Physiological Changes and Elemental Ratio of Scrippsiella trochoidea and Heterosigma akashiwo in Different Growth Phase

The elemental ratios in phytoplankton are important for predicting biogeochemical cycles in the ocean. However, understanding how these elements vary among different phytoplankton taxa with physiological changes remains limited. In this paper, we determine the combined physiological–elemental ratio changes of two phytoplankton species, Scrippsiella trochoidea (Dinophyceae) and Heterosigma akashiwo (Raphidophyceae). Our results show that the cell growth period of S. trochoidea (26 days) was significantly shorter than that of H. akashiwo (32 days), with an average cell abundance of 1.21 × 104 cells·mL−1 in S. trochoidea and 1.53 × 105 cells·mL−1 in H. akashiwo. The average biovolume of S. trochoidea (9.71 × 103 μm3) was higher than that of H. akashiwo (0.64 × 103 μm3). The physiological states of the microalgae were assessed based on elemental ratios. The average ratios of particulate organic nitrogen (PON) to chlorophyll-a (Chl-a) and particulate organic carbon (POC) to Chl-a in S. trochoidea (57.32 and 168.16) were higher than those of H. akashiwo (9.46 and 68.86); however, the ratio of POC/PON of the two microalgae was nearly equal (6.33 and 6.17), indicating that POC/Chl-a may be lower when the cell is actively growing. The physiological variation, based on the POC/Chl-a ratio, in different phytoplankton taxa can be used to develop physiological models for phytoplankton, with implications for the marine biogeochemical cycle.


Introduction
Phytoplankton is the basis of the marine food web, accounting for more than 50% of the primary production [1]. The cell size, chlorophyll-a (Chl-a) content, and elemental composition of microalgae (i.e., organic carbon and organic nitrogen) are important indicators for assessing biomass, as they have strong influences on the food web structure and the biogeochemical cycling of carbon, as well as cell growth rates [2][3][4]. However, these indicators vary with physical environmental parameters (i.e., irradiance and nutrient availability) and the growth phase of microalgae [5,6]. Such variations in physiological changes and elemental ratios depend on how efficiently phytoplankton biomass is remineralized by bacteria, deep ocean export flux, or utilization by consumers [7,8]. In such cases, these variations may provide a mechanistic basis for modeling phytoplankton growth, elemental ratios, and quantifying the flow energy among organisms in an ecosystem [9].
Estimation of chlorophyll is an easy technique to assess the fundamental photosynthetic performance (mainly in photosystems II) of microalgae under various environmental conditions, which reflects the photosynthetic performance of the microalgae [10]. Therefore, Water 2021, 13, 132 2 of 11 it can be used as a tool to estimate phytoplankton productivity. The elemental composition of cells and the content of their constituent organic matters are two important physiological indicators for phytoplankton [11]. Particulate organic matters (POM) can influence processes such as transport, transformation, and removal of elements, which subsequently determine biological life processes and primary productivity. Thus, it is an important parameter for evaluating marine productivity [12]. Therefore, we can provide a preliminary view for evaluating the productivity of the sea based on the analysis of physiological parameters and elemental ratios.
Phytoplankton produces organic carbon by absorbing nutrients from seawater and, in turn, nutrients are returned to the seawater through remineralization. It is well-understood that studies on particulate organic carbon and nitrogen (POC/PON) have great significance for productivity modeling and the quantification of carbon in the ocean [13]. However, particulate organic nitrogen (PON) and particulate organic carbon (POC) in microalgae cells cannot be distinguished from zooplankton and non-living organic matter, so their contents are not directly available in the ocean [14,15]. Therefore, previous studies have commonly used chlorophyll content to estimate changes in intracellular carbon content, as it accurately reflects microalgae growth and photosynthetic rates [16,17]. Interestingly, the ratio between POC and Chl-a varies. Earlier studies have demonstrated that POC/Chl-a ratios are influenced by nitrogen concentrations [5]. This ratio decreases when phytoplankton are exposed to low light levels, as well as nutrient-rich zones [18,19]. Furthermore, the POC/Chl-a ratio has been found to be lower in an estuary, compared to the open ocean [5]. Moreover, Sathyendranath et al. studied the POC/Chl-a of phytoplankton using high-performance liquid chromatography (HPLC) and observed that the ratio ranged from 15 to 176 [20]. Most recent studies have examined POC/Chl-a and elemental content changes in phytoplankton with respect to the changes in coastal ecosystems, especially focusing on seasonal variation. However, there is scant information regarding the changes in carbon and nitrogen content during phytoplankton growth [21].
S. trochoidea (Dinophyceae) and H. akashiwo (Raphidophyceae) are two widely distributed harmful algal species in Chinese coastal waters. They have significant physiological differences (e.g., cell size and growth) due to variation in their taxonomic positions. Most studies have focused on growth rates and biomass under different environmental conditions, such as temperature or nutrients. For example, Qi Yu et al. studied the life history of S.trochoidea and found that the emergence of cysts and its reproduction rate were closely related to nitrogen concentration [22]. Furthermore, Xiao et al. pointed out a relationship between the cysts of S. trochoidea and vegetative cells in Daya Bay, showing that formation of cysts led to a reduction in vegetative cells [23]. Futhermore, some investigations have explained the effects of physicochemical methods for S. trochoidea under environmental conditions. These methods include heating, ultraviolet (UV) radiation, and others [24]. Similarly, a variation in pH also influenced the carbon assimilation and acquisition of H. akashiwo, along with physiological changes of growth rate [25]. Despite the fact that these studies have provided significant contributions, very few studies have examined the elemental ratios of these two important species in different stages of their growth phase [26,27].
The objective of this study was to explore the variations in POC and PON during the entire growth cycles of S. trochoidea and H. akashiwo. To accomplish this, we estimated the physiological changes (cell abundance, cell volume, Chl-a), element ratios (POC and PON), and inferred POC/Chl-a ratios from S. trochoidea and H. akashiwo during different life phases. We hope that our results provide comprehensive information on how elemental composition varies with the size difference of phytoplankton. This may be useful for marine biogeochemical modeling.

