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研究生：	吳季芳 Wu, Ji-Fang
論文名稱：	以澱粉為碳源基質進行生物產氫 Biohydrogen production using starch as the carbon substrate
指導教授：	張嘉修 Chen, Jo-Shu
學位類別：	碩士 Master
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2006
畢業學年度：	94
語文別：	中文
論文頁數：	178
中文關鍵詞：	生物產氫、光合產氫、澱粉酶
外文關鍵詞：	Rhodopseudomonas palustris, Caldimonase taiwanensis, Clostridium, Biohydrogen
相關次數：	點閱：63 下載：6
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氫氣是目前世界各國積極開發的替代能源之一，而澱粉是自然界中植物所提供產量僅次於纖維素的有機資源，相當適合作為未來商業化生物產氫程序之基質來源。雖然厭氧暗醱酵產氫因具有高產氫速率而被認為具有商業化之可行性，然而，屬高分子多醣類之澱粉並不易為厭氧產氫菌所分解利用，且暗醱酵產氫所伴隨之溶解態代謝物(有機酸及醇類)仍面臨後續處理的負擔。有鑑於此，本研究分別進行澱粉水解（糖化）、糖化產物暗醱酵產氫及有機酸光醱酵產氫之相關研究，企圖發展創新之澱粉三階段生物產氫程序。
本研究首先探討環境因子對本土性嗜熱型澱粉水解菌株Caldimonase taiwanensis On1T水解澱粉之影響，以尋求最適培養條件。在各個不同培養溫度(45-60oC)、初始pH值 (5.5-9.5)、攪拌速率(100-200 rpm)及基質濃度(10-50 g/L)之澱粉水解批次試驗中，結果顯示溫度為55oC、初始pH為7.5、攪拌速率為150 rpm以及澱粉濃度為50 g/L時有最佳的澱粉水解速率，還原糖產生速率可達1.72 g/h/L。此外，於各種不同基質濃度之試驗，還原糖產生量隨澱粉濃度增加而增加，各試驗之還原糖產率為0.46-0.53 g reducing sugar/g starch，顯示並未發生基質抑制之情形。
在厭氧醱酵產氫試驗中，分別以五種Clostridium厭氧產氫菌株(Cl. butyricum CGS2、Cl. butyricum CGS5、Cl. pasteurianum CH1、Cl. pasteurianum CH5、Cl. pasteurianum CH7)及混菌培養進行澱粉糖化產物之批次產氫實驗，同時亦分別對澱粉進行直接產氫試驗以作為相對評比，結果顯示此五種菌株及混菌培養皆可有效利用澱粉糖化產物而轉化產氫，其中以Cl. butyricum CGS2及Cl. pasteurianum CH5有較佳之產氫效能，Cl. butyricum CGS2之最大產氫速率為165.33 mL/h/L，氫氣產率為6.40 mmol H2/g COD，總氫氣產率為5.74 mmol H2/g starch；Cl. pasteurianum CH5之最大產氫速率為175 mL/h/L，氫氣產率為6.66 mmol H2/g COD，總氫氣產率為6.36 mmol H2/g starch；在澱粉直接產氫方面，兩株 C. pasteurianum並無產氫現象發生，而混菌培養具有較佳之產氫表現，最大產氫速率為58 mL/h/L，氫氣產率為3.14 mmol H2/g starch，明顯低於水解/產氫分離之程序。接著以Cl. butyricum CGS2進行連續醱酵產氫的測試，其結果顯示在HRT＝12 h 下以澱粉水解產物為碳源時，其產氫速率0.51 L/h/L，氫氣濃度約51%，其氫氣產率為2.03 mol H2/mol glucose (10.57 mmol H2/g COD) ，比產氫速率0.14 (mmol/g VSS/d) ，總氫氣產率為9.63 mmol H2/g starch。
在光合產氫試程中，本研究分別探討紫色不含硫光合細菌Rhodopseudomonas palustris WP3-5以乙酸及丁酸為碳源之醱酵產氫行為。當乙酸濃度為3000 mg COD/L時，產氫速率隨HRT調降而增加，系統於HRT＝12 h 時產氫效果最佳，其產氫速率為42.05 ml/h/L，氫氣產率為1.39 mol H2/mol acetate。當進流基質為丁酸2500 mg COD/L，產氫速率並未隨HRT調降而增加，於 HRT＝48 h時，其產氫速率與氫氣產率分別為20.25 ml/h/L及2.54 mol H2/mol butyrate。
本研究成功展示殿粉三階段生物產氫之可行性，期許未來進一步整合此三程序以建立完善之澱粉醱酵產氫技術。

