Chinese Journal of Oceanology and Limnology   2016, Vol. 34 issue(6): 1158-1172     PDF       
http://dx.doi.org/10.1007/s00343-016-5169-z
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

LI Junlei(李俊磊), SUN Xiaoxia(孙晓霞)
Effects of different phosphorus concentrations and N/P ratios on the growth and photosynthetic characteristics of Skeletonema costatum and Prorocentrum donghaiense
Chinese Journal of Oceanology and Limnology, 34(6): 1158-1172
http://dx.doi.org/10.1007/s00343-016-5169-z

Article History

Received Jun. 3, 2015
accepted for publication Jul. 28, 2015
accepted in principle Sep. 24, 2015
Effects of different phosphorus concentrations and N/P ratios on the growth and photosynthetic characteristics of Skeletonema costatum and Prorocentrum donghaiense
LI Junlei(李俊磊)1,2, SUN Xiaoxia(孙晓霞)1,3        
1 Jiaozhou Bay Marine Ecosystem Research Station, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;
2 University of Chinese Academy of Sciences, Beijing 100049, China;
3 Laboratory of Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China
ABSTRACT: The effects of different phosphorus (P) concentrations (0.36, 3.6, and 36 μmol/L corresponding to low-, middle-, and high-P concentration groups, respectively) and nitrogen (N)/P ratios on the growth and photosynthetic characteristics of Skeletonema costatum and Prorocentrum donghaiense were studied. For both species, the high-P (HP) concentration group showed the greatest algal density and highest specific growth rate. Changes in the maximum efficiency of photosystem II (Fv/Fm) were monitored under the various P and N/P conditions. The largest decrease in Fv/Fm was in the low-P (LP) group in S. costatum and in the HP group in P. donghaiense. There were high rapid light curves and photochemical quantum yields (ΦPS II) for S. costatum in the HP group, while the actual photosynthetic capacity was higher in P. donghaiense than in S. costatum in the MP group. Under eutrophic but relatively P-restricted conditions, P. donghaiense had higher photosynthetic activity and potential, which could cause this dinoflagellate to increasingly dominate the phytoplankton community in these conditions. Under the same P concentration and N/P ratio, P. donghaiense had a larger relative maximum rate of electron transport and higher ΦPS II values than those of S. costatum. These differences between P. donghaiense and S. costatum may explain the interaction and succession patterns of these two species in the Changjiang (Yangtze) River estuary from a photosynthesis perspective.
Key words: Skeletonema costatum     Prorocentrum donghaiense     nutrients     algae growth     photosynthetic characteristics    
1 INTRODUCTION

Phytoplankton are among the most important primary producers in marine ecosystems and are a basic link in the food chain. Algal productivity accounts for about 45% of global gross primary productivity (Field et al., 1998). Phytoplankton community structure, such as species composition and abundance, is likely to change as a result of changes in environmental factors. This could lead to changes in the structure and function of the entire marine ecosystem (Sun et al., 2011). Therefore, an understanding of the long-term variations of the phytoplankton community is essential for research on the changes and trends in the structure and function of coastal ecosystems. Such studies will provide the scientific basis for explaining the evolution of coastal ecosystems during global change.

Phytoplankton growth and reproduction are affected by the biological characteristics of each species and various environmental factors such as light, temperature, salinity, and nutrients (Lalli and Parsons, 1993). Numerous studies have found that phytoplankton community structure and composition have changed considerably in many of the world's oceans in recent decades. For example, several studies detected declining proportions of diatoms and increasing amounts and types of dinoflagellates (Smayda, 1990; Zhou et al., 2008). These changes have also occurred in China's Changjiang (Yangtze) River estuary, Jiaozhou Bay, and other waters, and an imbalance in nutrient structure is the main reason for these changes (Huo et al., 2001; Wang, 2003). Generally, the nutrient content of seawater affects the phytoplankton growth rate and the biochemical composition of algae (Goldman et al., 1979). Nitrogen (N) and phosphorus (P) are the most important factors controlling plankton blooms (Hallegraeff, 1993). Hodgkiss and Chan (1987) suggested that when attempting to control algae outbreaks, controlling N and P inputs should be considered because long-term or short-term changes in N/P ratios are often accompanied by a large increase in non-diatom phytoplankton species. Different concentrations of N and P in the water might result in different species becoming dominant via phytoplankton competition (Hodgkiss and Ho, 1997). Therefore, a long-term nutrient structure imbalance not only leads to the outbreak of non-diatom algal blooms, but also causes non-diatoms to dominate instead of diatoms (Cosper et al., 1989).