Microalgal Species and Pre-Culture Conditions
S. trochoidea and H. akashiwo used in the present study were isolated, in 96-well microplates, from the coastal waters of Qinhuang Island, China. They were subsequently identified and maintained in our laboratory. Microalgal cells were cultured in f/2 medium [28] under the following culture conditions: 25 ± 1 • C; 12 h:12 h for light-dark ratio; and 200 ± 10 µmol photons m −2 s −1 for irradiance, which ensured that they were not limited by light intensity.

Determination of Microalgal Growth
Microalgae cells were collected in a sterile bench when they reached the exponential phase of growth. The collected microalgae cells were then centrifuged at 5000 rpm for 5 min and washed with sterile seawater for another 5 min for the same treatment [29]. In this way, we removed the interference of the original medium on the concentration of the new medium. The collected cells were then transferred into 2000 mL conical flasks containing 1500 mL of f/2 medium, again under the above-mentioned light conditions. The initial cell abundances of the two microalgal cell cultures were approximately 360 cells·mL −1 and 3000 cells·mL −1 at the beginning of the experiment, based on the consistent in vivo fluorescence values. Algal cells were then placed into an algal shaker with a speed of 180 rpm/min. Aliquots of approximately 100 µL of microalgae solution were transferred into a blood cell counting chamber and counted immediately using an inverted microscope (XS-213). The different growth phases of both of the considered algae were determined based on the in vivo fluorescence values and cell abundances.

Sample Analyses
Individual growth rates and doubling times of single species cultured in the laboratory were calculated based on the following equations [30]: Doubling time (T d , t) = ln 2/µ where N b and N a denote the algal cell abundance (cells·mL −1 ) at times t b and t a , respectively. Cell volume was determined by constructing a geometric simulation graph [31]. Micrographic photos are presented in Figure 1. Chl-a was determined using a CE Turner Designs Fluorometer [32,33]. The PON and POC contents of the microalgae were analyzed using a Costech Elemental Analyzer (ECS4010, Milan, Italy).

Microalgal Species and Pre-Culture Conditions
S. trochoidea and H. akashiwo used in the present study were isolated, in 96-well microplates, from the coastal waters of Qinhuang Island, China. They were subsequently identified and maintained in our laboratory. Microalgal cells were cultured in f/2 medium [28] under the following culture conditions: 25 ± 1 °C; 12h:12h for light-dark ratio; and 200 ± 10 μmol photons m −2 s −1 for irradiance, which ensured that they were not limited by light intensity.

Determination of Microalgal Growth
Microalgae cells were collected in a sterile bench when they reached the exponential phase of growth. The collected microalgae cells were then centrifuged at 5000 rpm for 5 min and washed with sterile seawater for another 5 min for the same treatment [29]. In this way, we removed the interference of the original medium on the concentration of the new medium. The collected cells were then transferred into 2000 mL conical flasks containing 1500 mL of f/2 medium, again under the above-mentioned light conditions. The initial cell abundances of the two microalgal cell cultures were approximately 360 cells·mL −1 and 3000 cells·mL −1 at the beginning of the experiment, based on the consistent in vivo fluorescence values. Algal cells were then placed into an algal shaker with a speed of 180 rpm/min. Aliquots of approximately 100 μL of microalgae solution were transferred into a blood cell counting chamber and counted immediately using an inverted microscope (XS-213). The different growth phases of both of the considered algae were determined based on the in vivo fluorescence values and cell abundances.

Sample Analyses
Individual growth rates and doubling times of single species cultured in the laboratory were calculated based on the following equations [30]: Specific growth rate (μ, d −1 ) = (ln Nb − ln Na)/(tb − ta) Doubling time (Td, t) = ln 2/μ where Nb and Na denote the algal cell abundance (cells·mL −1 ) at times tb and ta, respectively.
Cell volume was determined by constructing a geometric simulation graph [31]. Micrographic photos are presented in Figure 1. Chl-a was determined using a CE Turner Designs Fluorometer [32,33]. The PON and POC contents of the microalgae were analyzed using a Costech Elemental Analyzer (ECS4010, Milan, Italy).

Date Analyses
All measured data are presented as mean ± standard deviation, plotted using the GraphPad Prism 8.0.2 software. Furthermore, all parameters (cell abundance, cell volume, Chl-a, and elements) between different species and growth phases were statistically analyzed using the SPSS software (version 21.0). Student's t-test was applied to test the differences between S. trochoidea and H. akashiwo. One-way analysis of variance (ANOVA) was used to analyze the difference of growth phases (e.g., lag, exponential, stationary, and degradation) for the two species. The levels of significance of the difference were set as p < 0.05 and p < 0.01.

Scrippsiella trochoidea
Beta growth then decay

Chlorophyll-a Content of Two Microalgal Species
The Chl-a content of the two microalgae showed similar variation ( Figure 2e). In both species, Chl-a values increased initially and then decreased. S. trochoidea reached a maximum of 0.43 mg·L −1 at day 16, whereas the higher value (1.25 mg·L −1 ) for H. akashiwo was observed on day 15. Nevertheless, the Chl-a concentration of H. akashiwo was significantly higher than that of S. trochoidea (p < 0.05).  (Figure 3a,b).

