氫氣被視為下一世代的能源，是目前最理想的清潔燃料之一，因此產氫之技術成為一大研究領域。近年環保意識抬頭，環境汙染問題也是一大課題。產氫菌發酵系統不僅可以處理農工業之廢水，還能夠產生氫氣作為能量來源，是許多科學家研究之議題，然而因發酵系統為複雜之微生物反應，為了解並發展該技術需耗費大量時間、人力及資源。本研究期望發展縮小化之培養系統，使技術可同步測試多組參數。本研究利用連續式產氫發酵系統作為實驗之菌源(初始菌種：Clostridium butyricum CGS5、Clostridium pasteurianum CH4、Klebsiella sp. HE1)，以不同方式去追求縮小之發酵系統。實驗中證明可將油品覆蓋在12孔盤中的在培養基上幫助阻絕空氣中之氧氣，而溶液中之溶氧可透過添加還原劑於培養基中來消除，也可依靠混菌中兼性菌的代謝消耗液體內溶氧，但無添加還原劑之系統需更長的培養時間系統才能逐漸趨向厭氧狀態。本研究也用PDMS建構密閉式縮小型發酵槽，並可在其中培養產氫之混菌，且不需覆蓋油品或於培養基中添加還原劑，但培養時間需拉長至10天以上。以12孔盤和PDMS縮小型發酵槽做比較，密閉式培養系統相較之下，其優點在於可蒐集氣體產物，但培養體積小所以氣體產量少，影響數據可信度，因此需要搭配微型氫氣感測器做即時偵測，方能有潛力取代實驗室規模之實驗。

Hydrogen has been regarded as the fuel of the next generation due to its usefulness as a compact energy source in fuel cells and batteries. It has been discovered that hydrogen can be generated through natural biological processes. However, high production cost is the major challenge for the commercialization of biohydrogen. Scientists try to solve the problem by conducting numerous lab-scale experiments, which are high-cost, labor-intensive, and space-taking. In this study, we aim to reduce the cost of experiments by developing miniature culture system which can provide valuable information for optimizing larger scale experiments.

In this study, a 7 ml fermenter made by PDMS was tested with semi-batch cultivations. HPLC and GC were applied to evaluate the metabolites of a mixed culture bacteria. The results show that the air-tight PDMS device can successfully cultivate mixed culture bacteria of Clostridium butyricum CGS5, Clostridium pasteurianum CH4, and Klebsiella sp. HE1. Metabolites including 1.8 g/l acetate and 1.43 g/l butyrate were measured to be the main products of the system after 10 days of cultivation. However, the amount of gas sample was small and hard to collect, which made the quantification easily compromised by leakage. Therefore, we try to quantify hydrogen in real time by palladium nanoparticles coated with temperature-sensitive luminophores, EuTTA. Unfortunately, the results show no obvious relationship between hydrogen partial pressure and luminescence strength.

摘要 I
Extended Abstract II
致謝 X
目錄 XI
表目錄 XIII
圖目錄 XIV
第一章 緒論 1
1-1 研究動機與目的 1
1-2 實驗架構 2
第二章 文獻回顧 3
2-1 傳統產氫技術 3
2-2 生物產氫技術 5
2-3生物產氫技術挑戰 11
2-4 增進產氫效率之策略 13
2-5 微小化培養系統之需求 16
第三章 實驗材料與方法 18
3-1 連續式產氫發酵系統 18
3-1-1 實驗材料 18
3-1-2 實驗方法 20
3-2 微流道包菌 24
3-2-1 實驗材料 24
3-2-2 實驗方法 27
3-3 12孔盤培養 34
3-3-1 實驗材料 34
3-3-2 實驗方法 35
3-4 縮小型發酵槽培養 36
3-4-1 實驗材料 36
3-4-2 實驗方法 36
3-5 微型氫氣感測器 39
3-5-1 實驗材料 39
3-5-2 實驗方法 41
第四章 結果與討論 43
4-1 連續式產氫發酵系統 43
4-2 固定化細菌培養 47
4-2-1 微流道裝置生成包菌粒子 47
4-2-2 微流道凹槽固定化 49
4-2-3 高分子膠體固定化 51
4-3 12孔盤培養 56
4-3-1 培養時間 56
4-3-2 彌封油品 60
4-3-3 還原劑影響 65
4-4 縮小型發酵槽培養 67
4-5 微型氫氣感測器 70
4-5-1 製造鈀奈米粒子之結果 70
4-5-2 鈀奈米粒子附加螢光染劑感測氫氣 75
第五章 結論 80
參考文獻 81

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