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研究生: 曾啟銘
Tseng, Chi-Ming
論文名稱: 實驗室與室外環境下以藍綠菌Thermosynechococcus sp. CL-1 固定二氧化碳與生質組成之研究
A Study on Carbon Dioxide Fixation and Biomass Composition of Thermosynechococcus sp. CL-1 in the Laboratory and Outdoor Environments
指導教授: 申永輝
Shen, Yun-Hwei
雷大同
Ray, Dah-Tong
朱信
Chu, Hsin
學位類別: 博士
Doctor
系所名稱: 工學院 - 資源工程學系
Department of Resources Engineering
論文出版年: 2017
畢業學年度: 105
語文別: 英文
論文頁數: 141
中文關鍵詞: 生物能二氧化碳光合作用醣類類胡蘿蔔素藻藍蛋白C / N比平板光生物反應器藍細菌CO2固定亮/暗循環
外文關鍵詞: Bio-energy, Carbon dioxide, Photosynthesis, Carbohydrate, Carotenoid, Phycocyanin, C/N ratio, photobioreactor, CO2 fixation, light/dark cycle
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  • 利用光合微生物吸收二氧化碳的碳固定作用並自細胞組成產生之生質能,具有巨大的環境保護及能源再生潛力。本研究中利用自台灣地區溫泉中分離的嗜熱菌株Thermosynechococcus sp. CL-1 (TCL-1),吸收熱煙道氣洗滌塔廢水中溶解的無機碳(DIC)。培養系統為兩種類型(連續和分批)的管狀光生物反應器。在試驗中,以碳酸鹽/碳酸氫鹽,模擬洗滌塔吸收的C源(溶解的無機碳,DIC,50℃),而以硝酸鹽模擬作為N源(溶解的無機氮,DIN)。細胞組分中的脂質和碳水化合物,係做為生質能之前軀物,其含量以DIC / DIN摩爾比的函數表示。當DIC和DIN不受限制時,在0.06 h -1的稀釋速率下,二氧化碳吸收速率為917 mg L-1d-1時,可獲得的最大脂質生產率,約為150 mg L-1d-1。細胞質量和細胞組成不僅受N濃度的限制,而且還受批次培養試驗中的C/N比之影響。在N受限及高C/N比下,TCL-1在進行光合作用時,碳的固定的途徑從合成蛋白質轉變為合成碳水化合物而非脂質,最大碳水化合物含量為61%。
    TCL-1也使用在平板光生物反應器,在室內密集培養,以檢測光源和照明強度對生質體產生、二氧化碳固定及藻藍蛋白產生之影響。結果顯示平板光生物反應器對生質體產生、二氧化碳固定和藻藍蛋白產生能力,大為提高。以LED白光照明,12小時內的最大生質體生產率和二氧化碳固定率分別為0.82和1.29 g L-1。但是,LED紅光照明對生質體生產率及二氧化碳固定均無明顯效益。TCL-1以白光或紅光LED照明12小時,其藻藍蛋白含量約為2%。當亮循環與暗循環交替時,在12小時的暗循環期,TCL-1的生質量約降低0.1至0.2 gL -1,藻藍蛋白的含量約降低2.6%。以1,000μEm-2s-1 LED白光照明,TCL-1的藻藍蛋白最大含量為5.3%。TCL-1藻藍蛋白的收穫含量由適當的收穫時間而決定,最佳收穫時間是在暗循環之前的時間,TCL-1的藻藍蛋白在經濟上是非常有價值的。
    本項研究的另一個目標是創建一個藻藍蛋白,類胡蘿蔔素和碳水化合物(生質酒精前驅物)的戶外生產平台。 由前人實驗結果與培養實驗結果可知,室內培養之二氧化碳固定率(最大值5.316g CO2/L/d)優於室外培養情況之二氧化碳固定率(最大值3.41g CO2/L/d)。 然而,室外栽培的TCL-1,其碳水化合物含量可高達70%,遠優於室內栽培之61%。此外,藻藍蛋白及類胡蘿蔔素等高光敏色素的細胞含量,室內培養則比室外培養更加穩定。 TCL-1的高二氧化碳固定率以及碳水化合物,藻藍蛋白和類胡蘿蔔素的含量,在一些特定室外實驗條件下可達到60%,5%和1%的期望值。 因此實證,在室外培養TCL-1生產藻藍蛋白,類胡蘿蔔素和碳水化合物,是具有潛能的。

    There is a great potential to assimilate carbon dioxide and produce bio-energy from cellular component by utilizing carbon fixation of photosynthetic microorganisms. A thermophile strain, Thermosynechococcus sp. CL-1 (TCL-1) isolated previously from a hot spring in Taiwan, was feasible for using dissolved inorganic carbon (DIC) from the scrubber with regard to “hot” flue gas and used in the present study. Two types of culture system, continuous and batch, with tube photobioreactors were used and fed with carbonate/bicarbonate as C source (DIC, 50°C) and nitrate as N source (dissolved inorganic nitrogen, DIN) for simulating the absorbent from scrubber of flue gas. Lipid and carbohydrate of cellular component could be used as bio-energy precursors and their contents as function of various molar ratios of DIC/DIN was quantified. The maximal lipid productivity of