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研究生:郭育謙
研究生(外文):Yu-Chien Kuo
論文名稱:微藻生長階段與油脂合成指標之探討
論文名稱(外文):The study of the indicators of microalgae growth phase and lipid synthesis
指導教授:尤瓊琦
指導教授(外文):Chung-Chyi Yu
口試委員:洪滉祐陳俊明
口試日期:2011-07-21
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物產業機電工程學系所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:161
中文關鍵詞:粒徑分裂率特徵波長油脂含量生長指標
外文關鍵詞:cell sizedivision ratecharacteristic wavelengthslipid contentindicator of growth
相關次數:
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本研究目的主要是透過微藻之生長特性與狀況,建立快速且精確判別所處之生長階段與未來趨勢。首先探討以細胞粒徑作為判斷微藻生長階段、油脂含量、脂肪酸含量與組成之輔助指標的可行性,結果顯示大多數的微藻在延滯期時會累積油脂本研究目的主要是透過微藻生長相關參數(細胞粒徑、細胞分裂率及吸光值)建立快速且精確之生長指標,藉以判別所處之生長階段,同時可作為估計微藻密度、油脂含量及脂肪酸含量之模式。試驗首先探討五種微藻細胞(小球藻(Chlorella sp.)、擬球藻(Nannochloropsis oculata)、大溪地等鞭金藻(Isochrysis aff. galbana)、巴夫藻(Pavlova lutheri)及布朗葡萄球藻(Botrycocccus braunii))之粒徑與分裂情形對應實際微藻生長週期與密度,並以此作為判斷微藻生長階段、油脂及脂肪酸含量與組成之輔助指標的可行性,同時於試驗中分別檢測培養液中之pH值、硝酸態氮(NO3-)、磷酸鹽(PO43-)、吸光值(OD320-1000 nm)及微藻之油脂含量與組成,以進一步了解各因子之相關性,最後將試驗之數據進行迴歸分析,期能獲致較明確且容易量測之生長指標。
試驗結果獲致微藻細胞粒徑、細胞密度及細胞分裂率在各生長階段中,呈現相當高之相關性,其相關係數皆達0.8以上,顯示微藻細胞粒徑為相當合適之生長指標。且亦獲致微藻於生長階段之延滯期時會累積油脂,及驅使不飽和脂肪酸的合成;當進入指數增殖期時,微藻細胞快速增殖,水中營養鹽同時快速之吸收,導致pH值產生變化,多元不飽和脂肪酸亦呈現減少之趨勢;然進入停滯期後,脂肪酸之合成則因藻種有不同之變化。
最後探討使用微藻細胞特徵波長之吸光值與細胞粒徑二因子,預測微藻細胞密度、油脂含量及特定脂肪酸含量之可行性,獲致一階迴歸方程式之決定係數(R2)皆大於0.85且各因子之P-Value皆小於0.05,顯示此二因子為合適之油脂合成指標,並可作為培養微藻時能在最佳細胞活性、油脂含量及脂肪酸組成時進行收穫之參考依據。

The main propose of this study was to establish a rapid and accurate indicator of microalgae growth parameters such as microalgae cell size, cell division rate and absorption spectra. The cell size and cell division rate of five species (Chlorella sp., Nannochloropsis oculata, Isochrysis aff. galbana, Pavlova lutheri and Botrycocccus braunii ) were studied to correspond with microalgae growth phase and cell density, and to determine the feasibility of them as the assisted indicator for microalgae growth phase, lipid content, fatty acid content and composition. Moreover, the pH value, nitrate nitrogen, phosphate and absorption spectra (OD320-1000 nm) in the culture solution and the lipid content and composition of microalgae cells were also measured to inspect the correlation between each factor. Finally, the regression analysis was tested with the above date to expect that obtaining the indicators which is clear and easy measurement.
The experimental result obtained the high correlation between cell size, cell density and cell division rate of microalgae in each phase of growth stage, the correlation coefficients were all greater than 0.8. It demonstrate using microalgae cell size as an indicator for microalgae growth is suitable. Furthermore, microalgae can accumulate lipid and drive the synthesis of unsaturated fatty acid in the lag phase of growth stage; Microalgae cells growth rapid, Nutrients of culture solution have been absorbed quickly leading to changes in pH value and polyunsaturated fatty acids also showed a trend of reduction in the proliferative index phase; When entering the stagnation phase, there were different variations of fatty acid synthesis due to algal species.
