臺灣博碩士論文加值系統

摘 要
產氫因為具有潔淨無污染、可生生不息地循環使用及高能源轉換效率等三大特點，被認為是21世紀最有發展前景的替代能源之一。過去本實驗室所開發之厭氧顆粒污泥床(anaerobic granular sludge bed, AGSB)生物反應器可有效促進產氫菌之滯留而有利於低HRT (hydraulic retention time)之操作，因此產氫速率明顯優於傳統的CSTR (continuous flow stirred tank reactor)反應器。有鑑於此，本研究以蔗糖為碳源，進一步探討基質濃度及微量金屬對AGSB系統醱酵產氫之影響，以期提升該系統之產氫效能。
在基質濃度方面之探討，分別進行四種不同蔗糖濃度(20-35 g-COD/L)操作於各種不同HRT (4-1 h)之醱酵產氫試驗，藉以探討基質濃度及有機負荷(organic loading rate, OLR)對AGSB醱酵產氫之影響。結果顯示，當各種不同基質濃度之試程操作於HRT≧1 h時(OLR≦40 g-COD/L/h)，產氫速率隨基質濃度增加而增加，顯示並未有基質抑制的情形，而各試程操作於HRT = 0.5 h時 (OLR > 40 g-COD/L/h)，產氫速率並未隨基質濃度增加而增加，反而呈現表現相當的情形，推測系統之OLR的臨界點為40 g-COD/L/h。在各項試程中，當系統操作於基質濃度20 g-COD/L、HRT = 1 h (OLR = 20 g-COD/L/h)時，可獲最大氫氣產率3.75 mol-H2/mol-sucrose；而當基質濃度為35 g-COD/L、HRT = 0.5 h (OLR = 70 g-COD/L/h)時，可得最佳產氫速率8.53 L/h/L。關於溶解態代謝產物方面，各試程均顯示主產丁酸，其次為丁醇和乙酸，然而當系統操作於高OLR或高基質濃度之條件時，丁醇的含量有偏高的趨勢，若能進一步降低丁醇的含量，應有助於產氫效能的提升。
由於本實驗室過去的研究顯示AGSB之高徑比偏高易形成氣塊(slugging)現象而使顆粒污泥浮舉(flotation)，造成系統操作的不穩定，為改善此現象，本研究於高徑比12之床體中加裝攪拌裝置，企圖藉此促進生物氣體之釋放並增進系統之混合效果，實驗以蔗糖濃度20 g-COD/L進行測試，結果顯示系統操作於高HRT (4 & 2 h)時，產氫速率並未明顯提升，然而當系統操作於HRT = 0.5 h時，由於氣塊現象明顯改善，系統可穩態操作達20天，平均產氫速率及氫氣產率分別高達9.72 L/h/L及3.89 mol-H2/mol-sucrose，顯示攪拌裝置可有效提升AGSB之產氫效能。
在微量金屬方面之探討，本研究以回應曲面法(response surface methodology, RSM)企圖尋求最佳的微量金屬配方；依據文獻報告，本研究選擇醱酵產氫之重要因子鎂、鐵、鋅進行實驗(蔗糖濃度20 g-COD/L, HRT = 2 h)。結果顯示，當MgCl2•6H2O = 150 mg/L、FeSO4•7H2O = 30 mg/L、ZnCl2 = 20 mg/L時可獲最佳產氫速率及氫氣產率分別為2.23 L/h/L及3.62 mol-H2/mol-sucrose，然而以產氫速率及氫氣產率為反應變數進行反應曲面分析時呈現鞍點(saddle point)的現象，此需進行更進一步的脊線分析(ridge analysis)以求得局部最佳條件。

Abstract
For the past decades, the consumption rate of fossil fuels exponentially increased, leading to a visible crisis of the shortage of energy source in the near future. Therefore, searching new and effective alternative energy sources has become a critical issue. Among the energy alternatives being considered, hydrogen displays its unique advantages of being clean, sustainable, and efficient, and has been considered an ideal energy carrier in the future. Production of hydrogen from biological means, especially from anaerobic fermentative processes, has a great potential to become a cheap, clean and sustainable energy source to fossil duels, and has caught marked attention all over the world. As a result, tremendous research efforts have been devoted to the development of highly efficient and cost-effective biohydrogen production systems.
