1 Introduction

Nitrogen (N) is an important nutrient element for the growth of algal cells. Although dissolved inorganic nitrogen (DIN) is considered the most important nutrient source for microalgae, dissolved organic nitrogen (DON) also contributes significantly to the total nutrient pool, and often exceed the concentration of the inorganic forms (Sipler and Bronk, 2015; Glibert, 2017). Many researchers have demonstrated that phytoplankton are capable of utilizing organic nutrients to sustain their growth when they meet the limitation of inorganic nutrients (Pustizzi et al., 2004; Cucchiari et al., 2008; Glibert and Burford, 2017). Actually, up to 70% of DON in the marine environment may be bioavailable (Berman and Bronk, 2003; Sipler and Bronk, 2015). Concentration and proportion of different forms of N can lead to changes in phytoplankton community composition and abundance (Glibert et al., 2016; Moschonas et al., 2017).

phytoplankton have been demonstrated in both natural assemblages and laboratory cultures (Kudela and Cochlan, 2000; Berman and Bronk, 2003; Glibert et al., 2006). Of particular interest has been the linkage between DON and the growth of harmful algal bloom (HAB) species, including the dinoflagellates Karenia brevis (Steidinger et al., 1998), Pfiesteria piscicida (Lewitus et al., 1999), Lingulodinium polyedrum (Kudela and Cochlan, 2000), the diatoms Pseudonitzschia spp. (Loureiro et al., 2009), the raphidophytes Heterosigma akashiwo (Herndon and Cochlan, 2007), Fibrocapsa japonica (Cucchiari et al., 2008), and the brown tide alga Aureococcus anophagefferens (Lomas and Glibert, 2000; Pustizzi et al., 2004; Herndon and Cochlan, 2007). The use of organic compounds may serve as an important ecological strategy for harmful flagellates in the specific competition especially in organic nutrients rich coastal waters (Glibert et al., 2006; Glibert and Burford, 2017).

In this study, we have examined the N growth capabilities of the three different marine phytoplankton taxa, Skeletonema costatum (diatom, Bacillariophyceae), Prorocentrum micans (dinoflagellate, Dinophyceae), and Chattonella marina (Raphidophyceae) in laboratory conditions. Effects of different N sources, concentrations and ratios between nitrate (NO₃-N) and urea on the growth of algal cells were estimated. The Monod equation was applied to examine and compare the effect of ambient N concentrations on the growth of algal cells. The purpose of this study was to compare nitrogenous utilization of different microalgal species, and to understand their advantages in phytoplankton competition in the natural sea waters.

2 Materials and Methods 2.1 Algal Cultures

Uni-algal cultures of S. costatum, P. micans, and C. marina were isolated from Daya Bay, China. The stock cultures were maintained in autoclaved (121℃, 20 min) f/2 media (Guillard, 1975) at 20℃ ± 1℃ and salinity 30 and under 100 μmol photon m^-2 s^-1 of cool-white fluorescent illumination with a dark: light cycle of 12 h: 12 h. The cultures were treated with mixture antibiotics to destroy the external bacteria before the experiment. Firstly, 10 μg mL^-1 penicillin (final concentration) was added in 100 mL cultures in exponential phase, and incubated for 24 h, then 10 mL of the penicillin-treated culture was inoculated to the fresh f/2 medium with 10 μg mL^-1 streptomycin sulphate, and incubated for another 24 h, and followed by the treatment of 10 μg mL^-1 kanamycin sulfate. The multi-antibiotics treated algal cultures were maintained in the sterile f/2 medium. Before the experiments, the cultures were transferred to the modified f/2 medium, which contained 100 μmol N L^-1 (NaNO₃), 7 μmol P L^-1 (KH₂PO₄) and 70 μmol Si L^-1 (Na₂SiO₃), approximating the maximum nutrient concentrations found in Daya Bay (Wang et al., 2009). Clones were maintained in the exponential growth phase by serial transfer in the modified f/2 medium. The cells for inoculation were pre-incubated for two days in the N-free medium.

