臺灣博碩士論文加值系統

藻類具有很高之營養與經濟價值。台灣不論在海水或淡水水域中皆有豐富之藻類資源，目前主要應用於餌料、食品與化妝品之開發。油脂含量高之藻種稱為油脂藻，其油脂結構與柴油成份(C 13-18)相近，近年來有以油脂藻作為生質柴油料源之研究。本研究主要探討淡水油脂藻Botryococcus braunii與Chlorella vulgaris之生長特性，期望能大量培養作為生質柴油之料源。
B. braunii與C. vulgaris以光照循環(light:dark, h) 16:8及12:12，光照強度54-81 μmol/m2s和122-150 μmol/m2s之條件下進行靜置培養。結果顯示B. braunii以光循環16:8，照度54-81 μmol/m2s 下生長最佳，世代時間為5.3天。C. vulgaris則較適合生長於照度122-150 μmol/m2s，光循環為16:8之環境，其世代時間為1.2天。
C. vulgaris分別以Watanabe、BG11與BBM培養基培養，於BG11培養基中之生質量最高，乾重為0.3 g/l，藻細胞之比油脂量以Watanabe培養基為最高690±120 μg/mg。C. vulgaris於上述培養基中之世代時間差異不大為1.1-1.3天。B. braunii則是以3N-BBM+V 與mChu 13，以及利用田口方法尋找最適培養基進行藻細胞之培養，結果顯示以mChu 13培養基培養，B. braunii可獲得最高之葉綠素含量(424±121 μg/l)，和較短世代時間(約6天)，其油脂濃度為802±52 μg/ml。以比油脂量(單位葉綠素所含之油脂濃度)而言，B. braunii於上述三種培養基培養後之比油脂量皆相同。同時從田口方法中得知，氮源(NaNO3)對B. braunii生長影響最大，其次依序為鎂離子(MgSO4)、碳源(CH3COONa)，最低為磷源(K2HPO4)。
此外，本研究也分離出與B. braunii共同存在之細菌，初步得知為寡養性格氏陰性桿菌，屬於Acidovorax sp. 、Nevskia sp. 、α-Proteobacteria及β-Proteobacteria之兼性細菌。

中文摘要 i
英文摘要 iii
目錄 v
圖目錄 ix
表目錄 x
第一章 前言 1
第二章 文獻回顧 3
2-1油脂藻類之介紹 3
2-2 B. braunii與C. vulgaris的介紹 4
2-3影響油脂藻之生長因素 7
2-3-1光源、溫度 7
2-3-2碳源 8
2-3-3氮源 8
2-3-4磷源 9
2-3-5鹽分 9
2-4藻類與細菌之關係 10
2-5藻類油脂分析方法 11
2-6藻類生長量測方式 14
2-6-1顯微計數法(microscopic counting) 14
2-6-2濁度法(optical density) 14
2-6-3乾重(dry weight) 15
2-6-4葉綠素萃取法(chlorophyll a) 15
2-7田口方法 16
2-7-1田口直交表之介紹 16
2-7-2信號雜音比(S/N比) 17
第三章 材料與方法 20
3-1藻種來源與保存 20
3-2培養基 20
3-3實驗方法 26
3-3-1藻類培養條件之建立 26
3-3-1-1藻株之預培養 26
3-3-1-2光照強度與循環 26
3-3-2藻類生長量測 27
3-3-2-1顯微計數法 27
3-3-2-2冷凍乾燥 27
3-3-2-3濁度法 27
3-3-2-4葉綠素萃取法 28
3-3-3 生長曲線 28
3-3-4 田口方法之設計 28
3-3-5藻類油脂分析 29
3-3-5-1螢光顯微鏡之定性觀測 29
3-3-5-2螢光分光光度計定量 30
3-3-6細菌之分離與鑑定 30
3-3-6-1細菌分離 30
3-3-6-2純菌DNA之萃取與鑑定 31
3-3-7細菌於藻株培養期間之量測 31
3-3-8電子顯微鏡觀察 31
3-4通氣培養設備 32
3-5實驗流程 32
第四章 結果與討論 33
4-1藻類培養條件的建立 33
4-4-1 藻類生長曲線 33
4-1-2光照循環與光強度 36
4-2藻類油脂之量測 40
4-3 培養基對藻類生長及油脂含量之影響 43
4-3-1培養基對C. vulgaris生長之影響 43
4-3-2培養基對B. braunii生長及油脂含量之影響 46
4-4 B. braunii與細菌之關係 52
4-4-1細菌於B. braunii培養期間之生長情形 52
4-4-2 細菌之鑑定 58
第五章 結論與建議 60
5-1 結論 61
5-2 建議 62
第六章 參考文獻 62

