Growth and Nitrogen Uptake Kinetics in Cultured Prorocentrum donghaiense

We compared growth kinetics of Prorocentrum donghaiense cultures on different nitrogen (N) compounds including nitrate (NO3 −), ammonium (NH4 +), urea, glutamic acid (glu), dialanine (diala) and cyanate. P. donghaiense exhibited standard Monod-type growth kinetics over a range of N concentraions (0.5–500 μmol N L−1 for NO3 − and NH4 +, 0.5–50 μmol N L−1 for urea, 0.5–100 μmol N L−1 for glu and cyanate, and 0.5–200 μmol N L−1 for diala) for all of the N compounds tested. Cultures grown on glu and urea had the highest maximum growth rates (μm, 1.51±0.06 d−1 and 1.50±0.05 d−1, respectively). However, cultures grown on cyanate, NO3 −, and NH4 + had lower half saturation constants (Kμ, 0.28–0.51 μmol N L−1). N uptake kinetics were measured in NO3 −-deplete and -replete batch cultures of P. donghaiense. In NO3 −-deplete batch cultures, P. donghaiense exhibited Michaelis-Menten type uptake kinetics for NO3 −, NH4 +, urea and algal amino acids; uptake was saturated at or below 50 μmol N L−1. In NO3 −-replete batch cultures, NH4 +, urea, and algal amino acid uptake kinetics were similar to those measured in NO3 −-deplete batch cultures. Together, our results demonstrate that P. donghaiense can grow well on a variety of N sources, and exhibits similar uptake kinetics under both nutrient replete and deplete conditions. This may be an important factor facilitating their growth during bloom initiation and development in N-enriched estuaries where many algae compete for bioavailable N and the nutrient environment changes as a result of algal growth.


Introduction
Harmful algal blooms (HABs) have increased in coastal waters worldwide [1][2][3][4], exerting serious economic impacts on marine fisheries and aquaculture, and threatening public health and aquatic ecosystems [1,5]. In Chinese coastal waters, HABs have increased in frequency, intensity, and duration in recent decades [6][7][8]. Prorocentrum donghaiense is a HAB species that frequently blooms during late spring and early summer in coastal waters around the East China Sea, including the Changjiang River Estuary, and coastal waters adjacent to Zhejiang in China [9][10][11], and coastal areas near Japan and South Korea [9]. Between 2000 and 2006, about 120 P. donghaiense bloom events (maximum cell densities of up to 3.6610 8 cells?L 21 ) were reported from Chinese coastal waters (data from State Oceanic Administration, China) [2] and the spatial extent of these blooms ranged from several thousand to more than ten thousand km 2 . Blooms persisted several days to a month, during which time they caused serious economic impacts to marine fisheries, public health, and aquatic ecosystems [8,10,11]. Despite documentation regarding their impacts, the environmental factors promoting P. donghaiense bloom initiation and persistence are still unclear.
HABs are often linked to nutrient over-enrichment and consequent eutrophication of coastal waters including the East China Sea [5,[12][13][14]. The Changjiang River Estuary and East China Sea, where P. donghaiense blooms frequently occur, are hydrologically complex; they receive terrestrial nutrient inputs and freshwater through the watershed, but are also influenced by oceanic circulation that can affect particle transport in the estuary [15]. High nutrient loads to the watershed have been implicated as the proximate cause of P. donghaiense blooms in this system [16].
Dissolved inorganic N concentrations in the Changjiang River Estuary, mainly in the form of NO 3 2 , increased several-fold in the past few decades, reaching as high as 131.6 mmol N L 21 [15][16][17][18]. However, field studies have shown that NO 3 2 is rapidly consumed during the initiation of P. donghaiense blooms leading to NO 3 2depletion during blooms [16]. Our previous culture study determined that P. donghaiense can grow on multiple forms of N when they are supplied in saturating concentrations (50 mmol N L 21 ) [19]. However, it is not known whether P. donghaiense has a strong affinity for NO 3 2 or sources of recycled N (e.g., NH 4 + , urea, and amino acids) when they are present at lower concentrations, more realistic of environmental concentrations once blooms are established.
The dissolved N pool in aquatic systems is dynamic, consisting of varying concentrations of dissolved inorganic N (DIN) and dissolved organic N (DON) compounds, many of which remain to be identified [20][21][22]. In Chinese coastal waters DIN (NO 3 2 +N-O 2 2 +NH 4 + ) concentrations ranged 0-45.0 mmol N L 21 in the surface waters of Southern Yellow Sea during 2006-2007 [23] and 0-60.0 mmol N L 21 in waters adjacent to Hong Kong [24]. In the Changjiang River estuary and East China Sea, DIN (NO 3 2 , NO 2 2 and NH 4 + ) concentrations ranged 0-131.6 mmol N L 21 [15][16][17][18] over the same time period. In addition to DIN, urea can be an important component of the dissolved N pool and accounted for ,10-60% of the total dissolved N in Hong Kong waters in summer of 2008 [24]. In the East China Sea, DON concentrations ranged 5.78-25.26 mmol N L 21 , accounting for 44.0-88.5% of total dissolved nitrogen [16]. While concentrations of urea and DFAA were only a small component of the DON pool, 0.21-1.39 and 0.09-1.55 mmol N L 21 , respectively, these compounds are labile and rapidly regenerated and turned over in the environment [21,22].
To date, our understanding of the nitrogenous nutrition of P. donghaiense is limited. While previous research demonstrated that P. donghaiense can potentially utilize multiple inorganic and organic N compounds to grow [19], little is known about their affinity and capacity for growth on the diverse range of N compounds found in the environment. Therefore, we compared growth kinetics of P. donghaiense on medium supplied with NO 3 2 , NH 4 + , urea, glu, dialanine (diala) or cyanate as the sole source of N. In addition, we used stable isotopes as tracers to compare uptake kinetics for these compounds in cultures acclimated under NO 3 2 -replete anddeplete culture conditions, conditions likely present in the environment during bloom initiation and maintenance phases, respectively.

