微生物油脂具快速合成和積累、無需農業土地、不受天候影響、易大規模生產與性質相似於植物油等諸多優點，近幾十年來，被認定為具生產生物柴油潛力的非糧食類原料。油脂性微生物中酵母菌因具備高生長速率、無須光照且能更快速累積油脂等特點，使得以酵母菌作為替代能源生產者之研究比重逐漸攀升。粗甘油為生質柴油之主要副產物，隨著生質柴油的需求趨勢日益增加，勢必產生大量轉酯化後的廢棄物—粗甘油，因此本研究從生質柴油廠活性污泥槽篩選油脂性酵母菌，藉由酵母菌將廢棄甘油轉化成油脂累積於菌體內，累積的油脂可再進一步作為生質柴油的油料來源，以達到處理廢棄甘油與降低油料成本之雙贏目標。
影響酵母菌生長與油脂累積的培養因子有碳源種類、氮源種類、溫度、pH值與碳/氮比等。本研究探討酵母菌在各種不同培養條件下的菌體生長與油脂累積情形，以求得酵母菌生長與油脂生產的最佳條件。此外，為避免使用過量的有機溶劑進行油脂萃取進而增加環境污染風險與經濟上的損失，故本研究亦針對不同油脂萃取條件進行油脂萃取的最佳化，使有機溶劑達最大萃取效益。
結果顯示從生質柴油廠活性污泥槽中分離四株酵母菌，其中LP-1與LP-2在經18S rDNA序列比對下，得知菌名分別為Candida rugosa與Rhodotorula mucilaginosa。於油脂萃取試驗中，不同生質物含量顯著影響油脂萃取效率，兩株菌的萃取效率皆隨生質物含量降低而提升，所得最適萃取生質物含量為30 mg。輔助超音波震盪可提升油脂萃取效率，但時間過長反而使萃取效率下滑，所得最適震盪時間為30 min；其中酵母菌Candida rugosa LP-1可提升1倍以上油脂含量，酵母菌Rhodotorula mucilaginosa LP-2則可提升約65%。而凍融法的次數多寡對菌株Candida rugosa LP-1與Rhodotorula mucilaginosa LP-2油脂萃取並無顯著影響。
將所得油脂萃取最佳條件進一步應用於批次實驗中，可得知同時考慮細胞產量與油脂含量，菌株Candida rugosa LP-1最適溫度為27℃，菌株Rhodotorula mucilaginosa LP-2為33℃；脂肪酸組成含量受到溫度影響，其中又以Rhodotorula mucilaginosa LP-2所受之影響較為顯著。碳源試驗中最適Candida rugosa LP-1生長碳源為Crude glycerol，最適累積油脂為Pure glycerol，而以Crude glycerol為碳源可得最高油脂產量；最適Rhodotorula mucilaginosa LP-2生長與累積油脂碳源皆為Pure glycerol，因此能得最高油脂產量之碳源亦為Pure glycerol。另外，兩株菌之最適生長與累積油脂氮源皆為Yeast extract，(NH4)2SO4雖可降低油料所需成本，但因於消耗氮源的過程中釋出SO42-，使得pH驟降，進而影響菌體生長與油脂累積。碳/氮比試驗中則可得知兩株菌之細胞產量皆隨碳/氮比升高而降低，所得最適菌體生長碳/氮比為25，然油脂含量隨碳/氮比升高而增加，但過高的碳氮比不僅無法使菌體生長，同時也抑制油脂累積。其中碳/氮比45適合菌株Candida rugosa LP-1累積油脂，最適菌株Rhodotorula mucilaginosa LP-2的碳/氮比則為65，碳/氮比對Rhodotorula mucilaginosa LP-2脂肪酸的去飽和造成顯著的影響。

Microbial lipid has many advantages, such as short life cycle, no demand of large farm land, no influence by weather, easily large-scale production and similar fatty acids compositions with vegetable oils. Oleaginous yeast with high growth rate, no need of illumination and fast lipid accumulation has attracted much interest in recent decades. Crude glycerol is a major byproduct in the biodiesel manufacturing process. Along with increasing demand for biofuels,it consequentially produces the massive quantity of crude glycerol needed to be treated. To deal with crude glycerol and transform it into useful oil feed for biodiesel,in this study, oleaginous yeasts were isolated from activated sludge tank of a biodiesel plant, and the bioconversion of crude glycerol into cellular oil using isolated yeasts was investigated.
The factors affecting yeast growth and lipid accumulation include carbon source, nitrogen source, temperature, pH, and carbon/nitrogen ratio. This study used different incubation parameters to realize the cell growth and lipid accumulation. In addition, to avoid excess use of organic solvent to extract lipid, this study also established optimum lipid extraction conditions to receive efficient lipid yield.
The results showed that 4 yeast strains were isolated from the biodiesel plant. Strain LP-1 and LP-2 were identified as Candida rugosa and Rhodotorula mucilaginosa,respectively. Different biomass weight significantly influenced lipid extraction efficiency. Higher biomass amount caused lower extraction efficiency. The optimal biomass weight for lipid extraction was 30 mg. The lipid contents of strain Candida rugosa LP-1 and strain Rhodotorula mucilaginosa LP-2 could be improved more than 1 time and 65%, respectively, by ultrasonication when extracting lipid. However, long ultrasonication duration obtained decline of extraction efficiency. The optimal ultrasonication duration for lipid extraction was 30 min. The numbers of freeze-thaw cycles had no significant impact on lipid extraction.
The optimum temperatures of strain Candida rugosa LP-1 and strain Rhodotorula mucilaginosa LP-2 for maximum lipid productivity were 27oC and 33oC, respectively. The most suitable carbon sources for strain Candida rugosa LP-1 and strain Rhodotorula mucilaginosa LP-2 to accumulate lipid were crude glycerol and pure glycerol, respectively. Yeast extract was the most suitable nitrogen source for both strains. Although, (NH4)2SO4 was a cheap nitrogen source, it caused pH decrease so that cell growth and lipid accumulation were both influenced. Low carbon/nitrogen ratio was beneficial to biomass growth. In contrast, the cellular lipid content increased with the increase of the carbon/nitrogen ratio. Higher carbon/nitrogen ratio not only inhibited cell growth, but also oil accumulation. The most suitable carbon/nitrogen ratios for strain Candida rugosa LP-1 and strain Rhodotorula mucilaginosa LP-2 were 45 and 65, respectively. Carbon/nitrogen ratio caused a significant impact on the fatty acid desaturation, especially to strain Rhodotorula mucilaginosa LP-2.