about 150 mg L-1 d-1 was obtained while carbon dioxide uptake rate was 917 mg L-1 d-1 at the dilution rate of 0.06 h-1 when both DIC and DIN were not limited. Cell mass and cellular compositions were depended on not only limitation of N concentration but also C/N ratio in a batch culture test. The fixation of carbon during photosynthesis seemed to switch from the pathway of protein synthesis to forming carbohydrate not lipid under the N-limitation and high C/N ratio for TCL-1. The maximal carbohydrate content was 61%.
    TCL−1 was also cultivated in flat plate photobioreactors in this reearch with dense culture indoors to examine the effects of light source and illumination intensity on the biomass production, carbon dioxide fixation and phycocyanin production. It showed that biomass production, carbon dioxide fixation, and phycocyanin production can be increased in flat plate photobioreactors. The greatest biomass productivity and carbon dioxide fixation rate were 0.82 and 1.29 g L−1 respectively with white LED illumination for 12 hours. It showed no effort in biomass production and carbon dioxide fixation for red light LED. The content of phycocyanin of TCL−1 was about 2% with white or red light LED illumination for 12 hours. When light/dark cycle was applied, the biomass of TCL-1was reduced about 0.1 to 0.2 gL−1 and the content of phycocyanin was reduced about 2.6% during the same dark period of 12 hours. The maximum content of phycocyanin for TCL−1 was 5.3% with 1,000 μE m−2s−1 white LED illumination. The optimum phycocyanin content of TCL-1 can be determined by the appropriate harvest time. The best time for TCL-1 to harvest the phycocyanin is the time before darkness. The phycocyanin of TCL-1 is very valuable economically.
    Another goal of this research was to create an outdoor production platform of phycocyanin, carotenoid and carbohydrates which is the bio-ethanol precursor. The results of previous studies and present cultivation experiments indicated that the maxium carbon dioxide fixation rate for indoor case (5.316g CO2/L/d) is better than outdoor case (maxium 3.41g CO2/L/d). However, the carbohydrate content of TCL-1 cultivated outdoors was as high as 70% which was much better than indoor cultivation. In addition, the cellular contents of phycocyanin and carotenoid, both are high light sensitive pigments, were much stable when cultivated indoors than outdoors. The high carbon dioxide fixation rate and the content of carbohydrate, phycocyanin and carotenoid of TCL-1 were addressed to reach the experimental expectation of 60%, 5% and 1% under some specific outdoor conditions. Consequently, the production of phycocyanin, carotenoid and carbohydrate under outdoor cultivation is economically fessible.

