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研究生:易琮凱
研究生(外文):Yi, Tsung-Kai
論文名稱:青海菜以梭狀芽孢桿菌生產生質丁醇之發酵條件探討及其產業生命週期之評估
論文名稱(外文):Study on the Fermentation Conditions by Clostridia and Life Cycle assessment on Bio-butanol Production from Monostroma nitidum
指導教授:潘崇良
指導教授(外文):Pan, Chorng-Liang
口試委員:周正俊游若萩鄭光成蕭心怡林泓廷
口試委員(外文):Chou, Cheng-ChunYu, Roch-ChuiCheng, kuan-ChenHsiao, Hsin-ILin, Hong-Ting
口試日期:2015-07-23
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:128
中文關鍵詞:青海菜生質丁醇鹽濃度梭狀芽孢桿菌發酵耐鹽衍生菌株氣提回收冷凝劑生命週期評估分析生產成本碳足跡
外文關鍵詞:Monostroma nitidumbio-butanolsalt concentrationClostridiumfermentationsalt tolerant strainsgas stripping recoverycoolantlife cycle assessment (LCA)production costcarbon footprint
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本研究之目的為研發自綠藻青海菜 (Monostroma nitidum) 生產生質丁醇的發酵技術與對可行之生質丁醇生產程序進行生命週期評估分析。主要工作包括將青海菜或青海菜熱萃多醣以熱酸、市售纖維素酶、及菌株 Pseudomonas vesicularis MA103 (MA103)與Aeromonas salmonicida MAEF108 (MAEF108)所產粗酵素液水解,以批式與饋料批式厭氧發酵探討提升丁醇產率之生產條件,並進行不同製程之生產成本與碳足跡變化之評估。去鹽與未去鹽之5% (w/v)青海菜熱酸水解液中之還原糖量分別為0.25 g與1.50 g及鹽濃度為1.46 mg/mL與1.75 mg/mL。熱酸水解反應之青海菜(5 g)以3次添加與1次添加處理後,青海菜水解液分別產生1.92 g與 1.93 g的總糖及1.33 g與1.28 g的還原糖,故一次添加基質者可獲相近的水解效果與較低生產成本。100 mL 之5% (w/v)青海菜水解液中加入市售纖維素酶760 U與4,560 U於pH 4.5與26oC下水解1天,可分別產生2.39 g與2.97 g的總糖以及1.88 g與 2.04 g的還原糖,4,560 U市售纖維素酶雖可獲得較高的水解效率,但因成本偏高故後續實驗均以添加760 U市售纖維素酶進行。以粉末、新鮮、與去鹽青海菜水解液分別誘導MA103與MAEF108所產之 1 mL多重粗酵素液中,在第3天時去鹽青海菜水解液誘導者呈現較高之洋菜酶與聚木糖酶酵素活性,為1.11 U與0.07 U。Clostridium acetobutylicum BCRC10639與C. beijerinckii BCRC17950之耐鹽性衍生株篩選試驗中獲得2株具有4.5%食鹽耐受能力之菌株,命名為C. acetobutylicum BCRC10639-ST (Salt tolerant)與C. beijerinckii BCRC17950-ST。以濃度0.5%、1.5%、與35.0%之丁醇溶液進行-20oC氣提 (Ethanol) 與-80oC氣提 (Dry ice + Ethanol) 之回收效率測試時結果顯示,發酵液中丁醇之回收率分別為9.4%、9.7%、和35%以及18%、16%、和72%,經由殘留丁醇濃度換算之丁醇散失率分別為33%、32%、和64%以及25%、27%、和4%。製程Mn (Monostroma nitidum)-CL (Low cellulase)-Si (Salt intolerant strain)-B (Batch)與Mn-DS (Desalted)-CL-Si-B (100 mL) 分別以未去鹽 (Mn) 與去鹽青海菜(Mn-DS)經760 U纖維素酶水解後,接種不耐鹽丁醇菌酛 (BCRC10639/17950) 在37oC下厭氧批式發酵5天,結果顯示丁醇產率為3.49%與4.00%。製程Mn-CH (High cellulase)-Si-B 與Mn-DS-CH-Si-B (100 mL) 分別以未去鹽與去鹽青海菜為原料,經4,560 U纖維素酶水解後,接種不耐鹽丁醇菌酛,在37oC下厭氧批式發酵5天,結果顯示丁醇產率為3.92%與4.49%,二者之丁醇產率皆高於添加760 U纖維素酶水解之發酵液,其中去鹽之青海菜水解液具較高的丁醇產率。製程Mn-DS-CL-St (Salt tolerant strain)-B與Mn-DS-CH-St-B (100 mL) 以去鹽青海菜為原料,分別以760與4,560 U纖維素酶水解後,接種耐鹽菌酛(BCRC10639-ST/17950-ST),分別在第5天及第2天可呈現最高丁醇產率6.71%與6.31%。製程Mn-DS-CL-Si-B與Mn-DS-CL-St-B (100 mL) 分別使用使用不耐鹽與耐鹽丁醇菌酛,進行5%去鹽青海菜發酵試驗5天,分別在第3天達到最高丁醇產率3.49%與6.71%。製程Mn-DS-CL-St-B-GSE (Ethanol) (2 L) 配合-20oC氣提 (Ethanol) (GSE)回收進行發酵,2天時可得到丁醇產率7.02%。去鹽青海菜水解液在製程Mn-DS-CL-St-FB (Fed batch)-GSE (2 L) (-20oC氣提)及製程Mn-DS-CL-St-FB-GSDE (Dry ice + Ethanol) (2 L) (-80oC氣提) (GSDE) 中,在37oC下厭氧饋料批式發酵至第2天時之最大丁醇產率分別是6.66%與6.98%,此時之丁醇濃度分別為0.42%及0.44%。-80oC氣提在發酵第5天時之丁醇產率為2.93%,濃度為0.55%,產量自6.6 g提升至8.8 g。以0.5%丁醇濃度在-80oC氣提組(後者)反應中之散失率25%,推算去鹽青海菜經製程Mn-DS-CL-St-FB-GSDE,以耐鹽性丁醇菌酛經饋料批式發酵5天後,可得知理論上最高丁醇濃度及產率分別為0.68%與3.60%,較實際測得數據為高,故降低氣提過程中之丁醇散失量,可進一步提升最終之丁醇產量。經生命週期評估分析,製程Mn-DS-CL-St-FB-GSE與Mn-DS-CL-St-FB-GSDE產製每L丁醇成本分別為24,791 NTD (New Taiwan Dollar) 與23,279 NTD,較製程 Mn-DS-CL-St-B-GSE 增加 93.08% 與 78.77%,碳足跡分別較製程 Mn-DS-CL-St-B-GSE 增加 86.21% (63,802.29 kg CO2/1,000 L) 與 75.26% (60,066.27 kg CO2/1,000 L)。若Mn-DS-CL-St-FB-GSDE製程中使用自行製備之纖維素酶、MA103/MAEF108培養液、與丁醇菌酛培養液,產製每公秉丁醇成本分別為50890.59 NTD、350.39 NTD與534,243.04 NTD,共可將產製每L丁醇成本降至22,710 NTD。未來若可添加 10% 丁醇於市售汽油中則每年可衍生2,960,706,619 NTD 的環境效益。