POC and PON Content in Two Microalgal Species
The POC content per cell of S. trochoidea decreased significantly during the first six days of the experiment, with a minimum value of 0.83 × 10 3 pg·cell −1 (exponential) and a maximum value of 3.78 × 10 3 pg·cell −1 (degradation) ( Table 2). However, there was no significant difference between the exponential and stationary phase in terms of POC content per cell. Moreover, in terms of POC, the lag, exponential, and stationary phases were significantly lower than the degradation phase (p < 0.05) (Figure 3c). The lowest POC Water 2021, 13, 132 5 of 11 value per cell of H. akashiwo was 0.12 × 10 3 pg·cell −1 in the stationary phase, whereas the highest value was 0.50 × 10 3 pg·cell −1 in the degradation of incubation (Table 2). Therefore, it is obvious that the POC value per cell of H. akashiwo accumulated across the degradation phase. Moreover, although the POC content per cell of H. akashiwo in the first three phases (lag., exp., and stat.) declined gradually, the rate of decreases was very low. and H. akashiwo (1.52 ± 0.38 d −1 ) did not show any significant differences (p > 0.05 doubling times of the two microalgae, S. trochoidea (0.80 d −1 ) and H. akashiwo (0.45 d − had a statistically significant difference (p < 0.05). To simulate the growth curves two microalgae, the beta growth and decay model was applied in this study (Ta With this model, we estimated the environmental capacities of these two species as 10 4 cells·mL −1 and 2.59 × 10 5 cells·mL −1 , respectively ( Table 1). The R 2 values of th fitting equations were 0.87 and 0.97, respectively, illustrating good fitting results. T erage cell volumes of S. trochoidea and H. akashiwo were 9.71 × 10 3 μm 3 and 0.64 × 10 respectively. As shown in Figure 2c,d, a clear distinction was observed between t exponential, and stationary phases (p < 0.01). The PON content per cell of two species showed unusual differences during the experiment. The PON content per cell of S. trochoidea increased during the initial four days and remained relatively stable over the following 16 days, with an average value of 0.25 × 10 3 pg·cell −1 (Figure 3c). Similar to the POC content per cell of H. akashiwo, PON content accumulation occurred towards the end of the growth phase, with an average value of PON of 0.04 × 10 3 pg·cell −1 and a maximum of 56.66 pg·cell −1 ( Table 2).
The PON and POC densities of the two species also showed a consistent trend. For example, the PON density in exponential and stationary phases was lower than that in the degradation phase. Interestingly, the POC density was significantly higher in the degradation phase than the other three phases (p < 0.05, Figure 3e Table 2).
The PON/Chl-a ratios of S. trochoidea and H. akashiwo had different trends throughout the growth stage, with average values of 57.32 and 9.46, respectively. The PON/Chl-a ratio for S. trochoidea showed a maximum value on day 16, then decreased rapidly in the following days (Figure 4a). The PON/Chl-a ratio of H. akashiwo changed very little during the first 20 days and rapidly increased to 25.80 on day 26 (Figure 4b). The POC/Chl-a ratio of both microalgal species showed similar trends throughout the growth phases. Moreover, the POC/Chl-a ratio of S. trochoidea was significantly higher than that of H. akashiwo. Both S. trochoidea and H. akashiwo showed an increasing trend in POC/PON during the degradation phase, which may be due to the PON loss rate being higher than that of POC (Figure 4c). ater 2021, 13, x FOR PEER REVIEW 6 of 11 of both microalgal species showed similar trends throughout the growth phases. Moreover, the POC/Chl-a ratio of S. trochoidea was significantly higher than that of H. akashiwo. Both S. trochoidea and H. akashiwo showed an increasing trend in POC/PON during the degradation phase, which may be due to the PON loss rate being higher than that of POC (Figure 4c).

