周宏農臺灣大學：漁業科學研究所余昌憲Yu, Chang-ShianChang-ShianYu2007-11-282018-07-062007-11-282018-07-062006http://ntur.lib.ntu.edu.tw//handle/246246/59323藻類能夠利用水中的氮、磷等無機元素，配合光合作用而生長，而藻類成長所需的氮和磷這兩種營養鹽的供應與其間的濃度比例，對細胞的成長和密度的維持有著顯著的影響，並可能影響其天然物的生合成。渦鞭毛藻Prorocentrum lima的培養經常使用K培養基，其所含氮、磷營養鹽包括有883 uM的硝酸鹽，50 uM的氨鹽10 uM的磷酸鹽，氮磷比遠超過一般認知的最適氮磷比，4~16，以致於藻細胞密度難以提升。為改善培養提升細胞生長，首先比較了三種不同濃度，10 uM (低)、50 uM (中)、100 uM (高) 的磷酸鹽提供對P. lima生長的影響，並測定其培養基中營養鹽的變化。結果顯示P. lima對磷酸鹽的吸收快速，其培養基中磷酸鹽在消耗殆盡後，藻細胞仍能持續生長一段時間，顯示和其他藻類ㄧ樣存在一種奢華吸收的現象，同時也發現生長初期以10 uM磷鹽供應的培養最為快速; 然而10 uM的磷酸鹽供應相較於50 uM與100 uM的供應在成長末期並無法支持較高的藻細胞密度，同時實驗所採最高磷酸鹽 (100 uM) 的也無法支持50 uM磷酸鹽所獲的細胞密度。後續實驗以最終50 uM的磷酸鹽濃度為基準，在培養過程中採用不同週期的脈頻方式每次添加10 uM磷鹽培養P. lima，在靜置狀態培養下，不同週期的添加皆受到碳源的限制，添加點間的比成長率隨添加次數下降快速，在改以震盪狀態下培養後，培養的細胞達到更高的最終細胞密度，並分析添加點間的比成長率，發現密集式的補充磷鹽相較長間隔下能維持較高的生長速率。同時也發現在單位細胞內總磷最小於12 fg/cell時，細胞將不再增殖，推測其為渦鞭毛藻株PL08維持細胞存活的最低含磷量。在不同週期脈頻方式添加磷鹽的實驗下，PL08單位細胞所含天然物prorocentrolide的變化顯示在細胞生長指數期的末段有ㄧ定程度蓄積而後，下降再蓄積，培養基中磷鹽濃度的高低與添加模式並不會影響該物在細胞內的含量。Algae absorb inorganic elements, such as nitrogen, phosphorus, etc., in order to grow based on their photosynthesis. The supply of nitrogen and phosphorus nutrients and their relative abundance significantly affect the growth, cell concentration, or even the bioorganic components of the algae. K-medium, one of the commonly-used media for the dinoflagellate cultures, containing 883 uM nitrate, 50 uM ammonia and 10 uM phosphate was applied in this experiment as basal medium for Prorocentrum lima. Cells of P lima hardly reached the expected concentration in K-medium, since the N:P ratio of K medium is far beyond the known optimal value, 4~16. In order to improve the growth of P. lima for prorocentrolide and okadaic acid production, three levels of phosphate concentration, 10 uM (low), 50 uM (medium), and 100 uM (high) were tested with other ingredients of K-medium in P. lima cultures, in which the cell density and nitrogen and phosphorous concentration were analyzed along the growth period. Preliminary results showed that phosphate nutrients of all three levels depleted rapidly and the cells kept to grow for sometime while the medium phosphate were used up. This phenomenon indicated a luxurious consumption of phosphate in P. lima, same as in some other algal species. It was also found in the early stage that the algae had a better growth in 10 uM phosphate than in other groups of higher phosphate concentration. However, low phosphate concentration could only support the least cell density at the end of the