Photosynthesis is the most critical process in the primary production of phytoplankton. The level of photosynthetic activity strongly affects primary production and growth potential, community structure, and the population succession of phytoplankton. Research on the photosynthetic capacity and characteristics of phytoplankton can increase our understanding of primary production and material circulation in the marine ecosystem and the population dynamics of phytoplankton (Strzepek and Harrison, 2004; Jakob et al., 2005; on and Claquin, 2012). Several studies have shown that there is a very close interaction between nutrient status and the photosynthetic activity of phytoplankton. The Calvin cycle is affected by the lack of nutrients during outbreaks and disappearances of phytoplankton. Photosynthetic activity changes considerably due to nutrient limitation, which has large impacts on phytoplankton composition and community structure (Glibert et al., 1988). Skeletonema costatum is a eurythermal and euryhaline planktonic diatom that is widely distributed in coastal waters. This species has bloomed many times in China (Huo et al., 2001; Zhou et al., 2001). Prorocentrum donghaiense is mainly found in the Changjiang River estuary and the adjacent East China Sea, and has often formed largescale algal blooms (ca.10 000 km 2) during the spring in recent years (Lu et al., 2003; Zhou et al., 2003; Li et al., 2007). P. donghaiense blooms often follow S. costatum blooms. Although many researchers have tried to explain this phenomenon, its mechanisms remain unclear (Zhao et al., 2009).

The aim of this study was to explore the relationships among the nutritional, physiological, photosynthetic, and reproductive characteristics of phytoplankton. The purpose of these analyses was to improve our understanding of the mechanisms underlying changes in phytoplankton composition and community structure. In this study, we compared and analyzed the effects of different P concentrations and N/P ratios on the specific growth rate, Chl a relative content, and photosynthetic characteristics of S. costatum and P. donghaiense. Also, we determined the optimum P concentration and N/P ratio for growth and photosynthesis of the two species. The results of this study provide a scientific basis for the analysis of changes in phytoplankton community structure and for the prediction and control of algal blooms, which have important effects on the marine environment and marine ecosystem stability.

2 MATERIAL AND METHOD 2.1 Culture and growth conditions

The two phytoplankton species, S. costatum and P. donghaiense, were isolated from the Changjiang River estuary and preserved in the laboratory. The algae were grown in sterilized seawater enriched with f/2 medium (Guillard and Ryther, 1962) and maintained by routine subculture as a stock culture. The unialgal stock and experimental cultures were maintained at 20±0.5 ℃ under a 12-h light/12-h dark photoperiod with 80 μmol/(s·m2) photosynthetically active radiation.

2.2 Experimental design basis and method

All glassware used in the experiment was washed with 30% v/v HCl, then rinsed three times with distilled water and autoclaved at 121 kPa for 20 min. S. costatum and P. donghaiense, in their exponential phase, were centrifuged at 4 500 r/min for 10 min and the supernatant was discarded. The algal cells were washed with filtered sterilized seawater, centrifuged again, and then inoculated into f/2 medium without additional N and P (each 250 mL flask contained 200 mL seawater). The initial inoculation density, as determined by counting cells under a microscope, was approximately 5 000 cells/mL. After 48 h, the N and P in the original liquid was almost exhausted and algae cells were in a state of nutrient starvation (concentrations of PO43- and NO3- were 0.12 μmol/L and 1.19 μmol/L, respectively). At this stage, the cell density of S. costatum was about 5.23×104 cells/mL and of P. donghaiense was about 0.86×104 cells/mL. Then NaNO3, a N source, and NaH2 PO4 ·2H2O, a P source, were added. The nutritional conditions were as follows:

According to a site investigation of the Changjiang River estuary and adjacent East China Sea, the highest dissolved-N (DIN) values were >100 μmol/L in the Changjiang River estuary and 20 μmol/L in the adjacent East China Sea; the highest phosphate value was about 2 μmol/L, and there was a relative lack of P compared with N (Chai et al., 2009). Therefore, three levels of P, representing a low-P group (LP= 0.36 μmol/L), a middle-P group (MP=3.6 μmol/L), and a high-P group (HP=36 μmol/L), were established. The N/P ratios for each group were 1, 8, 16, 32, 64, and 128(Table 1, first N/P ratio in the LP group was 3 but approximated as 1 when processing the results and for ease of illustration). Trace elements and vitamins were added to the f/2 medium and the experimental culture conditions were the same as those used for stock cultures. To prevent algal cells from attaching to the bottle wall or sinking, each culture flask was gently manually agitated approximately two to three times and its position randomly changed each day. Each treatment was conducted in triplicate.