The objective of this study is to develop fermentation techniques for producing bio-butanol from green algae Monostroma nitidum and to conduct life cycle assessment (LCA) analysis for the potential bio-butanol production protocol. The main works including hydrolyzing M. nitidum or its hot water polysaccharides (PS) extracte with hot acid, commercial cellulases, and crude enzymes from strain P. vesicularis MA103 (MA103) and A. salmonicida MAEF108 (MAEF108). The hydrolysates of M. nitidum were used to produced bio-butanol through anaerobic batch and fed-batch fermentation to obtain further yield of biofuel. And the evaluations on production cost and carbon footprint of various product protocol are also conducted. The reducing sugar content of desalted or fresh 5% (w/v) M. nitidum after hot acid hydrolyzing were 0.25 and 1.50 g and these salt concentration were 1.46 and 1.75 mg/mL, respectively. After 3 times added (2%, 2%, 1%) and added in once of 5 g M. nitidum into 100 mL water for hot acid hydrolysis, the total sugars and reducing sugars are 1.92 and 1.93 g and 1.33 and 1.28 g, respectively. The later showed similar hydrolysis efficiency and lower cost than former. The 760 U or 4,560 U cellulases are reacted with 100 mL effect of M. nitidum (5%, w/v) solution at pH 4.5 and 26oC for 24 hr, the results showed total sugars and reducing sugars were 2.39 and 2.97 g and 1.88 and 2.04 g, respectively. The 4,560 U cellulase hydrolysis group is higher than 760 U group, but 760 U cellulase treatment was used in the following experiment based on the lower cost. Multiple crude enzymes from strains MA103 and MAEF108 were induced by 3 kinds M. nitidum: powder, fresh, and desalting. The results showed that strains MAEF108 and MA103 had better agarase and xylanase enzyme activities (1.11 U and 0.07 U) at the third day when the desalting M. nitidum used as an inducing substrate. Adaptation experiment on butanol producing strain Clostridium acetobutylicum BCRC10639 and C. beijerinckii BCRC17950 with 4.5% salt concentration broth, developed two salt tolerant strains which are named C. acetobutylicum BCRC10639-ST (salt tolerant) and, C. beijerinckii BCRC17950-ST. Gas-stripping systems (I) ethanol (-20 oC) and (II) dry ice + ethanol (-80oC) were tested for their recovery rate with 0.5%, 1.5%, and 35% butanol solution, results indicated that the recovery rates for system (I) were 9.4%, 9.7%, and 35%, as well as for system (II) were 18%, 16%, and 72%, respectively. The loss rates of these tested gas-stripping systems for system (I) were 33%, 32%, and 64%, and for system (II) were 25%, 27%, 4%, respectively. Hydrolysate of Mn (Monostroma nitidum)-CL (low cellulase