全球已開發及開發中之國家，隨著人口成長、都市化和工業化的發展，每日都會產出大量的生活污水，然而在污水中除了存在適合微藻培養的營養鹽外，還具有有機物質，係分為易分解與難分解有機物，會對於微藻去除污染物效力造成影響，使得成為微藻處理廢污水之棘手問題。因此，為了要得知污水中易分解與難分解有機物對微藻去除污染物的影響並提升效率同時克服難分解有機物之酚毒害，所以需要了解微藻異營性生長的特性與增加對有機物去除效率方法，期望可以處理污水同時生產的藻體可製成生質能源、飼料等環保及經濟效益。
本研究分為三大部分進行探討：(1)易分解有機物(葡萄糖)，首先以Chlorella sp.異營性培養為基礎，利用不同C/N濃度比探討其生長特性與污染物去除較佳操作條件，並加入固定化方式加入探討，了解與懸浮培養的差異性以及污染物去除影響；(2) 模擬生活污水，利用不同初始食微比在含有易分解有機物的環境中探討模擬生活污水與小球藻生物質量之比值圍以及其中污染物質去除的影響；(3)難分解有機物(酚)，將Chlorella sp.於不同酚濃度下培養，探討其生長與對酚忍受程度影響並加入固定化方式對酚去除影響加以比較，並針對藻菌共生方式探討，提升對難分解有機物去除的效率的影響，並同時利用動力學參數深入了解其中的相關性。
    結果顯示出，Chlorella sp.在C/N 8~12之間有最佳的碳與氮去除率，而C/N 32有最多的脂質累積量與總脂質量。發現在食微比為0.2和12.5 mg TOC*mg-1 biomass*d-1已達極限值，低於或高於此數值皆不適合Chlorella sp.持續生長，而較適合Chlorella sp.於模擬生活污水的F/M範圍為1.5~3 mg TOC*mg-1 biomass*d-1。Chlorella sp.在酚濃度300 mg/L有最佳比生長速率為0.61 d-1，且在酚濃度為800 mg/L時仍有生長。在藻菌共生培養時，顯示出A. vinelandii為主要酚消耗的微生物。Chlorella sp.固定化結合A. vinelandii懸浮培養系統裡有最佳的去除效率，其降解速率與比酚利用率分別為125.6 mg phenol*d-1和28.3 x 10-11 mg*cell-1d-1。

With the trend of population growth, urbanization and industrialization in global countries, tremendous of municipal wastewater are generated every day.   However, gradients in wastewater contain not only nutrients for microalgae to grow but uncertain organics to interfere with.  Those organics include easily degradable and recalcitrant components which may affect the digestible ability of microalgae to treat wastewater.  In order to distinguish the microalgae’s digestibility and growth condition in organic wastewater, experiments were conducted and compared for the better understanding of microalgae growth in water containing either easily degradable organic (glucose) or recalcitrant organic (phenol).  Hopefully, the results could give some clues of treating wastewater and producing biofuel from microalgae simultaneously for future environmental needs.
The objective to this study includes three subjects.  First, find out the suitable growth conditions of mixotrophic microalgae (Chlorella sp.) cultivated in different carbon to nitrogen ratio (C/N ratio) of nutritional water, and cooperated with free living and immobilization of algae.  Second, discuss of the suitable range of food to microorganism ratio for Chlorella sp. biomass and the effects of pollutants removement in synthetic sewage. Third, discuss the tolerance and survival-ship of microalgae cultivating freely or immobilized in variable concentration of phenol solution. Furthermore, confirm the synergistic relation with bacteria (Azotobacter) either in free living or immobilized phenol solution.
The results show that, Chorella sp. between C / N 8 ~ 12 has the best carbon and nitrogen removal, and C / N 32 has the most to total lipid accumulation in fat mass. Found in food micro ratio of 0.2 and 12.5 mg TOC * mg-1 biomass * d-1 has reached the limit, below or above this prime number neither for Chlorella sp. continue to grow, but more suitable for Chlorella sp. in synthetic sewage F / M range of 1.5 ~ 3 mg TOC * mg-1 biomass * d-1. Chlorella sp. at phenol concentration 300 mg / L have the optimum specific growth rate was 0.61 d-1, and a phenol concentration of 800 mg / L could still growing. When symbiotic algae culture as the main exhibit A. vinelandii phenol consumed microorganisms. Chlorella sp. Immobilized binding A. vinelandii suspension culture system has the best removal efficiency, the ratio of phenol degradation rate and utilization rate of 125.6 mg phenol * d-1 and 28.3 x 10-11 mg * cell-1d-1.

