LIU Lei, WANG Zhaohui, 2, WANG Chaofan, LI Weiguo, and NIE Xiangping, 2

Effects of Nitrogen Sources and Concentrations on the Growth of Different Phytoplankton Taxa

LIU Lei1), WANG Zhaohui1), 2), *, WANG Chaofan1), LI Weiguo1), and NIE Xiangping1), 2), *

1) Department of Ecology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China 2) Engineering Research Center of Tropical and Subtropical Aquatic Ecological Engineering, Ministry of Education, Guangzhou 510632, China

Effects of different nitrogen (N) compounds and concentrations on the growth of the three different phytoplankton taxa,(Bacillariophyceae),(Dinophyceae), and(Raphidophyceae), were investigated. The Monod equation was applied to examine effects of N concentrations on the growth of algal cells. Results showed that nitrate (NO3-N) and urea served as good N sources for the three phytoplankton taxa.grew well on all of the seven N sources.can effectively use the two amino acids, glycine (Gly) and serine (Ser), however cannot utilize alanine (Ala), threonine (Thr), and asparaginic acid (Asp).cannot grow in five amino acid substrates. All of the three phytoplankton taxa grew well under different proportions of urea-N, andgrew significantly better in medium with both NO3- and urea-N. The values of maximum growth rate (max) and half-saturation nutrient concentration (S) for NO3-N were 0.71 divisionsd−1and 53.55 μmolL−1for., 0.67 divisionsd−1and 23.31μmolL−1for, and 0.23 divisionsd−1and 17.57μmolL−1for, respectively. The results suggested thathad a high N demand for growth, and was capable of using wide ranges of N compounds. The strategy of N utilization formay make this species an advantage in N-enriched sea areas especially the dissolved organic nitrogen (DON) rich coastal waters, which might be the reason whywidely distributes in the cosmopolitan coastal and estuarine sea areas.

;;; DON; half-saturation constant; urea

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., 2004; Cucchiari., 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., 2016; Moschonas., 2017).

The importance of DON as N sources for growth of phytoplankton have been demonstrated in both natural assemblages and laboratory cultures (Kudela and Cochlan, 2000; Berman and Bronk, 2003; Glibert., 2006). Of particular interest has been the linkage between DON and the growth of harmful algal bloom (HAB) species, including the dinoflagellates(Steidinger., 1998),(Lewitus., 1999),(Kudela and Cochlan, 2000), the diatomsspp. (Loureiro., 2009), the raphidophytes(Herndon and Cochlan, 2007),(Cucchiari., 2008), and the brown tide alga(Lomas and Glibert, 2000; Pustizzi., 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., 2006; Glibert and Burford, 2017).

In this study, we have examined the N growth capabilities of the three different marine phytoplankton taxa,(diatom, Bacillariophyceae),(dinoflagellate, Dinophyceae), and(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.,., and.were isolated from Daya Bay, China. The stock cultures were maintained in autoclaved (121℃, 20min) f/2 media (Guillard, 1975) at 20℃±1℃ and salinity 30 and under 100μmolphotonm−2s−1of cool-white fluorescent illumination with a dark: light cycle of 12h:12h. The cultures were treated with mixture antibiotics to destroy the external bacteria before the experiment. Firstly, 10μgmL−1penicillin (final concentration) was added in 100 mL cultures in exponential phase, and incubated for 24h, then 10mL of the penicillin-treated culture was inoculated to the fresh f/2 medium with 10μgmL−1streptomycin sulphate, and incubated for another 24h, and followed by the treatment of 10μgmL−1kanamycin 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 100μmolNL−1(NaNO3), 7μmolPL−1(KH2PO4) and 70μmol SiL−1(Na2SiO3), approximating the maximum nutrient concentrations found in Daya Bay (Wang., 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, 1000μmolNL−1). The concentrations of P and Si were 7μmolPL−1and 70μmolSiL−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 250mL flasks with 150mL of test medium. According to the cell sizes of the three taxa, the initial cell densities were set at about 5×104cellsmL−1for., and about 103cellmL−1for.and.. 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 100μmolNL−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 0.1μm 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 ureaand 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 100μmolNL−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 0.05–0.1mL 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 count- ed 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 (, divisionsd−1) was calculated using the following equation:

where2 and1 are cell densities at times2 and1.2 and1 are cell densities at d2 and d0 in this study.