activity)-Si (salt intolerant strain)-B (Batch) and Mn-DS (Desalted)-CL-Si-B (100 mL) fermented by strains BCRC10639/BCRC17950 could produce 3.49% and 4.00% bio-butanol after anaerobically fermented at 37oC for 5 days. Butanol yields of procedures Mn-CH (high cellulase activity)-Si-B and Mn-DS-CH-Si-B were 3.92% and 4.49% after anaerobically fermented at 37oC for 5 days in 100 mL solution. Both of their butanol yields were higher than the M. nitidum that hydrolysed by 760 U cellulase, and the desalted M. nitidum showed better butanol yield than fresh M. nitidum. Procedure Mn-DS-CL-St (salt tolerant strain)-B and Mn-DS-CH-St-B (100 mL) used desalted M. nitidum as substrate and hydrolyzed by 760 U or 4,560 U cellulase, respectively, and then fermented by BCRC10639-ST/17950-ST, showed the highest butanol yield of 6.71% (day 5) and 6.31% (day 2), respectively. Procedure Mn-DS-CL-Si-B and Mn-DS-CL-St-B (100 mL) fermented by salt intolerant or salt tolerant strains for 5 days, both showed the highest butanol yields of 3.49% and 6.71% in day 3, respectively. Gas striping technique (-20oC Ethanol) were applied in procedure Mn-DS-CL-St-B to increase the fermentation yield of bio-butanol, when desalted M. nitidum used as substrate of batch fermentation, the butanol yield is 7.02% in day 2. In the fed-betch fermentation procedures Mn-Ds-CL-St-FB-GSE (-20oC) and Mn-Ds-CL-St-FB-GSDE (-80oC), the highest butanol yield achieved in day 2, which were 6.66% and 6.98%, and the butanol concentrations of fermentation solution were 0.42% and 0.44%, respectively. The later (-80oC, GSDE) showed higher butanol concentration in day 5, and its butanol production amount increased from 6.6 g in day 2 to 8.8 g. Based on the lost rate of gas stripping 25%, while -80oC (dry ice + ethanol) recovery condition and 0.5% (v/v) butanol was used as fermentation solution. The postulated butanol concentration and yield of butanol of protocol Mn-Ds-CL-St-FB-GSDE will be by 25%, 0.68% and 3.60%, which are higher than the collected data. Therefore, if the lost rate of gas stripping could lower down, the yield of collected butanol from various production protocol could be improved. After analyzed by LCA, the cost of butanol producting per L by protocols of Mn-Ds-CL-St-FB-GSE and Mn-Ds-CL-St-FB-GSDE were 24,791 and 23,279 NTD, which were 93.08% and 78.77% than production protocol Mn-Ds-CL-St-B-GSE had. As to the amount of carbon footprints, they both higher than the later, increased for 86.21% (63,802.29 kg) and 75.26% (60,066.27 kg), respectively. If self-made cellulase as well as MA103/MAEF108 and clostridia culturing broth used were replaced for commercial cellulase and media in the production, the cost of procedure Mn-Ds-CL-St-FB-GSDE cost could dropped to 50890.59 NTD, 350.39 NTD and 534,243.04 NTD. The cost to producing 1 L butanol could dropped to 22,710 NTD. In the future, if the addition of 10% bio-butanol into gasoline is applied, the amount of environmental benefits could reach 2,960,706,619 NTD.
