微藻是一個很好水質汙染的處理者，廢污水中的氮和磷皆是微藻生長必要的元素，藉由微藻的處理可快速的淨化水質並且也相對的有高的生物質量產生，因此，相對而言微藻也是很好的燃料生產者，而且微藻擁有高的光合效率以及生長速率比其他能源作物快。但是通常利用微藻處理廢水的其中一個主要問題是在於微藻的回收，然而微藻固定化是其中一種解決之方式。
    為了找出小球藻(Chlorella sp.)生長和營養鹽去除最佳操作參數，本研究先以不同C/N比濃度的光合異營培養為基礎，了解小球藻生長機制，接著利用這個最佳C/N比濃度，加入不同F/M的固定化顆粒與不同顆粒數，找出固定化培養的最佳生長條件。
    實驗結果顯示，在不同C/N比濃度的光合異營培養下，C/N比為10時，擁有高的比生長速率為2.44 1/day，以及對氮與碳的去除量最高，分別為16.71 mg/g‧hr和86.99 mg/g‧hr。另外，在固定化培養實驗中，發現到顆粒越多，食微比越低，並不會使小球藻生長更好或是去除效果更好。除此之外，也發現到空白顆粒(沒包覆任何藻體)對營養鹽的去除效果良好，硝酸鹽氮和總有機碳分別共去除141 mg/L和684 mg/L，這表示空白顆粒可以吸附這些營養鹽，因此，在這個實驗中，當食微比為0.2和投入的顆粒數為6:1(v/v)時，對硝酸鹽氮和總有機碳量最高，分別去除141 mg/g‧hr和684 mg/g‧hr。

Microalgae are useful material to treat wastewater. Nitrogen and phosphorus of the wastewater are the necessary elements of microalgae growth. Microalgae could quickly purify wastewater and also could generate a large amount of biomass. Microalgae are great producer for fuel because it has higher photosynthetic efficiency and growth rate than other energy crops. However, a big problem for using microalgae to treat the waste water is separation of the microalgae and liquid. Immobilization of microalgae is one of the solution methods for this problem.
   For finding out the optimum parameters of microalgae growth and nutrient removal, this study used the different C/N ratios of photosynthetic heterotrophic culture as basis to explore the mechanism of the microalgae growth. The different C/N ratios and F/M ratios of the immobilized particle number and particle size were tested to find out the optimum growth parameters of the immobilized culture.
   The results showed that the highest specific grower rate is 2.44 1/day in photosynthetic heterotrophic culture when the C/N ratio is 10. And the highest remove rate of N and C were 16.71 mg/g·hr and 86.99 mg/g·hr, respectively. Furthermore, in immobilized culture, the more of particle number and the less of the F/M ratio did not improve the Chlorella sp. growth and nutrient removal. In the blank experiment, the gel particle without seeding with microalgae, the nitrate nitrogen and total organic carbon were removed 141 mg/L and 684 mg/L, which indicated that the gel particle could absorb that nutrient. Therefore, in this study, when the F/M ratio is 0.2 and particle number is 6:1(v/v), the highest removed quantity of nitrate and total organic carbon were 141 mg/g·hr and 684 mg/g·hr, respectively.