The growth rate at the exponential growth phase was used to calculatemaxandsusing the Monod equation (1949):

whereis the specific growth rate (divisionsd−1) in the test groups,maxis the maximum specific growth rate (divisionsd−1),is the nutrient concentration (μmolL−1), ands, the half-saturation constant for growth, is the nutrient concentration atmax/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< 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 (0–1000μmolNL−1) are shown in Fig.1. Growth of.was observed at concentrations ≥10μmolL−1, with maximum yields increasing with increases in N concentrations from 10 to 1000μmolL−1(Figs.1A, 2A). The maximum yields occurred in day 5 after incubation, and reached 2.84×105cellsmL−1at the concentration of 1000μmolL−1.

Fig.1 Growth of the three phytoplankton taxa under different nitrate concentrations.

Fig.2 Maximum yields of the three phytoplankton taxa under different nitrate concentrations.

Fig.3 Relationships between growth rates (μ) and NO3-N concentrations of the three microalgal taxa. The curves were fitted to the Monod equation.

could not grow at low N concentrations (0–5 μmolL−1) (Figs.1B, 2B), and significant increases in growth were observed at concentrations more than 10 μmolL−1(<0.01). Best growth occurred at concentration of 100 μmolL−1with the maximum yield of 4.99Í 103cellsmL−1, which was significantly higher than those under other N concentrations (<0.01).

Growth of.was observed under nitrate concentrations of 5–1000μmolL−1(Figs.1C, 2C), and cell numbers in these treatments were significantly higher than that in N-free control (<0.01). The maximum yields increased with increasing concentration of NO3-N to 500 μmolL−1, which was 4.64Í103cellsmL−1(Fig.2C).

The relationships between specific growth rates () of the three microalgae and the concentrations of NO3-N were determined using the Monod equation (Fig.3). From the equations, the values ofmaxandsfor NaNO3were 0.71 divisionsd−1and 53.55 μmolL−1for.(=0.88), 0.67 divisionsd−1and 23.31 μmolL−1for(=0.95), and 0.23 divisionsd−1and 17.57μmolL−1for(=0.96), respectively.

3.2 Effects of Different Nitrogen Sources on the Growth

Of the seven compounds provided as N sources,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 (<0.05, or<0.01).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).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 (>0.05). The highest yields were obtained in the inorganic NO3-N treatment forand, and were recorded in both NO3-N and urea for(Fig.5).

Fig.4 Growth of the three phytoplankton taxa under different nitrogen compounds.

Fig.5 Maximum yields of the three phytoplankton taxa under nitrogen compounds.

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 ofincreased significantly after experiencing one day lag period, and reached to the maximum numbers of (1.73–2.26)×105cellsmL−1at day 4–6 (Fig.6A). The highest yield ofwas recorded in treatment with 75% urea-N (Fig.7A), which was significantly higher than those in the other treatments (<0.05). The growth ofshowed 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 ofamong all treatments (>0.05, Fig.7B).grew better in the mixture N sources than using NO3-N or urea as the sole N source (<0.05, or <0.01). Best growth ofoccurred in treatment with 25% urea-N, and growth decreased as increasing proportions of urea-N (Figs.6C, 7C).

Fig.6 Growth of the three phytoplankton taxa under different proportions (%) of urea-N.

Fig.7 Maximum yields of the three phytoplankton taxa under different proportions (%) of urea-N.