Table 1 Nutrient structure of different phosphorus concentrations and nitrogen/phosphorus ratios
2.3 Determination of growth curve

The experimental culture period was 7 days for S. costatum and 12 days for P. donghaiense. S. costatum was sampled every day while P. donghaiense was sampled every other day after adding nutrients to the concentrations shown in Table 1. For analyses, S. costatum cells were fixed in Lugol's solution and then manually counted under a microscope. Cells of P. donghaiense were counted using a Coulter counter. The specific growth rate was calculated by the exponential model: μ =(ln Nt -ln N0)/ t (Lobban et al., 1988), where N0 and Nt are the initial and final cell counts, respectively; μ is the specific phytoplankton growth rate (/d), and t is the incubation time (d).

2.4 Determination of photosynthetic activity

A series of photosynthetic activity parameters were determined by a Photo-PAM chlorophyll fluorometer (Walz, Effeltrich, Germany). The parameters were the maximum quantum yield of photosystem II (Fv / Fm), rapid light curve (RLC), photosynthesis-irradiance (P-I) curve, maximum potential relative electron transfer rate without photoinhibition (rETRmax), nonphotochemical quenching (NPQ), and photochemical quenching (qP). The Photo-PAM was controlled using Phytowin 2.13 software (Walz). A 2-mL sample was collected with a pipette and placed in the measuring cup. After 10 min dark adaptation, the initial fluorescence (F0) was measured by opening the measuring radiation and the maximum fluorescence (Fm) was measured by opening the saturation pulse. Then, Fv / Fm, which reflects the potential for photochemical reactions in plants, was calculated as follows: Fv / Fm =(Fm - F0)/ Fm (Kolber et al., 1988). The PAR of actinic light was increased by 100 μmol/(s·m2) every 20 s from 1 μmol/(s·m2) up to 2 000 μmol/(s·m2) to obtain the RLC by fitting the following formula (Platt et al., 1980):

where P represents the relative electron transfer rate (r ETR); Pm represents r ETRmax ; α is the initial slope of the P-I curve, which reflects light-use efficiency, and β is the photoinhibition parameter. The effective quantum yield of PS II (ΦPS II), NPQ, and qP were calculated from the measured fluorescence values using the following formulas (Bilger and Björkman, 1990):

where Fm ' is the stable maximum fluorescence value under a certain light intensity and Ft is the fluorescence value of phytoplankton in a steady state.

2.5 Determination of relative chlorophyll a content

Chlorophyll a content was determined using the Photo-PAM after calibration, because the instantaneous fluorescence yield is proportional to the Chl a content within a certain range. To avoid possible errors in the instrument calibration process, the Chl a content in this study is expressed as relative content (ratio of daily measured chlorophyll content to chlorophyll content at inoculation)(Yin et al., 2007).

2.6 Statistical analysis

All the figures were plotted using Origin 8.0 software (OriginLab Corp. Northampton, MA, USA) and one-way ANOVA and LSD tests were performed using SPSS 16(SPSS Inc., Chicago, IL, USA). Differences were considered significant at P<0.05.

3 RESULT 3.1 Effects of different P concentrations and N/P ratios on growth of S. costatum and P. donghaiense

The growth curves for S. costatum and P. donghaiense in media with different P concentrations and N/P ratios are shown in Figs. 1, 2. The statistical analyses showed that P concentration significantly affected the growth of the two algae. The HP group had the highest cell density, followed by the MP group and the LP group (P<0.05). The specific growth rate of S. costatum in each group decreased gradually, reaching a stable state after 6 days and declining on day 7(Table 2). The growth of P. donghaiense reached a stable state, but began declining on day 10 in the MP and LP groups (Table 2).

Figure 1 Effects of different phosphorus concentrations and nitrogen/phosphorus ratios on growth of S. costatum
Figure 2 Effects of different phosphorus concentrations and nitrogen/phosphorus ratios on growth of P. donghaiense
Table 2 Specific growth rate (/d) of Skeletonema costatum (upper) and Prorocentrum donghaiense (lower) in media with different phosphorus concentrations and nitrogen/phosphorus ratios

The average growth of S. costatum during 7 days of culture and the algal densities on day 7 at the different P concentrations and N/P ratios are shown in Fig. 3. The maximum cell densities of S. costatum were with N/P ratios of 128, 64, and 32 in the LP, MP, HP groups, respectively (32.5×104, 97.45×104 and 146.4×104 cells/ mL, respectively). These values corresponded to maximum average growth of 0.26, 0.42 and 0.48 in the LP, MP, and HP groups, respectively, during 7 days. Figure 4 shows the average growth of P. donghaiense during 12 days of culture and the algal densities on day 12 of culture with different P concentrations and N/P ratios. P. donghaiense showed maximum algal cell densities in the LP, MP, and HP groups with N/P ratios of 128, 16, and 16, respectively (2.08×104, 6.68×104 and 24.10×104 cells/mL, respectively). These values corresponded to maximum average growth during 12 days of 0.074, 0.171, and 0.278 in the LP, MP, and HP groups, respectively.

Figure 3 Average growth of S. costatum over 7 days of culture and algal density on day 7 of culture in media with different phosphorus concentrations and nitrogen/phosphorus ratios
Figure 4 Average growth of P. donghaiense over 12 days of culture and algal density on day 12 of culture in media with different phosphorus concentrations and nitrogen/phosphorus ratios
3.2 Effects of different P concentrations and N/P ratios on chlorophyll a relative content of S. costatum and P. donghaiense

Chlorophyll content is an important indicator of the physiological condition of algae, and is closely related to photosynthesis (Liang et al., 2007; Zhou et al., 2009). Figure 5 shows the Chl a relative contents of S. costatum at the different P concentrations and N/P ratios. Comparing results under the same N/P ratio, but different P concentrations over the culture period, the HP group showed the largest increase in Chl a relative content, followed by the MP group and LP group (P<0.05). The Chl a relative content decreased in the LP group from day 6 and in the MP group from day 7. In S. costatum, the maximum Chl a relative contents in the LP, MP, and HP groups were with N/P ratios of 128, 64, and 16, respectively. These maximum values were in the LP, MP, and HP groups were recorded on day 5, 6, and 7, respectively, and were 3.84 times, 11.18 times, and 13.19 times higher, respectively, than the initial content.

Figure 5 Effects of different phosphorus concentrations and nitrogen/phosphorus ratios on chlorophyll a relative content in S. costatum

Figure 6 shows the Chl a relative contents of P. donghaiense at the different P concentrations and N/P ratios. Comparing results under the same N/P ratio but at different P concentrations, the HP group had the largest increase in Chl a relative content, followed by the MP group and the LP group (P<0.05). The maximum Chl a relative contents in P. donghaiense in the LP, MP, and HP groups were with N/P ratios of 32, 64, and 16, respectively (2.01 times, 8.72 times, and 21.68 times higher, respectively, than the initial content).

Figure 6 Effects of different phosphorus concentrations and nitrogen/phosphorus ratios on chlorophyll a relative content in P. donghaiense
3.3 Effects of different P concentrations and N/P ratios on Fv / Fm of S. costatum and P. donghaiense

The Fv / Fm values for S. costatum at different P concentrations and N/P ratios are shown in Fig. 7. The Fv / Fm values for S. costatum in the different groups decreased by varying amounts over the culture period (initial value, 0.65). Comparing results for the same N/P ratio but different P concentrations, the Fv / Fm values showed the largest decreases in the LP group, followed by the MP group and the HP group. The largest decreases were when the N/P ratios were 64, 128, and 1 in the LP, MP, and HP groups, respectively. The final Fv / Fm values for S. costatum in the LP, MP, and HP groups were 0.04, 0.1, and 0.39, respectively.

Figure 7 Effects of different phosphorus concentrations and nitrogen/phosphorus ratios on Fv / Fm of S. costatum

Figure 8 shows the maximum quantum yields (Fv / Fm) for P. donghaiense at the different P concentrations and N/P ratios. The Fv / Fm values in the different groups decreased by varying degrees over the culture period (initial value, 0.64). Comparingconcentrations, the Fv / Fm value showed the largest decrease in the HP group, and smaller decreases in the MP and LP groups. The Fv / Fm values for P. donghaiense showed the largest decreases when the N/P ratios were 8, 1 and 16 for the LP, MP, and HP groups, respectively. The final Fv / Fm values for the LP, MP, and HP groups were 0.58, 0.58, and 0.49, respectively.

Figure 8 Effects of different phosphorus concentrations and nitrogen/phosphorus ratios on Fv / Fm of P. donghaiense
3.4 Effects of different P concentrations and N/P ratios on rapid light curve (RLC) and effective quantum yield of PS II (ΦPS II) of S. costatum and P. donghaiense

Figure 9 shows the RLC for S. costatum on day 7(left panel) and P. donghaiense on day 12(right panel) under different N/P ratios for the HP group. On day 7, S. costatum in the HP group had high RLC values when the N/P ratios were 32, 64, and 128, followed by 8 and 16. The lowest RLC was when the N/P ratio was 1. On day 12, the RLC of P. donghaiense in the HP group was highest when the N/P ratio was 1, followed by 8, 64, 128, 16, and 32. The r ETRmax (Table 3) also showed consistent trends, confirming that r ETRmax reflected the RLC and the photosynthetic rate to a large degree.

Figure 9 RLC of S. costatum on the 7th day and P. donghaiense on the 12th day under different nitrogen/phosphorus ratios of high-phosphorus group
Table 3 rETRmax (μmol/(s·m2)) of S. costatum (upper) and P. donghaiense (lower) under different phosphorus concentrations and nitrogen/phosphorus ratios

The N and P absorption ratio basically follows the Redfield ratio of 16:1 when the total nutrient level is high enough to satisfy phytoplankton growth. Therefore, this ratio is commonly used to determine relative nutrient limitations (Redfield, 1958). Given the importance of the Redfield ratio, photosynthesis in S. costatum and P. donghaiense at an N/P ratio of 16 was chosen as a typical example in this study. Figure 10 shows the RLC and ΦPS II of S. costatum at different P concentrations when N/P=16, on days 1 and 7. On days 1 and 7, when the N/P ratio was 16, the highest RLC and ΦPS II were in the HP group, followed by the MP group and the LP group. Due to nutrient consumption during the culture period, the RLC and ΦPS II value for each group were higher on day 1 than on day 7. The RLC and ΦPS II of P. donghaiense at the different P concentrations on days 2 and 12 are shown in Fig. 11. On day 2, the highest RLC and ΦPS II were in the HP group, and were lower in the MP and LP grouns, but did not differ much between them. On day 12, the highest RLC and ΦPS II were in the MP group, followed by the HP group and the LP group. Due to nutrient consumption, the RLC and ΦPS II of each group were higher on day 2 than on day 12. Further analysis of the data in Table 3 showed that over the culture period (1-7 days), at the same N/P ratio, S. costatum had a higher r ETRmax and photosynthesis rate in the HP group than in the MP and LP groups. In contrast, over the culture period (6-12 days), at the same N/P ratio, P. donghaiense had a larger r ETRmax and faster photosynthesis rate in the MP group than in the HP and LP groups. A comparison of Figs. 10 and 11 showed that P. donghaiense had a higher RLC and ΦPS II than those of S. costatum at the same P concentration and N/P ratio (N/P=16). Further analysis of the data in Table 3 showed that P. donghaiense had a higher rETRmax and photosynthesis rate, at the same P concentration and N/P ratio, than those of S. costatum.

Figure 10 RLC and ΦPS II of S. costatum (N/P=16) under different phosphorus concentrations on day 1(left panel) and day 7(right panel)
Figure 11 RLC and ΦPS II of Prorocentru m donghaiense (N/P=16) under different phosphorus concentrations on day 2(left panel) and day 12(right panel)
3.5 Effects of different P concentrations and N/P ratios on non-photochemical quenching (NPQ) and photochemical quenching (qP) of S. costatum and P. donghaiense

Figure 12 shows the NPQ and qP values for S. costatum at different P concentrations on days 1 and 7 of culture. At N/P=16, NPQ had an upward trend while qP had a downward trend as light intensity increased on day 1 and day 7 of culture. The NPQ and qP values were higher for the HP group than for the MP and LP groups.

Figure 12 Values of NPQ and qP for S. costatum (N/P=16) cultured under different phosphorus concentrations on day 1(left panel) and day 7(right panel) of culture

The NPQ and qP values for P. donghaiense cultured with different P concentrations on day 2 and day 12 of culture are shown in Fig. 13. There was little difference in NPQ and qP values among the different groups on day 2, but the NPQ and qP values were higher for the HP group than for the MP and LP groups on day 12.

Figure 13 Values of NPQ and qP for P. donghaiense (N/P=16) cultured under different phosphorus concentrations on day 2(left panel) and day 12(right panel) of culture
4 DISCUSSION 4.1 Photosynthetic characteristics and environmental factors

The coastal ecosystem has been seriously affected by human activities, which have led to ecological instability and a series of marine ecological disasters (Sun et al., 2011). Nutrients are essential for the normal growth of micro-algae, and changes in nutrient concentrations and ratios will lead to changes in phytoplankton community structure (Wu et al., 2005). Micro-algae have threshold nutrient requirements, which play a key role in community succession and in the outbreak of algal blooms in marine ecosystems. Furthermore, eutrophication can destroy the ecological balance in the water and cause structural changes to, and functional degradation of, the ecosystem (Diaz and Rosenberg, 1995; Capriulo et al., 2002). Photosynthetic characteristics are the most fundamental and important physiological and ecological characteristics of algae. Studies on the responses of photosynthetic characteristics to changes in environmental factors, such as nutrient levels, can help to identify suitable environmental conditions for algae growth and reproduction, and improve research into marine ecology. There are three ways that phytoplankton can consume absorbed light energy: through the production of oxygen and organic matter via photosynthesis; through heat dissipation; and through the re-release of light energy in the form of chlorophyll fluorescence (Papageorgiou et al., 2007). These three processes compete with each other so that any increase in the efficiency of one process can result in decreased yields of the other two. Information about changes in photochemical processes and heat dissipation efficiency can be obtained by measuring chlorophyll fluorescence yield. These data can also be used to analyze photosynthetic mechanisms, especially PS II function, and to evaluate the different effects of ecological factors on photosynthesis (Bolhar-Nordenkampf et al., 1989; Schreiber et al., 1994). Marine phytoplankton represent the first trophic level in the ocean. They are extremely sensitive to the external environment and are able to respond rapidly and accurately to environmental factors that inhibit photosynthesis. The maximum quantum yield, Fv / Fm, is often used as an indicator of the impact of external stress on algal photosynthesis (Häder et al., 1998). The value for algae is approximately 0.65(Kolber et al., 1988), which is normally stable, but decreases significantly when algae are under stress. The RLC, NPQ, and qP values are also good indicators of phytoplankton growth potential. The combination of changes in phytoplankton population biomass, the photosynthetic characteristics of different groups, and ecological factors do not fully explain the changes in the phytoplankton community, but are good early warning signals for algal blooms.

4.2 Effects of different P concentrations and N/P ratios on growth

The results of this study showed that different P concentrations and N/P ratios significantly affected the growth of S. costatum and P. donghaiense. In the HP group, the P levels were suitable for cell growth. The metabolic activity of the cells was strong, so the specific growth rate, biomass of the stable stage, and Chl a relative content of both species were higher in the HP group than in the MP and LP groups. Phytoplankton are sensitive to P limitation. Under the low-P conditions of the MP and LP groups, ATP synthesis, H + -ATP enzyme activity, and cell metabolism were limited, which seriously affected cell division and proliferation. Therefore, both algal species showed a shortened exponential growth phase and reached the stable stage early because of the lack of nutrients in the culture solutions. This slowed their specific growth rates and lowered their biomass and Chl a relative contents (Figs. 1-6).