Culture conditions
Prorocentrum donghaiense, was isolated from the East China Sea, and obtained from the Research Center of Hydrobiology, Jinan University, Guangzhou, China. Cultures were grown in artificial seawater enriched with, sterile, silicate-free f/2 trace metals and vitamins [25]. Concentrations of NO 3 2 were adjusted to conform to different experimental treatments (see below). Cultures were maintained in an environmental room at constant temperature (2360.5uC) and irradiance (6065 mmol quanta m 22 s 21 ) on a 12 h:12 h (light: dark) light cycle and transferred to fresh medium every week to ensure cells remained in exponential growth phase for the duration of the experiments.
For all experiments, dissolved nutrients were measured after filtering through a 0.2 mm Supor filter. Samples were stored frozen until analysis. NO 3 2 and urea were measured on an Astoria Pacific autoanalyzer according to the manufacturer's specifications and using colorimetric methods [26]. NH 4 + was determined using the manual phenol hypochlorite method [27]. Concentrations of dissolved free amino acids (DFAA) were measured by high performance liquid chromatography (HPLC) [28]. Cells were enumerated using a FACSCalibur flow cytometer (Becton Dickinson Instruments). Samples were also collected onto GF/F filters and frozen for analysis of chlorophyll a (Chl a). Chl a concentrations were measured fluorometrically within one week of sample collection after extraction of cells in 90% acetone [29] Cultures were maintained in the environmental chamber under conditions described above for at least three generations prior to experiments in order to ensure cells were acclimated to treatment conditions. In vivo fluorescence was monitored to estimate chlorophyll biomass using a Turner Designs AU-10 fluorometer at the same time each day. Growth rates, m (day 21 ), were calculated using a least squares fit to a straight line after logarithmic transformation of in vivo fluorescence data as described by Guillard [30] using the equation: where N 1 and N 0 were the biomass (in vivo fluorescence) at time T 1 and T 0 , respectively, during the linear portion of exponential phase growth. The relationship between specific growth rate and N concentration was fitted to a Monod growth kinetic model using the equation: where m was the specific growth rate (day 21 ) calculated during the linear portion of exponential phase growth (Equation 1), m m was the maximum specific growth rate (day 21 ), S was the N concentration (mmol N L 21 ), and K m (half saturation constant; mmol N L 21 ) was the N concentration at m m /2 [31].