目錄 -------------------------------------------------------------------------------- I
圖目錄 ---------------------------------------------------------------------------- V
表目錄 ---------------------------------------------------------------------------- X
第一章 前言 ---------------------------------------------------------------------- 1
1-1 研究緣起 ---------------------------------------------------------------- 1
1-2 研究目的 ------------------------------------------------------------------ 3
第二章 文獻回顧 ---------------------------------------------------------------- 4
2-1 藻類介紹 ------------------------------------------------------------------ 4
2-2 小球藻 (Chlorella sp.) --------------------------------------------------- 4
2-3 小球藻生殖方式 --------------------------------------------------------- 6
2-4 光合作用 ------------------------------------------------------------------ 8
2-5 小球藻生長方式 ------------------------------------------------------- 12
2-6 小球藻培養方式 ------------------------------------------------------- 13
2-7 影響小球藻成長因子 ------------------------------------------------- 15
2-7-1 小球藻生長與營養鹽的關係 ----------------------------------- 15
2-7-2 光照強度和週期的影響 ----------------------------------------- 17
2-7-3 小球藻與pH 的關係 --------------------------------------------- 19
2-8 固定化微生物技術的研究 ------------------------------------------- 20
2-8-1 固定化的特性 ----------------------------------------------------- 20
2-8-2 固定化的方法 ----------------------------------------------------- 21
2-8-3 固定化優缺點 ----------------------------------------------------- 25
2-8-4 固定化之擔體的使用 -------------------------------------------- 26
2-8-5 褐藻膠(alginate) --------------------------------------------------- 30
第三章 實驗材料與方法 ----------------------------------------------------- 34
3-1 藻種來源 ---------------------------------------------------------------- 34
3-2 培養基 ------------------------------------------------------------------- 34
3-3 實驗方法 ---------------------------------------------------------------- 35
3-4 實驗步驟 ---------------------------------------------------------------- 41
3-4-1 Chlorella sp.的前期培養 ----------------------------------------- 41
3-4-2 小球藻（Chlorella sp.）的自營性空白實驗 ------------------ 41
3-4-3 主要培養基碳氮成分濃度比實驗(C/N 比) ------------------- 42
3-4-4 小球藻（Chlorella sp.）固定化培養 -------------------------- 43
3-5 分析方法 ----------------------------------------------------------------- 45
3-5-1 懸浮培養分析項目 ---------------------------------------------- 45
3-5-1-1 藻體生長濃度(OD660 值)的測定 -------------------------- 45
3-5-1-2 藻體生物質量(biomass)的測定 --------------------------- 45
3-5-1-3 葡萄糖濃度的測定 ------------------------------------------ 46
3-5-1-4 藻類比生長速率計算 --------------------------------------- 46
3-5-2 固定化分析的項目 ---------------------------------------------- 47
3-5-2-1 顆粒溶解方法 ------------------------------------------------ 47
3-5-2-2 顆粒內藻體之生物質量測量 ------------------------------ 47
3-5-2-3 藻體油脂含量測定 ------------------------------------------ 47
3-5-2-4 顆粒內部觀察 ------------------------------------------------ 47
3-6 實驗設備 ---------------------------------------------------------------- 49
第四章 結果與討論 --------------------------------------------------------- 53
4-1 Chlorella sp.空白自營性實驗 -------------------------------------- 53
4-2 Chlorella sp.培養於不同碳氮比（C/N）的實驗結果 --------- 58
4-3 固定化與懸浮式培養對細胞生長的影響 ------------------------ 65
4-4 不同F/M 與顆粒數對細胞生長的影響 -------------------------- 70
4-5 不同F/M 顆粒內的小球藻對營養鹽去除結果 ----------------- 87
4-6 不同F/M 與顆粒數對油脂變化的影響 -------------------------- 92
第五章 結論 ------------------------------------------------------------------ 95
第六章 參考文獻 ------------------------------------------------------------ 97

