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研究生： 黃毓涵
Huang, Yu-han
論文名稱： 小球藻最適化連續式培養之研究
Studies on Continuous Cultivation of Chlorella sp. under Optimal Conditions
指導教授： 吳文騰
Wu, Wen-teng
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2009
畢業學年度： 97
語文別： 中文
論文頁數： 119
中文關鍵詞： 小球藻 、最適化 、連續式培養策略 、實驗設計法
外文關鍵詞： continuous culture process, Chlorella sp., optimization, design of experiments
相關次數： 點閱：106  下載：16 
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    • 微藻因生長速率快且可經由光合作用固定大氣中的二氧化碳並轉化生成生物質，是相當具有潛力的生質能源生產者。因此，本研究為獲得高產率的藻類之生物質量與油脂，採用實驗設計法探討調控小球藻生長的各項重要影響因子，再以迴歸分析決定出培養小球藻的最佳條件，並以連續式培養策略與最適化操作條件來量產小球藻，以獲得其生物質量與油脂。
    本研究以實驗設計法探討不同氮源濃度、光照強度、二氧化碳通氣量與鹽度對生物質量與油脂的影響。再以迴歸分析決定出小球藻之生物質量伴隨最高油脂產率的最佳培養條件：氮源濃度0.2 g/L、光照強度1200 μmol photon m-2 s-1、二氧化碳通氣量2.86％以及鹽度18.75 g/L。並由實驗獲致最高油脂產率與伴隨的生物質量產率分別為0.175 g/L-day和0.465 g/L-day。
    在上述的最適化培養條件下，以具有時延項的Logistic方程式和Luedeking-Piret方程式分別擬合小球藻之生長及油脂生成的動力模式，並依此決定出連續式培養策略的最佳稀釋率，以獲得最大的小球藻目標物產量。所推導出的連續式操作策略，使小球藻於化學恆定的狀態下穩定的生長與量產藻體，且實驗所獲致的藻體產率為0.773 g/L-day，與具有時延項之生長動力模式所預測的結果之間的相對誤差為-10.53%。因此證實此具有時延項模型對於該系統的優越掌握能力，與連續式操作策略於最適化操作條件下的可行性。

    Microalgae have received much attention as renewable energy resources, since the photoautotrophic mechanism can convert the atmospheric carbon dioxide into biomass. In order to harvest microalgae with the high productivities of biomass and lipid, design of experiments with regression analysis was applied to optimize the culture conditions of medium. Under the mentioned optimal culture conditions, a continuous culture process was purposed to carry out for harvesting microalgae continuously.
    In this study, the design of experiments was defined to investigate the effects of initial nitrogen concentration, light intensity, carbon dioxide aeration rate and salinity on the growth and lipid formation of Chlorella sp.. Using regression analysis, the optimal culture conditions for the highest lipid productivity were estimated as 0.2 g/L of nitrogen concentration, 1200 μmol photon m-2 s-1 of light intensity, 2.86% of carbon dioxide aeration rate and 18.75 g/L of salinity. These conditions were verified experimentally, since the productivities of the biomass and lipid achieved to 0.465 and 0.175 g/L-day, respectively.
    For knowing the growth kinetics and lipid formation of Chlorella sp., Logistic and Luedeking-Piret equations are considered as the models to approach the dynamics. These two kinetic models were further employed to predict the optimal dilution rate associated with the biomass and lipid concentration as 0.9253 and 0.7526 day-1, respectively. Under the optimal operating conditions, an experimental verification was carried out, and a reasonable good agreement between experimental data and model prediction is observed by the relative error of -10.53% and R squared of 0.9701. Therefore, the chemostat strategy with optimal operational condition is successfully employed for producing microalgal bioproduct.