Discussion
Our results showed a notable difference between the specific growth rate and cell abundance of two different phytoplankton taxa isolated from the Bohai sea. The specific growth rate results were relatively close to those of earlier reports by Xu et al. [34] and Ono et al. [35]. They noted that the maximum specific growth rate of S. trochoidea, under orthogonal test results, was 0.76 d −1 [34], while that of H. akashiwo did not exceed 2 d −1 under optimal conditions in the laboratory [35]. However, our results showed that the maximum growth rates of S. trochoidea and H. akashiwo were 0.863 d −1 and 1.524 d −1 , respectively. Interestingly, the highest abundances of S. trochoidea and H. akashiwo in this experiment were only 2.60 × 10 4 and 2.79 × 10 5 cells·mL −1 , respectively. Earlier studies have

Discussion
Our results showed a notable difference between the specific growth rate and cell abundance of two different phytoplankton taxa isolated from the Bohai sea. The specific growth rate results were relatively close to those of earlier reports by Xu et al. [34] and Ono et al. [35]. They noted that the maximum specific growth rate of S. trochoidea, under orthogonal test results, was 0.76 d −1 [34], while that of H. akashiwo did not exceed 2 d −1 under optimal conditions in the laboratory [35]. However, our results showed that the maximum growth rates of S. trochoidea and H. akashiwo were 0.863 d −1 and 1.524 d −1 , respectively. Interestingly, the highest abundances of S. trochoidea and H. akashiwo in this experiment were only 2.60 × 10 4 and 2.79 × 10 5 cells·mL −1 , respectively. Earlier studies have shown that, during "red tide", the cell abundances of S. trochoidea at Daya Bay and H. akashiwo at Dalian Bay reached 4.06 × 10 4 and 72 × 10 4 cells·mL −1 , respectively [23,36]. In these coastal ecosystems, the cell abundance is influenced by many environmental factors, such as the light intensity of surface water, temperature, and nutrients. Cell abundance reaches its maximum when these environmental factors are optimal [37]. Futhermore, Daya Bay and Dalian Bay have special geographical characteristics of semi-enclosed coastal inlets, which has led to increased nutrient loading by rapidly expanding mariculture and human population growth in these regions [37,38]. However, the nutrients were depleted by microalgae during batch culture in the lab. Therefore, the cell abundances of the two microalgae species in Daya Bay and Dalian Bay were higher than those under laboratory incubation. Compared with the larger-sized S. trochoidea in the same culture conditions, we observed that the smaller H. akashiwo presented high cell abundance values. A similar phenomenon was observed, by Marañón, for the relationship between cell abundance and cell size in the surface waters of a coastal site. It has been demonstrated that cell abundance decreased with increased cell volume in different microalgae species [2]. Past studies have shown that cell size can determine the nutrient diffusion and nutrient requirements of microalgae cells. Small cells can easily maintain their growth in low-nutrient conditions [39]. Therefore, the cell abundance under large biovolume was lower than that of smaller cells in similar conditions.
Microalgae can transform inorganic matter into organic matter through photosynthesis, which plays an important role in the ocean carbon sink. In addition to Chl-a, intracellular carbon and nitrogen content were also used as indicators to characterize the abundances of microalgae [23]. In this experiment, the maximum Chl-a values of S. trochoidea and H. akashiwo were preceded by the maximum cell abundance. A similar result has been found in the varied N/P ratios of microalgae growth by past studies. The trend could be explained by good adaptation of microalgae in new experiments. This adaptation, in advance, could supply cellular substances for further proliferation [40]. Furthermore, the Chl-a concentration is also an important parameter for characterizing algal blooms. As the two algae in this study were isolated from Qinhuangdao Island during blooming conditions, the Chl-a concentration of the natural population in the coastal water likely reached a maximum before the cell abundance. Therefore, the Chl-a concentration exceeding a certain threshold may serve as an indicator for microalgae blooms. POC and PON content per cell of two microalgae increased in the degradation phase. As observed previously, cell division slowed down in the stationary phase, and energy began to accumulate [21,41]. For microalgae in their natural environment, the accumulated POC remains in living and non-living organisms. Eventually, it is deposited on the seafloor, thus completing the biological carbon sink [1]. In this study, the carbon content per cell of