Our recent work showed that the anaerobic granular sludge bed (AGSB) is an excellent biohydrogen producing system. The AGSB system could effectively facilitate the biomass retention at low HRT (hydraulic retention time), resulting in a much higher hydrogen production efficiency than that of CSTR (continuous flow stirred tank reactor) systems. In order to further improve the performance of AGSB system, this study aimed to investigate the effect of substrate (sucrose) concentration and composition of trace elements on the hydrogen-producing efficiency of the AGSB system.
For the study associated with different substrate concentration, the AGSB bioreactor was conducted at different HRT (0.5-4 h) and substrate concentration (20-35 g-COD/L) to investigate the effect of substrate concentration and organic loading rate (OLR) on hydrogen production. The results show that the hydrogen production rate increased with substrate concentration when HRT ≧ 1 h (i.e., OLR ≦ 40 g-COD/L/h), indicating that substrate inhibition did not occur for the substrate concentration used. However, when the system was operated at HRT=0.5 h (i.e., OLR > 40 g-COD/L/h), the hydrogen production did not increase with substrate concentration, suggesting the presence of a critical OLR of 40 g-COD/L/h for the AGSB system. The highest hydrogen production rate of 8.53 L/h/L was achieved at an HRT of 0.5 h and a sucrose concentration of 35 g-COD/L (i.e., OLR=70 g-COD/L/h), while the best hydrogen yield (3.75 mol-H2/mol-sucrose) was obtained at HRT=1 h and sucrose concentration of 20 g-COD/L (i.e. OLR=20 g-COD/L/h). The soluble metabolites were dominated by butyric acid, followed by butanol and acetic acid. However, when the system was operated at high OLR or high substrate concentration, butanol production tended to increase. Hence, the performance might be improved if the production of butanol can be limited to a lower level.
On the other hand, the slugging phenomena occurred when a high height to diameter (H/D) ratio was designed for the AGSB system, causing floatation of granular sludge and lowering the operational stability. To avoid this problem, a mechanical agitation device was supplemented into the AGSB reactor with a H/D ratio of 12 to enhance the mixing efficiency. The results show that when the reactor was operated at high HRT (2 and 4 h), there was limited improvement in hydrogen production. However, a significant improvement was observed when the system was carried out at a HRT of 0.5, as the steady-state operation can be maintained for 20 days and the average hydrogen production rate and hydrogen yield boosted to 9.72 L/h/L及3.89 mol-H2/mol-sucrose, respectively. This indicates that implementation of mechanical agitation could marked enhance the hydrogen producing efficiency of the AGSB system.
As for the investigation regarding the impact of trace element composition on hydrogen production in AGSB, a response surface methodology (RSM) was applied to identify the optimal formulation of trace elements. The target trace elements used for this study were selected according to the literature survey. With a fixed sucrose concentration of 20 g-COD/L and HRT of 2 h, the best hydrogen production rate (2.23 L/h/L) and hydrogen yield (3.62 mol-H2/mol-sucrose) were attained when MgCl2•6H2O = 150 mg/L、FeSO4•7H2O = 30 mg/L、ZnCl2 = 20 mg/L. The experimental results showed that a highest hydrogen production rate of 2.23 L/h/L was achieved at an HRT of 2 h when concentration of MgCl2•6H2O, FeSO4•7H2O, ZnCl2 were kept at 150 mg/L, 30 mg/L, 20 mg/L. However, the RSM analysis showed a saddle point behavior, thereby requiring further ridge analysis to identify the local optimum values.

