| Abstract: | 本研究以利用菇蕈剩餘物進行水解，使纖維素轉化糖液添加於產乙醇微生物，進行生質乙醇生產，並選擇Kluyveromyces marxianus和Saccharomyces cerevisiae兩種菌株進行發酵培養，針對C級金針菇與金針菇基底進行初篩，探討其合適的發酵條件及如何提高生質乙醇產量。比較C級金針菇與金針菇基底之纖維素含量，金針菇基底的纖維素為27.44 mass%，半纖維素為47.96 mass%，其木質素為12.51 mass%，相較於 C 級金針菇具有優勢。再探討不同溫度及時間對菌株生長及乙醇產量的影響，藉以尋找出最佳培養條件以獲得較佳乙醇量。本研究固定接種量為5%，生長曲線結果顯示，產乙醇酵母在溫度25 ℃及轉速160 rpm下振盪培養於發酵培養基96小時，其菌量從快速上升到穩定增加的趨勢，而乙醇含量則都在第24小時達至最高後隨即下降，葡萄糖則是第0小時最高，之後則是供給產乙醇微生物轉化為乙醇。因K. marxianus耐熱性及離子液的耐受程度較好。因此，運用微量量熱卡儀(Isothermal calorimeter, TAM Air)分析中，選擇104 CFU/mL菌數進行K. marxianus發酵產生生質乙醇之等溫生長熱分析探討。在離子液體篩選中，[AMIM][Cl]、[BMIM][Cl]和[EMIM][Ac]水解金針菇基底的還原糖含量分別為9.99、6.84、4.25 mg/g，三種離子液都比控制組的效果好，而 [AMIM][Cl]有最佳的水解效果。在還原糖含量測定中，水解纖維素時添加比例越高，其還原糖含量越多；而水解金針菇基底，在離子液比例1:0.1-1:15間，以1:0.1有最佳的水解效果(13.60 ± 0.38 mg/g)，離子液[AMIM][Cl]在不同的作用時間下，離子液體濃度越高，其還原糖含量越高。當離子液濃度為1:20時，還原糖含量都達到8.25 mg/g以上。在濃度1:10、1:15及1:20時，反應5小時能獲得最高的還原糖含量，分別為5.04±0.08、7.60±0.01、9.76±0.01mg/g。Cellulose添加1:20之離子液[AMIM][Cl]在5小時與20小時都有較高之還原糖含量，分別為9.59 ± 0.11及9.46 ± 0.42 mg/g，以時間及經濟效益著眼，反應時間5小時的效果是最好的。運用微差掃描量熱卡儀(Differential scanning calorimetry, DSC)分析中，添加去離子水及離子液對金針菇基底的有結構變化。1:20進行5小時水解後的沉澱物的熱反應特性，可以看出升溫速率也會影響吸熱峰的最高溫度，升溫速率越快，其熱反應溫度越高，熱焓值也越大。不同添加量的起始溫度及吸熱峰的溫度依上升，說明離子液添加能提吸熱峰的溫度。在微量熱分析中，離子液含量10.00 mg/mL在不同溫度下酵母菌的生長皆被抑制，其中以45 ℃最為明顯。不同溫度(40、50、60 ℃)對纖維素及金針菇基底與離子液的影響中，在40和50 ℃下的熱變化並不明顯，在60 ℃下離子液的濃度越高，作用時間越短，而濃度低，作用時間則越長。離子液水解纖維素與金針菇基底在 40 ℃ 下對K. marxianus的影響，稀釋之水解液，使離子液濃度≦10.00 mg/mL，對酵母菌的生長並無抑制作用，乙醇濃度在離子液與纖維素比例為1:0和1:10有最高的乙醇含量9.26 ± 0.05及9.37 ± 0.06 uL/mL；離子液與金針菇基底比例為1:1和1:10時，有最高的乙醇含量9.29 ± 0.03及9.28 ± 0.01 uL/mL。總結上述，K. marxianus 比 S. cerevisiae 對離子液的耐受度較好，而離子液至少需要 60 ℃ 以上且作用時間大於 3 小時才有較好的水解木質纖維素效果，水解後的離子液殘留濃度也減少，因此在培養 K. marxianus 時並無明顯的抑制效果，微量量熱卡儀可以用來評估離子液轉化纖維素對 K. marxianus 發酵狀態的影響。
Using mushroom waste to hydrolyze, the cellulose-inverted sugar solution was added to ethanol-producing microorganisms for reproductive ethanol production, and two strains of Saccharomyces cerevisiae and Kluyveromyces marxianus were selected for fermentation and cultivation, and for the preliminary screening of C-grade Flammulina velutipes and the bottom of the mushroom and the surface of the culture medium in this study. To asses suitable fermentation conditions and how to improve the bioethanol yield. This experiment will focus on the cellulose content of C-grade Flammulina velutipes and the bottom of the mushroom and the surface of the culture medium. The cellulose of the base of Flammulina velutipes is 27.44%, the hemicellulose is 47.96%, and the lignin is 12.51%, which is more in line with the purpose of this study than the C-grade Flammulina velutipes. And then discuss the effects of different temperatures and times on strain growth and ethanol production. In order to find out the optimal culture conditions to obtain the preferred amount of ethanol yield, as a basis for a small amount of amplification of the fermentation. In this experiment, the fixed inoculation amount was 5%, and the growth curve results showed that the ethanol-producing yeast was cultured in a YPD medium at a temperature of 25 ° C and a speed of 160 rpm for 96 hours. The Colony count increased rapidly to a steady increase, while the alcohol content reached the maximum at the 24th hour and then decreased immediately, and the Glucose was the maximum at the 0th hour. After that, the ethanol-producing microorganisms were supplied to convert into ethanol. Because K. marxianus has better heat resistance and ionic liquid tolerance. Therefore, in the analysis of Isothermal calorimeter (TAM Air), the bacterial