[[alternative]]Studies on lipid production from microalgae using two-step cultivation strategy

碩士[[abstract]]地球能源隨著國際經濟的熱絡而有耗竭的現象，因此尋找替代能源成為迫切且緊要的事情。生質能源當中微藻是非常好的生產者，其光合效率特別高，生物質量與生長速率均優於其它能源作物 (Dote et al., 1994；Minowa et al., 1995)。在微藻種類裡，小球藻(Chlorella sp.)擁有高蛋白質含量以及高脂質累積量的潛能(Becker, 1994)並且取得容易，所以本研究以此為探討對象。 為了要讓細胞生物質量生產出高的柴油量，必須要先得到細胞內高的脂質含量與油脂量(Mutsumi et al., 2006)，本研究先以 Chlorella sp.於不同濃度CO2的自營性培養做探討，再以不同C/N比濃度的異營性培養為基礎，了解自營與異營培養之間的差異，接著透過異營與類自營二階段式的培養提高Chlorella sp.細胞胞內脂質含量和總油脂量。 實驗結果顯示，自營培養環境下，20% CO2的培養有最高的總油脂量與胞內脂含量，分別為0.45 gL-1與70%，生長速率為0.0242 day-1(培養1-12天之間)。而在異營培養實驗，C/N比為7或14對最大油脂量與胞內脂質含量影響不明顯，甚至對生物質量也是如此，分別為0.4 gL-1、20%和1.5 gL-1，生長速率為2.1 day-1(培養24-44小時間)。在異營結合類自營性二階段培養實驗結果，顯示此模式最大總油脂量增為初始值的3.25倍，明顯優於異營性培養(C/N比為7)之最大增加量為初始值的2倍，自營性培養則為原始的1.5倍。[[abstract]]The global fossil fuel consumption is demanding and supposed to be used up by A.D. 2050(Ginzburg, 1993) while economy and population are continuously growing. Substitution of the energy resources have been researched for decades including the wind-powered energy, solar energy and the production of biodiesel mainly by agricultural activities. The microalgae have been suggested as one of a good resource of bio-fuel production due to its higher photosynthesis efficiency, higher biomass production and higher growth rate comparing to other energy crops such as the sugar cane, beet and corn…etc. (Dote et al.,1994; Minowa et al., 1995).The objective of the research is to increase the lipid content of Chlorella sp., a kind of microalgae, by two-step (heterotrophic and stressed pseudo-autotrophic) cultivation strategy. The results of the autotrophic cultivation by applying two different concentrations of CO2 (0, 3%, 20%) come out that the maximum lipid production and lipid content in 20% CO2 culture, 0.45 gL-1 and 70% respectively. The result of the heterotrophic cultivation by applying two different carbon-to-nitrogen ratios (C/N = 7, 14) of nutrients come out that both the C/N = 7 and C/N = 14 have the maximum lipid production, lipid content and biomass production, which are 0.4 gL-1,20% and 1.5 gL-1 respectively. The results of the two-step cultivation by combining the nutrients of carbon-to-nitrogen ratios (C/N = 7) with CO2 (20%) come out that the lipid production and lipid content have the maximum ratio, which are 3.25 and 1.6 respectively. The lipid production and lipid content of the two-step cultivation are better then heterotrophic or autotrophic cultivation alone evidently.[[tableofcontents]]目錄.....................................................I 圖目錄 ................................................V 表目錄 ..............................................VII 第一章 前言.............................................1 1.1 緣起.................................................1 1.2 研究目的.............................................3 第二章 文獻回顧.........................................4 2.1 藻類的介紹...........................................4 2.2 小球藻(Chlorella sp.)................................4 2.3 小球藻(Chlorella sp.)的生殖方式......................7 2.4 光合作用.............................................8 2.5 藻類形成脂質的過程..................................12 2.6 影響藻類生長因子....................................14 2.6.1 CO2的影響.........................................14 2.6.2 氮的影響..........................................15 2.6.3 光照強度和週期的影響..............................16 2.6.4 溫度的影響........................................18 2.6.5 異營性培養之結果..................................19 2.6.6 環境衝擊的影響....................................19 2.7 脂質轉換成生化柴油之過程............................20 2.8 生質燃料的優缺點....................................23 2.9 國內外發展概況 ......................................25 2.9.1 目前國內生質柴油發展狀況..........................25 2.9.2 國外生質柴油發展狀況..............................25 第三章 材料與方法......................................29 3.1 藻種來源............................................29 3.2 培養基..............................................29 3.3 實驗流程(Fig. 3-1)..................................30 3.4 實驗方法............................................34 3.4.1 Chlorella sp. 於不同CO2濃度環境下培養實驗.........34 3.4.2主要培養基碳氮成分濃度比實驗(C/N比)................34 3.4.3 二階段培養模式實驗(異營結合類自營行培養)..........35 3.5 分析方法............................................36 3.5.1 藻體生長濃度(OD值)的測定..........................36 3.5.2 藻體油脂含量測定..................................36 3.5.3 細胞生物質量與硝酸鹽濃度的測量....................36 3.5.4 藻類生長速率計....................................37 3.5.5 葡萄糖濃度的測定..................................37 3.5.6 光照強度測定......................................37 3.5.7 實驗數據比例值之算法..............................37 3.5.8 實驗設備..........................................38 第四章 結果與討論......................................40 4.1 Chlorella sp.