本論文的主要目的是以固定化混菌和單一菌株Klebsiella sp.來生產一系列的生物燃料，包括氫氣、乙醇、1,3-丙二醇、2,3-丁二醇等。使用混菌配合固定化細胞製備之複合材料(CA + CH + TiO2)(藻酸鈣+幾丁聚醣+氧化鈦)在批次操作可得氫氣產速為21.3 mmol•l-1•h-1和氫氣產率為5.1 mol H2/mol substrate，由此結果得知使用該固定化細胞應可應用於連續式產氫操作。以(PMMA/collagen/activated carbon)(聚甲基丙烯酸甲酯/膠原蛋白/活性碳)細胞固定化進行批次與連續操作，展現出好的機械強度與產氫活性，可有效地進行高穩定性的連續操作於低有機負荷速率，並穩定與高效能的產生氫氣，於4至8小時的水利停留時間操作下，可得到最佳的氫氣產速為1.8 l h-1 l-1(為批次操作的七倍多)，且在水利停留時間為6小時，可得到2.0 mol H2mol sucrose-1的氫氣產率。
本研究並以以連續反應槽(CSTR)使用水利停留時間(HRT)的高低轉換來提升生物燃料氫氣和乙醇的產生效能，結果發現水利停留時間之增減對生物燃料的產出效能影響頗大，主要的原因是有機負荷速率與稀釋速率轉變時，微生物的菌相也產生變化，此一關鍵因素會影響微生物社會族群之功能與醱酵動力學。以葡萄糖為基質來進行水利停留時間連續轉換培養操作時，HRT操作轉高時，能量的產出量與效率均較高。
在生物反應器組態的研究中發現，由於流體化床的質量傳送效率較填充床好，因而產生的氫氣產速和乙醇產速較高，且以蔗糖為基質時有較佳之效能。而於填充床進行反應，以葡萄糖當基質，可有效的產出乙醇產速378 mmol l-1 h-1和乙醇產率0.65mol EtOH/mol substrate，此乃不同的反應器型式，在不同的基質操作時，微生物的菌相結構產生變化所致。
最後，本研究自厭氧污泥中篩選出產氫兼性菌種Klebsiella sp.，發現此菌株能同時產出氫氣、乙醇、1,3-丙二醇和2,3-丁二醇來當生物燃料與工業應用的生物化學品，並嘗試以批次醱酵方法提升該菌種之產氫與產醇效能。結果顯示，以甘油和蔗糖為基質，能有效的生產氫氣、乙醇、1,3-丙二醇和2,3-丁二醇。結果顯示，1,3-丙二醇產速為7.0 mmol l-1 h-1和1,3-丙二醇產率為0.37 mol 1,3-propanediol/mol glycerol與2,3-丁二醇產速為7.1 mmol l-1 h-1和2,3-丁二醇產率為0.59 mol 2,3-butanediol/mol sucrose。在生質能源工業應用上，當以低價位的生質柴油副產物甘油來進行厭氧醱酵生產，可得到高價位的生物化學品(如1,3-丙二醇和2,3-丁二醇)，此進行途徑是可行性高且有未來前景的。

In this dissertation, mixed and pure culture (i.e., Klebsiella sp. HE1) were immobilized to produce various biofuels, such as hydrogen, ethanol, 1,3-propanediol and 2,3-butanediol. A series of cell immobilization approaches were used to entrap mixed microflora for hydrogen production. We found that the cells immobilized with a composite matrix of AC + CH + TiO2 (Activated carbon + Chitosan + Titanium oxide) displayed a good H2 production rate of 21.3 mmol l-1 h-1and a hydrogen yield 5.1 mol H2/mol sucrose. The results from this work suggest the potential of using the immobilized-cell systems for continuous H2 production in practice. We also found that immobilized-cell system was suitable for batch and continuous H2 production giving a high stability and efficiency. The cells entrapped with novel composite polymeric matrix (PMMA/collagen/activated carbon) displayed good mechanical strength and H2-producing activity. Batch tests were conducted to explore favorable conditions for H2 production with the PMMA immobilized cells. Under optimal conditions, continuous H2 fermentation was conducted at a hydraulic retention time (HRT) of 4-8 h, giving the best H2-producing rate of 1.8 l h-1 l-1 (over 7 fold of the best batch result) at a HRT of 6 h and a H2 yield of 2.0 mol H2/mol sucrose. The outcome of this work suggests the potential of using this immobilized-cell system for continuous H2 production in practice.
Using a continuously stirred tank bioreactor (CSTR), production of the two biofuels (hydrogen and ethanol) was dependent on the sugar substrate used and also varied with the HRT shifting operation. It is of great interest to observe that sequential HRT decreasing and increasing had significant impact on the biofuels producing performance. The HRT shifting operation appeared to influence the abundance and composition of bacterial population in the culture, and also governed the organic loading rate, which is normally a critical factor affecting the fermentation kinetics. In contrast, energy generation from glucose substrate seemed to be more efficient during a HRT-increasing process.
For the feasible bioreactor systems for simultaneous production of H2 and ethanol as biofuels, fluidized bed reactors (FBR) were able to produce H2 and ethanol at a significant higher rate than packed bed reactors (PBR) due to better mass transfer efficiency in FBR. In PBR (packed bed bioreactor), glucose gave the best performance in terms of production rate and yield of the two biofuels, displayed a good EtOH production rate 378 mmol l-1 h-1and EtOH yield 0.65 mol EtOH /mol substrate. This difference in substrate preference could be due to variations in bacterial population structure resulting from different bioreactor configuration.
Finally, a Klebsiella sp. HE1 strain isolated from sewage sludge, using an indigenous Klebsiella sp. HE1 strain for simultaneous production of H2, ethanol, 1,3-propanediol and 2,3-butanediol as biofuels or industrially applicable biochemical products. The H2-producing HE1 strain successfully isolated from aerobic and anaerobic sludge exhibitied the ability to produce hydrogen gas, 1,3-propanediol, 2,3-butanediol, ethanol from glycerol and sucrose substrate. This study shows the efficiency of producing 7.0 mmol l-1 h-1 of 1,3-propanediol with a yield of 0.37 mol 1,3-propanediol/mol glycerol and of producing 7.1 mmol l-1 h-1 of 2,3-butanediol with a yield of 0.59 mol 2,3-butanediol /mol sucrose. Using Klebsiella sp. to convert abundant and low-cost glycerol generated during the production of biodiesel into higher value products (1,3-propanediol or 2,3-butanediol etc.) represents a promising route to achieve economic viability in the biofuels industry.

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