count of 104 CFU/mL was selected for the isothermal growth thermal analysis of K. marxianus fermentation to produce bioethanol.In the ionic liquid screening, the reducing sugar contents of [AMIM][Cl], [BMIM][Cl] and [EMIM][Ac] hydrolyzed Flammulina velutipes substrates were 9.99, 6.84 and 4.25 mg/g, respectively. The effect of the group was good, while [AMIM][Cl] had the best hydrolysis effect. Determination of reducing sugar content, the higher the addition ratio of cellulose hydrolyzed, the more reducing sugar content; and the hydrolysis of Flammulina velutipes base, in the ionic liquid ratio of 1:0.1-1:15, 1:0.1 has the best hydrolysis effect (13.60 ± 0.38 mg/g), under different action time of ionic liquid [AMIM][Cl], the higher the ionic liquid concentration, the higher the reducing sugar content. When the ionic liquid concentration is 1:20, the reducing sugar content is all above 8.25 mg/g. At the concentrations of 1:10, 1:15 and 1:20, the highest reducing sugar content was found at 5 hours, which were 5.04 ± 0.08, 7.60 ± 0.01, 9.76 ± 0.01 mg/g, respectively. Cellulose added 1:20 ionic liquid [AMIM][Cl] with higher reducing sugar content at 5 hours and 20 hours, respectively 9.59 ± 0.11 and 9.46 ± 0.42 mg/g. In terms of economic benefits, the action time 5 hours is the best. Applications of differential scanning calorimetry (DSC) analysis, the addition of deionized water and ionic liquid had structural changes on the substrate of Flammulina velutipes. The thermal reaction characteristics of the precipitate after hydrolysis at 1:20 for 5 hours, it can be seen that the heating rate also affects the maximum temperature of the endothermic peak. The faster the heating rate, the higher the thermal reaction temperature and the greater the enthalpy value. The initial temperature and the temperature of the endothermic peak increased with different addition amounts, indicating that the addition of ionic liquid can increase the temperature of the endothermic peak.Using isothermal microcalorimeter analysis, the growth of yeast was inhibited at different temperatures with ionic liquid content of 10 g/L, and the most obvious at 45℃. Among the effects of different temperatures (40, 50, and 60°C) on cellulose and Flammulina velutipes substrates and ionic liquids, the thermal changes at 40 and 50°C were not obvious. The higher the concentration of ionic liquid at 60°C, the longer the action time. The shorter the concentration, the longer the action time. The effect of ionic liquid hydrolyzed cellulose and Flammulina velutipes substrate on K. marxianus at 40℃. Dilute the hydrolyzate so that the concentration of ionic liquid is less than or equal to 10 g/L, and has no inhibitory effect on the growth of yeast. The highest alcohol content was 9.26 ± 0.05 and 9.37 ± 0.06 uL/mL when the ratio of cellulose was 1:0 and 1:10; the highest alcohol content was when the ratio of ionic liquid to Flammulina velutipes was 1:1 and 1:10. The content was 9.29 ± 0.03 and 9.28 ± 0.01 uL/mL, respectively.To sum up the above, K. marxianus has better tolerance to ionic liquid than S. cerevisiae, and the ionic liquid needs to be at least 60 ℃ above and the action time is more than 3 hours to have a good effect of hydrolyzing lignocellulose. The residual concentration was also reduced, so there was no obvious inhibitory effect when culturing K. marxianus. Microcalorimetry can be used to evaluate the effect of ionic liquid-converted cellulose on the fermentation state of K. marxianus. |