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(Pustizzi., 2004). Organic N compounds are considered as valuable N sources for the growth of many HAB species, such as dinoflagellates(Steidinger., 1998; Killberg-Thoreson., 2014),(Lewitus., 1999),(Kudela and Cochlan, 2000), the raphidophytes(Herndon and Cochlan, 2007),(Cucchiari., 2008), and the brown tide alga(Pustizzi., 2004). Our results demonstrated that NO3- N and urea served as good N sources for all of the three phytoplankton taxa (Figs.4, 5).cannot grow in medium of the five free amino acids.can effectively use the two amino acids Gly and Ser, how- ever 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 toincluding NH4-N, NO3-N, urea, and humic-N (Killberg-Thoreson., 2014). Some phytoplankton taxa cannot even grow on organic N compounds including urea, uric acid and various amino acids, for example(formerly, Nagasoe., 2010; Wang., 2017) and(Yamaguchi., 2008).grew well on a variety of N sources including NO3-N, urea and all the five free amino acids. The results suggested thathad the ability to use a diverse array of N compounds, whileandcan only utilize selective organic N sources. Other reports also demonstrated thathad high affinity for DON (Kwon and Oh, 2014; Li., 2017).

In natural aquatic environments, urea represents the majority of the organic N, and is important for phytoplankton as the N source (Moschonas., 2017). Dissolved amino acids are also utilized by many phytoplankton species (Hu., 2014; Moschonas., 2017). Shilova. (2017) found that urea addition resulted in the greatest increases in chlorophylland 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 asspp.,spp.,spp., andspp., showed significant correlations with urea and dissolved free amino acids (Moschonas., 2017). Therefore, the ability ofto use wide kinds of organic N compounds provide this species an advantage to predominate in DON rich coastal and estuarine waters. Actually,has been reported as a common do- minant diatom in the worldwide coastal sea areas (Wang., 2009; Moschonas., 2017; Zhou., 2017).

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

The Monod equation has been used to describe satura- tion kinetics in many field and laboratory studies (Taylor., 2006; Gobler., 2012). Maximum specific growth rates (max) and half saturation constants (s) are two important kinetic parameters derived from this model that quantify the growth responses to environmental nutrient concentrations. The maximum specific growth rates (max) and half-saturation constant (s) have been reported for the growth of a lot of phytoplankton taxa, however mostsvalues are assessed for N uptake rates fitted to the Michealis-Menten equation (Table 1).svalues for growth were generally <10μmolL−1, for example, 1.1μmolL−1for(Taylor., 2006), 2.06μmolL−1for(formerly, Gobler., 2012), 5.36 for(Oh., 2009), 8.98μmolL−1for, and 0.3μmolL−1for(Zhang., 2006). In comparison with these species,svalues obtained in this study were quite high, which were 53.55, 23.31, and 17.57μmolL−1for,, and, 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 maximumsvalue (Tantanasarit., 2013). The NO3-N uptake rate bysp., as reported by Lomas and Glibert (2000), demonstrates that at N concentrations between 0 and 40μmolL−1, the uptake rate reached a maximum plateau of 0.024pmol cell−1h−1. However, at higher N concentrations, 50 to 260μmolL−1, the uptake rate increased in a linear pattern with increasing N concentration in solution. Anyway, thesvalues obtained in our study were comparable withsinat the same N levels, 0 to 1000 μmolL−1(Nagasoe., 2010), and much lower thansinat higher N concentrations, 0 to 2110μmolL−1(Tantanasarit., 2013).

Table 1 Maximum specific growth rates (μmax) and half-saturation constants (Ks) of nitrogen for different phytoplankton taxa

Notes:+: The half-saturation constants (s) for growth rate fitted to the Monod equation.++: The half-saturation constants (s) for N uptake rate fitted to the Michealis-Menten equation.

Meanwhile, cell size and shape are known to define species-specific differences in N uptake rates andsvalues, and larger cells are suggested to have a lowersvalues than smaller cells (Malone, 1980). The maximum growth rates decreased with increasing cell size in this study, andsvalue was significantly higher forthan those for the other two species (Table 1). The result suggested thathad a high N demand for growth. As a small diatom,is characterized with high growth rate and short growth circle (Li., 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., 2006, 2014; Glibert, 2017). Results of this study showed thatis capable to utilize wide kinds of organic N compounds. Therefore, the strategy of N utilization formake 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., 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., 2008), mixotrophy (Burkholder., 2008), allelopathy (Graneli., 2008; Li., 2019), and the release of toxic compounds (Sole., 2006). Therefore, a proportional shift away from a diatom-dominated community toward flagellates might be expected as the increase of anthropogenic nutrient loadings (Heisler., 2008).

Acknowledgements

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

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