李篤中Lee, Duu-Jong臺灣大學:化學工程學研究所戴榮Tai, JungJungTai2010-06-302018-06-282010-06-302018-06-282009U0001-1008200910091500http://ntur.lib.ntu.edu.tw//handle/246246/186957本研究著重於使用Clostridium butyricum自含酚廢水中產氫。使用葡萄糖 (glucose) 和纖維二糖 (cellobiose) 為合成廢水中之主要碳源並且觀察其在含酚廢水中對於酚的耐受性和產氫能力。 在批次實驗中，發現C.butyricum在毒性環境中也能夠產生氫氣。氫氣產率對於以葡萄糖和纖維二糖為碳源分別為1.46 mole H2/mole glucose和2.11 mole H2/mole cellobiose而且在糖類發酵降解的過程中也觀察到酚有被降解掉。 在以cellobiose為碳源的批次實驗中發現最高的氫氣產率是發生在酚濃度為600 ppm的時候而不是無酚的環境，而漆氧化酶 (laccase) 的活性分佈與氫氣產率相同，在600 ppm時候最高，由於酚被漆氧化酶轉化成丙酮酸 (pyruvate) ，其在繼續被CoA轉化成acetyl-CoA時會釋放出氫氣，而較多的酚被轉化則在變成acetyl-CoA的過程中有較多的氫氣被釋放，這或許說明了所以在600 ppm時候氫氣產率最高的原因。而漆氧化酶的活性隨著酚濃度的更提高由於毒性對於C.butyricm的抑制造成活性開始下降。氫化酵素活性則是隨著酚濃度的提高而減少，在無酚濃度時的活性是最高的，但是若在培養液中有酚的存在則減少，在全部的產氫速度與氫化酵素的產速度中有一段差距，這段可能是由細胞膜上的氫化酵素 (membrane-bound hydrogenase) 所作用，而當酚濃度的提高，因為毒性對C.butyricum的抑制所以膜上的氫化酵素活性反而漸漸減少。This thesis focus on the research of producing hydrogen in phenol-containing synthetic wastewater using Clostridium butyricum which has very good ability of hydrogen production by degrading hydrocarbons. And we use glucose and cellobiose as the main carbon source its concentration to culture and observe its resistance to phenol and ability of hydrogen production.uring batch experiment, we discover C.butyricum had bood performace of hydrogen production in toxic surrounding. The highest hydrogen production of C.butyricum is 1.46 mole hydrogen．mole-1 glucose and 2.11 mole hydrogen．mole-1 cellobiose when using glucose and cellobiose as main carbon source. We also found phenol was degraded during glucose and cellobiose fermentation.n cellobiose fermentation batch test, we found C.butyricum can produce higher hydrogen yield in phenol-containing medium than no phenol and the highest hydrogen yield is 2.11 at 600 ppm. The activity of laccase also was highest at 600 ppm. Maybe more phenol was cracked and converted to pyruvate by laccase and pyruvate was converted to acetyl-CoA and released hydrogen. Therefore more hydrogen was produced at 600 ppm phenol concentration. As the phenol concentration over 600 ppm the hydrogen yield began to decrease because high phenol concentration inhibit C.butyricum growth. Hydrogenase activity was decrease as phenol concentration increase. The activity of ydrogenase was highest when no phenol inside and cut down immediately in the presence of phenol. But it had a gap between overall hydrogen production rate and enzymatic hydrogen production rate. The difference maybe from membrane-bound hydrogenase. Membrane-bound hydrogenase activity was low gradually because the inhibition of phenol.中文摘要.........................................................................................................................I文摘要.......................................................................................................................II錄..............................................................................................................................III目錄..........................................................................................................................VI目錄.......................................................................................................................VIII一章 前言................................................................................................................1二章 文獻回顧........................................................................................................22-1 生質產氫...........................................................................................................22-2 產氫之微生物群.................................................................................................4 2-2-1 Clostridium菌屬....................................................................................6 2-2-2 氫化酵素與漆氧化酶...........................................................................72-3 影響產氫的因素...............................................................................................11 2-3-1 碳源及氮源.........................................................................................13 2-3-2 酸鹼值(pH值).....................................................................................14 2-3-3 溫度.....................................................................................................152-4 酚的生物降解...................................................................................................15三章 實驗材料與方法..........................................................................................203-1 實驗材料...........................................................................................................20 3-1-1 微生物.................................................................................................20 3-1-2 培養基組成.........................................................................................203-2 實驗設計...........................................................................................................21 3-2-1 C.butyricum菌種保存.........................................................................21 3-2-2 厭氧消化實驗.....................................................................................223-3 實驗方法...........................................................................................................23 3-3-1 還原糖測定.........................................................................................23 3-3-2 總糖測定.............................................................................................24 3-3-3 蛋白質測定.........................................................................................25 3-3-4 生物產氣及氫氣組成測定.................................................................27 3-3-5 揮發性脂肪酸 (VFA) 