2.2 Experiment Design 2.2.1 Growth under different nitrate concentrations

The experiments were conducted in batch cultures. The growth of algal cells was examined at seven different concentrations of N (0, 5, 10, 50, 100, 500, 1000 μmol N L^-1). The concentrations of P and Si were 7 μmol P L^-1 and 70 μmol Si L^-1, respectively. Silica was removed in the media for non-diatoms. Elements other than N, P and Si were the same as those in f/2 medium. To reduce the background N and P concentrations, the medium was made with artificial sea salt (Red Coral Sea, nutrient free formula) with salinity 30-31 and pH 7.9 ± 0.1. Algal cells were inoculated into triplicate 250 mL flasks with 150 mL of test medium. According to the cell sizes of the three taxa, the initial cell densities were set at about 5×10⁴ cells mL^-1 for S. costatum, and about 10³ cell mL^-1 for P. micans and C. marina. The experiment was run for 8 days. To minimize cell sedimentation, cultures were shaken by hand three times a day. The incubation condition was the same as that of algal culture in Section 2.1.

2.2.2 Growth under different nitrogen compounds

Seven N compounds were used in this experiment as the sole sources of N supplied, including nitrate (NO₃), urea, alanine (Ala), glycine (Gly), threonine (Thr), serine (Ser), and asparaginic acid (Asp). Nitrogen concentration of all experimental groups was 100 μmol N L^-1, and P and Si concentrations were the same as those in Section 2.2.1. Test group N0P0 was set as both N- and P-free control, in which neither N nor P was added. N0Pi was the N-free control, in which no N compound was added and P and Si concentrations were the same as the other test groups. To avoid the decomposition of N and P compounds during autoclave treatment, all compounds were added individually to the autoclaved medium (N- and P-free f/2 medium) after filtration through a 0.1 μm pore size disposable syringe filter (Millipore Corporation, Bedford, MA, USA). The methods for pre-incubation and culture conditions were the same as the described in Section 2.2.1.

2.2.3 Growth under different ratios of urea to nitrate

Using urea and NO₃-N as N sources, five experimental groups were set with the relative urea-N proportions of 0, 25%, 50%, 75% and 100%, respectively. The sum concentration of urea and NO₃-N was 100 μmol N L^-1, and P and Si concentrations were the same in Section 2.2.1.

2.3 Cell Counting and Relative Growth

The cell number was counted every day during the incubation period. The cell counting was performed in a cell counting chamber by placing 0.05-0.1 mL of culture into the chamber, fixed with a drop of Lugol's fixative, and observed under an inverted microscope (Leica DM IRB) at a magnification of 200×. Each sample was counted more than three times until the differences in cell counts were less than 10%.

2.4 Specific Growth Rate (μ) and Half-Saturation Constant (Ks)

Specific growth rate (μ, divisions d^-1) was calculated using the following equation:

$ \mu\left(\text { divisions } \mathrm{d}^{-1}\right)=\frac{\ln N 2-\ln N 1}{t 2-t 1}, $

(1)

where N2 and N1 are cell densities at times t2 and t1. N2 and N1 are cell densities at d2 and d0 in this study.

The growth rate at the exponential growth phase was used to calculate μ_max and K_s using the Monod equation (1949):

$ \mu = \frac{{{\mu _{\max }} \times S}}{{{K_{\rm{s}}} + S}}, $

(2)

where μ is the specific growth rate (divisions d^-1) in the test groups, μ_max is the maximum specific growth rate (divisions d^-1), S is the nutrient concentration (μmol L^-1), and K_s, the half-saturation constant for growth, is the nutrient concentration at μ_max/2.