Hydrogen is a promising energy carrier of the future. Starch is well suited to act as a cost-effective substrate for biohydrogen production, since it is the second abundant organic resources (next to cellulose) from plants. Dark hydrogen fermentation from original starch encountered problems with poor hydrogen producing efficiency since hydrolysis of starch is often a rate-limiting step. Moreover, the soluble metabolites (e.g., volatile fatty acids, alcohols) produced during dark H2 fermentation required further treatment. Therefore, this study applied enzymatic hydrolysis step to hydrolyze starch and utilized hydrolyzed starch as the substrate of dark fermentation. Meanwhile, the soluble metabolites (e.g., volatile fatty acids) were decomposed by photosynthetic bacteria to produce more H2 via photo-fermentation. This three-step process allowed biological production of H2 from starch.
This study investigates the effect of environmental factors on starch hydrolysis of an indigenous strain Caldimonase taiwanensis On1T to identify the optimal operation conditions. The tested temperature, pH, agitation rate, substrate concentration were 45-60oC, 5.5-9.5, 100-200 rpm and 10-50 g/L, respectively. The results show that the best starch hydrolysis efficiency occurred when temperature, pH, agitation rate, substrate concentration were 55oC, 7.5, 150 rpm, and 50 g/L, respectively, resulting in a reducing sugar production rate of 1.72 g/h/L. Moreover, the reducing sugar production appeared to increase as the starch concentration increased from 10-50 g/L, suggesting that there was no substrate inhibition during the range of starch concentration examined. The reducing sugar yield was 0.46-0.53 g reducing sugar per g starch.
The original and hydrolyzed starch was used as the substrate to produce H2 through dark fermentation. In these batch H2 production experiments, five pure Clostridium isolates, namely, Cl. butyricum CGS2, Cl. butyricum CGS5, Cl. pasteurianum CH1, Cl. pasteurianum CH5, Cl. pasteurianum CH7, as well as mixed cultures were used. The results show that all of the pure and mixed cultures were able to utilize the hydrolyzed starch for H2 production, especially for Cl. butyricum CGS2 and Cl. pasteurianum CH5. The Cl. butyricum CGS2 strain exhibited the highest H2 production rate of 165.33 mL/h/L and a H2 yield of 6.40 mmol H2/g COD (overall H2 yield of 5.74 mmol H2/g starch). Cl. pasteurianum CH5 had the maximum H2 production rate of 175 mL/h/L and a H2 yield of 6.66 mmol H2/g COD (overall H2 yield of 6.36 mmol H2/g starch). For using original starch as the carbon substrate, the two Cl. pasteurianum strains did not produce H2, while H2 production was observed for Cl. butyricum and the mixed cultures. The latter gave a higher H2 production rate of 58 mL/h/L and a H2 yield of 3.14 mmol H2/g starch. Apparently, using hydrolyzed starch as the substrate led to much better H2 production efficiency. Furthermore, continuous H2 production from starch hydrolysate with Cl. butyricum CGS2 shows a H2 production rate of 0.51 L/h/L at HRT=12 h with a H2 content of 51%, H2 yield of 2.03 mol H2/mol glucose (10.56 mmol H2/g COD), and specific H2 production rate of 0.14 mmol/g VSS/d.
In photo H2 fermentation experiments using a photosynthetic bacterium Rhodopseudomonas palustris WP3-5, acetate and butyrate were used as the carbon substrate. When acetate concentration was 3000 mg COD/L, the H2 production rate increased with a decrease in HRT. The best H2-producing performance took place at HRT=12 h, giving a H2 production rate of 42.05 mL/h/L and a H2 yield of 1.39 mol H2/mol acetate. Moreover, when using butyrate (2500 mg COD/L) as the carbon substrate, the H2 production rate did not increase with decreasing HRT. When HRT was 48 h, the H2 production rate and yield were 20.25 mL/h/L and 2.54 mol H2/mol butyrate, respectively.
In summary, this study demonstrates the feasibility of bioH2 production from starch via a three-stage approach (i.e., enzymatic starch hydrolysis, dark fermentation, and photo-fermentation). More detailed and improved operation strategies will be identified to establish key technologies for the starch-to-H2 bioprocess.

目錄
中文摘要....................................................................Ⅰ
英文摘要....................................................................Ⅳ
致謝........................................................................Ⅵ
目錄........................................................................Ⅶ
表目錄....................................................................ⅩⅣ
圖目錄....................................................................ⅩⅧ

第一章 緒論..................................................................1
1-1 研究動機.................................................................1
1-2 研究目的.................................................................2

第二章 文獻回顧與產氫原理....................................................5
2-1 澱粉與澱粉水解酶.........................................................5
  2-1-1 澱粉.................................................................5
  2-1-2 澱粉水解酵素.........................................................7
  2-1-3 Caldimonas taiwanensis On1T.........................................12
2-2 傳統產氫的方法..........................................................13
  2-2-1 熱化法..............................................................13
  2-2-2 電化學法............................................................13
2-3 生物法產氫..............................................................15
  2-3-1 直接光合作用產氫....................................................19
  2-3-2 間接光合作用產氫....................................................22
  2-3-3 暗醱酵產氫..........................................................23
  2-3-4 Clostridium butyricum CGS2..........................................29
  2-3-5 光醱酵產氫..........................................................29
  2-3-6 Rhodopseudomonas palustris WP3-5....................................36
2-4 影響細胞生長的條件......................................................37
  2-4-1 溫度................................................................37
  2-4-2 酸鹼度(pH 值) ......................................................39
  2-4-3 氣體................................................................41
  2-4-4 氮源................................................................44
  2-4-5 光照強度............................................................45
2-5厭氧醱酵產氫與光醱酵產氫之結合...........................................47
2-6 回應曲面法(response surface methodology，簡稱RSM) ......................49
  2-6-1 回應曲面法之介紹....................................................49
  2-6-2 回應曲面法之原理....................................................50
  2-6-3 中心混成設計........................................................52
第三章 實驗材料及方法.......................................................55
3-1 實驗儀器裝置與方法......................................................55
  3-1-1 常規儀器設備........................................................55
  3-1-2 澱粉水解之實驗儀器..................................................55
  3-1-3 厭氧醱酵產氫之實驗儀器..............................................56
  3-1-4 光醱酵產氫之實驗儀器................................................56
3-2 藥品試劑................................................................56
  3-2-1 培養基試劑..........................................................56
  3-2-2 酚-硫酸法之藥品.....................................................57
  3-2-3 液態代謝產物之標準品................................................57
  3-2-4 DNS之藥品...........................................................57
  3-2-5 澱粉濃度檢測法之藥品................................................58
  3-2-6 氨氮濃度檢測法之藥品................................................58
3-3 菌種來源................................................................58
  3-3-1 澱粉水解菌..........................................................58
  3-3-2 厭氧產氫菌..........................................................58
  3-3-3 光合產氫菌..........................................................59
3-4 培養基組成..............................................................59
  3-4-1 澱粉水解菌基質配方..................................................59
  3-4-2 厭氧產氫菌基質配方..................................................60
  3-4-3 光合產氫菌基質配方..................................................61
3-5 分析儀器及方法..........................................................62
  3-5-1 菌量分析............................................................62
  3-5-2 還原醣濃度之定量....................................................62
  3-5-3 總糖濃度之量........................................................63
  3-5-4 澱粉濃度之定量......................................................63
  3-5-5 氨氮濃度之檢測法....................................................64
  3-5-6 氣體組成分析........................................................65
  3-5-7 液體組成分析........................................................66
  3-5-8 實驗數據分析........................................................67
3-6 實驗步驟及方法..........................................................70
  3-6-1 澱粉水解批次之搖瓶實驗..............................................70
    3-6-1-1 不同初始pH值之搖瓶實驗..........................................70
    3-6-1-2 不同恆溫培養箱轉速之搖瓶實驗....................................70
    3-6-1-3 不同瓶頂空間之搖瓶實驗..........................................70
    3-6-1-4 不同溫度之搖瓶實驗..............................................71
    3-6-1-5 不同澱粉濃度之搖瓶實驗..........................................71
    3-6-1-6 不同硫酸氨濃度之搖瓶實驗........................................72
    3-6-1-7 不同硫酸氨濃度之搖瓶實驗........................................72
    3-6-1-8 反應槽中進行不同轉速之測試......................................73
  3-6-2 厭氧醱酵產氫試驗....................................................73
    3-6-2-1 厭氧醱酵產氫之批次實驗..........................................73
    3-6-2-2 厭氧醱酵產氫之連續流實驗........................................74
  3-6-3 光醱酵產氫實驗......................................................75
    3-6-3-1 光醱酵產氫之批次實驗............................................75
      3-6-3-1-1 不同初始pH值對光合產氫的影響 (乙酸) ........................75
      3-6-3-1-2 不同yeast濃度對光合產氫的影響 (乙酸) .......................76
      3-6-3-1-3 不同氮源及其濃度對光合產氫的影響 (乙酸).....................76
      3-6-3-1-4 RSN實驗設計法尋找最佳的碳源(丁酸)、氮源及鐵離子濃度.........77
    3-6-3-2 光醱酵產氫之連續流實驗..........................................77
      3-6-3-2-1 不同乙酸濃度的測試..........................................77
      3-6-3-2-2 以乙酸為碳源進行不同HRT的測試...............................79
      3-6-3-2-3  不同丁酸酸濃度的測試.......................................80
      3-6-3-2-3  以丁酸為碳源進行不同HRT的測試..............................80