壹、前言 ......................................................................................................................1
貳、文獻整理 3
一、 生質能源 3
1-1. 生質能源介紹 3
1-2. 第三代生質燃料 3
1-3. 生質丁醇特性 4
1-4. 生質汽油應用 4
二、 丁醇之介紹 4
2-1. 丁醇之特性 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 6
三、 產丁醇菌株之特性 6
四、 青海菜 (Monostroma nitidum) 8
4-1. 青海菜之生態特性 8
4-2. 青海菜之組成成分 8
五、 發酵方式 8
5-1. 批次培養 (Batch culture) 8
5-2. 饋料批式培養 (Fed-batch culture) 8
5-3. 連續式培養 (Continuous culture) 9
六、 菌種突變與適應性測試 9
七、 饋料批式發酵與氣提裝置 9
八、 氣提裝置之冷凝劑 (Coolant) 10
九、 生命週期評估(Life cycle assessment, LCA) 10
9-1. 生命週期評估背景 10
9-2. 生命週期各階段定義 11
9-3. 生命週期評估 11
9-4. 生命週期評估之限制 12
參、實驗設計 ..13
一、 以青海菜生產丁醇之最佳條件 13
二、 以青海菜生產生質丁醇之產業生命週期評估 14
肆、實驗材料與方法 15
一、 實驗材料 15
1-1. 原料 15
1-2. 實驗菌株 15
1-2-1. 降解青海菜多醣之多重粗酵素生產菌株 15
1-2-2. 產丁醇菌株 15
1-3. 試驗藥品 15
1-3-1. 藥品 15
1-3-2. 酵素 16
1-3-3. 培養基組成 16
1-3-4. 青海菜熱萃多醣誘導酵素反應基質 18 1-3-5. 還原糖量反應試劑 18
1-4. 儀器設備 18
二、 實驗方法 19
2-1. 青海菜之前處理 19
2-2. 青海菜之一般成分分析 19
2-2-1. 水分含量 19
2-2-2. 粗脂肪含量 19
2-2-3. 粗蛋白含量 19
2-2-4. 灰分含量 20
2-3. MAEF108 與 MA103 粗酵素液之生產 20
2-3-1. 菌株保存 20
2-3-2. 菌株活化 20
2-3-3. 青海菜熱萃多醣誘導粗酵素液之製備 .20 2-4. 青海菜熱萃多醣誘導酵素之活性測定 21 2-4-1. 洋菜酶活性測定 21
2-4-2. 纖維素酶活性測定 21
2-4-3. 聚木醣酶活性測定 21
2-4-4. 澱粉酶酵素活性測試反應基質 21
2-4-5. Carrageenase 酵素活性測試反應基質 22
2-4-6. 酵素活性單位定義 22
2-5. 青海菜寡醣水解液之製備 22
2-5-1. 去鹽青海菜製備 22
2-5-2. 熱酸水解 23
2-5-3. 酵素水解 23
2-6. 產丁醇菌株之特性 23
2-6-1. 菌株保存 23
2-6-2. 菌株活化 24
2-6-3 20 g/L 葡萄糖液批式發酵 24
2-6-3-1. 攪拌批式發酵 (Agitating batch fermentation) 24
2-6-3-2. 靜置批式發酵 (Static batch fermentation) 24
2-6-4. 產丁醇菌株突變與適應性測試 24
2-7. 產丁醇菌株耐受性之探討 25
2-7-1. 食鹽濃度耐受性探討 25
2-7-2. 丁醇濃度耐受性探討 25
2-7-3. 糖類利用性探討 25
2-8. 批式發酵與饋料批式發酵 25
2-8-1. 氣提 (Gas stripping) 25
2-8-2. 批式發酵 26
2-8-3. 饋料批式發酵與氣提反應 27
2-9. 分析方法 28
2-9-1. 總糖量測定 28
2-9-2. 還原糖量測定 28
2-9-3. NaCl濃度測定 28
2-9-4. Clostridium sp. 菌量測定 29
2-9-4-1. 簡單生長量判定 29
2-9-4-2. 生長菌量測定 29
2-9-5. 丁醇濃度測定 29
2-9-6. 丁醇產率計算 29
