香蘭素對丁醇生產的影響與固定化細胞連續式丁醇生產的評估

Abstract

由於石化燃料供應有限造成價格通貨膨脹，以及面臨溫室效應和氣候變遷等問題，世界各國紛紛積極發展生質能源，以減緩石油消耗、節約能源和符合環保的重要性。目前已產業化發展的生質酒精與生質柴油之外，還可利用Clostridium菌株經由ABE發酵生產丁醇，具有比乙醇較高的能量密度，其辛烷值接近汽油可均勻混和且含水量低不需改裝車輛引擎，是具有前瞻性的一種生質燃料。 在台灣地區每年產出約兩百萬公噸的農業廢棄物，若透過生質能源技術將廢棄物精煉為可再生資源，可以減少農業廢棄物對環境造成的污染和降低溫室效應氣體的排放。木質纖維素生物質經過特定預處理過程後，會產生可溶解糖供給微生物作為發酵糖使用，而水解副產物中的香蘭素屬於酚類化合物的一種，其導致乙醇發酵有明顯抑制作用，但對ABE發酵則較少被探討；另外將微生物包埋於PVA-海藻酸鈉製備為固定化顆粒以提高細胞密度，並進行連續式ABE發酵操作於上流式生物反應槽，可減少停滯時間與減緩丁醇產物抑制問題，所以本實驗使用C. saccharoperbutylacetonicum N1-4經由ABE發酵過程生產丁醇，探討批次式懸浮細胞存在於香蘭素對細胞濃度、葡萄糖利用率和酸醇變化之影響，並利用Gompertz方程式進行分析結果；與連續式固定化細胞在不同稀釋率下獲得最佳丁醇產量和生產率之討論。 在懸浮細胞實驗中，批次基質包含25 g/L葡萄糖、2.5 g/L乙酸和2 g/L丁酸，若添加0.5 g/L香蘭素導致微生物延遲4天將葡萄糖消耗完全，而細胞濃度有20%抑制生長情形造成丁醇濃度下降33%，香蘭素藉由ABE發酵過程參與還原反應可轉換為香草醇，此物質可降低對微生物的毒害且生物轉換率為74%，而香蘭素濃度若提升至微生物無法負荷仍可能造成嚴重抑制。在固定化細胞實驗中，從細胞濃度與葡萄糖利用率之評估可得0.01 h-1稀釋率為最佳丁醇生產條件，而隨著稀釋率提升至0.03 h-1顯示總ABE產量和產率有逐漸增加趨勢，此時有最高丁醇產量和產率分別為0.28 g/g 和0.15 g/L/h，而批次式的丁醇產量和產率分別為0.24 g/g 和0.06 g/L/h，相較之下得知連續式固定化優於批次式懸浮細胞生產丁醇，當連續進料基質包含25 g/L葡萄糖、2.5 g/L乙酸和2 g/L丁酸時，操作於0.03 h-1稀釋率與丁酸濃度降為1 g/L所獲得之丁醇產量和產率差異不大，表示上流式生物反應槽具有穩定性且連續式操作可以減少停滯時間。

High demand and apply of fossil fuel has caused not only shortage problem, but also greenhouse effect and climate change. Therefore, the development and application of biomass energy has caught the attention around the world to reduce fossil fuel consumption and energy conservation to be eco-friendly. Beside bio-ethanol and bio-diesel that had industrialized developed, bio-butanol can be prospective biofuels product by Clostridium through ABE fermentation process. This is because bio-butanol has a higher energy density than ethanol, and can be uniformly mixed with gasoline due to their similar octane number. Low water content without modification of vehicle engine is not necessary as well because bio-butanol has low water content. This would be a high prospective biofuel. Taiwan's has annual output of two million tonnes agricultural wastes, these agricultural waste can become renewable resources through biomass technology so that the reduction greenhouse gas emission can be achieved. Lignocellulosic biomass pretreatment process will produce some degradation byproducts and soluble sugar that can be use as a fermentable sugar for microorganism. Vanillin in compound of hydrolysis byproducts belongs to a type of phenolic compound, which results in significantly inhibited ethanol fermentation, however the ABE fermentation was less been explored. Microorganisms was embedded in PVA- Alginate as immobilized cells to increase cell density, and ABE fermentation was occurred inside the upflow bioreactor operated in continuous condition which can help to reduce the dead time and reduce the inhibition of butanol production. Butanol will be produce by C. saccharoperbutylacetonicum N1-4 through ABE fermentation process in this experiment. Investigation of the influence of batch free cells with and without vanilla on the cell concentration, glucose utilization and changes in solvent will be carry out in this experiment. After that Gompertz equation will be used to analyze the results. During the free cell experiment, with the medium containing 25 g/L glucose, 2.5 g/L acetic acid and 2 g/L butyric acid, if 0.5 g/L vanillin was added, the complete glucose consumption by microbes will be delayed for four days. The alcohol concentration will be decreased by 33% due to the inhibition of the growth of cell concentration by 20%. Vanillin involved in the reduction reaction can be converted to vanillyl alcohol through ABE fermentation process, this substance can reduce the toxicity to microorganisms and biological conversion rate by 74%, but if the concentration of vanillin unable to load up to microbes may still cause severe suppression. In the immobilized cell experiment, at dilution rate of 0.01 h-1, the glucose utilization rate and cell concentration is the highest, which will provide best condition for butanol production. The continuous operation with dilution rate increased to 0.03 h-1 show the gradually increasing trend of ABE yield and ABE productivity. At dilution rate of 0.03 h-1, the butanol production and yield were highest, which are 0.28 g/g and 0.15 g/L/h respectively while in the batch operation, the butanol production and yield were 0.24 g/g and 0.06 g/L/h. Therefore, by comparison, the butanol productivity and yield of continuous operation is better than batch operation. On the other hand, when two continuous feed media with 0.03 h-1 dilution ratio, both containing 25 g/L glucose, 2.5 g/L of acetic acid with one containing 2 g/L butyric acid and another containing 1 g/L butyric acid, the difference in butanol productivity and yield is negligible, which represents that the up-flow bioreactor is stable and the continuous operation can reduce the dead time.