N uptake kinetic experiments.
For N uptake kinetic experiments, cultures of P. donghaiense were grown on f/2 medium modified to contain 50 mmol N L 21 as NO 3 2 in two, 5-liter bottles. Cell abundance and ambient NO 3 2 concentrations were monitored daily. N-deplete uptake kinetic experiments were initiated after NO 3 2 concentrations were below the detection limit (0.05 mmol N L 21 ) for 5 consecutive days. Uptake kinetics experiments were initiated by dispensing 35 ml of NO 3 2 -deplete culture into 50 ml Pyrex test tubes and adding 15 3 2replete uptake kinetic experiments, 35 ml of culture was dispensed into a series of 50 ml Pyrex test tubes and each was amended with an additional 50.0 mmol N L 21 as 14 NO 3 2 to ensure the cultures were NO 3 2 -replete. As for NO 3 2 -replete treatments, 15 NH 4 + , and 15 N-13 C dually labeled urea, glu, or an algal amino acid mixture were added to duplicate tubes at the same concentrations indicated above, however, NO 3 2 uptake kinetics could not be measured in these NO 3 2 -replete cultures. Uptake experiments were incubated at ,23uC and at an irradiance of ,60 mmol quanta m 22 s 21 . After ,1 hour, incubations were terminated by gentle filtration through precombusted (450uC for 2 hours) GF/F (nominal pore size ,0.7 mm) filters. Filters were immediately frozen in sterile polypropylene cryovials until analysis. Frozen samples were dried in an oven at 40uC for 2 days and pelletized into tin discs prior to analysis on a Europa Scientific 20-20 isotope ratio mass spectrometer equipped with an automated N and C analyzer preparation unit. Specific uptake rates were calculated using a mixing model [32]: where the source pool was the dissolved N pool that was enriched during uptake experiments. Specific uptake rates (V; h 21 ) at each N concentration for each N substrate tested were fitted to a Michaelis-Menten model using the following equation: where V m is the maximum specific uptake rate (h 21 ); S the substrate concentration (mmol N L 21 ); and K s the half saturation constant, the concentration (mmol N L 21 ) where V is equivalent to half of V m , for the N substrate tested.

Statistical analysis
All statistical tests were carried out using Origin 7.0 and Microsoft Excel 2007 with the level of significance set at an a equal to 0.05. Differences between growth and uptake kinetic parameters were compared using a one-way ANOVA test. Monod and Michaelis-Menten curves were fitted using Origin 7.0.

Growth kinetics of P. donghaiense
P. donghaiense exhibited standard Monod-type growth kinetics over N concentraions ranging from 0.5-500 mmol N L 21 for NO 3 2 and NH 4 + , 0.5-50 mmol N L 21 for urea, 0.5-100 mmol N L 21 for glu and cyanate, and 0.5-200 mmol N L 21 for diala ( Fig. 1). Maximum specific growth rates (m m ) ranged from 0.7160.04 to 1.5160.06 d 21 (Table 1). The highest maximum growth rates were observed in cultures growing with glu or urea as the sole N source; maximum growth rates were a factor of 2 lower in cultures grown with NO 3 2 , NH 4 + , cyanate, or diala supplied as the sole N source (Table 1). Half saturation constants (K m ) ranged from 0.2860.08 to 5.2560.40 mmol N L 21 . The lowest K m concentrations were observed in P. donghaiense cultures growing on media containing NO 3 2 , NH 4 + , and cyanate as the sole source of N (0.28-0.51 mmol N L 21 ) ( Table 1). In contrast, K m concentrations were an order of magnitude higher in cultures grown with urea, glu, or diala as the sole source of N suggesting higher affinities for cyanate, NO 3 2 , NH 4 + , and urea than for glu and diala (Table 1).

N uptake kinetics by P. donghaiense
Kinetics for NH 4 + , urea, and algal amino acids conformed to Michaelis-Menten kinetics in both NO 3 -deplete and -replete cultures of P. donghaiense ( uptake also conformed to Michaelis-Menten kinetics in NO 3deplete cultures (Fig. 2a), NO 3 2 uptake kinetics could not be measured in NO 3 2 -replete cultures because of the excess NO 3 2 in the growth media (Fig. 3a). Glu uptake did not conform to the Michaelis-Menten kinetic model in either NO 3 -deplete ( Fig. 2d) or -replete (Fig. 3d) cultures of P. donghaiense and higher glu uptake rates were observed at lower glu concentrations.
Uptake kinetics for NO 3 2 , NH 4 + , urea, and algal amino acids followed Michaelis-Menten type uptake kinetics in NO 3 2 -deplete batch cultures of P. donghaiense (Table 2; Fig. 2). