目前全球所使用的能源約有80%來自化石燃料。如此大量依賴單一型態的能源利用結構，是主要造成目前全球各種能源相關問題的主要因素。其衍伸的問題擴及經濟與環境層面。開發永續、潔淨的替代能源為各國關注的焦點。當前再生能源中，利用生物系統產氫為一同時具有經濟與環保價值的概念。

生物系統產氫中，厭氧生物產氫程序可以針對各種廢棄有機物，進行能源回收。此為一資源再利用的優勢，極具發展潛力。而針對厭氧生物產氫系統，目前在代謝機制上的分析，仍不明確。本研究試以代謝通量方法評估Clostridium tyrobutyricum 之生物產氫表現。

Clostridium tyrobutyricum 純菌植入連續流攪拌反應器 (Continuous-flow stirred tank reactor, CSTR)，以 12,000 mg/L 葡萄糖作為進流基質，溫度控制於 35℃，所有試程的 pH 皆在 5.95-6.05 間，將反應槽操作在不同水力停留時間 (HRT = 18, 12, 8, 6, 4, 3, 2.5, 2, hr，共 8 個試程)，並收集穩定狀態下的氣體及水質數據。C. tyrobutyricum 的最大產氫速率、比產氫速率與產氫濃度皆是在水力停留時間 2 小時的試程出現最大值，而氫氣產率則是在水力停留時間 18 小時出現最大值，分別為 5.63 mmol H2/L.hour、5.04 (H2mmoL/g dryweight*hr)、80% 和1.42 mol H2/mol-glu-applied 。
 

由碳平衡、COD、O/R、NADH流量四種平衡方式，確立細胞外部最終產物所表現方程式的正確性。碳平衡和COD平衡，其回收率約±10%之間。O/R值整體回收率約只能達到55%，有可能是因為部分產物無法計算，導致誤差放大；另外整體的NADH平衡則有97%的回收率。

另外利用CellNetAnalyzer 評估細胞內部中間產物的物質與能量流向，最後依據整體計量，建構整體系統的代謝路徑。利用代謝路徑計算ATP的生成和消耗後發現，計量的ATP確有不足的現象，並且經由與H2的yidld的比較，發現H2的yidld與計量的ATP不足成一高度正相關。而此一現象與產氫不利於熱力學相關。

80% of global utilized energy comes from fossil fuels, and the energy shortage mainly comes from this dependence. Since the energy issue affects both economy and environment, the governments paid much attention on finding a clean and Sustainability energy. Hydrogen is considered as a clean and efficient energy among renewable energies.

Hydrogen can be produced from organic materials by anaerobic microorganism but the mechanism of fermentative hydrogen production is not clear. Thus, our research will focus on evaluation of fermentative biohydrogen with metabolic flux consideration by Clostridium tyrobutyricum.

In our study, the influent substrates were 12,000 mg/L glucose, temperature was maintained at 35℃, and pH was controlled at 6±0.05 by computer. The reactor was operated at different hydraulic retention times (HRT 18, 12, 8, 6, 4, 3, 2.5, 2 hr), and we collected gas production and water quality data under steady state at each HRT. Our result shows that maximum hydrogen production rate, maximum specific hydrogen production rate and maximum hydrogen concentration, 5.63 mmol H2/L*hr, 5.04 (H2mmoL/g dry weight*hr) and 80%, were all occurred at HRT 2 hr. However, maximum hydrogen yield, 1.42 mol H2/mol-glucose-applied, was observed at HRT 18 hr.

In addition, we used 4 balance systems, e.g. carbon balance, COD, O/R value and NADH, to evaluate the end products. The experimental errors of carbon and COD recovery rates were ±10% respectively. The recovery rate of O/R value was only 55%, because a meager part of end products could not be included in this calculation, the error was enlarged. Besides, the average of NADH balance was 97%.

Finally, we used CellNetAnalyzer to evaluate the rates of metabolic pathways at each HRT. The difference between produced and utilized ATP shows that ATP is shortage in the cell and there was a relationship between the values of shortage ATP and H2. This situation had a relationship with hydrogen yield which is unfavorable based on thermodynamic analysis.

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