The results also showed that S. costatum in the LP, MP, and HP groups reached the maximum algal cell density and specific growth rate when the N/P ratios were 128, 64, and 32, respectively (Fig. 3). The maximum Chl a relative content in S. costatum was when the N/P ratios were 128, 64, and 16 in the LP group on day 5, in the MP group on day 6, and in the HP groups on day 7, respectively (Fig. 5). These results suggested that S. costatum grew better under high-N/P ratio conditions. Studies have shown that when there is a low NO3- concentration in the environment, the absorption of NO3- by S. costatum shows a saturation kinetics relationship. That is, the absorption capacity increases linearly as the NO3- concentration increases. This suggests that S. costatum has strong nitrogen storage ability and is able to adapt to changes in N concentrations in the environment (Lomas and Glibert, 2000). The optimum N/P ratio for the growth of most of red tide dinoflagellates is approximately 6-15, and the optimum N/P ratio for P. donghaiense is approximately 6-13(Hodgkiss and Ho, 1997). Other studies have shown that N/P ratios ranging from 12 to 20 are suitable for P. donghaiense and the best N/P ratio is 12(Lü and Ou, 2006). In this study, P. donghaiense reached its maximum algal cell density and specific growth rate (Fig. 4) when the N/P ratios were 128, 16, and 16 in the LP, MP and HP groups, respectively. The results of the MP and HP group were generally consistent with those of previous studies.

4.3 Effects of different P concentrations and N/P ratios on photosynthetic characteristics

Studies have shown that reductions in the supply of amino acids will limit protein synthesis (Geider et al., 1993). Furthermore, nutrient limitation can affect the PS II reaction center and algae cells may not be able to repair damage to PS II. This means that Fv / Fm will decline more rapidly if the cell total protein content decreases (Young and Beardall, 2003). When cells are subject to P limitation, ATP synthesis and the regeneration cycle for NADP + and NADPH are disrupted, which may further aggravate damage to the PS II reaction center (Fredeen et al., 1990; Jacob and Lawlor, 1991; Lippemeier et al., 2003, 2010) and lead to a sustained decline in Fv / Fm. In this study, the Fv / Fm values of the two species declined by different amounts under the different nutrient conditions as the incubation time increased. The largest decrease in Fv / Fm value for S. costatum was in the LP group, and the smallest decrease was in the HP group, indicating that the maximum photosynthetic potential of S. costatum was higher under HP conditions. The Fv / Fm values for P. donghaiense were higher in the MP and LP groups than in the HP group, suggesting that the maximum photosynthetic potential of P. donghaiense was higher under MP and LP conditions, and that high-nutrient concentrations may inhibit the Fv / Fm (Figs. 7 and 8).

In this study, on days 1 and 7 of culture, when the N/P ratio was 16, the highest RLC and ΦPS II values for S. costatum were in the HP group, followed by the MP and LP groups (Fig. 10). During the culture period (1-7 days), at the same N/P ratio, S. costatum showed higher r ETRmax and photosynthesis rates in the HP group than in the MP and LP groups (Table 3), suggesting that high-P conditions can promote growth and increase the photosynthetic capacity of S. costatum. This may be because S. costatum cells require more energy to take up N and P under highnutrient conditions. Analyses of P. donghaiense showed that on day 2, the RLC and ΦPS II were higher in the HP group, and showed little difference between the MP and LP groups. However, on day 12, the MP group showed the highest RLC and ΦPS II, followed by the HP and LP groups (Fig. 10). During most of the culture period (6-12 days), at the same N/P ratio, P. donghaiense had a higher r ETRmax and photosynthesis rate in the MP group than in the HP and LP groups, indicating that the MP conditions were more conducive to growth and improved the photosynthetic capacity of P. donghaiense. This may be because rapid cell division by P. donghaiense was not conducive to protein and carbohydrate accumulation, because photosynthesis and growth were not fully synchronized, or because high nutrient concentrations inhibited the photosynthetic activity of P. donghaiense. Therefore, only within a certain range can the nutrient concentration promote the photosynthesis of P. donghaiense and maintain a high PS II photochemical efficiency. The RLC and ΦPS II values were higher for P. donghaiense than for S. costatum at the same P concentration and N/P ratio (N/P=16)(Figs. 10, 11), while S. costatum had higher r ETRmax and photosynthesis rates than those of P. donghaiense at the same P concentration and N/P ratio (Table 3). These results showed that, under the same nutrient conditions, the photosynthetic capacity of P. donghaiense was greater than that of S. costatum.

