臺灣博碩士論文加值系統

目前由於環保意識抬頭，以及全球面臨石油短缺與能源不足等問題，因此開發一種新的替代能源來代替日益短缺的石油是必然的。近年來生化工業上以微生物進行生產製程已慢慢成為趨勢，如使用紅酵母菌屬之一的Rhodotorula glutinis，其優勢在於能夠使用廢棄物作為碳源累積油脂，是一種兼具環保與成本的可靠生產製程。紅酵母同時被視為酵素、酯類及維生素的良好來源，R. glutinis中所累積的油脂中不飽和脂肪酸和脂肪酸所佔之比例高達70%，且其成分類似於植物油，因此相當適合作為生質柴油之原料，具有極高發展潛力。此外，若是使用廢棄食用油(WCO)作為其碳源，不僅僅能夠提高R. glutinis菌體濃度以及油脂含量，還能夠產出一種商業價值也很高的酵素，脂肪酶，其能夠運用在乳品工業、個人護理用品、油脂化學工業等等產業上。因此如何利用WCO來進行R. glutinis的高濃度培養以及如何有效將脂肪酶從發酵液當中萃取出來將是本論文之研究主題。
本研究探討WCO作為碳源之可能性，並改變其濃度、發酵策略等不同條件來找出最適合R. glutinis生長之環境。其中以搖瓶所得出的實驗結果表明使用界面活性劑Tween 80濃度為10 ml/L為最適合之濃度，以及WCO濃度為90 ml/L時，其菌體濃度及脂質含量最高，分別為7.27 g/L以及42.63%。而以5L攪拌式發酵槽進行放大培養後，其中以利用180 ml/L的WCO濃度所得到的菌體濃度以及脂質含量最高，分別為25.8 g/L以及64.1%。而使用Fed-batch作為其發酵策略時，且加入WCO的濃度為90+90 ml/L，能夠得到較高的脂質含量，但其菌體濃度會略為下降，分別為菌量16.5 g/L以及油脂含量67.4%。最後，因考慮到攪拌速率及通氣量不能過高，故使用60 ml/L的WCO放大至30 L攪拌式發酵槽，其結果最大菌體濃度得到38.1 g/L，最大脂質含量為67.96%，故可以證實將此發酵條件進行放大之可行性。
本研究利用二水相萃取系統(ATPE)來進行脂肪酶的萃取，因其操作簡單，分層時間短，且能在常溫常壓下進行，因此較適合作為分離萃取酵素的手段。通過實驗結果發現，使用發酵時間較長的上清液能夠產出更多的脂肪酶酵素，本實驗使用草酸鉀及PEG作為ATPE之溶劑，並透過改變其上下層濃度、PEG的分子量、反應溫度等不同條件來找出萃取之最佳條件。根據實驗結果表明其溫度在超過40℃之後酵素活性開始下降，所以使用37℃為較適當萃取溫度，萃取效果最好之實驗條件為加入 2.15 g的PEG 1000，以及1.67 g的草酸鉀，利用ddH2O充分溶解後定量至8.15 ml，再加入1.85 ml的發酵上清液並混合均勻，並混合均勻後得到二水相系統，靜置分層後測量其上下層的脂肪酶活性，其Ke值為5.34，表示原有84.2%的脂肪酶被分配至上層。上述數據表明由於其操作成本及原料成本較低，使用ATPE來做為將發酵上清液中的脂肪酶之萃取方法是有相當高可能性，在未來工業化的發展上具有相當大的潛力。

Currently due to environmental awareness, and the world is facing problems such as the shortage of energy and the oil crisis. It is inevitable to develop a new alternative energy to replace the increased shortage oil. Recently, there is a trend that using microorganism to produce in biochemical industry. Like using one of the red yeast species, Rhodotorula glutinis, its advantage is that it can decompose the carbon-containing industrial waste by using its own metabolism. And it can increase biomass and target product as well. It is a both environmentally friendly and cheap cost process production. It is also considered as great source of enzymes, esters and vitamins. R. glutinis can accumulate fat content in saturated and unsaturated fats as high as 70%, which is similar to vegetable oil. Therefore, it is very suitable as the raw material of biodiesel, and it has extremely high development potential. Besides, if we use waste cooking oil (WCO) as its carbon source. WCO can not only increase the biomass and lipid content of R. glutinis, but also produce a high economic value enzyme, lipase. Lipase can use in dairy industry, personal care products and oil chemical industry, etc. Thence, the research direction in this study is that how to use WCO for high-concentration cultivation of R. glutinis and how to effectively extract lipase from fermentation broth.
