為了避免引起與民爭糧之爭議，目前第二代生質酒精之生產將以非糧食作物 (即含木質纖維素之農林業廢棄物; 如稻桿、蔗渣、木材等）為料源。木質纖維素主要由纖維素、半纖維素及木質素所組成。而大部分自然界存在的酒精醱酵菌株不能直接利用木質纖維素進行醱酵產酒精，因此纖維素水解糖化過程通常是整個纖維酒精製程之速率決定步驟。因此，木質纖維素之前處理及醣化製程十分重要。常用的纖維酒精生產製程主要可分成兩種，分別為兩階段水解醣化醱酵程序（SHF）及同步醣化醱酵程序（SSF），其中又因SSF製程因具有設備成本低廉、減低產物抑制及減低污染可能性之優點而較受到重視。另外，本研究也試圖利用饋料批次（fed-batch）及原位酒精產物移除（in-situ ethanol removal）來克服基質抑制（substrate inhibition）及產物抑制（product inhibition）之問題，以提升整體酒精醱酵效能。
　　本研究利用固定化Zymomonas mobilis進行酒精醱酵，發現於20%(w/v) 固定化細胞量在葡萄糖濃度20 g/L下有較高的酒精濃度及酒精產生速率，分別為9 g/L 及1.18 g/L/h，且在重複批次試驗及磷酸緩衝溶液組成的醱酵培養基中該固定化細胞皆能穩定地維持相似的酒精產量及酒精產生速率，因而大幅降低其生產成本。另外，本研究也採取兩種碳源進料策略，分別為Constant feeding及Cyclic feeding；其中，以Cyclic feeding的模式可得較高的酒精產率，達理論轉化率的99.45%，顯示固定化菌株確實能將葡萄糖幾近完全轉換為酒精。為了降低酒精抑制，本研究試圖利用薄膜蒸餾（membrane distillation, MD）進行同步酒精移除，其結果顯示酒精抑制現象確實得以消除。在纖維酒精生產程序上，可以分成酵素水解醣化及酒精醱酵兩階段。在酵素水解方面，本研究主要以本實驗室所篩選出的Pseudomonas sp. CL3進行纖維素分解酵素之生產。在該酵素纖維水解醣化之測試方面，於酵素量為3.0 FPU/mL時，可得最大的葡萄糖產生速率（Vmax）及Michaelis constant（Km），分別為1.05 g/L/h 及1.79 g/L。而本研究也試圖針對同步醣化醱酵程序（SSF）及兩階段水解醣化醱酵程序（SHF）進行探討。結果發現，以蔗渣（20 g/L）為碳源時，在SHF系統下有較高的酒精產率，可達理論產率的58.36%，而在SSF系統下僅有30.48%的酒精產率，其原因可能是部分酵素吸附在固定化細胞，造成水解效率下降。藉由添加界面活性劑（Tween-80）確實可以提升整體酵素水解效率，並使SSF系統之酒精產率提升約16.94%。由上述結果顯示，本研究所開發之纖維酒精生產系統確實可穩定操作並得到高酒精產率，但其酒精生產效率仍須再提昇，才能達到商業化之目標。

Bioethanol is the most promising and commercially feasible biofuel which is available at present. To avoid competition with food supply and land for crops, the second generation of bioethanol is to be produced from lignocellulosic feedstock, such as agricultural wastes (rice straw, bagasse, and etc.) and forestry residues (wood, leaves, and etc.). However, most ethanol-producing microorganisms cannot directly utilize cellulose or hemicellulose as carbon source to grow and produce ethanol, leading to the major limitation of biofuels production by lignocellulosic materials. Therefore, pretreatment and hydrolysis of the lignocellulosic feedstock are often required prior to cellulosic bioethanol production. The cellulosic ethanol could be produced by either simultaneous saccharification and fermentation (SSF) or separate hydrolysis and fermentation (SHF) processes. SSF is usually more favorable due to the merits of lower costs, fewer product inhibition, and lower risk of contamination. This study also tried to use fed-batch strategy and in-situ ethanol removal to improve ethanol production efficiency and to overcome the problems with substrate and product inhibition.
In this work, saccharified sugars were used to produce ethanol by immobilized Zymomonas mobilis cells, resulting in a ethanol concentration and productivity of 9 g/L and 1.18 g/L/h, respectively, with 20% (w/v) of immobilized cells and a glucose concentration of 20 g/L. The immobilized resting-cells could produce ethanol on a phosphate buffer containing enzymatic hydrolysate of lignocellulosic feedstock to reduce the cost of bioethanol production. There were two fed-batch strategies used for ethanol production, including constant and cyclic feeding. The ethanol produced from immobilized cells by cyclic feeding could convert glucose into ethanol completely, resulting in a higher yield of 99.45% (against the theoretical value) than that obtained by constant feeding (46.09%). Using membrane distillation (MD) could remove ethanol in-situ, thereby alleviating the product inhibition caused by ethanol and concentrating ethanol in the permeate.
Cellulosic bioethanol was produced by enzymatic hydrolysis and simultaneous fermentation of the resulting sugars. For the aspect of enzymatic hydrolysis, cellulases were produced from Pseudomonas sp. CL3 and used to hydrolyzed pretreated lignocellulosic feedstock. The maximum glucose production rate (Vmax) and Michaelis constant (Km) with enzyme dosage of 3.0 FPU/mL were 1.05 g/L/h and 1.79 g/L, respectively. The enzymatic cellulose hydrolysates were used to produce ethanol with immobilized Z. mobilis cells by SHF and SSF processes. The SHF process could obtain higher ethanol concentration and yield (4.61 g/L and 58.36% of theoretical yield) than those by SSF process (2.41 g/L and 30.48% of theoretical yield). This is due mainly to the insufficient enzyme dosage in SSF process, in which some of the cellulase enzymes added to the fermentation medium adsorbed on immobilization matrix leading to inefficient saccharification of cellulosic substrate. The ethanol yield increased by 16.94% in SSF process when adding 0.5% Tween-80 in the fermentation medium, suggesting that the surfactant could decrease adsorption of the enzyme on the immobilized matrix.
The results of this work are expected to provide useful information on assessing the feasibility of the proposed ethanol production system. The efficiency of bioethanol production should still be enhanced to reach the goal of commercialization.

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