磷營養鹽供應模式在利瑪原甲藻生長及生產大環內酯天然物的影響

Abstract

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.