目錄	I
圖目錄	IV
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	Chlorella sp.及Azotobacter vinelandii介紹	4
2-1-1 Chlorella sp.培養生長機制	5
2-1 -2 A. vinelandii ATCC 12837生長機制	11
2-1-3 Chlorella sp.與Azotobacter vinelandii培養方式	13
2-2	反應動力學介紹	16
2-2-1 微生物生長動力學-Monod equation (一階反應)	16
2-2-2 微生物基質利用動力學-Michaelis-Menten equation (一階反應)	17
2-3	微藻對有機物降解的現象	17
2-3-1 易分解有機物(葡萄糖)	17
2-3-1-1 不同碳氮比對微藻的影響	17
2-3-2 難分解有機物(酚)	17
2-3-2-1 應用分解之微生物	18
2-3-2-2 細菌與微藻降解酚之現象	19
2-4	藻菌共生對有機物降解之現象	23
2-4-1 一般共生簡介	23
2-4-2 藻菌共生系統	23
2-5	固定化技術簡介	25
2-5-1 固定化技術應用在廢水處理	25
2-5-2 Ca-alginate固定化材料介紹	27
第三章 材料與方法	29
3-1	實驗材料	29
3-1-1 Chlorella sp.	29
3-1-2 Azotobacter ATCC 12837	29
3-2	實驗流程	29
3-3	實驗方法	31
3-3-1 實驗設備設置	31
3-3-2 易分解污染物之探討	31
3-3-3 模擬生活污水之探討	33
3-3-4 難分解污染物之探討	33
3-3-5 固定化顆粒之製備與溶解	35
3-4	分析方法	37
3-4-1 生物質量與細胞數的分析	37
3-4-2 水質分析方法	37
3-4-3 酚的分解活性測定(Wesley et al., 1961)	38
3-4-4 微藻及細菌固定化顆粒之內部觀察	39
3-4-5 微藻及菌類固定化顆粒之物理性質分析	39
3-4-6 Monod equation和Michaelis-Menten equation計算	40
3-4-7脂質分析	41
3-4-8光照強度之測定	41
3-5	實驗儀器與設備	42
第四章 結果與討論	43
4-1	易分解污染物之探討	43
4-1-1 Chlorella sp.懸浮生長的影響	43
4-1-1-1不同碳源的生長影響	43
4-1-1-2 不同碳氮比生長的影響	45
4-1-2	Chlorella sp.固定化生長的影響	48
4-1-2-1固定化顆粒基本性質	48
4-1-2-2 Chlorella sp.固定化不同顆粒濃度配比與不同食微比之生長影響	55
4-1-3 Chlorella sp.懸浮與固定化生長培養之綜合討論	61
4-1-4	污染物去除的影響	63
4-1-4-1 Chlorella sp.懸浮培養之污染物去除影響	63
4-1-4-2 Chlorella sp.固定化不同顆粒數與食微比之影響	63
4-2	模擬生活污水	67
4-2-1 Chlorella sp.不同食微比之生長影響	67
4-2-2 Chlorella sp.不同F/M比之汙染物去除影響	70
4-3	難分解污染物之探討	75
4-3-1 Chlorella sp.生長的影響	75
4-3-1-1 不同酚濃度懸浮培養	75
4-3-1-2 Chlorella sp.固定化培養並與懸浮比較	82
4-3-1-3 A. vinelandii 懸浮與固定化生長之比較	86
4-3-2 藻菌共生克服酚毒害之生長影響	89
4-3-3難分解有機物之去除影響	95
4-3-3-1不同酚濃度懸浮培養去除情形	95
4-3-3-2 固定與懸浮去除酚之比較	97
4-3-3-3 藻菌共生克服酚毒害之去除比較	98
第五章 結論與建議	99
5-1 結論         99
5-2 建議	100
參考文獻	101