    目錄 摘要......................................................I Abstract.................................................II 誌謝....................................................III 目錄 ....................................................IV 圖目錄 ................................................VIII 表目錄 ...................................................X 符號 ...................................................XII 第一章 緒論...............................................1 1-1 前言..................................................1 1-2 研究目的與動機 ........................................3 第二章 文獻回顧...........................................4 2-1 藻類簡介..............................................4 2-2 綠藻生理介紹..........................................7 2-2-1 光合作用............................................7 2-2-2 呼吸作用............................................9 2-2-3 影響生長的調控因子.................................12 2-3 綠藻培養介紹.........................................18 2-3-1 培養系統...........................................19 2-3-2 培養策略...........................................27 第三章 研究材料與方法....................................31 3-1 藻種.................................................31 3-2 培養基組成...........................................33 3-2-1 碳源...............................................33 3-2-2 氮源與其他營養鹽...................................33 3-3 培養條件.............................................36 3-3-1 微藻種源保存.......................................36 3-3-1-1 包埋固定法.......................................36 3-3-1-2 繼代培養.........................................37 3-3-2 前培養.............................................37 3-3-3 主培養.............................................37 3-4 實驗設備.............................................38 3-4-1 光生物反應器.......................................38 3-4-2 培養系統...........................................38 3-4-3 分析系統...........................................39 3-5 實驗分析方法.........................................40 3-5-1 光照強度測定.......................................40 3-5-2 酸鹼值測定.........................................40 3-5-3 培養液尿素含量分析.................................41 3-5-4 微藻濃度測定.......................................42 3-5-4-1 藻體乾重測定.....................................42 3-5-4-2 分光光度計測定法.................................43 3-5-5 油脂分析...........................................45 3-5-5-1 氣相層析儀分析法.................................45 3-5-5-2 螢光測定法.......................................48 3-6 生長動力學模型.......................................50 3-6-1 微藻之生長動力學模型...............................50 3-6-2 微藻油脂之生成動力學模型...........................50 3-6-3 微藻消耗氮源之動力學模型...........................51 3-6-4 具有時延項之動力學模型.............................52 第四章 實驗結果與討論....................................53 4-1 批次實驗.............................................53 4-1-1 不同氮源濃度之影響.................................53 4-1-2 不同光照強度之影響.................................56 4-1-3 不同二氧化碳通氣量之影響...........................59 4-1-4 不同鹽度之影響.....................................61 4-2 最適化培養條件之探討.................................64 4-2-1 實驗參數設計.......................................64 4-2-2 迴歸分析簡介.......................................68 4-2-3 迴歸模型建構.......................................69 4-2-4 迴歸模型檢定.......................................72 4-2-5 迴歸模型之診斷.....................................75 4-2-6 最佳培養條件之計算與實驗驗證.......................78 第五章 生長與產物生成之動力學研究........................80 5-1 最佳培養條件之穩定性與再現性.........................80 5-2 微藻之生長動力學模型建立.............................82 5-2-1 動力學模型的建立與係數決定.........................82 5-2-2 模型可信度檢驗.....................................82 5-3 微藻油脂之生成動力學模型建立.........................84 5-3-1 動力學模型的建立與係數決定.........................84 5-3-2 模型可信度檢驗.....................................84 5-4 微藻消耗氮源之動力學模型建立.........................86 5-4-1 動力學模型的建立與係數決定.........................86 5-4-2 模型可信度檢驗.....................................86 5-5 連續式培養策略.......................................88 5-5-1 