長期自營培養的實驗結果.................40 4.2 自營性空白實驗的實驗結果............................42 4.3 異營性結合類自營性培養之二階段培養模式結果..........44 4.3.1 異營性結合類自營性培養對細胞生長的影響............44 4.3.2 異營性結合類自營性培養對油脂量的影響..............47 4. 4 Chlorella sp.培養在不同濃度CO2的實驗結果...........51 4.4.1 Chlorella sp.在不同CO2濃度下的自營生長情況........51 4.4.2 CO2濃度對藻類油脂含量的影響.......................59 4.5 Chlorella sp.培養於不同C/N比的實驗結果..............64 4.5.1 不同C/N比對細胞生長的影響.........................64 4.5.2 不同C/N比培養對油脂量變化的影響...................67 4.6 微藻的自營性異營性培養之差異........................71 4.7 樣品存儲溫度對實驗的影響............................77 第五章 結論與建議.......................................81 5.1 結論................................................81 5.2 建議................................................82 第六章 參考文獻.........................................83 圖目錄 Fig. 2-1 The cell of Chlorella sp. ....................5 Fig. 2-2 The growth of Chlorella sp. .............8 Fig. 2-4 The pathway of dark reaction via photosynthesis. ................................................10 Fig. 2-5 The lipid which is composed of fatty acid and glycerin. ................................................12 Fig. 2-7 Transesterification of triglycerides with alcotjol. ................................................21 Fig. 2-8 The transesterification reactions of vegetable oil with alcohol to esters and glycerol. ............21 Fig. 2-9 The mechanism of alkali-catalyzed transesterification of triglycerides with alcohol ..22 Fig. 4-1 Variations of OD660 in long term (50 days) batch culture. ................................................41 Fig. 4-2 Variation of OD660、biomass、lipid and lipid content within autotrophic cultured system. ............43 Fig. 4-3 Effect of different culture on (A) biomass and (B)growth rate of biomass. ..............................46 Fig. 4-4 Effect of different culture on (A) lipidt and (B)growth rate of lipid. .............................49 Fig. 4-5 Effect of different culture on (A) lipid content and (B)growth rate of lipid content. ....................50 Fig. 4-6 The effects of variable CO2 concentration on (A)number of cells (B)growth rate of cell numbers. ..53 Fig. 4-7 Effect of variable CO2 concentration on (A)OD660 (B)growth rate of OD660. .............................56 Fig. 4-8 Effect of variable CO2 concentration on (A)biomass (B)growth rate of biomass. .....................57 Fig.4-9 Photomicrograph of Chlorella sp. under variable CO2 concentraiton. .......................................58 Fig. 4-10 Effect of variable CO2 concentration on (A)lipid content (B)growth rate of lipid content. ...........62 Fig. 4-11 Effect of variable CO2 concentration on (A)lipid (B)growth rate of lipid. ..............................63 Fig. 4-12 Effect of variable C/N ratio on OD660. ..66 Fig. 4-13 Effect of variable C/N ratio on biomass. ..66 Fig. 4-14 Effect of variable C/N ratio on lipid. ..69 Fig. 4-15 Effect of variable C/N ratio on lipid content. ................................................70 Fig. 4-16 Effect of variable C/N ratio on NO3-N concentration. .......................................70 Fig. 4-17 Photograph of the autotrophic cultivation of Chlorella sp. (A)apperance of sample (B) microscopy (400X) ................................................73 Fig. 4-18 Photograph of the heterotrophic cultivation of Chlorella sp. (A)apperance of sample (B) microscopy (400X) ...............................................73 Fig. 4-19 Variation of biomass in heterotrophic and autotrophic cultured system. ...................74 Fig. 4-20 Variation of GRbiomass in heterotrophic and autotrophic cultured system. ...................74 Fig. 4-21 Variation of GRlipid in heterotrophic and autotrophic cultured system. ...................75 Fig. 4-22 Variation of lipid content in heterotrophic and autotrophic cultured system. ....................75 Fig. 4-23 Variation of GRlipid content in heterotrophic and autotrophic cultured system. ....................76 Fig. 4-24 Effect of blank culture stored in refrigerator on biomass. .....................................78 Fig. 4-25 Effect of blank culture stored in refrigerator on lipid. ..............................................79 Fig. 4-26 Effect of blank culture stored in refrigerator on lipid content. .....................................79 Fig. 4-27 Effect of blank culture stored in refrigerator on lipid content. .....................................80 表目錄 Table 2-1藻類產品的商業應用 .............................6 Table 2-2 光合作用反應簡表 .............................11 Table 2-3國外生質柴油發展目標與現況 ...................28 Table 3-1 培養基成分表 ............................38 Table 3-2 實驗儀器設備表 ............................39 Table 4-1 Chlorella sp.經過儲存後的胞內脂質含量 ..80[[note]]學號: 695480276, 學年度: 9