Finally, the feasibility study of microalgae cell density, lipid content and fatty acid content forecast demonstrated that using two factors of microalgae characteristic wavelengths of absorbance and cell size, coefficient of determination (R2) were greater than 0.85 and P-Value were less than 0.05 of each factors of first-order regression equation was obtained, and the indicator for lipid synthesis was suitable. The results presented show the reference on cultivation of microalgae when harvesting in the best cell activity, lipid content and fatty acid composition.


目錄
摘要 I
ABSTRACT II
目錄 IV
表目錄 XI
圖目錄 XIV
第一章 緒論 1
1-1前言 1
1-2研究目的 3
第二章 文獻探討 4
2-1微藻簡介 4
2-1-1小球藻(Chlorella sp.) 4
2-1-2擬球藻(Nannochloropsis oculata) 4
2-1-3 大溪地等鞭金藻(Isochrysis aff. galbana) 5
2-1-4巴夫藻(Pavlova lutheri) 5
2-1-5布朗葡萄球藻(Botryococcus braunii) 6
2-2油脂 6
2-2-1 脂肪酸 7
2-2-2 油脂合成途徑 10
2-3藻類環境生長因子對細胞增殖、油脂及脂肪酸合成之影響 17
2-3-1溫度 17
2-3-2 pH值 17
2-3-3鹽度 18
2-3-4光源 18
2-4培養基成分對細胞增殖、油脂及脂肪酸合成之影響 20
2-4-1碳源 20
2-4-2氮源 22
2-4-3 無機鹽 23
2-4-3-1大量元素 23
2-4-3-2微量元素 24
2-4-3-3絡合劑(EDTA) 25
2-4-4輔助生長物質 25
2-4-5游離脂肪酸 26
2-4-6 f/2 培養液 26
2-5生長階段 27
2-5-1微藻生殖方式 28
2-5-1-1分裂生殖 29
2-5-1-2孢子生殖 29
2-5-1-3營養生殖 30
2-5-2 微藻的生活史(life cycle) 30
2-6微藻細胞粒徑變化 31
2-7微藻細胞光合色素 32
第三章 材料與方法 35
3-1試驗設計概念 35
3-2試驗材料 36
3-3試驗量測方法 36
3-3-1 水中溫度與鹽度量測方法 36
3-3-2 水中pH值量測方法 37
3-3-3 曝氣量 37
3-3-4 光照強度 37
3-3-5 水質量測方法 37
3-3-6微藻細胞密度量測方法 38
3-3-7 吸收光譜 38
3-3-8 油脂含量與脂肪酸組成分析量測方法 39
3-3-8-1油脂萃取 39
3-3-8-2油脂皂化 39
3-3-8-3 油脂甲基化(甲酯化) 40
3-3-9 統計分析 40
3-4 實驗設計 41
第四章 結果與討論 42
4-1試驗結果 42
4-1-1 小球藻 42
4-1-1-1小球藻細胞密度之變化 42
4-1-1-2 小球藻細胞粒徑 44
4-1-1-3 小球藻培養液濃度試驗 45
4-1-1-4小球藻單位細胞乾物重與油脂量之變化 47
4-1-1-5小球藻脂肪酸含量之變化 49