圖目錄
Fig. 2-1 The cell of Chlorella sp.	5
Fig. 2-2 The pathway of light reaction via photosynthesis.	6
Fig. 2-3 The pathway of dark reaction via photosynthesis	7
Fig. 2-4 Heterotrophic metabolism in microalgae.	9
Fig. 2-5 Simplified model of metabolism.	12
Fig. 2-6 The cells of A. vinelandii ATCC 12837.	13
Fig. 2-7 The cells in batch culture for time course of microbes.	14
Fig. 2-8 The different of specific growth rate by substrate concentration curve.	16
Fig. 2-9 The chemical structure of phenol.	18
Fig. 2-10 Ortho pathway of phenol degradation.	21
Fig. 2-11 Meta pathway of phenol degradation.	22
Fig. 2-12 The synergistic of microalgae and bacteria.	24
Fig. 2-13 Chemical structures of G-block, M-block, and alternating block in alginate.	27
Fig. 2-14 Alginate hydrogels prepared by ionic cross-linking (egg-box model).	28
Fig. 3-1 Experimental flow-chart.	30
Fig. 3-2 Beads preparation device.	35
Fig. 3-3 Preparation Process Particles.	36
Fig. 4-1 Biomass concentration of cultures for different sources of carbon.	44
Fig. 4-2 Removal of C and N and biomass growth with different initial C/N ratios.	46
Fig. 4-3 Lipid concentration and increment of lipids at the initial and after 96h of culture time and for different C/N ratios.	47
Fig. 4-4 Lipid/cell changes for different C/N ratios at the initial and after 96h of culture time.	47
Fig. 4-5 High (left) and low (right) magnifications of SEM photos of immobilized Chlorella sp. alginate beads at three sodium alginate (SA)-to-calcium chloride ratios with cultivation for 168 h.	53
Fig. 4-6 The effect of bead’s swelling ratio by different concentrations of SA and CaCl2.	54
Fig. 4-7 Variation of biomass by F/M (A) 0.4; (B) 0.2; (C) 0.1 (mg*mg-1*d-1) within immobilization beads cultured system.	56
Fig. 4-8 Increase of biomass by different of F/M in Chlorella sp. bead ratio 6:1 (v/v).	57
Fig. 4-9 The SEM of Chorella sp. cells in beads when F/M 0.4	57
Fig. 4-10 The SEM of Chorella sp. cells in beads when F/M 0.2	58
Fig. 4-11 The SEM of Chorella sp. cells in beads when F/M 0.1	59
Fig. 4-12 The SEM of Chlorella sp. leaking when beads broken up.	60
Fig. 4-13 Effect of variable F/M ratio (A) 0.4; (B) 0.2; (C) 0.1 on NO3--N concentration by Chlorella sp. beads.	65
Fig. 4-14 Effect of variable F/M ratio (A) 0.4; (B) 0.2; (C) 0.1 on TOC concentration by Chlorella sp. beads.	66
Fig. 4-15 Effect of variable F/M ratio onbiomass by Chlorella sp..	68
Fig. 4-16 Effect of variable F/M ratio on specifitic growth rate by Chlorella sp..	69
Fig.4-17 Effect of variable F/M ratio on TOC concentration by Chlorella sp..	71
Fig.4-18 Effect of variable F/M ratio on NH4+-N concentration by Chlorella sp..	74
Fig. 4- 19 Variation of biomass in different concentrations of phenol by Chlorella sp..	76
Fig. 4- 20 Variation of biomass in different concentrations of phenol (A) 200; (B) 600; (C) 1000 mg/L by Chlorella sp..	77
Fig. 4-21 Calculate of μmax and Ks by Monod equation.	79
Fig. 4-22 Calculate of μmax and Ks by Haldane equation.	80
Fig. 4-23 Campare of Monod, Haldane and experiment.	81
Fig. 4-24 Dry weight and cell density (A) nitrate-N and phosphate-P concentration in liquid culture (B) for time course of individually cultivated Chlorella sp., free or immobilized in beads 	84
Fig. 4-25 Dry weight and cell density (A), nitrate-N and phosphate-P concentration in liquid culture (B) for time course of individually cultivated A. vinelandii, free and immobilized in beads	88
Fig. 4-26 Effect of synergistic relationships on dry weight (total) over time course for co-culturing 	92
Fig. 4-27 Variation in specific growth rate and cell density of Chlorella sp. and A. vinelandii in different synergistic culturing systems. (A) Both in suspension, (B) co-immobilized, (C) free Chlorella sp. and immobilized A. vinelandii, (D) immobilized Chlorella sp. and free A. vinelandii	93
Fig. 4-28 Variation of the concentrations of phenol witnout microalge.	95
Fig. 4-29 Removal of phenol by Chlorella sp. with different initial concentration of phenol.	96
Fig. 4-30 The biodegration rate of phenol by Chlorella sp..	96
Fig. 4-31 Phenol reduction over time course with individually cultivated or co-cultured cells, free or immobilized. (A) Individually cultivated Chlorella sp., (B) individually cultivated A. vinelandii, (C) co-culture of Chlorella sp. and A. vinelandii.	98

表目錄
Table 2-1 Summary of pH change and energy and carbon sources in autotrophic, heterophic and mixotrophic metabolic pathways 	10
Table 3-1 The RO concentration of the solar energy industry wastewater treatment.	31
Table 3-2 The original name v.s. simple name.	34
Table 3-3 The equipments by microalgae cultivation.	42
Table 3-4 Analytical instruments and equipments.	42
Table 4-1 The characteristics of beads by the fixed concentrations of SA and the different concentrations of CaCl2.	51
Table 4-2	Effects of alginate and calcium chloride concentrations in bead-immobilized conditions on Chlorella sp. growth as cell density and dry weight	…………………………………………………...………52
Table 4-3 Chlorella sp. specific growth ratio in suspension cultured…………62
Table4-4 The consumption ratio by the concentration of carbon and nitrogen in different stages.	62
Table 4-5 The remove of TOC in different F/M ratio.	72
Table 4-6 The remove of NH4+-N in different F/M ratio.	74
Table 4-7 Comparison of growth rates between free and immobilized Chlorella and Azotobacter cultures in 100 mg/L phenol	85
Table 4-8 Growth rates, reaction kinetic coefficients, and yield coefficients for four synergistic co-culturing systems	94

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