測定...............................................................29 3-3-6 C.butyricum菌量分析.........................................................................29 3-3-7 化學需氧量 (Chemical Oxygen Demend, COD) 測定.....................30 3-3-8 酚測定.................................................................................................30 3-3-9 酸鹼值 (pH) 測定..............................................................................31 3-3-10 DNA萃取..........................................................................................31 3-3-11 聚合酶連鎖反應...............................................................................32 3-3-12 酵素測定...........................................................................................33 3-3-12-1 酵素活性定義...............................................34 3-3-12-2 氫化酵素萃取...............................................35 3-3-12-2-1 胞內氫化酵素萃取…………………………………...35-3-12-2-2 胞上氫化酵素萃取…………………………………...36 3-3-12-3 氫化酵素活性分析.......................................38 3-3-12-4 漆氧化酶萃取............................................39 3-3-12-5 漆氧化酶活性分析.......................................40 3-3-13 碳源測試...........................................................................................41四章 結果與討論..................................................................................................424-1 菌種鑑定...........................................................................................................424-2 碳源測試...........................................................................................................434-3 以葡萄糖為碳源在不同酚濃度中的產氫測試...............................................47 4-3-1 不同酚濃度中葡萄糖、pH和菌體濃度變化趨勢..............................47 4-3-2 氫氣產率、產氫速率與酚的生物降解.......................................52 4-3-3 代謝物與化學需氧量.........................................................................54-4 以纖維二糖 (Cellobiose) 為碳源並在不同酚濃度中的產氫測試...............58-4-1 纖維二糖的水解、pH值變化及生物產氣..........................................58-4-2 最大產氫量、產氫速度、氫氣產率與化學需氧量.............................63-4-3 揮發性脂肪酸、酵素作用、產氫機制與酚降解…….........................67-4-4 產氫機制.............................................................................................74五章 結論..............................................................................................................76六章 參考文獻......................................................................................................77目錄2-1 有機物厭氧代謝示意圖................................................................................32-2 Clostridium butyricum厭氧發酵代謝途徑…...............................................72-3 漆氧化酶對酚類化合物的反應機制............................................................82-4 中間介質與漆氧化酶的作用機制................................................................92-5 Clostridium屬的代謝酚途徑......................................................................162-6 脫硝細菌之酚厭氧代謝途徑......................................................................172-7 benzoic acid降解途徑..................................................................................182-8 甲烷化方式之酚厭氧代謝..........................................................................193-1 C.butyricum的TEM影像............................................................................203-2 125 ml血清瓶照..........................................................................................223-3 以葡萄醣為基質，DNSA法測得之還原醣標準曲線.................................243-4 以纖維二榶為基質，Anthrone法所測得之總糖標準曲線........................253-5 以BSA為標準，Lowry法測得之蛋白質標準曲線....................................273-6 以GC量測之氫氣檢量線............................................................................283-7 細菌生長階段和收集細胞萃取酵素最好時機..........................................343-8 硫酸銨飽和濃度對於蛋白質析出之影響..................................................364-1 經DNA萃取，PCR放後之電泳結果..........................................................424-2 EcoPlate之碳源分佈...................................................................................454-3 在37℃、150 rpm、葡萄糖為碳源之不同酚濃度的葡萄糖變化趨勢........494-4 在37℃、150 rpm、葡萄糖為碳源之不同酚濃度的pH變化圖..................504-5 在37℃、150 rpm、葡萄糖為碳源之不同酚濃度的菌體濃度變化............514-6 在37℃、150 rpm、葡萄糖為碳源之不同酚濃度C.butyricum的產氫速率圖..............................................................................................................534-7 在37℃、150 rpm、葡萄糖為碳源之不同酚濃度C.butyricum的氫氣產率圖..............................................................................................................534-8 在37℃、150 rpm、葡萄糖為碳源不同時間點之酚濃度化........................544-9 在37℃、150 rpm、葡萄糖為碳源之不同酚濃度COD變化.......................564-10 在37℃、150 rpm、不同酚濃度之纖維二糖水圖........................................594-11 在37℃、150 rpm、不同酚濃度之纖維二糖水解程度................................604-12 在37℃、150 rpm、不同酚濃度之pH值變化趨勢......................................614-13 在37℃、150 rpm、不同酚濃度之生物產氣量 (biogas)圖.........................624-14 在37℃、150 rpm、不同酚濃度之氫氣最大產量........................................634-15 在37℃、150 rpm、不同酚濃度之產氫速度................................................644-16 在37℃、150 rpm、不同酚濃度之氫氣產率................................................644-17 在37℃、150 rpm、不同酚濃度之COD圖...................................................664-18(a) 在37℃、不同酚濃度之氫氣產率氫化酵素氧化路徑活性...................674-18(b) 胞內 (Kameyama et al. 1957) 與胞上 (Schink et al. 1979) 氫化酵素萃取法所作之氫化酵素還原路徑活性……………………….……….684-18(c) 胞內與胞上 (Kow et al. 1984) 氫化酵素萃取法所作之氫化酵素還原路徑活性………………………………………………………………..684-19 在37℃、150 rpm之酚代謝圖......................................................................714-20 在37℃、不同酚濃度下漆氧化酶活性分佈圖…………………………....724-21 Membrane-bound hydrogenase電子傳遞產氫示意圖................................741328634 bytesapplication/pdfen-US氫氣廢水Clostridium butyricumhydrogenwastewater[SDGs]SDG7thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/186957/1/ntu-98-R96524056-1.pdf