目 錄
摘要.................................................................................................................................Ⅰ
Abstract………………………………………………………………………................Ⅲ
目錄………………………………………………………………………………...…. VI
表目錄………………………………………………………………………………….Ⅹ
圖目錄………………………………………………………………………………….XI


第一章 緒論....................................................................................................................1
1-1 研究動機..................................................................................................................1
1-2 研究目的..................................................................................................................2
第二章 文獻回顧及原理……………….……………………………………….……..3
2-1 文獻回顧……………………………………..……………………………….…...3
2-2 生物氫氣的價值及利用…………………..………..……………………………..4
2-3 製氫氣的方法………………………………..………………..……………….….5
2-3-1 熱化學法……………………………………………………………………...5
2-3-2 電化學法……………………………..…..……………………………….......5
2-3-3 生物法…………………………………...………………………….…….......6
2-4 細菌的生長與生長曲線………………..………………………………….…….12
2-5 影響細胞生長的條件……………..……………..………………………….…...14
2-5-1 溫度..…………………………….……………………………………..……14
2-5-2 酸鹼度(pH値)……………….………………..…………………………......16
2-5-3 溼度……..………………………………………..…………………….……16
2-5-4 氧氣……………..……………………………………..……………….……17
2-5-5 攪拌之影響………………………………………………………………….18
2-5-6 有機物負荷之影響………………………………………………………….19
2-5-7 無機營養鹽之影響………………………………………………………….19
2-6 內孢子菌……………..…………………………………………..………………20
2-6-1 激活……..……………………………………..……………………….……21
2-6-2 萌發孢子…..…………………………………..……………………….……21
2-6-3 生出菌體…..…………………………………..……………………….……21
2-6-4 芽孢桿菌屬(Bacillus)………………………………………………….……23
2-6-5 梭孢桿菌屬(Clostridium)…………..……………………………….…........24
2-7 氧化-還原的平衡…..……………..………………………………………….......27
2-8 回應曲面設計法(簡稱RSM )原理……………………………………………...29
2-9 活性污泥添加活性炭的特點................................................................................34
第三章 實驗材料及方法………………………………………………………..……35
3-1 藥品試劑與培養基………..………………………..……………………………35
3-1-1 碳源…………………………………………..…………..…………….……35
3-1-2 緩衝鹽類………………………………...………..………..…………….….35
3-1-3 無機鹽類………………………………...…………..……..………………..35
3-1-4 其它…………………………………..……………………..…………….…35
3-2 培養基濃度………………………………..…………………..…………….…...36
3-2-1 不同基質濃度的配方……………………………………………………….36
3-2-2 不同試程的微量金屬配方………………………………………………….36
3-3 污泥來源………………………………..………………………….……….……38
3-4 分析儀器及方法……………………………………...……..…..………….……38
3-4-1 氣體組成分析………………………………...……………....…….……….38
3-4-2 液體組成分析………………………………………...…..…..……….…….39
3-4-3 菌量分析……………………………………...…………..…..…….……….39
3-4-4 總糖定量………………………………………….…………..…….……….40
3-5 高解析可變真空掃描式電子顯微鏡(VVSEM)觀察樣本之製備………...…....40
3-6 氫氣產率…………………………………………………………………………41
3-7 連續式產氫實驗儀器裝置與方法………………………………….....………...41
3-7-1 實驗儀器……………………….……………………………………………41
3-7-2 厭氧顆粒污泥床(anaerobic granular sludge bed, AGSB)生物反應器連續式
實驗操作步驟……………………………………….....……………………42
3-7-2-1 不同HRT之連續醱酵產氫(基質濃度20 g-COD/L)…..…….42
3-7-2-2不同進料濃度之連續產氫實驗……………………………..42
3-7-2-3改變不同無機鹽濃度之連續產氫實驗…………………….42
3-7-2-4添加攪拌葉片之連續產氫實驗……………………………..43
第四章 第四章結果與討論……………………………………………….…….……46
4-1 不同基質濃度對產氫之影響………………………….…..….…………………46
4-1-1 蔗糖濃度20 g-COD/L之醱酵產氫行為.......................................................46
4-1-2 蔗糖濃度25 g-COD/L之醱酵產氫行為.......................................................46
4-1-2-1 粒徑分布……………………………………………………………….47
4-1-3 蔗糖濃度30 g-COD/L之醱酵產氫行為.......................................................47
4-1-4 蔗糖濃度35 g-COD/L之醱酵產氫行為.......................................................47
4-1-5 基質濃度對顆粒污泥形成及產氫之效應.....................................................48
4-1-6 溶解態代謝產物分析、基質利用率與氫氣產率比較...................................48
4-1-7 掃瞄式電子顯微鏡(VVSEM)菌相觀察……………………………………48
4-1-8 總結………………………………………………………………………….48
4-2 利用回應曲面設計法尋找AGSB系統最佳無機鹽配方....................................67
4-2-1 試程15-(0,0,0)之醱酵產氫行為....................................................................67
4-2-2 試程16-(0,0,0)之醱酵產氫行為……………………………………………67
4-2-3 試程17-(0,0,0)之醱酵產氫行為……………………………………………67
4-2-4 試程(-1,-1,-1)之醱酵產氫行為......................................................................68
4-2-5 試程(1,-1,-1)之醱酵產氫行為.......................................................................68
4-2-6 試程(-1,1,-1)之醱酵產氫行為……………………………………………...68
4-2-7 試程(1,1,-1)之醱酵產氫行為……………………………………………….68
4-2-8 試程(-1,-1,1)之醱酵產氫行為……………………………………………...69
4-2-9 試程(1,-1,1)之醱酵產氫行為……………………………………………….69
4-2-10 試程(-1,1,1)之醱酵產氫行為……………………………………………...69
4-2-11 試程(1,1,1)之醱酵產氫行為………………………………………………69
4-2-12 試程(2,0,0)之醱酵產氫行為………………………………………………70
4-2-13 試程(-2,0,0)之醱酵產氫行為……………………………………………...70
4-2-14 試程(0,2,0)之醱酵產氫行為………………………………………………70
4-2-15 試程(0,-2,0)之醱酵產氫行為……………………………………………..70
4-2-16 試程(0,0,2)之醱酵產氫行為…………………………..………………….71
4-2-17 試程(0,0,-2)之醱酵產氫行為……………………………………………..71
4-2-18 不同無機鹽試程之產氫速率與氫氣產率綜何比較……………………...71
4-2-19 利用電腦程式做出最佳的統計分析……………………………………...71
4-2-20 溶解態代謝產物分析……………………………………………………...72
4-2-21 掃瞄式電子顯微鏡(VVSEM)菌相觀察…………………………………..72
4-3 加裝攪拌葉片以增進AGSB系統效能………………………………………..104
4-3-1 加裝攪拌葉片的AGSB系統之連續產氫操作…………………………...104
4-3-2 溶解態代謝產物分析、基質利用率與氫氣產率比較……………………104
4-3-3 掃瞄式電子顯微鏡(VVSEM)菌相觀察…………………………………..104
第五章 結論……………………………………………………….………….…......109
參考文獻………………………………………………………………….…………..110








表 目 錄
表2-1 厭氧醱酵產氫之文獻報告評比……………………………………………......3
表2-2 產氫微生物的種類Broad classification………………………………………...7
