由於pH控制是丁醇醱酵之重要影響參數，本研究首先探討分別使用添加化學藥品及使用自動控制裝置維持發酵液pH值對於丁醇生產之影響。結果顯示，當添加100 mM醋酸緩衝溶液時，能將pH控制在4.3，並有效地將丁醇生產濃度從2.0 g/l (無pH控制) 提升至5.5 g/l。添加碳酸鈣對pH控制有較佳的效果，當添加8 g/l以上之碳酸鈣時，能將發酵液pH值維持在4.8以上，此pH值為較適合丁醇生產的pH，因此丁醇產量可提升至10.4 g/l。此外，使用自動控制裝置維持發酵液pH為4.5則是最佳之丁醇生產方式，其丁醇濃度可達11.1 g/l。
接著本研究利用不同料源(稻桿、蔗渣、微藻)為料源進行丁醇生產。結果發現當使用二階段水解發酵(SHF)時，以稻桿作為料源其丁醇產量為9.1 g/l，以蔗渣作為料源時，其丁醇產量為8.4 g/l。當使用微藻作為料源時，當藻體濃度大於120 g/l時，必須使用3%硫酸進行水解才可達到較高之葡萄糖回收率，但藻體在此酸濃度下進行水解會伴隨著抑制物的生成，導致菌體無法生長。因此本研究直接利用未經過水解的藻體作為料源進行丁醇之生產之。結果發現，在藻體濃度為60, 120, 180 g/l時，其丁醇產量分別為0.34, 3.20, 4.36 g/l。
為了提升丁醇生產速率，本研究以連續式生產系統進行丁醇生產之探討。結果發現在HRT 為24小時，其丁醇濃度、產率、生產速率分別為8.7±0.9 g/l, 0.42±0.03 mol-butanol/mol-glucose, 0.36±0.04 g/l/h。當連續式丁醇生產系統結合真空薄膜蒸餾裝置進行產物同步移除後，其丁醇濃度、產率、生產速率分別為11.5±0.2 g/l, 0.48±0.03 mol-butanol/mol-glucose, 0.51±0.09 g/l/h，顯見結合產物同步移除裝置確實能提升整體丁醇生產效率。然而，在低HRT (12小時)情況下，因水力滯留時間過短，導致其丁醇產量仍然偏低且無法長時間操作。
在操作連續式丁醇生產系統時，通常存在著穩定性的問題，即當操作時間達2-3星期時會有菌體失活的情形，使得無法於低HRT下操作。因此，本研究採用PVA固定化菌體提升菌體添加量以提升丁醇產量。在批次實驗中，當固定化菌體添加量為20.63 g/l時有最佳的丁醇生產效果。使用固定化細胞可提高菌體添加量，此時基質中的昂貴的營養成分(如酵母萃取物yeast extract)可完全移除，以降低成本。接著，本研究採用饋料批次系統結合產物移除裝置以固定化菌體進行丁醇生產，結果發現其丁醇產量及產率可達29 g/l及0.44 mol-butanol/mol-glucose。最後本研究以固定化菌體進行連續式丁醇生產，在HRT 6小時的情況下，其最佳丁醇產量及生產速率為8.8±0.8 g/l 及 1.47±0.14 g/l/h，且穩定操作時間可長達52天。

This study first investigated the feasibility of using two kinds of pH control methods (i.e., chemical addition and auto-titration) for bio-butanol production with Clostridium acetobutylicum ATCC 824. Addition of 100 mM acetate buffer could maintain the pH at a constant level and exhibiting an improvement in the butanol concentration from 2.0 g/l to 5.5 g/l. Calcium carbonate was also used to maintain the pH of the ABE fermentation. When the concentration of calcium carbonate was greater than 8 g/l, the pH could effectively be maintained at around 4.8, which is an appropriate pH for ABE fermentation. Finally, the pH of ABE fermentation was controlled at 4.5 via auto-titration, resulting in better butanol production of 11.1 g/l.
Next, the renewable feedstock such as rice straw, bagasse, starch, and microalgae biomass was used for butanol fermentation. The results showed that using separated hydrolysis and fermentation (SHF) process, the butanol concentration and butanol productivity were 9.1 g/l and 0.79 g/l/h, respectively, for rice straw, and 8.4 g/l and 0.80 g/l/h, respectively, for bagasse. On the other hand, microalgal biomass was also used as feedstock for butanol production. Because some inhibitors were formed during acidic hydrolysis of microalgal biomass, butanol production was conducted directly using non-hydrolyzed microalgae biomass, which contained high carbohydrate content (mainly in the form of starch). The results showed that butanol concentration reached 0.34, 3.20, and 4.36 g/l with a microalgal biomass concentration of 60, 120, and 180 g/l (equivalent to 17.4, 34.8, and 52.2 g/l of starch), respectively.
The butanol yield and productivity found in the continuous mode at the HRT of 24 hr were 0.42±0.03 mol-butanol/mol-glucose and 0.36±0.04 g/l/h, respectively. In the integrated process of continuous fermentation combined with in-situ butanol removal system by vacuum membrane distillation, the butanol yielded and productivity were elevated to 0.48±0.03 mol-butanol/ mol-glucose and 0.51±0.09 g/l/h, respectively.
The feasibility of using PVA-immobilized Clostridium acetobutylicum was also examined for batch, fed-batch, and continuous butanol production. The immobilized cells were operated on the batch mode with different kinds of cells loading and the results showed that the optimum cell loading was 20.63 g-cells/l. When using the high cells loading, the costly nutrient, yeast extract, could be completely removed from the medium with similar butanol producing performance on batch mode. By integrated with VMD, the fed-batch butanol production and yield were 29 g/l and 0.44 mol-butanol/mol-glucose, respectively. However, when continuous butanol fermentation was operated with yeast extract-free medium, butanol production cannot maintain steady-state after long-term operation. Therefore, the optimum yeast extract concentration of 1.25 g/l was used for continuous butanol production and a stable continuous culture was achieved. At a HRT of 6 h, the butanol concentration and productivity were 8.8±0.8 g/l and 1.47±0.14 g/l/h, respectively, and the steady-state operation could be maintained for more than 52 days.

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