2.5 Data Analysis

The mean and standard deviation were calculated for each treatment from three independent replicate cultures. The means and standard deviations (SD) of all data were calculated and graphed. One-way ANOVA was performed to compare the test groups with the controls and the significant difference among groups using SPSS 19.0 for Windows. Differences are termed significant when P < 0.05. The relationships between growth rate and nutrient concentration were fitted with the Monod equation using Originpro 2019.

3 Results 3.1 Growth of the Three Phytoplankton Taxa Under Different NO₃-N Concentrations

The growth curves of the three microalgae obtained under different nitrate concentrations (0-1000 μmol N L^-1) are shown in Fig. 1. Growth of S. costatum was observed at concentrations ≥ 10 μmol L^-1, with maximum yields increasing with increases in N concentrations from 10 to 1000 μmol L^-1 (Figs. 1A, 2A). The maximum yields occurred in day 5 after incubation, and reached 2.84×10⁵ cells mL^-1 at the concentration of 1000 μmol L^-1.

P. micans could not grow at low N concentrations (0-5 μmol L^-1) (Figs. 1B, 2B), and significant increases in growth were observed at concentrations more than 10 μmol L^-1 (P < 0.01). Best growth occurred at concentration of 100 μmol L^-1 with the maximum yield of 4.99×10³ cells mL^-1, which was significantly higher than those under other N concentrations (P < 0.01).

Growth of C. marina was observed under nitrate concentrations of 5-1000 μmol L^-1 (Figs. 1C, 2C), and cell numbers in these treatments were significantly higher than that in N-free control (P < 0.01). The maximum yields increased with increasing concentration of NO₃-N to 500 μmol L^-1, which was 4.64×10³ cells mL^-1 (Fig. 2C).

The relationships between specific growth rates (μ) of the three microalgae and the concentrations of NO₃-N were determined using the Monod equation (Fig. 3). From the equations, the values of μ_max and K_s for NaNO₃ were 0.71 divisions d^-1 and 53.55 μmol L^-1 for S. costatum (r = 0.88), 0.67 divisions d^-1 and 23.31 μmol L^-1 for P. micans (r = 0.95), and 0.23 divisions d^-1 and 17.57 μmol L^-1 for C. marina (r = 0.96), respectively.

3.2 Effects of Different Nitrogen Sources on the Growth

Of the seven compounds provided as N sources, S. costatum grew in both inorganic and the six organic N sources (Figs. 4A, 5A), however the maximum yields in all organic N sources were significantly lower than that obtained in treatment with NO₃-N (P < 0.05, or P < 0.01). P. micans grew only in treatments with NO₃-N and urea, and cell numbers in the other five amino acids (Ala, Gly, Thr, Ser, and Asp) and the N-free controls (N0P0 and N0Pi) never exceeded the initial inoculation numbers (Figs. 4B, 5B). C. marina grew well in inorganic and three organic N compounds (urea, Gly, and Ser, Figs. 4C, 5C), and cell numbers in the other three amino acids (Ala, Thr, and Asp) increased but showed no significant differences with the N-free controls and the initial inoculation numbers (P > 0.05). The highest yields were obtained in the inorganic NO₃-N treatment for S. costatum and P. micans, and were recorded in both NO₃-N and urea for C. marina (Fig. 5).

3.3 Varying Urea: Nitrate on the Growth

In the experiments with various urea: nitrate ratios, algal cells grew well in all cultures (Figs. 6, 7). Cell numbers of S. costatum increased significantly after experiencing one day lag period, and reached to the maximum numbers of (1.73-2.26)×10⁵ cells mL^-1 at day 4-6 (Fig. 6A). The highest yield of S. costatum was recorded in treatment with 75% urea-N (Fig. 7A), which was significantly higher than those in the other treatments (P < 0.05). The growth of P. micans showed similar patterns under different treatments, and cell number increased rapidly at day 1 after incubation, and then dropped at day 5-6 (Fig. 6B). There were no significant differences in maximum yields of P. micans among all treatments (P > 0.05, Fig. 7B). C. marina grew better in the mixture N sources than using NO₃-N or urea as the sole N source (P < 0.05, or < 0.01). Best growth of C. marina occurred in treatment with 25% urea-N, and growth decreased as increasing proportions of urea-N (Figs. 6C, 7C).

