Effect of different phosphorus concentrations on the growth and 

alkaline phosphatase activity of free-living zooxanthellae

LI Yue1,2, WANG Yunlong2, OUYANG Longling2

(1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai201306, China; 

2. Key laboratory of East China Sea Fishery Resources Explotation and Utilization, Ministry of Agriculture and Rural Affairs, 

East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai200090, China)

Abstract: Symbiodinium sp. provides coral with oxygen and synthetic organic matter, while CO2, nitrate and phosphate produced by coral metabolism are used by Symbiodinium sp. to maintain its growth. As a beneficial reservoir of functional diversity, freeliving zooxanthellae can potentially repopulate highly impacted reef ecosystems. However, phosphorus is severely restricted in many coastal and estuarine waters, which could affect phytoplankton growth. Freeliving zooxanthellae is in a natural environment with constantly changing phosphate, and the effect of different phosphate concentrations on zooplankton algae is unknown. To understand how Symbiodinium sp., a species of freeliving zooxanthellae, responded to the dynamic change of dissolved inorganic phosphorus (DIP), we designed short term test and long term test to examine the effects of different phosphate concentrations on the growth, alkaline phosphatase activity (APA), chlorophyll a (chl a) and effective photochemical efficiency (Fq′/Fm′). Symbiodinium sp. was cultured under four different DIP initial concentrations (0.15, 10.00, 20.00,35.00 μmol·L-1PO43-) for 7 d in short term test, while the algal cells were cultured under a high DIP initial concentration (35.00 μmol·L-1PO43-) for 55 d in long term test. Subsequently, the cell density, DIP concentration in the culture medium and APA of algal cells were measured on 0, 1, 2, 4, 7 d in short term test, and every 5 d in long term test, respectively. In addition, chl a and Fq′/Fm′ were also measured in long term test. Results revealed that there were significant differences in the growth of Symbiodinium sp. under different initial concentrations of phosphate. The cell density of high initial concentration group was significantly higher compared with the other three groups (P＜0.05). In particular, when the initial concentration of phosphate was 0.15 μmol·L-1, although the initial concentration was low, the phosphate concentration decreased to 0.05 μmol·L-1 after 1 d, followed by no significant change (P>0.05), indicating that the growth of freeliving zooxanthellae was positively correlated with phosphate concentration. APA of algal cells in the test group of initial concentration of phosphate of 10.00, 20.00, 35.00 μmol·L-1was significantly lower than that of 0.15 μmol·L-1 test group (P<0.05), indicating that it was negatively correlated with the concentration of inorganic phosphorus in the environment. It could be seen that low inorganic phosphorus could induce the increase of alkaline phosphatase (AP) activity. Two days after inoculation, the algal cell density of each test group increased slowly or showed a downward trend, showing a growth retardation period of at least two days. On the 7th day of culture, the density of algal cells varied with the initial concentration of phosphate, which was shown as follows: the initial concentration of phosphate was 35.00 μmol·L-1, the density of algal cells was the highest and significantly higher than that of other groups (P<0.05); the initial concentration was 10.00 μmol·L-1 and 20.00 μmol·L-1, the density of algal cells was the second, and there was no significant difference between two groups (P<0.05); the initial concentration of phosphate was 0.15 μmol·L-1, the density of algal cells was the lowest, which was only 1/3 of that of the highest experimental group. When the initial concentration of phosphate was 35.00 μmol·L-1, the cell concentration in the medium began to increase rapidly, and reached the highest after 20 days, which was 2.34×108 L-1, and then there was a fluctuating decline. Under high concentration of phosphate (35.00 μmol·L-1), changes in the content of chl a in Symbiodinium sp. were consistent with the trend of algal cell density. However,changes in cell growth and chl a showed a 5day lag trend. The chl a content of algal cells increased exponentially in the first 15 d, and reached the maximum value of 3 034.27 μg·L-1 on the 15th day, then began to decline, and basically remained stable after the 40th day, which was 807.71873.23 μg·L-1. Results of Fq′/Fm′ analysis of algal cells showed that Fq′/Fm′ increased 5 d before culture, then decreased significantly, and the change trend changed after the 25th day, showing a fluctuating downward state. Along with the consumption of phosphate, the specific growth rate (μ) and Fq′/Fm′ of Symbiodinium sp. decreased, suggesting that a positive correlation existed between phosphorus limitation and photosynthetic efficiency. At the beginning of longterm culture experiment, the phosphate concentration in the culture medium decreased rapidly, and decreased to 0.04 μmol·L-1 on the 5th day, and then remained at a low level, showing a small fluctuation, ranging from 0.04 μmol·L-1 to 0.53 μmol·L-1. In contrast, the APA was significantly enhanced (P<0.05) after 20 d of culture at the phosphate initial concentration of 35.00 μmol·L-1, increased to the maximum on the 30th day, indicating that the DIP content was negatively correlated with APA, which was also presented when Symbiodinium sp. was cultivated under low phosphate initial concentration (0.15 μmol·L-1). During 2055 d, the change trend of algal cell APA was opposite to that of the number. It was speculated that the increase of algal cell number after the decline was due to the decline of algal cells, which released organic phosphorus to provide a substrate for AP, making algal cells regain inorganic phosphorus and proliferate. Alkaline phosphatase catalyzes dissolved organic phosphorus to DIP, which could enable Symbiodinium sp. to keep growing under low DIP concentration. In the shortterm and longterm culture experiments, the enhancement of AP activity did not cause the increase of DIP in the culture medium, which may be due to the fact that AP hydrolysis and DIP absorption were carried out at the same time. These results implied that like other dinoflagellates, Symbiodinium sp. could adapt to low phosphorus condition, and might help understand the mechanism of phosphorus utilization in this dinoflagellate in further research.

Keywords: freeliving zooxanthellae; dissolved inorganic phosphorus; alkaline phosphatase; photosynthesis