In this study, we use WCO as a carbon source and changes the concentration of WCO, fermentation strategy and other different conditions to find the most suitable environment for the growth of R. glutinis. Among them, the experimental results obtained in shakers show that the concentration of surfactant Tween 80 is 10 ml/L, which is the most suitable concentration. And when the concentration of WCO is 90 g/L. It has the highest biomass and lipid content as well, 7.27 g/L and 42.63% respectively. However, using a 5 L stirred fermenter for scale-up culture. The experimental results show that when the concentration of WCO is 180 ml/L, it has the highest biomass and lipid content, 25.5 g/L and 64.1% respectively. When we use fed-batch as the fermentation strategy, we can get higher lipid content. But the biomass of R. glutinis will drop slightly, 16.5 g/L and 67.4% respectively. Finally, considering that the stirring rate and the ventilation volume cannot be too high. The best condition obtained are scaled up to a 30 L stirred fermenter. The experimental result shows that the maximum biomass is 38.1 g/L and the maximum lipid content is 67.96%. Thus, it can be confirmed that the feasibility of scaling up the fermentation conditions.
In addition, in this study, the aqueous two-phases extraction (ATPE) is used to extract lipase. Because of its simple operation, short stratification time, and can be carried out at normal temperature and pressure, it is more suitable as a method for separating and extracting enzymes. The experimental results show that the use of supernatant with longer fermentation time can produce more lipase. In this experiment, potassium oxalate and polyethylene glycol (PEG) are used as ATPE solvents. And by changing its top and bottom layer concentration, PEG molecular weight, reaction temperature and other different conditions to find the best extraction conditions. According to the experimental result, the enzyme activity begins to decrease after its temperature exceeds 40℃, so 37℃ is the best extraction temperature. The best extraction conditions are adding 2.15 g of PEG 1000 and 1.67 g of potassium oxalate, and using ddH2O to quantify the volume to 8.15 ml. After fully dissolving and mixing it to obtain an ATPE system, then add 1.85 ml of fermentation supernatant to mix well, measure the lipase activity of the upper and lower layers after standing for one hour. The Ke value is 5.34. This means that 84.2% of the lipase is distributed to the top layer. The above data shows that it is possible to use ATPE as an extraction method for lipolytic enzymes in the fermentation supernatant. Due to its low operating cost and raw material cost, it also has considerable potential for future industrial development.

