Effects of phosphate supplying mode on the growth and prorocentrolide productivity of Prorocentrum lima

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.