化學恆定之穩態條件推導.............................88 5-5-1-1 最大藻體生長速率.................................89 5-5-1-2 最大油脂生成速率.................................91 5-5-2 穩態點之控制與穩定性分析...........................93 5-5-2-1 比例迴饋控制.....................................93 5-5-2-2 穩定性分析.......................................94 5-5-3最大藻體生長速率的連續式操作之模擬與實驗驗證........96 5-5-3-1 微藻濃度恆定.....................................96 5-5-3-2具有時延項的生長動力學模型........................99 5-5-3-3 具有時延現象的穩態點操作與差異比較..............101 5-5-4 最大藻體油脂生成速率的連續式操作之模擬............104 第六章 結論與未來展望...................................107 6-1 結論................................................107 6-2 未來展望............................................109 參考文獻................................................110 自述....................................................119 圖目錄 圖2- 1 光合作用之光反應與暗反應示意圖.....................7 圖2- 2 光合反應速率與光強度的關係圖.......................9 圖2- 3 呼吸作用的脂肪酸氧化過程示意圖....................11 圖2- 4 以光強度500 μmol photon m-2照射濃度0.1 g/L之裸藻培養系統的反應器內光分佈情形.................................14 圖2- 5 開放式藻類培養系統示意圖 (a)大型淺池 (b)開放式槽體 (c)圓型培養池 (d) 跑道型培養池...........................21 圖2- 6 密閉式藻類培養系統示意圖 (a)發酵槽 (b)培養袋 (c)平板光生化反應器 (d)管型光生化反應器.........................24 圖2- 7 連續式培養系統示意圖..............................30 圖3- 1 小球藻（Chlorella sp.）的光學顯微鏡圖（放大400倍).32 圖3- 2 固定化藻珠製備流程圖..............................36 圖3- 3 藻體乾重對應其吸光值之檢量線......................44 圖4- 1 小球藻培養於不同氮源濃度下之氮源濃度時間趨勢圖與生長曲線。(○,●) 0.025 g/L，(▽,▼) 0.05 g/L，(□,■) 0.10 g/L，(◇,◆) 0.15 g/L和(△,▲) 0.30 g/L，空心符號代表氮源濃度，實心符號代表藻體濃度。...............................55 圖4- 2 小球藻培養於不同光照強度下之生長曲線..............58 圖4- 3 於不同二氧化碳通氣量下的培養系統之培養液酸鹼值時間趨勢圖與小球藻生長曲線。(○,●) 0.03 %，(▽,▼) 2 %，(□,■) 5 %和(◇,◆) 10 %，空心符號代表培養液的酸鹼值，實心符號代表藻體濃度。...............................................60 圖4- 4 小球藻培養於不同鹽度下之生長曲線..................62 圖4- 5 進行中心點實驗之小球藻培養系統的氮源濃度時間趨勢圖與生長曲線。（中心點實驗的培養條件：初始氮源濃度0.1 g/L、光照強度600 μmol photon m-2 s-1、二氧化碳通氣量2 %與鹽度16.7 g/L）。..................................................67 圖4- 6 藻體產率預測模型之殘差分析 (a)常態機率圖 (b) y殘差圖 (c)時序殘差圖.........................................76 圖4- 7 油脂含量預測模型之殘差分析 (a)常態機率圖 (b) y殘差圖 (c)時序殘差圖.........................................77 圖5- 1 小球藻於初始氮源濃度0.2 g/L、光照強度1200 μmol photon m-2 s-1、二氧化碳通氣量2.86 %以及鹽度18.75 g/L的培養條件下，重複三次批次實驗所獲得之生長曲線。...............81 圖5- 2 小球藻於最適化培養條件下之藻體濃度隨時間變化的過程83 圖5- 3 小球藻於最適化培養條件下之藻體油脂濃度隨時間變化的過程.......................................................85 圖5- 4 小球藻培養於最適化條件下之培養液內氮源濃度隨時間變化的過程...................................................87 圖5- 5 操作於最大藻體生長速率的連續式小球藻培養系統中之藻體濃度隨時間變化的過程.....................................98 圖5- 6 操作於最大藻體生長速率，且具有時延現象的連續式小球藻培養系統中之藻體濃度隨時間變化的過程....................103 圖5- 7 操作於最大油脂生成速率，且具有時延現象的連續式小球藻培養 系統中之藻體濃度、氮源濃度與油脂濃度隨時間變化的模擬過程。..................................................106 表目錄 表2- 1 藻類的商業化應用...................................6 表2- 2 商業化大規模藻類培養系統與所培養的藻種............22 表2- 3 開放式系統與密閉式系統之分類與設計技術關鍵........25 表2- 4 藻類培養於開放式系統與密閉式系統之比較............26 表3- 1 Walne’s Medium-Nutrient solution組成.............35 表3- 2 Walne’s Medium-Trace metal solution（TMS）組成...35 表3- 3 Walne’s Medium-Vitamin solution組成..............35 表3- 4 小球藻培養系統的實驗儀器設備表....................38 表3- 5 小球藻分析系統的實驗儀器設備表....................39 表4- 1 小球藻培養於不同氮源濃度下之比生長速率、最終藻體濃度以及油脂 含量和濃度......................................56 表4- 2 小球藻培養於不同光照強度下之比生長速率、最終藻體濃度以及油脂含量和濃度.......................................58 表4- 3 小球藻培養於不同二氧化碳通氣量下之比生長速率、最終藻體濃度以及油脂含量和濃度.................................61 表4- 4 小球藻培養於不同鹽度下之比生長速率、最終藻體濃度以及油脂含量和濃度...........................................63 表4- 5 實驗參數設計之各組實驗的培養條件與結果............66 表4- 6 藻體產率迴歸模型之變異數分析表....................74 表4- 7 藻體油脂含量迴歸模型之變異數分析表................74 表4- 8 於最佳培養條件下的小球藻培養系統實驗值與預測值的相對誤差.....................................................79 表5- 1 小球藻培養於連續式系統中的穩定性條件..............96 表5- 2 操作於最大藻體生長速率之連續式小球藻培養系統的藻體產率之實驗值與預測值的相對誤差.............................98 表5- 3 操作於最大藻體生長速率，且具有時延現象的連續式小球藻培養系統的藻體產率之實驗值與預測值的相對誤差............105

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