化石燃料的大量使用造成全球暖化與各種能源問題，因此尋找經濟、環保與可永續利用的替代能源日趨重要。多種再生能源中，厭氧生物產氫可以針對各種廢棄有機物進行能源再生與資源再利用，為一同時具有經濟與環保價值的再生能源，極具發展潛力。
先前之研究以生質酒精廢液做為基質進行生化產氫試驗，發現試驗之菌群具有兩階段式降解產氫之現象：麥芽糖降解生成乳酸、乙酸及其他有機酸，而氫氣則伴隨著乳酸與乙酸濃度的下降生成。許多文獻中已提及乳酸與乙酸降解生成氫氣的代謝反應，然而尚未有以乳酸乙酸作為基質之氫氣研究被提出，此產氫機制也尚未透過實驗證實，僅有部分研究以批次實驗觀察產氫現象，未有以連續流生物反應器進行完整之產氫效能評估。
因此，此研究設計連續流生物反應器，以乳酸與乙酸優勢化產氫菌群進行產氫試驗並評估其產氫效能。五個反應槽試驗操作在pH = 6，溫度為35°C ，以基質濃度15000 mg/L乳酸與45000 mg/L乙酸進行HRT = 23、18、12 hrs，基質濃度為30000 mg/L乳酸與9000 mg/L乙酸進行HRT=18、12 hrs產氫試驗。各試程主要的代謝產物為丁酸，提供乳酸與乙酸濃度分別為30000/9000 mg/L，操作在HRT = 18hrs時，有最大的濃度40%與最大的氫氣產率0.42 mol-H2/mol-HLa。評估電子流向，此試程有最少電子利用於微生物生成，而最多的電子利用於氫氣的生成。各試程微生物與氫氣的電子分配呈負相關。
進行批次實驗，探討pH、起始微生物濃度(X0)與S0/X0對產氫之影響。結果顯示pH = 5.5有最大產率與產氫速率，在三個不同S0/X0的條件下， pH = 6在氫氣產率與產氫速率皆優於 pH = 7的產氫表現。起始微生物濃度越高 (X0=1500 mg-VSS/L)有越小的比產氫速率和較短的lag phase，但不同X0在相同基質條件下氫氣產率沒有明顯差異。另一個S0/X0的批次結果則顯示，隨著初始微生物濃度的提升 (X0 = 300-1700 mg-VSS/L)，較多的電子被利用於氫氣生成 (6.4%-10.4%)，而較少電子被利用於微生物生成 (9.6%-1.5%)。推測在無高濃度基質抑制產氫的情況下，當提供的初始微生物濃度達一足夠值，微生物傾向於生成氫氣而不傾向於生成微生物。
利用功能性基因選殖、定序與限制片段長度多型性分析等分子生物技術分析優勢化微生物族群。結果顯示以乳酸與乙酸馴養之微生物族群中，優勢產氫菌為Clostridium tyrobutyricum。連續流與批次產氫試驗之代謝路徑與C. acetobutylicum相似，以乳酸與乙酸做為基質生成氫氣、二氧化碳、丁酸，乙酸可能為反應中的電子接受者。

The importance of renewable energy sources increases as the issues of fossil fuel exhaustion and global warming become serious. Considering our dependence on energy and the global environment, there is an urgent need in developing a clean and renewable energy source such as hydrogen. In our previous study, wasted residues from a bioethanol fermentation process were found to contain large amount of volatile fatty acids including lactate and acetate. In this study, a continuous-flow stirred tank reactor (CSTR) fed with lactate and acetate was operated to enrich hydrogen-producing bacteria, and the biohydrogen production from utilizing lactate and acetate was evaluated.
The CSTR was maintained at pH 6 and 35°C, and three hydraulic retention time (HRT= 23, 18 and 12 hrs) and two substrate concentration (HLa/HAc = 15000/4500 mg/L, 30000/9000 mg/L) were employed during this study. At 30000 mg/L of lactate and 9000 mg/L of acetate in the influent feed, a stable biohydrogen yield was attained at 0.42 mmol-H2/mmol-HLa at 18 hrs of HRT, with a hydrogen composition of 40%. In addition to hydrogen, the major metabolite in the CSTR from lactate and acetate consumption was butyrate. Batch experiments were conducted to evaluate fermentative biohydrogen production potential under different pH (5-6.5) and initial substrate-to-biomass (S0/X0=6.7-100) conditions using enriched cultures. The results indicated that pH 5.5 was optimum for achieving the highest hydrogen production rate and yield simultaneously, and pH 6 has higher hydrogen performance under 3 different S0/X0. Furthermore, a higher X0 (lower S0/X0) condition achieved a lower specific hydrogen production rate (SHPR) and a lower lag phase, but the hydrogen yields were similar. Moreover, another batch tests showed that when X0 were from 300 to 1700 mg-VSS/L, the hydrogen yield increased and the biomass yield decreased, in addition, the electron distribution fractions of hydrogen (6.4%-10.4%) and biomass (9.6%-1.5%) also had the same trend.
Finally, molecular methods including cloning/sequencing and restriction fragment length polymorphism (RFLP) were performed to investigate microbial ecology of lactate/acetate consuming and hydrogen-producing bacteria enriched in the CSTR. The results confirmed that Clostridium tyrobutyricum was the dominant hydrogen producing bacteria enriched in the CSTR. The pathway of hydrogen production in this study is similar with the metabolic pathway of C. acetoutylicum.

任維傑(2006)，「探討厭氧產氫純菌Clostridium在不同pH 下之反應動力機制」， 國立成功大學環境工程研究所碩士論文。
林哲安(2010)「以代謝通量方法評估 Clostridium tyrobutyricum 之生物產氫表現」，國立成功大學環境工程研究所碩士論文。
莊崇柏(2009)「利用生質酒精發酵殘渣產氫程序之研究」，國立成功大學環境工程研究所碩士論文。
劉怡君(2006)「Clostridium tyrobutyricum 在不同水力停留時間下之代謝表現與產氫行為之研究」，國立成功大學環境工程研究所碩士論文。
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