圖目錄
圖2- 1 小球藻剖析圖 .............................................................................. 5
圖2- 2 光學顯微鏡下的小球藻（400X） ............................................. 6
圖2- 3 Chlorella sp. 生長圖 ................................................................... 7
圖2- 4 光合作用之光反應流程圖 ......................................................... 9
圖2- 5 光合作用之暗反應流程圖 ....................................................... 10
圖2- 7 固定化技術一般分類 ............................................................... 23
圖2- 8 固定化技術圖 ........................................................................... 23
圖2- 9 海藻酸鈉的結構 ........................................................................ 31
圖3- 1 實驗流程圖…………………………………………………………….40
圖3- 2 小球藻固定化操作流程圖 ......................................................... 44
圖3- 3 小球藻固定化操作圖 ................................................................. 45
圖3- 4 高壓滅菌釜 ................................................................................ 49
圖3- 5 無菌操作台 ................................................................................ 49
圖3- 6 培養箱 ......................................................................................... 50
圖3- 7 燈管 ............................................................................................. 50
圖3- 8 分光光度計 ................................................................................. 50
圖3- 9 高速微量離心機 ......................................................................... 51
圖3- 10 細胞計數器 .............................................................................. 51
圖4- 1 Chlorella sp.自營培養中總有機碳與硝酸鹽氮變化情形..…...56
圖4- 2 Chlorella sp.自營培養中硝酸鹽氮和藻體生物量變化情形 . 56
圖4- 3 Chlorella sp.自營培養中藻體濃度和細胞數變化情形 ......... 57
圖4- 4 Chlorella sp.生長變化 .............................................................. 57
圖4- 5 Chlorella sp.異營培養中不同C/N 之藻體濃度變化情形 ..... 60
圖4- 6 Chlorella sp.異營培養中不同C/N 之藻體生物質量變化情形60
圖4- 7 Chlorella sp.異營培養中不同C/N 之比生長速率。 ............. 61
圖4- 8 Chlorella sp.異營培養中C/N 為4 之硝酸鹽氮和總有機碳變化
情形 ....................................................................................................... 61
圖4- 9 Chlorella sp.異營培養中C/N 為8 之硝酸鹽氮和總有機碳變化
情形 ....................................................................................................... 62
圖4- 10 Chlorella sp.異營培養中C/N 為10 之硝酸鹽氮和總有機碳變
化情形 ................................................................................................... 62
圖4- 11 Chlorella sp.異營培養中C/N 為20 之硝酸鹽氮和總有機碳變
化情形 ................................................................................................... 63
圖4- 12 Chlorella sp.異營培養中不同C/N 之單位藻體去除量情形63
圖4- 13 固定化培養和空白顆粒培養 ............................................... 67
圖4- 14 Chlorella sp.自營培養中懸浮式和固定化之比生長速率。 67
圖4- 15 固定化顆粒與空白顆粒和懸浮培養於自營培養中硝酸鹽氮變
化情形 ................................................................................................... 68
圖4- 16 固定化顆粒與空白顆粒和懸浮培養於自營培養中總有機碳變
化情形 ................................................................................................... 68
圖4- 17 固定化顆粒培養和懸浮培養之但為藻體去除總有機碳和硝酸
鹽氮的量 ............................................................................................... 69
圖4- 18 Chlorella sp.固定化培養中食微比為0.4 時硝酸鹽氮變化情形 75
圖4- 19 Chlorella sp.固定化培養中食微比為0.2 時硝酸鹽氮變化情形 75
圖4- 20 Chlorella sp.固定化培養中食微比為0.1 時硝酸鹽氮變化情形 76
圖4- 21 Chlorella sp.固定化培養中食微比為0.4 時總有機碳變化情形 76
圖4- 22 Chlorella sp.固定化培養中食微比為0.2 時總有機碳變化情形 77
圖4- 23 Chlorella sp.固定化培養中食微比為0.1 時總有機碳變化情形 77
圖4- 24 Chlorella sp.固定化培養中食微比為0.4 之不同顆粒數比藻體
生物量變化情形 ................................................................................... 78
圖4- 25 Chlorella sp.固定化培養中食微比為0.2 之不同顆粒數比藻體
生物量變化情形 ................................................................................... 78
圖4- 26 Chlorella sp.固定化培養中食微比為0.1 之不同顆粒數比藻體
生物量變化情形 ................................................................................... 79
圖4- 27 Chlorella sp.固定化培養中顆粒比為6：1 之不同食微比藻體生物增加量 ............................................................................................... 79
圖4- 28 食微比為0.4 時，膠體顆粒內Chlorella sp.細胞SEM 圖 . 80
圖4- 29 食微比為0.2 時，膠體顆粒內Chlorella sp.細胞SEM 圖 . 81
圖4- 30 食微比為0.1 時，膠體顆粒內Chlorella sp.細胞SEM 圖 . 82
圖4- 31 固定化顆粒崩解造成微藻液流出來，造成所謂洩漏之SEM 圖
............................................................................................................... 83
圖4- 32 Chlorella sp.固定化培養中食微比為0.4 時單位藻體去除量變
化情形 ................................................................................................... 84
圖4- 33 Chlorella sp.固定化培養中食微比為0.2 時單位藻體去除量變
化情形 ................................................................................................... 84
圖4- 34 Chlorella sp.固定化培養中食微比為0.1 時單位藻體去除量變
化情形 ................................................................................................... 85
圖4- 35 Chlorella sp.固定化培養中顆粒比為6:1 v/v 與不同食微比之單
位藻體去除硝酸鹽氮和總有機碳量變化情形 ................................... 85
圖4- 36 Chlorella sp.固定化培養中食微比為0.4 與不同顆粒數比之小
球藻（不包含顆粒）以及空白顆粒對硝酸鹽氮去除情形............... 89
圖4- 37 Chlorella sp.固定化培養中食微比為0.2 與不同顆粒數比之小
球藻（不包含顆粒）以及空白顆粒對硝酸鹽氮去除情形............... 89
圖4- 38 Chlorella sp.固定化培養中食微比為0.1 與不同顆粒比之小球
藻（不包含顆粒）以及空白顆粒對硝酸鹽氮去除情形 ................... 90
圖4- 39 Chlorella sp.固定化培養中食微比為0.4 與不同顆粒數比之小
球藻（不包含顆粒）以及空白顆粒對總有機碳去除情形............... 90
圖4- 40 Chlorella sp.固定化培養中食微比為0.2 與不同顆粒數比之小
球藻（不包含顆粒）以及空白顆粒對總有機碳去除情形............... 91
圖4- 41 Chlorella sp.固定化培養中食微比為0.1 與不同顆粒數比之小
球藻（不包含顆粒）以及空白顆粒對總有機碳去除情形 .............. 91

表目錄
表2- 1 光合作用反應簡表 ..................................................................... 11
表2- 2 各種固定化方法之優缺點比較 ................................................. 24
表2- 3 不同世代固定化之優缺點比較 ................................................. 25
表2- 4 固定化常使用的擔體基質 ......................................................... 28
表2- 5 固定化藻類之相關文獻 ............................................................. 29
表3- 1 培養基成分 ................................................................................. 35
表3- 2 實驗儀器設備表 ......................................................................... 52
表4- 1 Chlorella sp.異營培養中不同C/N 之單位藻體去除量 ........... 64
表4- 2 Chlorella sp.固定化培養中顆粒比為6:1 v/v 與不同食微比之不
同階段單位藻體去除量之消耗比例 ..................................................... 86
表4- 3 Chlorella sp.固定化培養中食微比為0.4 與不同顆粒的藻體生物
量和脂質增加量 .................................................................................... 93
表4- 4 Chlorella sp.固定化培養中食微比為0.2 與不同顆粒的藻體生物
量和脂質增加量 .................................................................................... 93
表4- 5 Chlorella sp.固定化培養中食微比為0.1 與不同顆粒的藻體生物
量和脂質增加量 .................................................................................... 94

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