臺灣博碩士論文加值系統

Rhodotorula glutinis（BCRC 22360）是株能迅速累積高油脂的酵母菌，其生
長快速、可進行高密度細胞培養，，且能以廢棄物粗甘油作為碳源，兼具環保價
值，因此具有相當高發展潛力。本研究利用此菌株快速生長特性，改變滅菌策略
以及將培養基配製由純水置換成海水，並選用氣舉式發酵槽進行培養，以探討對
Rhodotorula glutinis生長及油脂含量的影響性。
生質柴油在歐美國家以普遍應用於交通工具中，大部分料源來自甘蔗、大豆
等糧食作物，造成需求面積廣大的問題，台灣土地面積狹小、人口密度高等環境
限制下更凸顯出困境，若能以海水培養富含高油脂的R. glutinis，便可解決土地
利用和成本過高的問題。
本實驗中所使用的5 L 氣舉式發酵槽中，將一般培養微生物的純水換成全海
水，R. glutinis濃度可達到15.9 g/L，最大油脂濃度也可達12.4 g/L 與使用純水相
差無幾，顯示其可在全海水下的環境生存，所以海水是可以取代RO水的。然而
在發酵工程中，高溫滅菌以保持單一菌種生長環境，是一個必要但相當耗能的過
程，所以本研究進一步探討改變培養基酸鹼值以取代高溫滅菌，降低發酵培養過
程的能耗，結果顯示黏紅酵母菌進行不滅菌培養(pH 5.5)，在39 hr 即受到抑制
無法繼續增加濃度，於是改其生長pH 由5.5 調降至4.0 且不滅菌；R. glutinis最
終濃度能達到11.7 g/L，但降低pH卻會導致油脂含量下降，此實驗發現在48 hr，
R. glutinis濃度為5.8 g/L，在此菌體濃度下，R. glutinis有機會成為槽體內優勢菌
種，於是改採用兩階段pH控制策略，起始pH 4.0 不滅菌並在48 hr後將pH調
升為5.5，兩階段pH的發酵策略雖可以有效增加菌體濃度與油脂含量，但其效
果與控制組R. glutinis濃度相距達5 g/L 之多，在完全不滅菌下，R. glutinis菌體
濃度將遠低於控制組。雖然此種做法可以降低生產成本，但大幅的降低了菌體最
終濃度，因此採用不滅菌的做法在商業化生產上較為不可行

Rhodotorula glutinis（BCRC 22360）is an oleaginous that can accumulate
copious quantities of lipid. As compared to plant seed oil, R. glutinis grew fast with
high lipid content biomass, therefore it is a potential alternative oil resource for
biodiesel production. In this study, the effects of seawater and sterilization strategy to
enhance the growth of R. glutinis and lipid production in airlift bioreactor.
Biodiesel as the renewable energy been applied to vehicle in USA and Europe
currently being produced from cane or soybean etc. But the problem of land usage
and densely populated in Taiwan, maybe it can be solved by using seawater to
cultivate R. glutinis.
In a 5 L airlift bioreactor, the experiments by using the medium of seawater can
lead to the biomass of 15.9 g/L and the max total lipid of 12.4 g/L. It mains that R.
glutinis can grow in the environment of seawater. In the fermentation engineering,
sterilization is an important and necessary process to avoid the contamination.
However, it also demands lots of energy consumption. So the use of non-sterile
fermentation would also effectively decrease the fermentation process cost. The pH
control at low level was adopted to replace the heat sterilization process. As a result
was found that non-sterilization at pH 5.5 hours was contaminated by germ after 39
hours cultivation. Therefore, a decrease in the growth pH from 5.5 to 4.0 led to an
increase in the biomass of R. glutinis at 11.7 g/L with the medium non-sterilized.
Nevertheless, the low pH could reduce the lipid content. In this study, a two-stage of
pH control was proposed, which the pH was controlled at 4.0 and adjusted to 5.5 after
the growth stepping into the stable phase. Conclusively, the results of this study
indicated that the two-stage pH control can significantly enhance the lipid content, but
the total lipid was still lower than that of control. Non-sterilization can solve the
problem for the high cost but it seriously influenced the production of biomass and
lipid content. It was not available.

