臺灣博碩士論文加值系統

本研究之目的為探討自紅藻龍鬚菜 (Gracilaria sp.) 生產生質丁醇的發酵技術，分別測試二階段氣提處理、提升Clostridium sp. 的丁醇耐受性、以及固定化細胞對丁醇產率之影響，並對可行之生質丁醇生產程序進行生命週期評估分析。主要工作包括將龍鬚菜以熱酸、市售纖維素酶、及菌株 Pseudomonas vesicularis MA103 (MA103) 與 Aeromonas salmonicida MAEF108 (MAEF108) 所產多重粗酵素液水解，以二階段氣提處理配合厭氧批式與厭氧饋料批式發酵，探討提升丁醇產率之生產條件。100 mL 5% (w/v) 去鹽與未去鹽之龍鬚菜熱酸水解液中之還原糖量分別為 1.77 g 與 2.39 g 及鹽濃度為 2.12% 與 2.74 %。100 mL 5% (w/v) 龍鬚菜水解液中加入市售纖維素酶 760 U 與 4,560 U 於 pH 4.5 與 37oC 下水解 1 天，可分別產生 6.27 g 與 6.66 g 的總糖量、3.55 g 與 3.70 g 的還原糖量，顯示 4,560 U 市售纖維素酶可獲得較高的水解效率，但因成本偏高，後續實驗仍以添加 760 U 市售纖維素酶進行。MA103 及 MAEF108 經以龍鬚菜熱酸萃與粉末熱萃多醣液之人工海水培養基 (AM-Gra-AP) 進行粗酵素液誘導，於第 2 天分別可得較佳 2.91 U 洋菜酶與 0.28 U 與澱粉酶素活性。以濃度 6% 與 0.5% 之丁醇溶液進行二階段氣提時回收率分別為 57.3% 與 38.5%，而經由溶液中殘留丁醇量換算可知過程中丁醇散失率分別為 9.9% 與 40.3%。接種 2 株具有 4.5% 食鹽耐受能力之菌株 Clostridium acetobutylicum BCRC10639-ST (Salt tolerant) 與 C. beijerinckii BCRC17950-ST，以 60 g/L Glucose 為發酵液厭氧批式發酵 120 hr，丁醇濃度與丁醇產率分別是 0.91% 與 12.16% 以及 0.95% 與 12.64%。以未去鹽龍鬚菜為基質接種 2 株耐鹽菌株在 37oC 下厭氧批式發酵 120 hr，結果顯示至發酵第 96 hr 之丁醇產率為 7.04% (7.04 g/100 g) 及丁醇濃度為 0.44%。以去鹽之龍鬚菜水解液為基質接種 2 株耐鹽菌株在 37oC 下厭氧批式發酵至第 96 hr 丁醇產率為 7.52%，此時丁醇濃度為 0.47%。以去鹽龍鬚菜為原料配合二階段氣提處理進行 120 hr 厭氧批式發酵，在發酵第 72 hr 加計氣提散失率丁醇產率為 8.49%，與丁醇濃度為 0.53%。C. acetobutylicum BCRC10639-ST 與 C. beijerinckii BCRC17950-ST 經紫外線誘導突變試驗後於不同丁醇濃度 (0.0-2.5%) 下厭氧培養，篩選獲得 2 株於 2% Butanol 時 OD600nm 分別達 0.61 與 0.84 之丁醇耐受能力之衍生菌株 C. acetobutylicum BCRC10639-ST-BT (Butanol tolerant) 與 C. beijerinckii BCRC17950-ST-BT。此 2 株耐丁醇實驗株以 60 g/L Glucose 為發酵液厭氧批式發酵 120 hr 後，發酵液中之丁醇濃度為 1.29% 與 1.22%，經計算其丁醇產率為 17.24% 與 16.22%。以去鹽龍鬚菜為原料，接種 2 株耐丁醇菌株在 37oC 下厭氧批式發酵 120 hr，結果顯示至發酵第 96 hr 之丁醇產率為 7.68% 及丁醇濃度為 0.48%。去鹽龍鬚菜水解液以 2 株耐丁醇菌在 37oC 下配合二階段氣提處理厭氧饋料批式發酵 144 hr，期間在發酵第 72 hr 有最大丁醇產率 9.76% 此時丁醇濃度為 0.61%。生命週期評估，去鹽龍鬚菜水解液進行厭氧批式發酵製程 (Gra-DS-CL-St-B) 丁醇產製 1 公秉丁醇之成本為 11,618,437.12/kL NTD (New Taiwan Dollar)，各製程之吸收量皆未大於釋放量，但可將碳足跡減少約46%。製程 Gra-DS-CL-St-B-DSDE 與 Gra-DS-CL-St/Bt-FB-DSDE 產製 1 公秉丁醇丁醇成本分別為 10,298,829.70 NTD 與 6,334,223.85 NTD，較製程 Gra-DS-ST-B 減少 11.36% 與 45.48%。未來若可添加 10% 丁醇於臺灣地區的市售汽油中，則每年可衍生 2,832,801,406 NTD 的環境效益。

The objective of this study is to develop potential fermentation techniques for producing bio-butanol from red algae Gracilaria sp., to exam the effect of 2 stages gas-stripping system, upgrading the butanol tolerance of Clostridium sp., and immobilized cell on bio-butanol production, respectively, and to conduct life cycle assessment (LCA) analysis for the potential bio-butanol production protocol. The main work including hydrolyzing Gracilaria sp. and its hot water extracted polysaccharides (PS) with acid, commercially available cellulase, and multiple crude enzyme from strain Pseudomonas vesicularis MA103 (MA103) and Aeromonas salmonicida MAEF108 (MAEF108). Prepare 5% (w/v) desalted or not desalted Gracilaria sp. powder in 100 mL distilled water treated with hot acid hydrolysis. After hot acid hydrolysis, 100 mL hydrolysate of desalted and not desalted