中文摘要 i
Abstract iii
圖目錄 x
表目錄 xii
第一章 緒論 1
第二章 文獻回顧 2
2.1 酵母菌 2
2.1.1 紅酵母菌 (Rhodotorula) 2
2.1.2 黏紅酵母菌 (Rhodotorula glutinis) 3
2.2 生質柴油 (Biodiesel) 3
2.3 廢棄食用油 (Waste cooking oil，WCO) 6
2.4 微生物的產油機制 6
2.5 微生物突變 (Mutant) 10
2.5.1 自然選育 10
2.5.2 人工誘變[10] 10
2.5.3 物理誘變 10
2.5.3.1 紫外照射 10
2.5.3.2 電離輻射 10
2.5.3.3 雷射法 11
2.5.3.4 微波法 11
2.5.4 化學誘變 12
2.5.4.1 烷化劑 12
2.5.4.2 鹼基類似物 12
2.5.4.3 無機化合物 12
2.6 界面活性劑 (Surfactants) 13
2.6.1 陰離子界面活性劑 (Anionic surfactant) [12] 13
2.6.2 陽離子界面活性劑 (Cationic surfactant) [12] 13
2.6.3 兩性界面活性劑 (Amphoteric surfactant)[13] 14
2.6.4 非離子界面活性劑 (Non-ionic surfactant)[14] 14
2.7 脂肪酶 (Lipase) 16
2.7.1 非特異性脂肪酶 (Non-specific lipase) 16
2.7.2 1,3 特異性脂肪酶 (1,3-specific lipase) 16
2.7.3 脂肪酸特異性脂肪酶 (Fatty acid specific lipase)： 17
2.7.4 脂肪酶的應用 17
2.7.4.1 家用清潔劑 17
2.7.4.2 乳品工業 17
2.7.4.3 油脂化學工業 18
2.7.4.4 合成三酸甘油酯 18
2.7.4.5 合成化妝品或保養品的添加物 18
2.7.4.6 合成界面活性劑 18
2.7.4.7 製藥工業 19
2.8 二水相萃取(Aqueous two-phase extraction, ATPE) 19
2.8.1 二水相萃取的特點 19
2.8.2 ATPE 在萃取脂肪酶之應用 20
2.9 碳源 (Carbon source) 21
2.10 發酵策略 21
2.10.1 批次發酵(Batch fermentation)[25] 21
2.10.2 連續式發酵(Continuous fermentation)[26] 22
2.10.3 饋料批次發酵(Fed-batch fermentation)[27] 22
第三章 實驗材料與方法 23
3.1 實驗材料 23
3.1.1 菌株 23
3.1.2 實驗藥品 25
3.2 實驗儀器 28
3.3 實驗架構 30
3.4 原始菌種保存 32
3.5 培養基組成 32
3.5.1 培養皿培養基 32
3.5.2 種子培養基 (Seed culture medium，SM) 32
3.5.3 發酵培養基 (Fermentation medium，FM) 32
3.6 接菌 33
3.7 實驗設計 33
3.7.1 搖瓶之發酵程序 33
3.7.1.1 菌株進行突變前後之影響 33
3.7.1.2 不同碳源之影響 34
3.7.1.3 不同廢棄食用油濃度之影響 35
3.7.1.4 不同界面活性劑濃度之影響 35
3.7.2 5L 攪拌式發酵槽之發酵程序 36
3.7.2.1 菌株進行突變前後之影響 36
3.7.2.2 不同廢棄食用油濃度之影響 37
3.7.2.3 利用不同發酵程序之影響 37
3.7.3 30 L 攪拌式發酵程序 38
3.7.4 二水相萃取法(ATPE) 38
3.7.4.1 不同濃度之鹽類 38
3.7.4.2 不同濃度之聚合物 39
3.7.4.3 不同分子量之聚合物 39
3.7.4.4 不同體積之發酵上清液 39
3.8 分析方法 40
3.8.1 菌體濃度分析方法 40
3.8.2 總脂質濃度分析方法 40