4-1-1-6小球藻吸收光譜波長 52
4-1-1-7小球藻綜合討論 53
4-1-2 擬球藻 56
4-1-2-1擬球藻細胞密度之變化 56
4-1-2-2 擬球藻細胞粒徑 58
4-1-2-3 擬球藻培養液濃度試驗 59
4-1-2-4擬球藻單位細胞乾物重與油脂量之變化 62
4-1-2-5擬球藻脂肪酸含量之變化 63
4-1-2-6擬球藻吸收光譜波長 68
4-1-2-7擬球藻綜合討論 70
4-1-3 大溪地等鞭金藻 73
4-1-3-1大溪地等鞭金藻細胞密度之變化 73
4-1-3-2大溪地等鞭金藻細胞粒徑 76
4-1-3-3大溪地等鞭金藻培養液濃度試驗 77
4-1-3-4大溪地等鞭金藻細胞乾物重與油脂量之變化 79
4-1-3-5大溪地等鞭金藻脂肪酸含量之變化 81
4-1-3-6大溪地等鞭金藻吸收光譜波長 84
4-1-3-7大溪地等鞭金藻綜合討論 86
4-1-4 巴夫藻 89
4-1-4-1巴夫藻細胞密度之變化 89
4-1-4-2巴夫藻細胞粒徑 91
4-1-4-3巴夫藻培養液濃度試驗 92
4-1-4-4巴夫藻細胞乾物重與油脂量之變化 94
4-1-3-5巴夫藻脂肪酸含量之變化 96
4-1-4-6巴夫藻吸收光譜波長 102
4-1-4-7巴夫藻綜合討論 103
4-1-5 布朗葡萄球藻 107
4-1-5-1布朗葡萄球藻細胞密度之變化 107
4-1-5-2布朗葡萄球藻細胞粒徑 109
4-1-5-3布朗葡萄球藻培養液濃度試驗 109
4-1-5-4布朗葡萄球藻細胞乾物重與油脂含量之變化 111
4-1-5-5布朗葡萄球藻脂肪酸含量之變化 113
4-1-5-6布朗葡萄球藻吸收光譜波長 116
4-1-5-7布朗葡萄球藻綜合討論 118
4-2結果討論 121
4-2-1細胞粒徑與生長階段之相關性 121
4-2-2 細胞粒徑與脂肪酸之相關性分析 121
4-2-3細胞粒徑與吸收波長之迴歸分析 124
第五章 結論與建議 129
5-1結論 129
5-2建議 131
第六章 參考文獻 132
附錄 155
附錄一 硝酸鹽量測方法-分光光度計 155
附錄二 正磷酸鹽量測方法-分光光度計/維生素丙法 157
附錄三 脂肪酸分析 159


表目錄
表2-1 F/2培養液 27
表4-1小球藻特徵波長之相關係數分析表 53
表4-2 小球藻(CHL.)各時間點試驗結果相關變數彼此間之相關係數分析表 54
表4-3 小球藻(CHLORELLA SP.)延滯期各試驗結果相關之相關係數分析表 55
表4-4 小球藻(CHLORELLA SP.)指數增殖期各試驗結果相關之相關係數分析表 55
表4-5小球藻(CHLORELLA SP.)停滯期各試驗結果相關之相關係數分析表 56
表4-6擬球藻特徵波長之相關係數分析表 69
表4-7 擬球藻(NANNO.)各時間點試驗結果相關變數彼此間之相關係數分析表 70
表4-8擬球藻(NANNO.) 延滯期各試驗結果相關變數之相關係數分析表 72
表4-9擬球藻(NANNO.) 指數增殖期各試驗結果相關變數之相關係數分析表 72
表4-10擬球藻(NANNO.) 停滯期各試驗結果相關變數之相關係數分析表 73
表4-11大溪地等鞭金藻特徵波長之相關係數分析表 85
表4-12 大溪地等鞭金藻(ISO.)各時間點試驗結果相關變數彼此間之相關係數分析表 86
表4-13大溪地等鞭金藻(ISO.)延滯期各試驗結果相關變數之相關係數分析表 88
表4-14大溪地等鞭金藻(ISO.)指數增殖期各試驗結果相關變數之相關係數分析表 88
表4-15大溪地等鞭金藻(ISO.)停滯期各試驗結果相關變數之相關係數分析表 89
表4-16巴夫藻特徵波長之相關係數分析表 103
表4-17 巴夫藻(PAVLOVA)各時間點試驗結果相關變數彼此間之相關係數分析表 104
