本研究探討以厭氧醱酵技術將木質纖維素料源轉化為生質丁醇。本研究使用之丁醇生產菌種乃由廢水污泥所馴化出之優勢菌群，經由變性梯度電泳分析與16S rDNA序列比對發現，該菌群中主要是四株厭氧菌，分別為Clostridium saccharoperbutylacetonicum, Clostridium butylicum, Clostridium sp., 與Clostridium acetobutylicum。本研究以葡萄糖濃度、FeSO4.7H2O濃度、和酵母萃取物濃度為主要變因，利用Box–Behnken法實驗設計和反應曲面法進行培養基最適化，以改善生質丁醇的生產效率。結果發現，在葡萄糖濃度為60 g/L、FeSO4.7H2O濃度為0.65g/L以及酵母萃取物濃度為5.13 g/L時，有最大的丁醇生產速率為3.03±0.18 mM/h。此外，由於丁酸為丁醇醱酵之前驅物，故本研究亦探討丁酸添加對於丁醇生產的影響。結果顯示，當添加丁酸濃度為6.0 g/L時可以大幅提升丁醇的生成，其產量可達約17.51±0.49 g/L。接著，本研究再利用產氣加壓迴流的醱酵策略，以提升氫分壓的方式抑制產氫，進而使得丁醇產量進一步提升到約21.31 g/L，產率為0.8 mol butanol/mol glucose，而生產速率達到16.92 mM/h，這些數據都優於相關之文獻報導。接著，本研究成功地將農業廢棄物(稻桿與蔗渣)轉化為生質丁醇；將蔗渣和稻桿經由氫氧化鈉前處理後，以自產纖維素分解酶進行纖維素酵素水解，在24小時內可以達到80%的水解效果並分別得到7.91±0.02和8.11±0.02 g/L的還原糖。以稻桿生產生質丁醇其丁醇產率(0.44±0.02 mol/mol reducing sugar)且產丁醇速率(84.16±1.93 mg/h)皆明顯高於蔗渣(丁醇產率為 0.32±0.02 mol/mol reducing sugar、丁醇速率為70.5±9.5 mg/h)，顯示以本研究開發的技術，稻桿是較適合生產丁醇的料源。

In this study, we utilized anaerobic fermentation to produce sustainable liquid biofuel (i.e., butanol) from renewable feedstock. An effective butanol-producing bacterial microflora was obtained from sewage sludge. Analysis with denaturing gradient gel electrophoresis (DGGE) followed by 16S rDNA sequence comparison, the major players in the bacterial microflora were identified as Clostridium saccharoperbutylacetonicum, Clostridium butylicum, Clostridium sp., and Clostridium acetobutylicum. Optimal medium composition for enhanced biobutanol production was obtained with the aid of Box–Behnken design and response surface methodology (RSM) using concentrations of glucose, FeSO4.7H2O and yeast extract as the key parameters. A maximum butanol production rate of 3.03±0.18 mM/h was obtained under the optimum condition of glucose concentration, 60 g/L; FeSO4.7H2O, 0.65 g/L; yeast extract concentration, 5.13 g/L. In addition, to improve butanol production efficiency, butyric acid was added to the fermentation broth as a precursor to improve the butanol production. The results show that addition of 6.0 g/L butyric acid significantly enhanced butanol production, leading to a final butanol concentration of 17.51±0.49 g/L. In addition, pressurized fermentation strategy was used in a 2 L fermentor to increase the partial pressure of hydrogen so that hydrogen generation was inhibited, thereby butanol production becomes metabolically favorable. Using the optimal dosage of butyric acid and pressurized fermentation strategy, the butanol production can reach up to 21.31 g/L culture with a yield of approximately 0.8 mol butanol/mol glucose and a maximum butanol production rate of 16.92 mM/h.
Butanol was also successfully produced from enzymatically hydrolyzed bagasse and rice straw, which were pretreated with alkaline (sodium hydroxide). Using cocktail cellulases/xylanase enzymes produced from bacterial strains isolated from our laboratory, nearly 80% of pretreated bagasse and rice straw was converted to reducing sugar, giving reducing sugar production of 7.91±0.02 and 8.11±0.02 g/L within 24 h of enzymatic hydrolysis. The butanol yield (0.35±0.03 mol/mol reducing sugar) and butanol rate 84.16±1.93 mg/h from rice straw was higher than that (0.32±0.01 mol/mol reducing sugar and 70.5±9.5 mg/h) from bagasse. This indicates that using the technology developed in this study, rice straw was a more efficient feedstock for cellulosic biobutanol production.

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