表2-3 微生物產氫過程的優點和缺點…………………………………………….......8
表2-4 細菌生長溫度範圍……………………………………………………….……15
表2-5 各種細菌的營養型別……………………………………………………….…19
表2-6 產生內孢子的細菌屬……………………………………………………….…23
表2-7 芽孢桿菌屬代表種的特徵……………………………………………….……24
表2-8 部份梭孢桿菌屬的特徵…………………………………………………….…25表2-9 22因子設計實例………………………………………………………………30
表2-10 2n-1部分因子設計表…………………………………………………………...32
表2-11 變異數分析表…………………………………………………………………33
表3-1 Endo厭氧醱酵配方…………………………………………………….…….36
表3-2 不同試程的微量金屬配方之一……………………………………………….37
表3-3 不同試程的微量金屬配方之二……………………………………………….37
表3-4 不同試程的微量金屬配方之三……………………………………………….38
表4-1 各試程之溶解態代謝物組成………………………………………………….55
表4-2 各種不同濃度於各項HRT試程之產氫效能評比…………………………….56
表4-3 不同微量金屬試程之產氫速率與氫氣產率綜何比較……………………...102
表4-4 各試程之溶解態代謝物組成………………………………………………...104
表4-5 各項HRT試程之產氫效能評比……………………………………………...108
表4-6 各試程之溶解態代謝物組成………………………………………………...108






圖 目 錄
圖2-1 光合作用產氫之代謝途徑圖..............................................................................9
圖2-2 傳統厭氧消化流程圖…………………………………………..……………...11
圖2-3 厭氧氫氣醱酵代謝途徑簡圖…………………………………………..……...13
圖2-4 細菌細胞的生長曲線與總細胞濃度曲線.........................................................12
圖2-5 相同消化程度下消化溫度與消化時間之關係……………………….………16
圖2-6 pH値的範圍。pH 7.0開始向上斜線指示嗜酸性，向下斜線指示嗜鹼性….16
圖2-7 四種細菌在深層瓊脂培養管中之生長分布情形…………………….………17
圖2-8 細胞內孢子之形成……………………………………………………….……21
圖2-9 顯示不同內孢子著生位置的各種梭孢桿菌(Clostridium)……………..……..23
圖2-10 梭孢桿菌丁酸群形成醱酵產生的途徑。“2H”代表來自一分子NADH的兩
個電子………………………………………………….……………………..26
圖2-11 生孢梭孢桿菌(Clostridium sporogenes)體內丙氨酸與甘氨酸間的氧化還原
偶合反應(史提克蘭德反應，Stickland reaction)……………..………………27
圖2-12 從丙酮酸產生氫分子的情形…………………………………………….…..28
圖2-13 實施RSM設計的步驟流程圖……………………………………………….29
圖3-1 AGSB連續式產氫反應器裝置圖……………………………………….…..44
圖3-2 加裝攪拌葉片的AGSB連續式產氫反應器裝置圖…………………………45
圖4-1 蔗糖濃度20 g-COD l-1之AGSB系統醱酵產氫行為……….………………50
圖4-2 蔗糖濃度25 g-COD l-1之AGSB系統醱酵產氫行為……….………………51
圖4-3 蔗糖濃度30 g-COD l-1之AGSB系統醱酵產氫行為……………………….52
圖4-4 蔗糖濃度35 g-COD l-1之AGSB系統醱酵產氫行為……………………….53
圖4-5 基質濃度與水力停留時間對AGSB系統醱酵產氫之影響…………………54圖4-6a 20 g-COD/L，HRT=4 h時VVSEM的菌相圖……………………………..57
圖4-6b 20 g-COD/L，HRT=2 h時VVSEM的菌相圖…………………………….57
圖4-6c 20 g-COD/L，HRT=1 h時VVSEM的菌相圖……………………………..58
圖4-6d 20 g-COD/L，HRT=0.5 h時VVSEM的菌相圖…………………………..58
圖4-7a 25 g-COD/L，HRT=4 h時VVSEM的菌相圖…………………………….59
圖4-7b 25 g-COD/L，HRT=2 h時VVSEM的菌相圖…………………………….59
圖4-7c 25 g-COD/L，HRT=1 h時VVSEM的菌相圖……………………………...60
圖4-7d 25 g-COD/L，HRT=0.5 h時VVSEM的菌相圖…………………………...60
圖4-8a 30 g-COD/L，HRT=4 h時VVSEM的菌相圖……………………………..61
圖4-8b 30 g-COD/L，HRT=2 h時VVSEM的菌相圖……………………………..61
圖4-8c 30 g-COD/L，HRT=1 h時VVSEM的菌相圖……………………………..62
圖4-8d 30 g-COD/L，HRT=0.5 h時VVSEM的菌相圖…………………………..62
圖4-9a 35 g-COD/L，HRT=4 h時VVSEM的菌相圖……………………………..63
圖4-9b 35 g-COD/L，HRT=2 h時VVSEM的菌相圖…………………………….63