表4-18巴夫藻(PAVLOVA)延滯期各試驗結果相關變數之相關係數分析表 105
表4-19巴夫藻(PAVLOVA)指數增殖期各試驗結果相關變數之相關係數分析表 106
表4-20巴夫藻(PAVLOVA)停滯期各試驗結果相關變數之相關係數分析表 106
表4-21布朗葡萄球藻特徵波長之相關係數分析表 117
表4-22 布朗葡萄球藻(BRAUNII)各時間點試驗結果相關變數彼此間之相關係數分析表 118
表4-23布朗葡萄球藻(BRAUNII)延滯期各試驗結果相關變數之相關係數分析表 120
表4-24布朗葡萄球藻(BRAUNII)指數增殖期各試驗結果相關變數之相關係數分析表 120
表4-25布朗葡萄球藻(BRAUNII)停滯期各試驗結果相關變數之相關係數分析表 121
表4-26微藻細胞粒徑與脂肪酸在延滯期之相關係數 122
表4-27微藻細胞粒徑與脂肪酸在指數增殖期之相關係數 123
表4-28微藻細胞粒徑與脂肪酸在停滯期之相關係數 124
表4-29小球藻試驗數據分析結果 126
表4-30擬球藻試驗數據分析結果 126
表4-31大溪地等鞭金藻試驗數據分析結果 127
表4-32巴夫藻試驗數據分析結果 127
表4-33布朗葡萄球藻試驗數據分析結果 128


圖目錄
圖2-1 不飽和脂肪酸結構圖 9
圖2-2 光反應機制 11
圖2-3 卡爾文循環 12
圖2-4 粒線體合成脂肪酸前驅物之作用方式 13
圖2-5 長鍊脂肪酸前驅物之合成途徑 15
圖2-6脂肪酸合成路徑圖 15
圖2-7 內質網合成油脂 16
圖2-8葉綠體中的色素在不同光波長的吸收情形 34
圖3-1 試驗設計流程圖 35
圖4-1小球藻總藻細胞密度之變化趨勢圖 42
圖4-2分裂中的小球藻細胞密度之變化趨勢圖 43
圖4-3小球藻的細胞分裂率之變化趨勢圖 44
圖4-4 小球藻細胞粒徑之變化趨勢圖 45
圖4-5 小球藻硝酸態氮(NO3-)濃度之變化趨勢圖 46
圖4-6 小球藻PH值之變化趨勢圖 47
圖4-7小球藻磷酸鹽(PO43-)濃度之變化趨勢圖 47
圖4-8 小球藻單位細胞乾物重之變化趨勢圖 48
圖4-9 小球藻單位細胞內油脂量之變化趨勢圖 49
圖4-10 飽和脂肪酸棕櫚酸(C16:0)含量變化趨勢圖 50
圖4-11 不飽和脂肪酸棕櫚油酸(C16:1N-7)含量變化趨勢圖 50
圖4-12 不飽和脂肪酸油酸(C18:1N-9)含量變化趨勢圖 51
圖4-13 不飽和脂肪酸Γ-次亞麻油酸(C18:3N-6)含量變化趨勢圖 51
圖4-14 小球藻在不同時間波長與吸光值之關係圖 52
圖4-15擬球藻總藻細胞密度之變化趨勢圖 57
圖4-16分裂中的擬球藻細胞密度之變化趨勢圖 57
圖4-17 擬球藻的細胞分裂率之變化趨勢圖 58
圖4-18 擬球藻細胞粒徑之變化圖 59
圖4-19 擬球藻硝酸態氮(NO3-)濃度之變化圖 60
圖4-20 擬球藻PH值之變化趨勢圖 61
圖4-21擬球藻磷酸鹽(PO43-)濃度之變化圖 61
圖4-22 擬球藻單位細胞乾物重之變化趨勢圖 62
圖4-23 擬球藻單位細胞內油脂量之變化趨勢圖 63
圖4-24飽和脂肪酸棕櫚酸(C16:0)含量變化趨勢圖 64
圖4-25不飽和脂肪酸棕櫚油酸(C16:1N-7)含量變化趨勢圖 65
圖4-26不飽和脂肪酸油酸(C18:1N-9)含量變化趨勢圖 65
圖4-27不飽和脂肪酸亞麻油酸(C18:2N-6)含量變化趨勢圖 66
圖4-28不飽和脂肪酸Α-次亞麻油酸(C18:3N-3)含量變化趨勢圖 66
圖4-29不飽和脂肪酸Γ-次亞麻油酸(C18:3N-6)含量變化趨勢圖 67
圖4-30不飽和脂肪酸花生四烯酸(C20:4N-6)含量變化趨勢圖 67
圖4-31不飽和脂肪酸EPA(C20:5N-3)含量變化趨勢圖 68
圖4-32擬球藻在不同時間波長與吸光值之關係圖 69
