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研究生:余依嬛
研究生(外文):Yi-Huan Yu
論文名稱:以工業廢棄物粗甘油與酒糟水做為培養基探討Rhodotorula glutinis的生長影響
論文名稱(外文):The Fermentation Process Development Of Rhdotorula glutinis By Using Crude Glycerol and Thin Stillage
指導教授:顏宏偉
指導教授(外文):Hong-Wei Yen
口試委員:顏宏偉楊芳鏘劉永銓
口試委員(外文):Hong-Wei YenFan-Chiang YangYung-Chuan Liu
口試日期:2012-06-15
學位類別:碩士
校院名稱:東海大學
系所名稱:化學工程與材料工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:85
中文關鍵詞:粗甘油酒糟水生質能源Rodotorula glutinis
外文關鍵詞:Crude GlycerolThin StillageBiofuelsRodotorula glutinis
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Rhodotorula glutinis (BCRC 22360) 為一株油脂性酵母菌,可累積大於70 %脂質含量且油脂成分類似於植物油,其細胞生長迅速、可用高密度細胞培養,因此具有相當高發展潛力,可作為生質柴油生產之替代性油料的來源之一。本研究主要探討利用粗甘油及酒糟水提升R. glutinis生長及累積油脂,進而提高整體油脂生產速率。
隨著時油價格逐年上升,當生質柴油產量大幅提高的同時,亦會大量累積生質柴油副產物-粗甘油,因含有過量之甲醇、未反應之催化劑與皂化物被認定為有害廢棄物,價值低廉且需要繁複的純化手續才能再售予精煉甘油業者,因此尋找粗甘油的用途已成了全球研究的重要課題。米酒是台灣常見的料理酒,台中酒廠在製造過程中,每天會產生兩百萬公升的廢棄酒糟水,是酒廠處理廢水之主要汙染負荷來源其BOD值高達25000 ppm,COD值則有50000 ppm,雖然已有好氧汙泥消化及厭氧消化等方法做為汙水處理的步驟,但基於回收再利用的環保觀點,建立一個有效利用廢酒糟水的系統是值得探討的。
在固定各種碳源 (葡萄糖、純甘油、粗甘油) 濃度為30 g/L之搖瓶實驗中,粗甘油作為碳源其菌體濃度可達5.4±0.4 g/L,油脂濃度為1.2±0.2 g/L,高於其他兩者。固定粗甘油濃度30 g/l進行探討氮源影響之發酵槽實驗,以酒糟水當氮源之菌體濃度為10.8±0.4 g/L,油脂含量為26.0±3.0% ,分別為使用標準氮源-酵母萃取物的2.0倍及1.1倍。由實驗結果可知,因為粗甘油中含有多種細胞生長所需的微量元素因此可增加菌體濃度,而酒糟水中含有豐富的有機酸提高了培養基之C/N,因此可以增加油脂含量。

Rhodotorula glutinis (BCRC 22360) is an oleaginous yeast that can accumulate copious quantities of lipids. R. glutinis grew fast to obtain high biomass concentration, and comparable with plant seed oil, therefore it is all potential alternative oil resources for biodiesel production. This investigation used different fermentation strategies to enhance the growth of R. glutinis and accumulation of oil, thereby increasing overall oil productivity.
With oil price increasing, a large of crude glycerol accumulates as biodiesel productivity rising. Biodiesel by product-crude glycerol containing glycerin、soap methanol and unreacted catalyst was identified as hazardous industrial waste. The low price waste crude glycerol was sold to glycerol refined manufactories and purified by complicate process. To find out a new usage of crude glycerol has become an important issue for global research. Rice wine is a common cooking wine in Taiwan. Taiwan winery, can generated 2 million liters waste thin stillage with pollution load up to 25000 ppm BOD and 50000 ppm COD. Although there are a lot of treatments to process the distillery waste water, like activated sludge or anaerobic digestion methods but from the view of resource recovery, these methods are not economical. Therefore, the development of an effective system for the utilization of distillery wastewater is desirable.
In flask batch experiments we fixed the carbon source ( glucose、 pure glycerol、crude glycerol) concentration in 30 g/l, the crude glycerol can lead to the highest biomass of 5.4±0.35 g/l and total lipid of 1.24±0.17 g/l. In the 5-L batch experiments, by using the thin stillage as the nitrogen source, we can get the biomass of 10.8±0.35 g/l and total lipid of 26.0±2.98 % higher than the standard nitrogen source- yeast extract 2.0 and 1.14 times respectively. From the results, the crude glycerol can provide lots of trace elements for cell to increase the biomass, and thin stillage also contained glycerol lead to high C/N ratio environment to let the cells accumulate more lipids.

