRC10639 stirring fermentation in 60 g/L glucose solution were 10%, 27%, and 3% (g/g glucose), respectively. Inoculating 10% C. beijerinckii BCRC17950 into 60 g/L glucose solution for stirring batch fermentation. After 168 hr, the consumption of glucose was 40.99 g/L,and produced 4.11 g/L acetone, 10.38 g/L butanol, and 1.53 g/L ethanol. The ABE yield of C. beijerinckii BCRC17950 were 10%, 25%, and 4% (g/g glucose), respectively. Then, using 3-fold dilution SPSH as substrate for stirring fermentation, inoculated with 10% C. acetobutylicum BCRC10639,after 72 hr the total sugars and reducing sugars decreased form 14.10 and 9.95 mg/mL to 5.61 and 3.72 mg/mL, respectively. The acetone and butanol concentrations in fermentation solution were 0.40 and 0.49 g/L.The yield of acetone and butanol were 0.80% and 0.98% (g/g dry biomass), respectively. The 3-fold dilution SPSH inoculated with 10% C. acetobutylicum BCRC10639 for standing fermentation at 120 hr obtained 0.01% acetone and 0.09% butanol, and the yield of acetone and butanol were 0.06% and 0.46% (g/g dry biomass). Inoculating 10% C. beijerinckii BCRC17950 into 3-fold dilution SPSH for standing fermentation, at 96 hr obtained 0.01% acetone and 0.04% butanol, and the yields were 0.04% and 0.21% (g/g dry biomass), respectively.
論文目次: | 中文摘要    I
| 英文摘要 (Abstract)    II
| 目錄    IV
| 圖目錄    VIII
| 表目錄    IX
| 附錄目錄    XI
| 壹、前 言    1
| 貳、文獻整理    3
| 一、生質能源    3
| 二、丁醇之介紹    4
| 2-1. 丁醇之特性與應用    4
| 2-2. 丁醇之生產方法    5
| 2-2-1. 化學合成法    5
| 2-2-2. 微生物發酵法    5
| 三、產丁醇菌株之特性    6
| 3-1. 梭狀芽孢桿菌 (Clostriidum spp.)    6
| 3-2. 其他產丁醇菌株    8
| 四、馬尾藻 (Sargassum sp.)    9
| 4-1. 馬尾藻之生態特性    9
| 4-2. 馬尾藻之成分組成    9
| 五、褐藻膠裂解酶 (Alginate lyase)    9
| 5-1. 褐藻膠之組成結構    9
| 5-2. 褐藻膠裂解酶之生產菌株    10
| 5-3. 褐藻膠裂解酶之生化特性    10
| 六、Acetone-butanol-ethanol (ABE) 發酵生產生質丁醇    11
| 6-1. 批式發酵 (Batch fermantation) 生產生質丁醇    11
| 6-2. 以藻類生物質 (Algal biomass) 為原料生產生質丁醇    11
| 參、實驗設計    13
| 肆、實驗材料與方法    13
| 一、實驗材料    14
| 1-1. 原料    14
| 1-2. 實驗菌株    14
| 1-2-1. 褐藻膠水解酵素生產菌株    14
| 1-2-2. 產丁醇菌株    14
| 1-3. 試驗藥品    14
| 1-3-1. 藥品    14
| 1-3-2. 酵素    16
| 1-3-3. 培養基組成    16
| 1-3-4. 褐藻膠裂解酶酵素活性反應基質    18
| 1-3-5. 還原醣量反應試劑    18
| 1-3-6. 總醣量反應試劑    18
| 1-4. 儀器設備    18
| 二、實驗方法    20
| 2-1. 馬尾藻之前處理    20
| 2-2. 馬尾藻之一般成分分析    20
| 2-2-1. 水分含量    20
| 2-2-2. 粗脂肪含量    20
| 2-2-3. 粗蛋白含量    20
| 2-2-4. 灰分含量    21
| 2-3. 馬尾藻多醣誘導 MAEF108 與 MA103 粗酵素液之生產    21
| 2-3-1. 菌株保存    21
| 2-3-2. 菌株活化    21
| 2-3-3. 馬尾藻多醣誘導之粗酵素液製備    21
| 2-3-4. 誘導粗酵素液中褐藻膠裂解酶 (Alginate lyase) 之酵素活性    22
| 2-4. 馬尾藻多醣水解液之製備    22
| 2-4-1. 以不同濃度稀鹽酸與檸檬酸進行熱酸水解    22
| 2-4-2. 酵素水解    22
| 2-5. 產丁醇菌株之特性    22
| 2-5-1. 菌株保存    22
| 2-5-2. 菌株活化    23
| 2-5-3. 革蘭氏染色 (Gram stain)    23
| 2-5-4. 耐鹽性    23
| 2-5-5. 醣類利用性    23
| 2-6. 60 g/L 葡萄糖液批式發酵    23
| 2-6-1. 攪拌批式發酵 (Agitating batch fermentation)    23
| 2-6-2. 靜置批式發酵 (Static batch fermentation)    24
| 2-7. 馬尾藻多醣水解液 (3 倍稀釋) 批式發酵    24
| 2-7-1. 攪拌批式發酵    24
| 2-7-2. 靜置批式發酵    24
| 2-8. 分析方法    24
| 2-8-1. 總醣量測定    24
| 2-8-2. 還原醣量測定    24
| 2-8-3. Clostridium spp. 菌量測定    25
| 2-8-4. 有機溶劑產物 (丙酮、丁醇、乙醇) 濃度測定    25
| 2-9. 馬尾藻多醣水解液與發酵期間之醣類組成分析    25
| 2-9-1. 薄層色層分析 (Thin layer chromatography, TLC)    25
| 2-9-2. 高效液相層析 (High performance liquid chromatography, HPLC)    25
| 三、統計分析    26
| 伍、結果與討論    27
| 一、乾燥莢托馬尾藻粉末之一般成分分析    27
| 二、以不同濃度稀鹽酸與檸檬酸進行熱酸水解    27
| 2-1. 以不同濃度稀鹽酸進行熱酸水解    27
| 2-2. 以不同濃度檸檬酸進行熱酸水解    27
| 三、馬尾藻多醣水解液之製備與醣類組成分析    28
| 3-1. 馬尾藻多醣水解液 (Sargassum polysaccharide hydrolysates, SPSH)    28
| 3-2. 薄層色層分析 (TLC)    28
| 3-3. 高效液相層析分析 (HPLC)    28
| 四、Clostridium spp. 之生長曲線    29
| 4-1. Clostridium acetobutylicum BCRC10639 之生長曲線    29
| 4-2. Clostridium beijerinckii BCRC17950 之生長曲線    29
| 五、Clostridium spp. 之耐鹽性試驗與醣類利用性試驗    29
| 5-1. 耐鹽性試驗    29
| 5-2. 醣類利用性試驗    30
| 六、以 60 g/L 葡萄糖發酵液批式發酵    30
| 6-1. 攪拌批式發酵    30
| 6-2. 靜置批式發酵    31
| 七、以馬尾藻多醣水解液 (SPSH) 批式發酵    32
| 7-1. SPSH 攪拌批式發酵    32
| 7-2. SPSH 靜置批式發酵    32
| 八、馬尾藻多醣水解液 (SPSH) 靜置批式發酵之殘醣組成分析    32
| 陸、結 論    33
| 陸、參考文獻    35
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