氫能源為未來替代能源的重要一環，利用微生物進行廢水或廢棄物中有機物的氫發酵，為生物處理回收能源的新趨勢，廢水一般含複雜成分(如碳水化合物及蛋白質)，且各具不同氫發酵特性，本研究探討厭氧氫發酵生物反應器於不同水力停留時間(HRT)下，表現的氫氣生成情形、微生物生長及複合基質所扮演角色，並以模式進行動力探討，及以分子生物學角度切入微生物生態的變遷，期望作為未來整體程序設計上參考依據。
　　本研究植種污泥來自食品廠處理廢水UASB污泥，經熱處理並批次馴養，植入連續流攪拌反應器(Continuous-Flow Stirred Tank Reactor, CSTR)，以20,000 mg/L葡萄糖及水解蛋白質(3：2(w/w))作為基質，溫度控制於35℃，進行七試程水力停留時間分別為18、12、8、6、4、2及1.45小時。水力停留時間長於1.45小時的試程，產氫表現及微生物生長皆可維持一穩定狀態。當水力停留時間為2小時，程序表現出最佳氫氣生成速率1,247 mmol H2/L/day，比產氫速率9.5 L H2/g VSS/day及氫氣產率(hydrogen yield)5.0 mmol H2/g-CODapplied。
　　在不同水力停留時間下，葡萄糖主要利用於產氫且可降解完全，蛋白質所扮演的角色則不盡相同，在較長的水力停留時間下，蛋白質可被利用於發酵及進行生物體合成，隨著水力停留時間縮短，其作用主要轉為生物質體合成用，或仍以有機氮的型式存在，只有較少部分被代謝至氨氮型式。產物主要以丁酸、乙酸甲酸及氨氮為主，隨著水力停留時間與負荷的改變，微生物代謝途徑有移轉的現象。利用雙基質定恆狀態模式(dual-substrate steady-state model)可描述程序的表現，並預測當稀釋率大於0.75 hr-1，微生物會有洗出(wash-out)的現象。
　　另利用分子生物技術進行16S rDNA的變性梯度明膠電泳指紋圖譜分析，觀察到產氫菌族群的多樣性隨著水力停留時間的縮短而愈趨單純，存在系統中優勢菌種主要為Clostridium sporogenes-like (99%)及Clostridium celerecrescens-like(99%)，二者分別為典型的clostridia氨基酸代謝菌及纖維素分解菌，其生理特性與程序表現吻合，另利用多管發酵法探討菌群結構在不同稀釋倍數下的變動情形，於文中會有詳加討論；經由掃描式電子顯微鏡觀察，中溫厭氧產氫微生物以懸浮狀桿菌為主，菌相隨著水力停留時間的縮短，由複雜愈趨單純。

　　Hydrogen energy plays an important role in future energy system. Hydrogen is an energy carrier which can be produced from various sources. One of which is to convert organic matters to mixed gas (CO2 and hydrogen) by anaerobic fermentation of waste organics. Generally, compositions of wastewater are varied and complex. Different compounds may play different roles on hydrogen fermentation. In this study, biohydrogen process performances fed with dual substrates containing glucose and peptone at different loading (HRT) was evaluated.
　　Seeding microorganisms of the biohydrogen process were obtained from the upflow anaerobic sludge blanket (UASB) bioprocess of the food industry and were pretreated by boiling for over 30 minutes. Multiple substrates containing glucose (12 g/L) and peptone (8 g/L) were fed into an anaerobic hydrogen fermentation continuous-flow stirred tank reactor (CSTR). The temperature of the fermentor was maintained at 35°C. The CSTR was operated at different hydraulic retention times (HRT) which were 18, 12, 8, 6, 4, 2 and 1.45 hours, respectively. In the conditions of HRT longer than 1.45 hours, hydrogen production and cell growth would be maintained stable. Maximum hydrogen production rate, specific hydrogen production rate and hydrogen yield were 1,247 mmol H2/L/day, 9.5 L H2/g-VSS/day, and 5.0 mmole H2/g-CODapplied, respectively when the CSTR was operated at 2 hours of HRT.
　　Glucose was utilized for hydrogen production and degraded completely at different HRTs. But the role of peptone utilized by microorganisms changed at different HRTs. Peptone was used for biosynthesis and fermented to ammonia and organic acids when HRT was long. When HRT was short, most of the peptone, however, was utilized for biosynthesis or was remained in its organic nitrogen form, and only a slight amount of it was fermented into the ammonia. The fermented products were butyrate, acetate, formate and ammonia mainly. Shift of metabolic pathway was observed when HRT and loading were changed. In addition, a dual-substrate kinetic model was developed to describe performance of this process. Base on this model, complete wash-out would occur while dilution rate approaches 0.75 hr-1.
　　The microbial diversity was investigated by the molecular biotechnology of genomic DNA fingerprint analysis on 16S-rDNA. The results reveled that the diversity of hydrogen-producing bacteria became simpler as HRT was shortened. Clostridium sporogenes-like (99%) and Clostridium celerecrescens-like (99%) were dominant in the mesophilic biohydrogen process. They are typical proteolytic and cellulolytic species of clostridia, respectively. Furthermore, most probable number method was used for investigating microbial structure at different dilution ratios. Scanning electron micrographs of microbial populations revealed that rod suspension cells were dominant in mesophilic biohydrogen fermentor. It also showed that the microbial populations became simpler with decrease of HRT.

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