3.8.3 脂肪酶活性分析方法 40
3.8.3.1 磷酸鹽緩衝液配製 41
3.8.3.2 脂肪酶顯色劑配製 41
3.8.3.3 脂肪酶吸光度標準曲線制定 41
3.8.3.4 脂肪酶活性測定 41
3.8.3.5 利用甲苯終止反應 42
3.8.3.6 溫度對於脂肪酶反應的影響 42
3.8.4 蛋白質總量分析方法 43
3.8.5 可溶性蛋白質分離與鑑定 44
3.8.5.1 SDS-PAGE膠體配製 44
3.8.5.2 發酵液準備 44
3.8.5.3 SDS-PAGE下膠配製 44
3.8.5.4 SDS-PAGE上膠配製 44
3.8.5.5 Instant Blue (Coomassie blue)染色 45
3.8.6 乙醇沉澱法 45
3.9 發酵培養裝置圖 47
3.9.1 搖瓶發酵培養裝置圖 47
3.9.2 5L 攪拌式發酵槽培養裝置圖 47
3.9.3 30L攪拌式發酵槽培養裝置圖 48
第四章 結果與討論 49
4.1 搖瓶發酵實驗 49
4.1.1 使用WCO作為碳源時菌體突變前後之影響 49
4.1.1.1 UV燈照射時間之影響 49
4.1.1.2 使用WCO作為碳源時菌體突變前後之菌體濃度 51
4.1.1.3 使用WCO作為碳源時菌體突變前後之pH值 52
4.1.1.4 使用WCO作為碳源時菌體突變前後之脂質含量 53
4.1.2 不同碳源之影響 54
4.1.2.1 改變碳源對菌量之影響 54
4.1.2.2 改變碳源對pH值之影響 55
4.1.2.3 改變碳源對Lipid content之影響 57
4.1.3 使用WCO作為碳源時改變不同濃度之影響 58
4.1.3.1 使用WCO作為碳源時改變不同濃度對菌體濃度之影響 58
4.1.3.2 使用WCO作為碳源時改變不同濃度對pH值之影響 59
4.1.3.3 使用WCO作為碳源時改變不同濃度對脂質含量的影響 60
4.1.4 使用WCO作為碳源時改變界面活性劑濃度之影響 61
4.1.4.1 使用WCO作為碳源時改變界面活性劑濃度對於菌體濃度之影響 61
4.1.4.2 使用WCO作為碳源時改變界面活性劑濃度對於pH值之影響 62
4.1.4.3 使用WCO作為碳源時改變界面活性劑濃度對於脂質含量之影響 63
4.2 5 L 攪拌式發酵槽發酵實驗 64
4.2.1 使用WCO作為碳源時菌體突變前後之影響 65
4.2.1.1 使用WCO作為碳源時菌體突變前後之菌體濃度 65
4.2.2 使用WCO作為碳源時濃度之影響 66
4.2.2.1 使用WCO作為碳源時濃度對菌體濃度之影響 67
4.2.2.2 使用WCO作為碳源時濃度對最大脂質含量之影響 68
4.2.3 使用WCO作為碳源時利用Fed-batch的影響 69
4.2.3.1 使用WCO作為碳源時利用Fed-batch對菌體濃度的影響 70
4.2.3.2 使用WCO作為碳源時利用Fed-batch對最大脂質含量的影響 71
4.3 30 L 攪拌式發酵槽發酵實驗 72
4.3.1 菌體濃度 73
4.3.2 脂質含量 74
4.4 不同條件萃取脂肪酶 75
4.4.1 不同時間點的發酵上清液之比較 76
4.4.2 利用不同濃度之發酵上清液進行ATPE萃取脂肪酶 77
4.4.3 二水相(ATPE)中不同濃度鹽類對於萃取脂肪酶之影響 78
4.4.4 二水相(ATPE)中不同濃度的PEG 4000對於萃取脂肪酶之影響 79
4.4.5 不同分子量之PEG對於萃取脂肪酶之影響 80
4.4.6 苯與甲苯作為終止試劑之比較 81
4.4.7 不同溫度下脂肪酶之活性 82
4.5 ATPE上下層總蛋白質濃度之測定 83
4.6 SDS-PAGE 84
4.7 文獻比較 85
4.7.1 菌體濃度與脂質含量之發酵實驗 85
4.7.2 脂肪酶活性之分析實驗與文獻比較 87
第五章 結論與未來展望 88
5.1 結論 88
5.2 未來展望 89
第六章 參考資料 90
第七章 附錄 94

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