中文摘要....................................................................................................................... II
Abstract ........................................................................................................................ 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 .................................................................. 4
2.1.2 9-胡蘿蔔素（9-carotene） ............................................................... 6
2.2 產油微生物................................................................................................... 7
2.2.1 微生物油脂........................................................................................ 9
2.3 微生物產油機制......................................................................................... 11
2.4 影響微生物油脂產量因子.......................................................................... 15
2.4.1 溫度.................................................................................................. 15
2.4.2 pH值 ................................................................................................ 15
2.4.3 光...................................................................................................... 15
2.4.4 碳源和氮源...................................................................................... 16
2.4.5 碳源.................................................................................................. 17
2.4.6 氮源.................................................................................................. 17
2.4.7 無機鹽和微量元素.......................................................................... 17
2.5 紅酵母菌（Rhodotorula） ........................................................................ 18
2.5.1 菌種選擇-Rhodotorula glutinis ....................................................... 19
2.6 海水............................................................................................................. 20
2.7 幫助溶氧物質（Oxygen-vector）............................................................. 22
2.7.1 界面活性劑 Tween-80 .................................................................... 22
2.8 發酵策略..................................................................................................... 23
2.8.1 批次微生物油脂發酵...................................................................... 23
2.8.2 兩階段培養策略.............................................................................. 23
第三章 材料與方法.................................................................................................. 24
3.1 實驗材料..................................................................................................... 24
3.1.1 菌株.................................................................................................. 24
3.1.2 實驗藥品.......................................................................................... 25
3.2 實驗儀器..................................................................................................... 27
3.3 分析方法..................................................................................................... 29
3.3.1 菌體濃度分析方法.......................................................................... 29
3.3.2 葡萄糖濃度分析方法...................................................................... 29
3.3.3 總脂質濃度分析方法...................................................................... 29
3.3.4 甘油分析方法.................................................................................. 29
3.4 實驗方法..................................................................................................... 31
3.4.1 原始菌種保存.................................................................................. 31
3.4.2 培養基組成...................................................................................... 31
3.4.3 搖瓶發酵培養基（Fermentation medium, FM） .......................... 32
3.4.4 接菌.................................................................................................. 33
3.5 實驗架構..................................................................................................... 34
3.6 實驗培養條件............................................................................................. 35
3.6.1 搖瓶批次發酵程序（Flask Experiments） .................................... 35
3.6.1.1 不同pH值之影響 ........................................................................ 35
3.6.1.2 不同pH值且不滅菌之影響 ........................................................ 35
3.6.1.3 改變氯化鈉濃度之影響............................................................... 35
3.6.1.4 不同海水含量培養基之影響....................................................... 36
3.6.1.5 添加幫助溶氧物質之影響........................................................... 36
3.6.2 5 L 氣舉式批次發酵程序 (Fermentation Experiments) ............... 37
3.6.2.1 不滅菌之影響............................................................................... 37
3.6.2.2 兩階段培養................................................................................... 37
3.6.2.3 在90℃下滅菌之影響.................................................................. 38
3.6.2.4 全海水培養基之影響................................................................... 38
3.6.2.5 添加界面活性劑之影響............................................................... 39
3.6.3 50 L 氣舉式批次發酵程序 (Fermentation Experiments) .............. 39
3.6.4 100 L 氣舉式批次發酵程序 (Fermentation Experiments) ............ 40
3.7 實驗裝置圖................................................................................................. 40
3.7.1 搖瓶發酵培養裝置圖...................................................................... 41
3.7.2 5 L 氣舉式批次發酵培養裝置圖 ................................................. 42
3.7.3 50 L 氣舉式饋料發酵培養裝置圖 ................................................ 43
3.7.4 100 L 氣舉式饋料發酵培養裝置圖 .............................................. 44
第四章 結果與討論.................................................................................................. 45
4.1 不滅菌發酵程序......................................................................................... 45
4.1.1 搖瓶批次發酵程序.......................................................................... 45
4.1.1.1 滅菌並降低pH值影響 ................................................................ 45
4.1.1.2 不滅菌並降低pH值影響 ............................................................ 48
4.1.2 5 L 氣舉式發酵槽批次發酵程序 ................................................... 50
4.1.2.1 改變酸鹼值之影響性................................................................... 50
4.1.2.2 兩階段控制酸鹼值之影響........................................................... 56
4.1.2.3 在90℃下滅菌之影響性.............................................................. 58
4.1.3 5 L 氣舉式發酵槽綜合探討 ........................................................... 61
4.2 海水培養基發酵程序................................................................................. 64
4.2.1 搖瓶批次發酵程序.......................................................................... 64
4.2.1.1 鹽度之影響................................................................................... 64
4.2.1.2 不同海水比例之影響................................................................... 66
4.2.2 5 L 氣舉式發酵槽批次發酵程序 ................................................... 68
4.2.2.1 海水培養基之影響性................................................................... 68
4.2.3 50 L 氣舉式發酵槽批次發酵程序 ................................................. 72
4.2.3.1 海水培養基之影響性................................................................... 72
4.2.4 海水培養基之成本影響性.............................................................. 75
4.3 添加幫助溶氧物質之發酵程序................................................................. 77
4.3.1 搖瓶批次發酵程序............................................................................ 77
4.3.1.1 添加物質之影響............................................................................. 77
4.3.1.2 添加界面活性劑之影響............................................................... 79
4.3.2 5 L 氣舉式發酵槽批次發酵程序 ................................................... 81
4.3.2.1 添加界面活性劑之影響............................................................... 81
4.3.3 體積溶氧係數kLa ........................................................................... 84
4.4 100 L 氣舉式發酵槽批次發酵程序 .......................................................... 85
4.5 海水培養基與添加界面活性劑及100 L 之氣舉式生物反應器之綜合探
討.......................................................................................................................... 86
第五章 結論與未來展望.......................................................................................... 89
5.1 結論............................................................................................................. 89
5.2 未來展望...................................................................................................... 90
參考文獻...................................................................................................................... 91
附錄.............................................................................................................................. 95
作者簡歷...................................................................................................................... 96

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