圖4-9c 35 g-COD/L，HRT=1 h時VVSEM的菌相圖……………………………..64
圖4-9d 35 g-COD/L，HRT=0.5 h時VVSEM的菌相圖……………………...........64
圖4-10 有機負荷速率對AGSB系統醱酵產氫之影響…………………………….65
圖4-11 黃色透明顆粒菌的重量分布情形…………………………………………..66
圖4-12 黃色顆粒菌的重量分布情形……………………………………………….66
圖4-13 試程15-(0,0,0)之AGSB系統醱酵產氫行為……………………………….73
圖4-14 試程16-(0,0,0)之AGSB系統醱酵產氫行為……………………………….74
圖4-15 試程17-(0,0,0)之AGSB系統醱酵產氫行為……………………………….75
圖4-16 試程(-1,-1,-1)之AGSB系統醱酵產氫行為………………………………...76
圖4-17 試程(1,-1,-1)之AGSB系統醱酵產氫行為………………………………....77
圖4-18 試程(-1,1,-1)之AGSB系統醱酵產氫行為…………………………………78
圖4-19 試程(1,1,-1)之AGSB系統醱酵產氫行為…………………………………..79
圖4-20 試程(-1,-1,1)之AGSB系統醱酵產氫行為…………………………………80
圖4-21 試程(1,-1,1)之AGSB系統醱酵產氫行為…………………………………..81
圖4-22 試程(-1,1,1)之AGSB系統醱酵產氫行為…………………………………..82
圖4-23 試程(1,1,1)之AGSB系統醱酵產氫行為…………………………………...83
圖4-24 試程(2,0,0)之AGSB系統醱酵產氫行為…………………………………...84
圖4-25 試程(2,0,0)之AGSB系統醱酵產氫行為…………………………………...85
圖4-26 試程(2,0,0)之AGSB系統醱酵產氫行為…………………………………...86
圖4-27 試程(2,0,0)之AGSB系統醱酵產氫行為…………………………………..87
圖4-28 試程(0,0,2)之AGSB系統醱酵產氫行為…………………………………..88
圖4-29 試程(2,0,0)之AGSB系統醱酵產氫行為…………………………………...89
圖4-30 FeSO4•7H2O和MgCl2•6H2O對產氫速率之3D立體圖...............................90
圖4-31 FeSO4•7H2O和ZnCl2對產氫速率之3D立體圖.........................................90
圖4-32 MgCl2•6H2O和ZnCl2對產氫速率之3D立體圖...........................................91
圖4-33 FeSO4•7H2O和MgCl2•6H2O對氫氣產率之3D立體圖.................................91
圖4-34 FeSO4•7H2O和ZnCl2對氫氣產率之3D立體圖...........................................92
圖4-35 ZnCl2和MgCl2•6H2O對氫氣產率之3D立體圖..........................................92
圖4-36 試程15-(0,0,0)之VVSEM的菌相圖………………………………………..93
圖4-37 試程16-(0,0,0)之VVSEM的菌相圖………………………………………..93
圖4-38 試程17-(0,0,0)之VVSEM的菌相圖………………………………………..94
圖4-39 試程(-1,-1,-1)之VVSEM的菌相圖…………………………………………94
圖4-40 試程(1,-1,-1)之VVSEM的菌相圖…………………………………….........95
圖4-41 試程(-1,1,-1)之VVSEM的菌相圖…………………………………….........95
圖4-42 試程(1,1,-1)之VVSEM的菌相圖…………………………………………..96
圖4-43 試程(-1,-1,1)之VVSEM的菌相圖………………………………………….96
圖4-44 試程(1,-1,1)之VVSEM的菌相圖…………………………………………..97
圖4-45 試程(-1,1,1)之VVSEM的菌相圖…………………………………………..97
圖4-46 試程(1,1,1)之VVSEM的菌相圖……………………………………………98
圖4-47 試程(2,0,0)之VVSEM的菌相圖……………………………………………98
圖4-48 試程(-2,0,0)之VVSEM的菌相圖…………………………………………..99
圖4-49 試程(0,2,0)之VVSEM的菌相圖…………………………………………....99
圖4-50 試程(0,-2,0)之VVSEM的菌相圖…………………………………………100
圖4-51 試程(0,0,2)之VVSEM的菌相圖…………………………………………..100
圖4-52 試程(0,0,-2)之VVSEM的菌相圖…………………………………………101
圖4-53 加裝攪拌葉片的AGSB系統醱酵產氫行為………………………………105
圖4-54 HRT=4 h時VVSEM的菌相圖……………………………………………106
圖4-55 HRT=2 h時VVSEM的菌相圖……………………………………………106
圖4-56 HRT=1 h時VVSEM的菌相圖……………………………………………107
圖4-57 HRT=0.5 h時VVSEM的菌相圖………………………………………….107

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