The parameter NPQ reflects the proportion of light energy that cannot be used for photosynthetic electron transport and dissipates in the form of heat. In this study, the NPQ of the two species increased with increasing light intensity. Excess light energy absorbed by photosynthetic apparatus was dissipated in the form of heat, which protected the algal photosynthesis organs from damage by high intensity irradiation. This process may be related to algal light protection mechanisms such as the xanthophyll cycle and reversible inactivation of the reaction center (Olaizola and Yamamoto, 1994; Kashino et al., 2002). The qP value reflects the fluorescence quenching caused by the conversion of excitation energy captured by the opening reaction center into chemical energy. Therefore, the higher the qP, the wider the opening of the PS II reaction center and the higher the electron transport activity (van Kooten and Snel, 1990). In this study, the qP of both species decreased as the light intensity increased, and the photochemical reaction and electron transport activity of PS II became gradually weaker. The light energy absorbed was instead used for heat dissipation to protect the algal photosynthetic system from damage. On days 1 and 7 of culture, when the N/P ratio was 16, the NPQ and qP values for S. costatum were higher in the HP group than in the MP and LP groups. On day 12 of culture, the highest NPQ and qP values for P. donghaiense were in the HP group, indicating that the cells may require more energy for N and P uptake under high-nutrient conditions (Figs. 12 and 13).

4.4 Relationships between algal photosynthetic characteristics and phytoplankton community

In the last decade, studies on the effects of environmental factors on the marine environment have shown that both inshore and offshore areas are P-limited (Dyhrman et al., 2006). The inorganic N nitrogen content is very high in the breeding waters of the northeastern Jiaozhou Bay, and phytoplankton blooms often cause phosphate depletion (Huo et al., 2001). The nutrient concentration in the East China Sea is increasing every year because of nutrient inputs via diluted water from Changjiang (Shen et al., 2001). The highest recorded phosphate value was about 2 μmol/L and has been rising over recent years, but there is still a relative lack of phosphate compared with N, and a serious imbalance in the N/P ratio in that area (Chai et al., 2009). Studies have shown that S. costatum accumulates much lower levels of lipids and carbohydrates than does P. donghaiense, so the latter is more likely to survive in nutrient-limited environments (Zhao et al., 2009). When P is abundant, S. costatum is the dominant species and forms algal blooms, but when P is inadequate, S. costatum is still dominant during the initial stages, but P. donghaiense becomes dominant as P is consumed (Lü and Li, 2006). This explains the frequent occurrence of P. donghaiense algal blooms in eutrophic but P-restricted waters, such as the Changjiang River estuary and the Zhoushan sea area (Chen et al., 2000; Wang et al., 2001; Li, 2006).

In this study, we conducted a comprehensive analysis of the two representative algae species found in the Changjiang River estuary, and concluded that under the same nutrient conditions, the photosynthetic capacity of P. donghaiense was stronger than that of S. costatum. The cell density and Fv / Fm of S. costatum declined rapidly after reaching their saturation concentration. Therefore, we can speculate that there is a rapid decline in S. costatum algal blooms in natural seawater and that the S. costatum red tide is replaced by other red-tide species. P. donghaiense in the MP group showed higher r ETRmax and Fv / Fm values. Therefore, we can speculate that in the natural environment, the photosynthetic activity of P. donghaiense is further enhanced after the outbreak of diatoms and the subsequent nutrient depletion. This is because P. donghaiense can maintain a high cell density for a long period, even when there is a lack of nutrients. This growth pattern may explain why P. donghaiense blooms occur widely and do not to disappear as quickly as the S. costatum red tide. It also provides a scientific explanation for the interaction and succession rules between the two species from a photosynthesis perspective. Under eutrophic but relatively P-restricted conditions, P. donghaiense has higher photosynthetic capacity, activity, and potential, which could lead to its increasing dominance in the phytoplankton community in these waters.

Different phytoplankton species have different nutrient requirements, which affect the coexistence and competition among various phytoplankton species and maintain the biodiversity of the marine ecosystem. Certain algae species that can become dominant are well adapted to the local environment, and are usually strongly competitive in natural seawaters. This explains why S. costatum and P. donghaiense can survive competition from other phytoplankton species to become the dominant species and rapidly proliferate under suitable environmental conditions. This paper describes a preliminary study on the long-term changes in phytoplankton structure from the point of view of nutrients and photosynthesis. More research is required to fully understand the mechanism behind changes in the phytoplankton community and the outbreak of algal blooms.

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