    摘要......................................................................................................................................Ⅳ ABSTRACT.........................................................................................................................Ⅶ ACKNOWLEDGEMENT....................................................................................................Ⅸ CONTENT...........................................................................................................................Ⅹ LIST OF TABLES.........................................................................................................ⅩⅤ LIST OF FIGURES..........................................................................................................ⅩⅥ NOMENCLATURE.........................................................................................................ⅩⅨ CHAPTER 1 INTRODUCTION............................................................................................1 1.1 CO2 Emission and Globle Warming.................................................................................1 1.2 Scope of Investigation......................................................................................................7 CHARTER 2 LITERATURE SURVEY.................................................................................9 2.1 Greenhouse Effect.....................................................................................................9 2.2 Introduction of Carbon Dioxide Treatment.............................................................11 2.2.1 Physical Treatment..........................................................................................12 2.2.2 Chemical Treatment........................................................................................13 2.2.3 Biological Treatment.......................................................................................16 2.3 Alternative Energy...................................................................................................19 2.3.1 Solar Energy....................................................................................................20 2.3.2 Wind Energy....................................................................................................21 2.3.3 Hydro Energy..................................................................................................22 2.3.4 Ocean Energy..................................................................................................24 2.3.5 Biomass Energy...............................................................................................26 2.3.6 Geothermal Energy..........................................................................................28 2.4 Acid-Base Balance..................................................................................................29 2.4.1 Calculation of Acid-Base Balance...................................................................29 2.4.2 Graphical Approach of Acid-Base Balance.....................................................29 2.5 Photosynhtic Microorganism..................................................................................31 2.5.1 Introduction of Microalgae..............................................................................31 2.5.2 Thermosynechococcus sp. CL-1......................................................................32 2.5.3 Previous Studies of the TCL-1........................................................................33 2.5.4 Previous Studies of The Other Alage...............................................................34 2.6 Photosynthesis........................................................................................................35 2.6.1 Light Reaction.................................................................................................36 2.6.2 Calvin-Benson Cycle.......................................................................................37 2.6.3 The Total Reaction..........................................................................................39 2.7 The Environmental Factors Affecting Photosynthetic Microbial Growth............40 2.7.1 Light Source....................................................................................................40 2.7.2 Temperature.....................................................................................................40 2.7.3 Medium Composition......................................................................................41 2.7.4 Salinity.............................................................................................................41 2.7.5 Carbon Dioxide...............................................................................................42 2.7.6 pH Value..........................................................................................................42 2.7.7 Dissolved Oxygen Concentration....................................................................43 2.8 Observation of Cell Viability.................................................................................43 2.8.1 Application of Fluorescent Dye to Cells.........................................................43 2.8.2 Flow Cytometry...............................................................................................43 2.9 Growth Curves and Determination.........................................................................45 2.9.1 Introduction of Growth Mode.........................................................................46 2.10 Culture System.....................................................................................................47 2.11 Major Product of Microalgae...............................................................................50 2.12 Common Toxins of Algae.....................................................................................51 2.13 Phycobiliproteins..................................................................................................54 2.13.1 Introduction of Phycobiliproteins..................................................................54 2.13.2 Impact Factor of Phycobiliproteins...............................................................57 2.13.3 Application of Phycobiliproteins...................................................................58 2.14 Carotenoids............................................................................................................59 2.14.1 Introduction of Carotenoids..........................................................................59 2.14.2 Structure of Carotenoids................................................................................59 2.14.3 Application of Carotenoids............................................................................61 CHAPTER 3 MATERIALS AND METHODS.................