中文摘要........................................I
英文摘要........................................II
謝誌........................................... IV
第一章 緒論.....................................1
第二章 文獻回顧.................................2
2.1 生質柴油....................................2
2.1.1 生質柴油副產物-粗甘油(Crude glycerol).....4
2.1.2 酒糟水 (Thin stillage)....................8
2.2 產油微生物..................................11
2.2.1 微藻......................................13
2.2.2 細菌......................................15
2.2.3 酵母菌與真菌..............................15
2.4 菌種選擇....................................16
2.4.1 菌種介紹-Rhodotorula glutinis............16
2.5 微生物產油機制..............................18
2.6 影響微生物油脂產量因子......................21
2.6.1 pH值......................................21
2.6.2 光........................................21
2.6.3 碳源和氮源................................22
2.6.4 碳源......................................22
2.6.5 氮源......................................23
2.6.6 無機鹽和微量元素..........................23
2.7 微生物油脂發酵策略介紹......................25
2.7.1 批次微生物油脂發酵........................25
2.7.2 兩階段培養策略............................25
第三章 材料與方法...............................26
3.1 實驗材料....................................26
3.1.1 菌株......................................26
3.1.2 實驗藥品..................................27
3.2 實驗儀器....................................28
3.3 分析方法....................................30
3.3.1 光照強度測定方法..........................30
3.3.2 菌體乾重 (Dry cell weight, DCW) 分析方法..30
3.3.3 總脂質濃度分析方法........................30
3.3.4 甘油分析方法..............................31
3.4 實驗方法....................................32
3.4.1 原始菌種保存..............................32
3.4.2 種子培養基 (Seed Medium, SM)..............32
3.4.3 搖瓶批次發酵之培養基......................33
3.4.4 攪拌式發酵槽之發酵培養基..................34
3.4.5 接菌......................................35
3.5 實驗架構....................................36
3.6 實驗培養條件................................37
3.6.1 搖瓶批次發酵程序(Flask Experiments).....37
3.6.3 兩階段培養................................40
3.6.4 15-L攪拌式發酵槽批次程序 (batch)..........41
第四章 結果與討論...............................44
4.1 Rhodotorula glutinis之脂肪酸分析結果 ......44
4.2 搖瓶批次發酵程序(Flask Experiment)........45
4.2.1 不同碳源之影響............................45
4.2.2 粗甘油預處理方法之影響....................47
4.2.3 不同氮源之影響............................49
4.2.4 酒糟水加不同粗甘油濃度之影響..............51
4.3 5-L 攪拌式發酵槽批次程序 (Batch)............53
4.3.1 粗甘油與純甘油對R. glutinis生長之影響 ....53
4.3.2 酒糟水對R. glutinis之影響.................56
4.3.3 照光對R.glutinis生長及累積油脂之影響 .....59
4.3.3.2 利用酒糟水作為氮源進行照光/不照光對於R. glutinis生長及累積油脂之影響 ................................................59
4.4 攪拌式發酵槽饋料批次程序 (fed-batch)........64
4.5 15-L 攪拌式發酵槽批次程序 (batch)...........66
第五章 結論與未來展望...........................71
5.1 結論........................................71
5.2 未來展望....................................72
參考文獻........................................73
附錄 ..........................................76
附錄 A.1.......................................76
附錄 B.1.......................................77
附錄 B.2.......................................78
附錄 B.3.......................................79
附錄 B.4.......................................80
附錄 B.5.......................................81
附錄 B.6.......................................82
附錄 B.7.......................................83
附錄 B.8.......................................84
作者簡歷........................................85