第四章 結果與討論...........................................................81
4-1 Caldimonase taiwanensis sp. nov水解澱粉生產還原醣之測試.................82
  4-1-1 初始pH對澱粉水解之影響..............................................82
  4-1-2 恆溫培養箱轉速對澱粉水解之影響......................................85
  4-1-3 瓶頂空間對澱粉水解之影響............................................87
  4-1-4 溫度對澱粉水解之影響................................................89
  4-1-5 澱粉濃度對澱粉水解之影響............................................91
  4-1-6 硫酸氨濃度對澱粉水解之影響..........................................93
  4-1-7 不同澱粉及滅菌方式對Cal. taiwanensis On1T水解澱粉之影響.............96
  4-1-8 反應槽中攪拌速率對澱粉水解之影響....................................99
  4-1-9 小結...............................................................101
4-2 厭氧醱酵之批次產氫測試.................................................102
  4-2-1 Cl. butyricum以原澱粉及澱粉水解產物為碳源之產氫試驗................102
  4-2-2 Cl. pastuerium以原澱粉及澱粉水解產物為碳源之產氫試驗...............108
  4-2-3 混菌以原澱粉及澱粉水解產物為碳源之產氫測試.........................113
  4-2-4 Cl. butyricum CGS2以澱粉水解產物為碳源之連續醱酵產氫...............116
  4-2-5 小結...............................................................118
4-3 光合產氫之批次實驗.....................................................122
  4-3-1 不同初始pH值對光合產氫之影響.......................................122
  4-3-2 不同氮源對光合產氫的影響...........................................124
  4-3-3 Yeast extract對光合產氫的影響......................................129
  4-3-4 RSM................................................................132
    4-3-4-1 產氫速率(Rmax)之最適化條件.....................................132
    4-3-4-2 總產氫速率(Roverall)之最適化條件...............................133
    4-3-4-3 各變因對遲滯期(λ)的影響........................................133
    4-3-4-4 各變因對氫氣產率(YH2)的影響....................................133
  4-3-5 小結...............................................................154
4-4 光合產氫之連續流實驗...................................................155
  4-4-1 乙酸進料濃度對光合產氫的影響.......................................155
  4-4-2 以乙酸為碳源探討不同HRT光合產氫的響................................157
  4-4-3 丁酸進料濃度對光R. palustris WP3-5合產氫的影響.....................159
  4-4-4 以丁酸為碳源探討不同HRT對光合產氫的影響............................160
  4-4-5 小結...............................................................163

第五章 結論................................................................164

參考文獻...................................................................168

自述.......................................................................178

                                    

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