圖4-33大溪地等鞭金藻總藻細胞密度之變化趨勢圖 74
圖4-34 分裂中的大溪地等鞭金藻細胞密度之變化趨勢圖 75
圖4-35 大溪地等鞭金藻的細胞分裂率之變化趨勢圖 76
圖4-36 大溪地等鞭金藻細胞粒徑之變化圖 77
圖4-37 大溪地等鞭金藻硝酸態氮(NO3-)濃度之變化趨勢圖 78
圖4-38 大溪地等鞭金藻PH值之變化趨勢圖 78
圖4-39大溪地等鞭金藻磷酸鹽(PO43-)濃度之變化趨勢圖 79
圖4-40大溪地等鞭金藻單位細胞乾物重之變化趨勢圖 80
圖4-41大溪地等鞭金藻單位細胞內油脂含量之變化趨勢圖 80
圖4-42飽和脂肪酸棕櫚酸(C16:0)含量變化趨勢圖 82
圖4-43不飽和脂肪酸棕櫚油酸(C16:1N-7)含量變化趨勢圖 82
圖4-44不飽和脂肪酸油酸(C18:1N-9)含量變化趨勢圖 83
圖4-45不飽和脂肪酸亞麻油酸(C18:2N-6)含量變化趨勢圖 83
圖4-46不飽和脂肪酸DHA(C22:6N-3)含量變化趨勢圖 84
圖4-47大溪地等鞭金藻在不同時間波長與吸光值之關係圖 85
圖4-48巴夫藻總藻細胞密度之變化趨勢圖 90
圖4-49 分裂中的巴夫藻細胞密度之變化趨勢圖 91
圖4-50 巴夫藻的細胞分裂率之變化趨勢圖 91
圖4-51 巴夫藻細胞粒徑之變化趨勢圖 92
圖4-52 巴夫藻硝酸態氮(NO3-)濃度之變化趨勢圖 93
圖4-53 巴夫藻PH值之變化趨勢圖 93
圖4-54巴夫藻磷酸鹽(PO43-)濃度之變化趨勢圖 94
圖4-55 巴夫藻單位細胞乾物重之變化趨勢圖 95
圖4-56巴夫藻單位細胞內油脂含量之變化趨勢圖 96
圖4-57飽和脂肪酸棕櫚酸(C16:0)含量變化趨勢圖 98
圖4-58不飽和脂肪酸棕櫚油酸(C16:1N-7)含量變化趨勢圖 98
圖4-59不飽和脂肪酸油酸(C18:1N-9)含量變化趨勢圖 99
圖4-60不飽和脂肪酸亞麻油酸(C18:2N-6)含量變化趨勢圖 99
圖4-61不飽和脂肪酸Α-次亞麻油酸(C18:3N-3)含量變化趨勢圖 100
圖4-62不飽和脂肪酸Γ-次亞麻油酸(C18:3N-6)含量變化趨勢圖 100
圖4-63不飽和脂肪酸花生四烯酸(C20:4N-6)含量變化趨勢圖 101
圖4-64不飽和脂肪酸EPA(C20:5N-3)含量變化趨勢圖 101
圖4-65不飽和脂肪酸DHA(C22:6N-3)含量變化趨勢圖 102
圖4-66巴夫藻在不同時間波長與吸光值之關係圖 103
圖4-67布朗葡萄球藻總藻細胞密度之變化趨勢圖 107
圖4-68 分裂中的布朗葡萄球藻細胞密度之變化趨勢圖 108
圖4-69 布朗葡萄球藻的細胞密度分裂率之變化趨勢圖 108
圖4-70 布朗葡萄球藻細胞粒徑之變化趨勢圖 109
圖4-71布朗葡萄球藻硝酸態氮(NO3-)濃度之變化趨勢圖 110
圖4-72布朗葡萄球藻PH值之變化趨勢圖 110
圖4-73布朗葡萄球藻磷酸鹽(PO43-)濃度之變化圖 111
圖4-74 布朗葡萄球藻單位細胞乾物重之變化趨勢圖 112
圖4-75布朗葡萄球藻單位細胞內油脂量之變化趨勢圖 113
圖4-76飽和脂肪酸棕櫚酸(C16:0)含量變化趨勢圖 114
圖4-77不飽和脂肪酸棕櫚油酸(C16:1N-7)含量變化趨勢圖 115
圖4-78不飽和脂肪酸油酸(C18:1N-9)含量變化趨勢圖 115
圖4-79不飽和脂肪酸亞麻油酸(C18:2N-6)含量變化趨勢圖 116
圖4-80不飽和脂肪酸Α-次亞麻油酸(C18:3N-3)含量變化趨勢圖 116
圖4-81布朗葡萄球藻在不同時間波長與吸光值之關係圖 117
附錄圖1硝酸鹽標準品濃度對吸光值之檢量線 156
附錄圖2 正磷酸鹽標準品濃度對吸光值之檢量線 158



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