both microalgae species was reduced at the beginning of culture. Microalgae have two fundamental mechanisms of physiological characteristics in the environment, including absolute and relative adaptation [42]. Culture studies have shown that microalgae have a relative adaptation period when nutrients are not limited in a new environment, which leads to an ability to synthesize organic matter slowly for algae at the beginning of the culture [40]. Interestingly, the POC content per unit cell of S. trochoidea was higher than that of H. akashiwo, while the POC and PON density content of S. trochoidea were lower than those of H. akashiwo. We hypothesize that such variations lead to the contributions of different species to algal blooms. According to a study by Finkel et al., small cells have more elemental stoichiometry density content, which has a larger side effect than larger individuals [39]. However, Emilio et al. concluded that blooms are dominated by large, rather than small, cells [2]. Due to the large size of the cell, it has higher metabolic rates than small individuals [43]. Some observations have implied that resource acquisition and assimilation rates can be maximized by microalgae with higher biovolume. Meanwhile, they can minimize loss and material transport rates in order to survive in pelagic ecosystems [44]. Compared with large individuals, small cells have a longer growth cycle and are more easily preyed upon by consumers [45]. Based on earlier studies regarding algal bloom occurrence in the Qingdao sea, we observed that the frequency of algal blooms in this area was more severe with respect to S. trochoidea compared to H. akashiwo [46]. Therefore, compared with H. akashiwo, we can further extend this explanation and conclude that S. trochoidea may dominate the algal bloom.
The amount of carbon in phytoplankton is influenced by the cell size, as well as environmental factors [47]. However, it is difficult to directly determine the carbon biomass of phytoplankton, as zooplankton and non-living organic matter may interfere in its measurement. Therefore, it is important to estimate the biomass of phytoplankton by using the ratio of carbon to Chl-a content [20]. Our results suggest that the POC/Chl-a values of microalgae increased in the degradation phase. This is due to the depletion of nutrients while the microalgae were growing. This led to a decrease in biological activity and the accumulation of carbon content. Several studies support our findings that low values of POC/Chl-a exist in active microalgae cells [48]. Interestingly, we found that the POC/Chl-a value of S. trochoidea was higher than that of H. akashiwo. In our results, the cell biovolume tended to decrease during the growth period. In eukaryotes, renewable components, such as membranes and nucleic acids, are increased with microalgal growth. This leads to a decline in other renewable and catalytic components of the cytoplasmic proportion, such as enzyme activity and the synthesis of organic matter. Therefore, compared to larger cells, small individuals transfer nutrients to biomass at a slow rate and have low POC/Chl-a values [49].
The POC/PON ratio of the phytoplankton community in seawater has been found to be 106:16 [50]. However, this ratio is not consistent between lab cultures and during the process of microalgae blooming in the ocean. Although the POC/PON values for the two microalgae decreased in the exponential phase, they were close to the Redfield ratio in the degradation phase. The chemometrics of deviation states have been associated with changes in the structural elements of microalgae. Different intracellular chemometrics have different properties, which reflect differences in their ability to store intracellular nutrients during the metabolic process [51]. Mature cells can increase their allocation of nutrients in the exponential phase. Under nutrient-restricted conditions, they resynthesize substances from other parts of the cell [52]. Therefore, we observed high values during the degradation phase.

Conclusions
Our findings demonstrated both physiological and elemental variations in two different phytoplankton, which can serve to strengthen efforts to mechanistically quantify and assess biogeochemical processes. The similar variation of the growth curve, biomass, and elements in this study may present a useful constraint for predicting variability of phytoplankton POC, PON, POC/Chl-a, and PON/Chl-a values in cellular trait-based models. We also demonstrated taxonomic variation in POC and PON accumulation mechanisms, highlighting the impacts that biochemical traits and the biogeography of Dinophyceae and Raphidophyceae may have on ocean element patterns, which may be valuable for marine biogeochemical modeling based on Dinophyceae and Raphidophyceae.