4 Discussion

The ability to use various nutrient components for growth differs among phytoplankton species, which may be one of the major factors accounting for species succession in situ (Pustizzi et al., 2004). Organic N compounds are considered as valuable N sources for the growth of many HAB species, such as dinoflagellates K. brevis (Steidinger et al., 1998; Killberg-Thoreson et al., 2014), P. piscicida (Lewitus et al., 1999), L. polyedrum (Kudela and Cochlan, 2000), the raphidophytes H. akashiwo (Herndon and Cochlan, 2007), F. japonica (Cucchiari et al., 2008), and the brown tide alga A. anophagefferens (Pustizzi et al., 2004). Our results demonstrated that NO₃-N and urea served as good N sources for all of the three phytoplankton taxa (Figs. 4, 5). P. micans cannot grow in medium of the five free amino acids. C. marina can effectively use the two amino acids Gly and Ser, however cannot utilize the other three amino acids. Though amino acids are small organic N sources that would be easy to be used by phytoplankton for growth, amino acids are the least prioritized N substrates to K. brevis including NH₄-N, NO₃-N, urea, and humic-N (Killberg-Thoreson et al., 2014). Some phytoplankton taxa cannot even grow on organic N compounds including urea, uric acid and various amino acids, for example Levanderina fissa (formerly Gyrodinium instriatium, Nagasoe et al., 2010; Wang et al., 2017) and Chattonella ovata (Yamaguchi et al., 2008). S. costatum grew well on a variety of N sources including NO₃-N, urea and all the five free amino acids. The results suggested that S. costatum had the ability to use a diverse array of N compounds, while P. micans and C. marina can only utilize selective organic N sources. Other reports also demonstrated that Skeletonema had high affinity for DON (Kwon and Oh, 2014; Li et al., 2017).

In natural aquatic environments, urea represents the majority of the organic N, and is important for phytoplankton as the N source (Moschonas et al., 2017). Dissolved amino acids are also utilized by many phytoplankton species (Hu et al., 2014; Moschonas et al., 2017). Shilova et al. (2017) found that urea addition resulted in the greatest increases in chlorophyll a and productivity compared to those of nitrate and ammonium in the oligotrophic waters of the North Pacific Ocean. Results from an annual survey in Loch Creran of Scotland indicated that the abundances of dominant phytoplankton species, mostly small diatoms such as Skeletonema spp., Thalassiosira spp., Pseudo-nitzschia spp., and Chaetoceros spp., showed significant correlations with urea and dissolved free amino acids (Moschonas et al., 2017). Therefore, the ability of S. costatum to use wide kinds of organic N compounds provide this species an advantage to predominate in DON rich coastal and estuarine waters. Actually, S. costatum has been reported as a common dominant diatom in the worldwide coastal sea areas (Wang et al., 2009; Moschonas et al., 2017; Zhou et al., 2017).

High ratios of DON to DIN have been suggested to favor the growth of the brown tide alga A. anophagefferens (Pustizzi et al., 2004), and increase loadings of urea from coastal runoff may be a determining factor in the blooms of Pseudo-nitzschia (Howard et al., 2007). All of the three phytoplankton taxa grew well under different proportions of urea-N in our study, and C. marina grew significantly better in cultures with both NO₃- and urea-N (Figs. 6, 7). The better growth in mixture N sources of C. marina thus make this species an advantage in interspecific competition in natural sea waters.