experiments, then the highest phosphate concentration, 100 uM, could not support a higher cell concentration than 50 uM either. So, in the continuing 10 uM pulse-fed experiments of phosphate and the control were based on a 50 uM phosphate in total of K-medium. The pulse-fed experiments of 10 uM phosphate were designed by the different intervals of 3, 5, 7, and 9 days, in a condition with or without shaking. Generally cultures without shaking had lower growth rates and less cell densities at the end of experiments than cultures with shaking. The specific growth rate between each pulse-fed declined along with the culturing period. The older culture had the lower specific growth rate, the shorter interval of pulse-fed phosphate had higher specific growth rate. A minimal content of phosphorous in P lima was found to be 12 fg/ml. At this status the cell remained dormant without the increase of cell number. Productivity of procentrolide in P. lima showed two peaks during the experiment period, one is at the end of log phase the other one is at the start of stationary phase. Procentrolide was once accumulated at the end of log phase and declined to the minimum in the following days before stationary phase and then continued accumulation during stationary phase.目錄 1 中文摘要 3 英文摘要 5 第一章 前言: 6 第二章 材料與方法 16 一、基本方法 16 1. 藻株與培養基 16 2. 培養液中氮磷含量分析 16 2-1培養基中硝酸氮的檢測 16 2-2培養基中磷酸鈉的檢測 17 3. 細胞內氮磷含量分析 17 3-1 細胞內總氮含量分析 17 3-2 細胞內總磷含量分析 18 4. 藻毒的萃取與分析 18 5. prorocentrolide檢量線的製作 19 6. 淨產毒率的算 19 7. 磷鹽淨吸收的計算 20 8. 薻培養比成長率的計算 20 二、實驗 20 1. 不同濃度磷鹽處理下的培養模式 20 2. 脈頻式添加磷鹽處理下的培養模式 20 第三章 結果與討論 22 ㄧ、磷鹽濃度對細胞生長的影響 22 1. 藻細胞的生長速率與密度 22 2. 藻細胞吸收硝酸鈉與磷酸鈉的速率 25 二、脈頻式添加磷鹽處理下的培養模式 27 1. 藻細胞的生長速率 27 2. 藻細胞吸收磷酸鈉的速率 29 3. 細胞生長與磷鹽供需關係 29 4. 細胞內氮磷鹽含量 31 5. prorocentrolide毒素含量變化 34 第四章 結論: 36 附圖 38 圖一、Michaelis-Menten酵素動力公式 38 圖二、不同濃度磷酸鹽培養下的細胞生長曲線 38 圖三、培養基中硝酸氮變化 39 圖四、不同磷鹽濃度培養下細胞吸收磷鹽 39 圖五、脈頻添加磷鹽實驗於靜置下的細胞生長曲線 40 圖六、脈頻添加磷鹽實驗於震盪下的細胞生長曲線 40 圖七、靜置培養下各添加點間的細胞生長速率 41 圖八、震盪培養下各添加點間的細胞生長速率 41 圖九、脈頻添加磷鹽實驗中培養基的磷酸鈉濃度變化 42 圖十、比成長率與細胞對磷的淨吸收量關係 43 圖十一、磷鹽濃度對細胞生長與吸收營養鹽速率影響示意圖 44 圖十二、prorocentrolide檢量線 45 圖十三、PL08藻株內prorocentrolide成分的高效液相層析圖 45 圖十四、單位細胞含毒量 46 圖十五、淨產毒率 47 附表 48 表一、K培養基 48 表二、整理文獻中Prorocentrum lima的培養 49 表三、不同磷酸鈉濃度培養下細胞吸收磷酸鈉的速率 51 表四、脈頻添加實驗平均比成長率和單位細胞獲磷量 51 表五、脈頻添加實驗之細胞內總磷量 52 表六、細胞內氮磷元素比 52 表七、不同磷酸鈉濃度培養下細胞吸收硝酸鈉的速率 52 參考資料 53516499 bytesapplication/pdfen-US原甲藻渦鞭毛藻磷營養鹽大環內酯prorocentrolidephosphateprorocentrum limanutrientotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/59323/1/ntu-95-R92b45022-1.pdf