2-9-7. 高效能液相層析分析 29
2-9-8. 超過濾 (Ultrafiltration, UF) 30
2-9-9. 薄層層析 (TLC) 分析 30
2-10. 殘醣分析 30
2-10-1.發酵後之殘留醣量 30
2-10-2.發酵後之殘留醣組成 30
2-11. 生命週期評估 30
2-11-1.系統範圍界定 30
2-11-1-1. 功能單位 31
2-11-1-2. 研究相關假設限制 31
2-11-2.盤查分析 31
2-11-3.環境效益評估.......................................................................31
2-11-4.比較評估 31
2-11-5.綜合討論 32
三、 統計分析 32
伍、結果與討論 33
一、 青海菜之一般成分分析 33
二、 青海菜添加於人工海水(Artificial sea water, ASW) 中誘導 P. vesicularis MA103與A. salmonicida MAEF108生產可降解 青海菜多醣之粗酵素液 33
三、 青海菜寡醣水解液之製備 33
3-1. 青海菜最佳去鹽條件分析 33
3-2. 一次添加與分次添加青海菜基質之水解條件探討 34
3-3. 酵素水解液之製備 34
3-3-1. TLC分析 34
3-3-2. HPLC分析 35
四、 產丁醇梭狀芽孢桿菌 35
4-1. 接種菌量與振盪條件探討 35
4-2. 適應性突變試驗 35
4-2-1. 食鹽濃度耐受性探討 35
4-2-2. 丁醇濃度耐受性探討 36
4-2-3. 糖類利用性 37
五、 青海菜同步糖化發酵與氣提試驗 38
5-1. 青海菜以產丁醇菌批式厭氧發酵生產生質丁醇 38
5-2. 去鹽青海菜以產丁醇耐鹽突變菌株批式厭氧發酵
生產生質丁醇 39
5-3. 氣提裝置效率測試 39
5-4. 去鹽青海菜以產丁醇菌搭配-20oC氣提裝置 (Ethanol)
批式厭氧發酵生產丁醇 40
5-5. 去鹽青海菜以產丁醇菌搭配-20oC氣提 (Ethanol)
饋料批式厭氧發酵生產丁醇 41
5-6. 去鹽青海菜以產丁醇菌搭配-80oC氣提 (Dry ice + Ethanol)
饋料批式厭氧發酵生產丁醇 41
5-7. 青海菜以產丁醇菌批式厭氧發酵搭配-20oC氣提 (Ethanol) 生產丁醇之理論最大產率 42
5-8. 青海菜以產丁醇菌饋料批式厭氧發酵搭配
-20oC氣提 (Ethanol) 生產丁醇之理論最大產率 42
5-9. 青海菜以產丁醇菌饋料批式厭氧發酵搭配
-80oC氣提 (Dry ice + Ethanol) 生產丁醇之理論最大產率 42
六、 以青海菜提煉生質丁醇之生命週期評估 43
6-1. 系統範圍界定 43
6-2. 不同青海菜發酵生產丁醇製程之盤查分析 43
6-2-1. 青海菜種植階段 43
6-2-1-1. 青海菜吸收之碳足跡 43
6-2-1-2. 青海菜養殖 43
6-2-2. 青海菜運輸階段 44
6-2-3. 青海菜處理階段 44
6-2-3-1.青海菜去鹽 44
6-2-3-2. 青海菜乾燥與磨粉 45
6-2-4. 丁醇水解階段 45
6-2-4-1. 熱酸水解(水) 45
6-2-4-2. 熱酸水解(電) 45
6-2-4-3. 熱酸水解(HCl) 46
6-2-4-4. 酸鹼中和 46
6-2-4-5. 纖維素酶水解 47
6-2-4-6. 粗酵素液水解 47
6-2-5. 青海菜多醣丁醇發酵階段 48
6-2-5-1. 梭狀芽孢桿菌培養 48
6-2-5-2. 饋料液濃縮 49
6-2-5-3. 發酵與氣提 49
6-2-6. 丁醇收集與其他成本 50
6-2-6-1. 丁醇蒸餾 50
6-2-6-2. 丁醇運輸 50
6-2-6-3. 設備折舊 50
6-2-6-4. 行政管銷初步估算 51
6-2-6-5. 青海菜藻渣收集再利用 51
6-3. 比較評估 51
6-4. 綜合討論 52
6-4-1. 移動源單位減量成本 52
6-4-2. 各汙染排放量減量 52
陸、結論 53
柒、參考文獻 55
捌、圖表 63
玖、附錄 102

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