本研究探討以厭氧醱酵技術將生產生質柴油的副產物－廢棄甘油轉化為生質丁醇之可行性應用。本研究企圖以自行篩選之厭氧產氫菌株Clostridium pasteuriaunm CH4探討生產丁醇之最適化條件與策略。結果顯示，在甘油濃度為100 g/L、FeSO4.7H2O濃度為25 mg/L以及酵母萃取物濃度為4 g/L時，可得最佳之丁醇濃度為12.6 g/L。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，可以促進丁醇的生成效能，其平均丁醇生產速率為0.12 g/L/h、丁醇產率為0.307 mol butanol/mol glycerol。然而當甘油濃度增加為100 g/L時，甘油利用率只有81.9%，丁醇生產效率也大幅下降，這可能是由於甘油濃度提高時，產生高濃度之丁醇，造成對菌體的毒害，形成產物抑制的現象。有鑑於此，本研究利用薄膜蒸餾(membrane distillation)技術，進行同步產物(丁醇)移除，以促進丁醇之生產效能。結合添加丁酸策略與薄膜蒸餾技術後，丁醇產量大幅提升至29.8 g/L，其產率也增加至0.39 mol butanol/mol glycerol。
雖然添加丁酸可促進丁醇之生產，但丁酸價格昂貴，造成丁醇生產經濟效益之降低。基於成本之考量，本研究改以源於木質纖維素的葡萄糖為碳源，讓菌體在醱酵初期先行生產丁酸，以取代直接添加丁酸之策略。並嘗試使用two-stage策略(先以葡萄糖為碳源再以甘油為碳源)與dual-substrate (同時以葡萄糖與甘油為碳源)策略，比較兩者之效能。結果發現利用dual-substrate策略，不僅操作方便，而且其碳源利用的遲滯期也較短。接著，進一步探討在dual-substrate策略下，甘油與葡萄糖之最佳比例。結果發現，當葡萄糖濃度為20 g/L、甘油濃度為60 g/L，有最佳之丁醇產量、丁醇生產速率與丁醇產率，分別為13.3 g/L、0.14 g/L/h與0.33 mol butanol/mol glycerol。此外，本研究也以木質纖維素料源與廢棄甘油為碳源，以降低丁醇生產之成本。結果顯示，以25 g/L蔗渣以及60 g/L廢棄甘油為碳源時，其丁醇產量可達11.8 g/L、丁醇產率為0.33 mol butanol/mol glycerol、丁醇生產速率為0.14 g/L/h。
另外，本研究也採取饋料批次進料策略，並與薄膜蒸餾技術結合，以同時改善基質抑制與產物抑制之現象。相較於批次醱酵的結果，結合饋料批次與薄膜蒸餾產物移除技術進行丁醇醱酵，其丁醇生產速率(0.3 g/L/h)可以提高250%，丁醇產量(49.1 g/L)也增加了將近四倍，而其丁醇產率(0.31 g butanol/g glycerol)可達到理論產率的80%。

Butanol was produced from glycerol by Clostridium pasteurianum CH4 isolated from effluent of H2-producing bioreactor. The effects of glycerol concentration (0-200 g/L), iron ion (II) concentration (0-50 mg/L) and yeast extract concentration (0-5 g/L) on butanol production were investigated on batch system. The maximum butanol production (12.6 g/L) was obtained under the optimum glycerol, yeast extract and iron ion (II) concentration of 100 g/L, 4.0 g/L, and 25 mg/L, respectively. Furthermore, to enhance butanol production efficiency, butyrate, a precursor for butanol formation,was added to the fermentation broth to investigate its effect on butanol production. The results show that addition of 6.0 g/L butyrate could improve butanol production yield (from 0.200 to 0.307 mol butanol/mol glycerol), average butanol production rate (from 0.08 to 0.12 g/L/h), and results in a maximum butanol production rate of 0.788 g/L/h. Meanwhile, addition of 6.0 g/L butyrate also leads to a decrease in 1, 3-propandiol concentration from 16.9 to 7.5 g/L.
When glycrol concentration was increased to 100 g/L, the glycerol utilization was only 81.9% due probably to product inhibition arising from an increase in butanol concentration. To avoid the toxicity effect of butanol, vacuum membrane distillation (VMD) was adopted to specifically remove butanol from the culture broth during batch fermentation. The employment of VMD could not only alleviate the inhibitory effect of butanol but also concentrate butanol concentration via condensation, thereby making downstream processing easier. The combination of VMD and fermentation strategy (e.g., butyrate addition) could enhance maximum butanol concentration from 12.6 to 29.8 g/L and improve the yield from 0.307 to 0.39 mol butanol/mol glycerol.
Although adding butyrate could improve the butanol production performance, the high cost of butanol makes the strategy economically infeasible. An alternative approach was to use glucose as carbon source to stimulate cell growth and butyrate formation of C. pasteurianum CH4. After that, glycerol was used for the fermentation to produce butanol in the presence of butyrate produced due to assimilation of glucose. Comparing two-stage (using glucose as carbon source first and then adding glycerol) and dual-substrate (both glucose and glycerol were added in the beginning) operation strategies, the dual-substrate system is easier to operate with a shorter lag phase for carbon source utilization and better butanol production performance. Hence, the dual-substrate system was used for further studies. The optimum glucose to glycerol ratio in dual-substrate system was 20 g/L to 60 g/L, giving a maximum butanol titer of 13.3 g/L, a productivity of 0.19 g/L/h, and a yield of 0.38 mol butanol/mol glycerol. Moreover, we used cellulose (from bagasse) and raw glycerol as the substrate to reduce the cost of carbon source. Under the optimum condition, using enzymatic bagasse hydrolysates (25 g/L) and raw glycerol (60 g/L) as the carbon sources, maximum butanol production can reach 11.8 g/L with a yield of 0.33 mol butanol/mol glycerol and a productivity of 0.14 g/L/h, respectively.
Butanol was also produced via integration of fed-batch fermentation with VMD product-recovery system to avoid both substrate and product inhibition. In this process, butanol productivity (0.3 g/L/h) was improved about 250% of that obtained from batch fermentation, while effective butanol concentration (49.1 g/L) enhanced approximately 4-fold when compared with that obtained from batch fermentation. The butanol yield was 0.31 g butanol/g glycerol, which represents for 80% of the theoretically maximum yield.

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