The Monod equation has been used to describe saturation kinetics in many field and laboratory studies (Taylor et al., 2006; Gobler et al., 2012). Maximum specific growth rates (μ_max) and half saturation constants (K_s) are two important kinetic parameters derived from this model that quantify the growth responses to environmental nutrient concentrations. The maximum specific growth rates (μ_max) and half-saturation constant (K_s) have been reported for the growth of a lot of phytoplankton taxa, however most K_s values are assessed for N uptake rates fitted to the Michealis-Menten equation (Table 1). K_s values for growth were generally < 10 μmol L^-1, for example, 1.1 μmol L^-1 for Prorocentrum minimum (Taylor et al., 2006), 2.06 μmol L^-1 for Margalefidinium polykrikoides (formerly Cochlodinium polykrikoides, Gobler et al., 2012), 5.36 for C. marina (Oh et al., 2009), 8.98 μmol L^-1 for Chattonella subsalsa, and 0.3 μmol L^-1 for H. akashiwo (Zhang et al., 2006). In comparison with these species, K_s values obtained in this study were quite high, which were 53.55, 23.31, and 17.57 μmol L^-1 for S. costatum, P. micans, and C. marina, respectively. These values were even 2.45-3.36 times greater than those obtained for the same species reported (Table 1). Differences in kinetic parameters are often observed in cells growing under different N sources and concentrations (Lomas and Glibert, 2000). Phytoplankton are able to take up more nutrients at higher nutrient concentrations and reach another maximum K_s value (Tantanasarit et al., 2013). The NO₃-N uptake rate by Chaetoceros sp., as reported by Lomas and Glibert (2000), demonstrates that at N concentrations between 0 and 40 μmol L^-1, the uptake rate reached a maximum plateau of 0.024 pmol cell^-1h^-1. However, at higher N concentrations, 50 to 260 μmol L^-1, the uptake rate increased in a linear pattern with increasing N concentration in solution. Anyway, the K_s values obtained in our study were comparable with K_s in L. fissa at the same N levels, 0 to 1000 μmol L^-1 (Nagasoe et al., 2010), and much lower than K_s in Chaetoceros calcitrans at higher N concentrations, 0 to 2110 μmol L^-1 (Tantanasarit et al., 2013).

Meanwhile, cell size and shape are known to define species-specific differences in N uptake rates and K_s values, and larger cells are suggested to have a lower K_s values than smaller cells (Malone, 1980). The maximum growth rates decreased with increasing cell size in this study, and K_s value was significantly higher for S. costatum than those for the other two species (Table 1). The result suggested that S. costatum had a high N demand for growth. As a small diatom, S. costatum is characterized with high growth rate and short growth circle (Li et al., 2017). The high ambient N concentration stimulated rapid growth of diatoms, and spring diatom blooms in coastal systems typically develop under conditions of high NO₃-N concentrations and a well-mixed water column (Lomas and Glibert, 2000). Nitrogen enrichment has been increasing constantly in the worldwide coastal sea areas due to the high anthropogenic loadings, especially for urea, which is widely used in nitrogenous fertilisers (Glibert et al., 2006, 2014; Glibert, 2017). Results of this study showed that S. costatum is capable to utilize wide kinds of organic N compounds. Therefore, the strategy of N utilization for S. costatum make this species an advantage in N enriched sea areas especially the DON-rich coastal waters. However, diatoms require silicon for their growth, and the rapid growth of diatoms may exhaust dissolved silicate in the water column, and thus lead to the collapse of diatom blooms (Wang et al., 2009). By this time, the flagellate species might predominate in the phytoplankton community due to their multiple ecological strategies, such as utilization of organic nutrients (Heisler et al., 2008), mixotrophy (Burkholder et al., 2008), allelopathy (Graneli et al., 2008; Li et al., 2019), and the release of toxic compounds (Sole et al., 2006). Therefore, a proportional shift away from a diatom-dominated community toward flagellates might be expected as the increase of anthropogenic nutrient loadings (Heisler et al., 2008).

Acknowledgements

This study was supported by the Science & Technology Basic Resources Investigation Program of China (No. 2018FY100200), and the National Natural Science Foundation of China (No. 42076141).