表目錄
表2.1 粗甘油微量元素分析表......................6
表2.2 微量元素對細胞生理功能之影響..............6
表2.3 Compositions of thin stillage.............10
表2.4 Oil content of some microorganisms........12
表3.1 實驗藥品清單..............................27
表3.2 實驗儀器清單..............................28
表3.3 種子培養基 (Seed Medium, SM) 組成.........32
表3.4 搖瓶批次發酵培養基之組成 (I)..............33
表3.5 搖瓶批次發酵培養基之組成 (II).............33
表3.6 搖瓶批次發酵培養基之組成 (III)............34
表3.7 攪拌式發酵槽培養基之組成 (I)..............34
表3.8 攪拌式發酵槽培養基之組成(II)..............35
表4.1 不同碳源對R.glutinis生長及累積油脂之影響..46
表4.2 預處理方法對R.glutinis生長及累積油脂之影響48
表4.3 不同氮源對R.glutinis生長及累積油脂之影響..50
表4.4 酒糟水加不同濃度粗甘油對R.glutinis生長及累積油脂之影響
................................................52
表4.5 粗甘油對R.glutinis生長及累積油脂之影響....55
表4.6 酒糟水對R.glutinis生長及累積油脂之影響....58
表4.7 照光之批次發酵策略下,R. glutinis 生長及累積油脂之變化
................................................63
表4.8 發酵槽饋料批次程序對R. glutinis 生長及累積油脂之變化..............................................65
表4.9 15-L 攪拌式發酵槽批次程序對R. glutinis 生長及累積油脂之變化..............................................67
表4.10 各實驗之動力學參數整理...................68
表4.11 Rhodotorula glutinis文獻與本研究成果比較(I)
................................................69
表4.12 Rhodotorula glutinis文獻與本研究成果比較(II)
................................................70

圖目錄
圖2.1 實驗室自製生質柴油.......................3
圖2.2 工業級粗甘油.............................7
圖2.3 不同甘油比較圖...........................7
圖2.4 阿米洛法米酒製程示意圖...................9
圖2.5 藻類戶外培養池...........................14
圖2.6 Rhodotorula glutinis的細胞型態...........17
圖2.8 A diagram to show the organisation of a hypothesised lipogenic metabolon.......................................20
圖3.1 Rhodotorula glutinis 在agar plate上之外觀26
圖3.2 實驗裝置圖 (Ι)..........................42
圖3.3 實驗裝置圖 (II)..........................43
圖4.1 不同碳源對R.glutinis生長及累積油脂之影響.46
圖4.2 不同預處理方法對R.glutinis生長及累積油脂之影響
................................................48
圖4.3 不同氮源對R.glutinis生長及累積油脂之影響.50
圖4.4 酒糟水加不同濃度粗甘油對R.glutinis生長及累積油脂之影響 ................................................52
圖4.5 140 g/l粗甘油批次發酵策略下,R. glutinis 生長及累積油脂之變化
................................................54
圖4.6 140 g/l純甘油批次發酵策略下,R. glutinis 生長及累積油脂之變化
................................................54
圖4.8 Y.E.為氮源之批次發酵策略下,R. glutinis 生長及累積油脂之變化
................................................57
圖4.9 140 g/l粗甘油加Yeast extract照光之批次發酵策略下,R. glutinis 生長及累積油脂之變化......................................60
圖4.10 140 g/l粗甘油加Yeast extract無光之批次發酵策略下,R. glutinis 生長及累積
................................................60
圖4.11 30 g/l粗甘油加酒糟水之批次發酵策略下,照光對R. glutinis 生長及累積油脂之變化............................................61
圖4.12 30 g/l粗甘油加酒糟水之批次發酵策略下,不照光對R. glutinis 生長及累積油脂之變化..........................................61
圖4.13 粗甘油加Yeast extract之批次發酵策略下,照光與不照光對R. glutinis 生長及累積油脂之變化..................................62
圖4.14 粗甘油加酒糟水之批次發酵策略下,照光與不照光對R. glutinis 生長及累積油脂之變化..........................................62
圖4.15 發酵槽饋料批次程序對R. glutinis 生長及累積油脂之變化 ................................................65
圖4.16 15-L 攪拌式發酵槽批次程序對R. glutinis 生長及累積油脂之變化 ................................................67


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