…...............................................63 3.1 Research Framework...............................................................................................63 3.2 Experimental Chemicals, Apparatus and Analytical Devices.................................64 3.2.1 Experimental Chemicals..................................................................................64 3.2.2 Apparatus.........................................................................................................65 3.2.3 Analytical Devices...........................................................................................66 3.3 Experiment Flowchart.............................................................................................68 3.4 Introduction of TCL-1 Strain..................................................................................70 3.4.1 Algal Sources...................................................................................................70 3.4.2 Algal Strains Preservation...............................................................................71 3.4.3 Algal Strain characteristics of TCL-1..............................................................72 3.4.4The Absorptive Characteristic and Wavelength Selection of Strains of TCL-1..............................................................................................................73 3.5 Bioreactor Design....................................................................................................73 3.6 Algal Preservation and Photobioreactors of Cultivation.........................................74 3.6.1 Algal Cryopreservation....................................................................................74 3.6.2 General Algal Preservation..............................................................................74 3.6.3 A Small Scale of Active Cultivation................................................................74 3.6.4 Indoor Cultivation with Tube photobioreactor................................................75 3.6.5 Indoor Cultivation with Plate Photobioreactor................................................76 3.6.6 Outdoor Cultivation.........................................................................................76 3.6.7 Nutrition Medium’s Preparation and Amendment..........................................78 3.7 Analysis of Algal Cells............................................................................................78 3.7.1 The Measure of Algal Cell Mass.....................................................................78 3.7.2 The Measure of Algal Cell Number................................................................79 3.7.3 Measure of Algal OD680 nm............................................................................79 3.7.4 Preparation of Dry Algal Cell mass.................................................................80 3.7.5 Analysis of Algal Lipid....................................................................................80 3.7.6 Analysis of Algal Carbohydrate.....................................................................81 3.7.7 Analysis of Algal Protein...............................................................................81 3.7.8 Extraction of Phycocyanin..............................................................................82 3.7.8.1 Preparation of Phycocyanin Extraction Purification Solution.............82 3.7.8.2 Discussion of Optimum Extraction Method.......................................82 3.7.8.3 Preparation of Crude Extract Solution of Phycocyanin.......................83 3.7.8.4 Extraction of Phycocyanin...................................................................83 3.7.8.4.1 Separation with Ammonium Sulfate...........................................84 3.7.8.4.2 Ion Exchange Chromatography...................................................85 3.7.8.5 Quantification of phycocyanin............................................................85 3.7.9 Extraction of Carotenoids................................................................................88 3.7.9.1 Purification of Carotenoids..................................................................88 3.7.9.2 Quantification of Carotenoids..............................................................88 3.7.10 Analysis of Algal Toxicity.............................................................................89 CHAPTER 4 RESULTS AND DISCUSSION............................................……...............91 4.1 Continuous Cultivation Under Various Feeding DIC Concentrations.....................91 4.2 Batch Cultivation Under Various Initial DIN Concentrations..................................94 4.3 Effects of Illumination Intensity and Quality on Biomass Productivity, CO2 Fixation Rate and Phycocyanin Productivity for Indoor Culture with Plate Photobioreactor.............................................................................................100 4.3.1 Indoor Cultivation of TCL-1 in Flat Photobioreactor with Various Illumination Sources and Intensities.............................................................100 4.3.2 Effects of 12/12 Light/Dark Cycle on Biomass Productivity and Phycocyanin Productivity………………………………………………………...............103 4.4 Batch Culture of TCL-1 Outdoors with Tube and Plate Photobioreactors ............105 4.4.1 Effects of Nitrogen Concentration on Batch Cultivation of TCL-1 within Plate Photobioreactors............................................................................................106 4.4.2 Effects of Aeration Rate on Outdoors Cultivation of TCL-1 in Plate Photobioreactors............................................................................................109 4.4.3 Effects of Different Carbon Source Conditions on TCL-1 Cultivated Outdoors in Plate Photobioreactors...............................................................................110 4.4.4 Effects of Different Photobioreactors on TCL-1 in Outdoor Cultures..........112 4.4.5 Algal Outdoor Cultivation with Low Carbon and Low Nitrogen Source......114 4.4.6 Outdoor Culture Experiments of TCL-1 in Different Conditions.................115 4.5 The culture strategy of TCL-1................................................................................119 CHAPTER 5 CONCLUSTIONS AND SUGGESTIONS..............................................121 5.1 Conclusions............................................................................................................121 5.2 Suggestions............................................................................................................122 REFERENCES...........................................................................................……................124

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