中文摘要 i
英文摘要 iii
目錄 v
圖目錄 ix
表目錄 x
第一章 前言 1
第二章 文獻回顧 3
2-1油脂藻類之介紹 3
2-2 B. braunii與C. vulgaris的介紹 4
2-3影響油脂藻之生長因素 7
2-3-1光源、溫度 7
2-3-2碳源 8
2-3-3氮源 8
2-3-4磷源 9
2-3-5鹽分 9
2-4藻類與細菌之關係 10
2-5藻類油脂分析方法 11
2-6藻類生長量測方式 14
2-6-1顯微計數法(microscopic counting) 14
2-6-2濁度法(optical density) 14
2-6-3乾重(dry weight) 15
2-6-4葉綠素萃取法(chlorophyll a) 15
2-7田口方法 16
2-7-1田口直交表之介紹 16
2-7-2信號雜音比(S/N比) 17
第三章 材料與方法 20
3-1藻種來源與保存 20
3-2培養基 20
3-3實驗方法 26
3-3-1藻類培養條件之建立 26
3-3-1-1藻株之預培養 26
3-3-1-2光照強度與循環 26
3-3-2藻類生長量測 27
3-3-2-1顯微計數法 27
3-3-2-2冷凍乾燥 27
3-3-2-3濁度法 27
3-3-2-4葉綠素萃取法 28
3-3-3 生長曲線 28
3-3-4 田口方法之設計 28
3-3-5藻類油脂分析 29
3-3-5-1螢光顯微鏡之定性觀測 29
3-3-5-2螢光分光光度計定量 30
3-3-6細菌之分離與鑑定 30
3-3-6-1細菌分離 30
3-3-6-2純菌DNA之萃取與鑑定 31
3-3-7細菌於藻株培養期間之量測 31
3-3-8電子顯微鏡觀察 31
3-4通氣培養設備 32
3-5實驗流程 32
第四章 結果與討論 33
4-1藻類培養條件的建立 33
4-4-1 藻類生長曲線 33
4-1-2光照循環與光強度 36
4-2藻類油脂之量測 40
4-3 培養基對藻類生長及油脂含量之影響 43
4-3-1培養基對C. vulgaris生長之影響 43
4-3-2培養基對B. braunii生長及油脂含量之影響 46
4-4 B. braunii與細菌之關係 52
4-4-1細菌於B. braunii培養期間之生長情形 52
4-4-2 細菌之鑑定 58
第五章 結論與建議 60
5-1 結論 61
5-2 建議 62
第六章 參考文獻 62
附錄一 藻類通氣培養裝置 72
附錄二C. vulgaris藻細胞數與濁度值之相關性 73
附錄二B. braunii濁度與葉綠素之相關性 74
圖目錄
圖2-1 Botryococcus braunii 6
圖2-2 Chlorella vulgaris 6
圖2-3 田口方法流程圖 19
圖3-1 實驗流程圖 32
圖4-1 B. braunii團聚之現象 34
圖4-2 油脂藻之生長曲線(a) B. braunii (b) C. vulgaris 35
圖4-3 光照度對C. vulgaris生長之影響 39
圖4-4 油脂藻細胞油脂之分佈(a) B. braunii (b) C. vulgaris 41
圖4-5 標準油品與相對螢光值之檢量線 42
圖4-6 培養基對C. vulgaris生長之影響 43
圖4-7 培養基對B. braunii 生長之影響 47
圖4-8 B. braunii以3N及mChu 13通氣培養下與其共存之細菌 53
圖4-9 與B. braunii共存之細菌 58


表目錄
表2-1 Botryococcus之特性 5
表2-2 藻類油脂萃取方法 13
表2-3 直交表L4 (23) 17
表3-1 3N-BBM+V培養基成分 21
表3-2 mChu 13培養基成分 22
表3-3 Watanabe培養基成分 23
表3-4 BG11培養基成分 24
表3-5 BBM培養基成分 25
表3-6 各因子代表之濃度 29
表3-7 直交表L9 (34) 29
表4-1 光照與光循環對油脂藻生長之影響 37
表4-2 C. vulgaris生長與油脂含量 44
表4-3 培養基成分對B. braunii生長之影響 49
表4-4 培養基對B. braunii生長之影響 50
表4-5a 與B. braunii共存細菌之分佈(mChu 13培養基) 55
表4-5b 與B. braunii共存細菌之分佈(3N-BBM+V培養基) 55
表4-6 細菌分離株DNA比對結果 60

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