摘要 i
Abstract iii
目錄 v
表目錄 viii
圖目錄 ix
第一章 緒論 1
1.1 研究緣起 1
1.2 研究目的及內容 2
1.3 研究架構 3
第二章 文獻回顧 4
2.1 能源現況 4
2.2 生質柴油 5
2.2.1 生質柴油簡介 5
2.2.2 生質柴油發展 5
2.2.2.1 第一代生質柴油 7
2.2.2.2 第二代生質柴油 7
2.2.2.3 第三代生質柴油 8
2.2.3 轉酯化反應 8
2.2.4 生質柴油使用現況 9
2.3 產油微生物 11
2.3.1 微藻 13
2.3.2 細菌 13
2.3.3 真菌和酵母菌 14
2.4 酵母菌產油機制 14
2.5 影響酵母菌油脂產量的因素 16
2.5.1 溫度 16
2.5.2 pH值 17
2.5.3 碳源 17
2.5.4 氮源 19
2.5.5 碳/氮比 20
2.6 再利用廢棄物特性介紹 21
2.6.1 粗甘油 21
2.6.2 糖蜜 23
2.6.3 生乳廢液 24
2.7 超音波輔助油脂萃取 25
2.7.1 超音波作用原理 25
2.7.2 超音波應用 26
2.8 聚合酶連鎖反應-變性梯度膠體電泳原理與應用 27
2.8.1 聚合酶連鎖反應 27
2.8.2 變性梯度膠體電泳 28
第三章 材料與方法 29
3.1實驗架構 29
3.2污泥來源 31
3.3 廢棄物特性 31
3.4培養基配製 32
3.4.1 篩選菌株培養基 32
3.4.2 油脂累積培養基 33
3.5實驗藥品與儀器設備 34
3.6 油脂性酵母菌之分離與篩選 38
3.6.1 菌株分離與保存 38
3.6.2 Nile red螢光染劑快篩與菌種鑑定 40
3.7生質柴油廠活性污泥槽之菌相分析 40
3.7.1 Total Genomic DNA之萃取 40
3.7.2 聚合酶連鎖反應-變性梯度膠體電泳(PCR-DGGE)法 41
3.7.2.1 聚合酶連鎖反應(PCR) 41
3.7.2.2瓊脂糖凝膠電泳分析分析 43
3.7.2.3變性梯度膠體電泳(DGGE) 43
3.8油脂萃取最佳化 44
3.8.1 不同生質物含量萃取試驗 44
3.8.2 不同超音波震盪時間試驗 46
3.8.3 不同凍融次數試驗 46
3.9油脂累積之批次實驗 47
3.9.1 不同培養溫度試驗 48
3.9.2 酵母菌累積油脂-不同碳源種類試驗 48
3.9.3 酵母菌累積油脂-不同氮源種類試驗 48
3.9.4 酵母菌累積油脂-不同碳/氮比試驗 48
3.10分析方法 49
3.10.1 細胞量分析 49
3.10.1.1 O.D.與細胞乾重檢量線 49
3.10.1.2 O.D.值與菌數檢量線 49
3.10.1.3 細胞量 50
3.10.2 單細胞油脂分析 50
3.10.2.1 油脂含量分析 50
3.10.2.2 油脂組成分析 51
3.10.3 水質分析 52
3.10.3.1 pH值 52
3.10.3.2 化學需氧量(COD)分析 52
3.10.3.3 總氮含量分析 53
3.10.3.4 甘油濃度 54
第四章 結果與討論 55
4.1 Nile red螢光染劑快篩生質柴油廠活性污泥槽中之油脂性酵母菌 55
4.1.1 油脂性酵母菌之分離 55
4.1.2 Nile red螢光染劑快篩與菌種鑑定 57
4.2生質柴油廠活性污泥槽之菌相結構 59
4.2.1 活性污泥槽菌相分析結果 59
4.3酵母菌於不同生質物含量、超音波震盪時間、凍融次數下進行油脂萃取之效率 59
4.3.1 最適油脂萃取之生質物量 61
4.3.2 最適油脂萃取之超音波震盪時間 63
4.3.3 最適油脂萃取之凍融次數 65
4.4酵母菌於不同溫度、碳源種類、氮源種類、碳/氮比等環境條件下之菌體生長與油脂累積之變化情形 67
4.4.1 溫度對油脂累積與菌株生長之影響 67
4.4.2 碳源種類對油脂累積與菌株生長之影響 73
4.4.3 氮源種類對油脂累積與菌株生長之影響 84
4.4.4 碳/氮比對油脂累積與菌株生長之影響 94
第五章 結論與建議 100
5.1 結論 100
5.2 建議 102
參考文獻 103

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