Gracilaria sp. contained reducing sugar 1.77 g and 2.39 g, and salt 2.12% and 2.74%, respectively. After the hot acid hydrolysis process, 100 mL 5% Gracilaria sp. hydrolysate was divided into 2 groups, 760 U or 4,560 U cellulases were reacted with hydrolysate at pH 4.5 and 37oC for 24 hr, results showed that in 100 mL of hydrolyzed Gracilaria sp. contained total sugars 6.27 g and 6.66 g as well as reducing sugars 3.55 g and 3.70 g, respectively. Although 4,560 U cellulase group showed better RS yield, but its cost is higher, 760 U cellulase was choosed for following experiments. Multiple crude enzymes from strain P. vesicularis MA103 (MA103) and A. salmonicida MAEF108 (MAEF108) had better enzyme activities at the two day and the third day when the “hot acid hydrolysate + powder” Gracilaria sp. used as inducing substrate. 2 stages gas-stripping system was tested for its recovery rate with 6% and 0.5% butanol solution, results indicated that the recovery rate is about 57.3% and 38.5, and loss rate is about 9.9% and 40.3%, respectively. Inoculating salt tolerant strains BCRC10639-ST (Salt tolerant) and BCRC17950-ST into 60 g/L glucose solution for batch fermentation, butanol yield is 12.16% and 12.64% as well as butanol concentration is 0.91% and 0.95%. Hydrolysate of Gracilaria sp. fermented by BCRC10639-ST + BCRC17950-ST achieve a yield of butanol 7.04%, and desalting Gracilaria sp. fermented by BCRC10639-ST + BCRC17950-ST could achieve a yield of butanol as 7.52%. As GS technique is applied to the batch fermentation with desalting Gracilaria sp. used as substrate, the max. butanol yield performed by BCRC10639-ST + BCRC17950-ST is 8.49% at the 72 hr, and the butanol concentration is 0.53%. Inoculating butanol tolerant strains BCRC10639-ST-BT (Butanol tolerant) and BCRC17950-ST-BT into 60 g/L glucose solution for batch fermentation, butanol yield is 17.24% and 16.22% as well as butanol concentration is 1.29% and 1.22%. Hydrolysate of Desalting Gracilaria sp. fermented by BCRC10639-ST-BT + BCRC17950-ST-BT could achieve a yield of butanol as 7.68% and its butanol concentration is 0.48%. While 2 stages GS technique is applied with the fed-batch fermentation using desalting Gracilaria sp. as substrate, the max. butanol yield produced by BCRC10639-ST-BT + BCRC17950-ST-BT reaches 9.76% at the 72 hr, and its butanol concentration is 0.61%. After analyzed by LCA, the cost of butanol produced per L by protocols of Gra-DS-CL-St-B-GSDE and Gra-DS-CL-St/Bt-FB-GSDE were 10,298 and 6,334 NTD, which were decreased 11.36% and 45.48% on the production cost of protocol Gra-DS-CL-St-B. As to the amount of carbon footprints, they both higher than protocol Gra-DS-ST-B, increased for 10.32% and 13.04%. If the addition of 10% Bio-butanol into gasoline is applied in Taiwan area, the amount of environmental benefits could reach 2,832,801,406 NTD per year.

壹、前言 1
貳、文獻整理 3
一、生質能源 3
1-1. 生質能源簡介 3
1-2. 生質乙醇特性 3
1-3. 生質丁醇特性 4
1-4. 第三代生質燃料 4
1-5. 生質汽油應用 4
二、臺灣海藻 5
2-1. 分佈與組成 5
2-2. 大型海藻簡介 5
2-3. 龍鬚菜 5
2-3-1. 龍鬚菜之來源與產量 5
2-3-2. 龍鬚菜之組成分析 6
2-3-3. 海藻之固碳 6
三、產丁醇菌株之特性 6
四、菌種突變與適應性測試 8
五、發酵方式 8
5-1. 批式培養 (Batch culture) 8
5-2. 饋料批式培養 (Fed-batch culture) 9
5-3. 連續式培養 (Continuous culture) 9
5-4. 重複批式培養 (Repeated batch culture) 9
六、固定化細胞 9
6-1. 擔體鍵結法 (Binding to carriers) 10
6-1-1. 自然吸附 (Natural adsorption) 10
6-1-2. 靜電吸引力 (Electrostatic forces) 10
6-1-3. 共價鍵結法 (Covalent binding) 10
6-2. 膠體包埋法 (Matrix entrapment) 10
6-3. 交聯法 (Cross-linking binding) 11
6-4. 薄膜包覆法 (Membrane enclosure) 11
6-5 PVA 固定化法 11
6-6. 微管束陣列膜 (Microtube array membrane, MTAM) 11
七、固態發酵 (Solid state fermentation) 12
7-1. 固態發酵的定義 12
7-2. 固態發酵的應用 12
7-3. 影響固態發酵的因素 12
7-4. 厭氧固態發酵 12
八、生命週期評估 (Life cycle assessment, LCA) 13
8-1. 生命週期評估背景 13
8-2. 生命週期各階段定義 13
8-3. 生命週期評估 14
8-4. 生命週期評估之限制 14
參、實驗設計 15
肆、實驗材料與方法 17
一、實驗材料 17
1-1. 原料 17
1-2. 實驗菌株 17
1-2-1. 降解龍鬚菜多醣之多重粗酵素生產菌株 17
1-2-2. 產丁醇菌株 17
1-3. 試驗藥品 17
1-3-1. 藥品 17
1-3-2. 酵素 18
1-3-3. 培養基組成 19
1-3-4. 酵素反應基質 21
1-3-4-1. 洋菜酶反應基質 21
1-3-4-2. α-澱粉酶反應基質 22
1-3-4-3. 紅藻膠酶反應基質 22
1-3-4-4. 纖維素酶反應基質 22
1-3-4-5. 聚木糖酶反應基質 22
1-3-4-6. 龍鬚菜熱萃多醣誘導酵素反應基質 22
1-3-5. 還原糖量反應試劑 22
1-4. 儀器設備 22
二、實驗方法 24
2-1. 龍鬚菜之前處理 24
2-2. 龍鬚菜之一般成分分析 24
2-2-1. 水分含量 24
2-2-2. 灰分含量 24
2-2-3. 粗蛋白含量 24
2-2-4. 粗脂肪含量 25
2-2-5. 粗纖維含量 25
2-3. 龍鬚菜誘導 P. vesicularis MA103 與 A. salmonicida
MAEF108 粗酵素液 25
2-3-1. 菌株保存 25
2-3-2. 菌株活化 25
2-3-3. 龍鬚菜粉末誘導 P. vesicularis MA103 與
A. salmonicida MAEF108 之粗酵素液製備 26
2-3-4. 酵素活性測定方法 26
2-3-4-1. 洋菜酶活性測定 26
2-3-4-2. α-澱粉酶活性測定 26
2-3-4-3. 紅藻膠酶活性測定 26
2-3-4-4. 纖維素酶活性測定 27
2-3-4-5. 聚木醣酶活性測定 27
2-3-4-6. 酵素活性單位定義 27
2-4. 龍鬚菜熱萃多醣水解液製備 27
2-4-1. 去鹽龍鬚菜製備 27
2-4-2. 熱酸水解 27
2-4-3. 酵素水解 28
2-5. 產丁醇菌株之特性 28
2-5-1. 菌株保存 28
2-5-2. 菌株活化 28
2-5-3. 菌株固定化 28
2-5-3-1. PVA-SA (Polyvinyl alcohol- Sodium alginate)
固定化顆粒製作 29
2-5-3-2. 微管束陣列膜製作 29
` 2-5-3-3. 固定化細胞之包覆率 29
2-5-4. 產丁醇菌株突變試驗 29
2-5-5. 丁醇濃度耐受性探討 30
2-6. 60 g/L 葡萄糖液批式發酵 30
2-6-1. 批式發酵 30
2-6-2. 重複批次發酵 30
2-7. 60 g/L 半乳糖液批式發酵 30
2-7-1. 批式發酵 30
2-8. 龍鬚菜酵素水解液發酵 30
2-8-1. 氣提 (Gas stripping) 30
2-8-2. 批式發酵與氣提反應 31
2-8-3. 饋料批式發酵與氣提反應 31
2-9. 固態發酵 31
2-9-1. 龍鬚菜酵素水解基質 31
2-9-1-1. 經熱酸處理之龍鬚菜酵素水解基質 31
2-9-1-2. 龍鬚菜酵素水解基質 32
2-9-2. 厭氧固態發酵生產丁醇 32
2-10. 分析方法 32
2-10-1. 總糖量測定 32
2-10-2. 還原糖量測定 32
2-10-3. NaCl 濃度測定 33
2-10-4. 菌量測定 33
2-10-4-1. 簡單生長量判定 33
2-10-4-2. 產丁醇之梭狀芽孢桿菌菌量測定 33
2-10-5. 丁醇濃度測定 33
2-10-6. 丁醇產率計算 34
2-10-7. 龍鬚菜水解液及發酵液殘醣分析-薄層色層分析
(Thin layer chromatography, TLC) 34
2-10-8. 龍鬚菜水解液及發酵液殘醣分析-高壓液相層析
(High performance liquid chromatography, HPLC) 34
2-11. 生命週期評估 34
2-11-1. 生命範圍界定 34
2-11-1-1. 功能單位 35
2-11-1-2. 研究相關假設限制 35
2-11-2. 盤查分析 35
2-11-3. 環境效益評估 35
2-11-4. 比較評估 35
2-11-5. 綜合討論 36
伍、統計分析 36
 
陸、結果討論 37
一、龍鬚菜之一般成分分析 37
二、龍鬚菜添加於人工海水 (Artificial sea water, ASW) 中誘導
P. vesicularis MA103 與 A. salmonicida MAEF108 生產可
降解龍鬚菜多醣之粗酵素液 37
三、龍鬚菜酵素水解液之製備 38
3-1. 龍鬚菜基質之水解條件探討 38
3-2. 酵素水解液之製備 38
四、產丁醇梭狀芽孢桿菌 39
4-1. 丁醇耐受菌株篩選試驗 39
五、龍鬚菜厭氧發酵與二階段氣提試驗 40
5-1. 去鹽或未去鹽龍鬚菜以耐鹽產丁醇菌批式厭氧發酵 40
5-2. 去鹽龍鬚菜以耐丁醇突變菌株批式厭氧發酵 41
5-3. 二階段氣提裝置效率測試 42
5-4. 去鹽龍鬚菜以產丁醇耐鹽菌搭配二階段氣提系統
批式厭氧發酵 42
5-5. 去鹽龍鬚菜以耐丁醇菌搭二階段氣提系統進行
饋料批式厭氧發酵生產丁醇 43
六、固定化之丁醇菌株 43
6-1. 探討丁醇菌株經 PVA-SA 固定化後是否增加丁醇產率 44
6-2. 探討丁醇菌株經微管束陣列膜固定化後是否增加丁醇產率 44
6-3. 丁醇菌株分別以游離細胞、MTAM 固定化、與
PVA-SA 固定化後以 60 g/L 葡萄糖發酵液進行批式發酵 45
七、固態發酵 46
7-1. 龍鬚菜酵素水解基質 46
7-2. 厭氧固態發酵生產丁醇 46
八、以龍鬚菜提煉生質丁醇之生命週期評估 47
8-1. 系統範圍界定 47
8-2. 不同龍鬚菜發酵生產丁醇製程之盤查分析 47
8-3. 比較評估 54
8-4. 綜合討論 55
柒、結論 56
捌、參考文獻 58
玖、圖表 69
拾、附錄 107

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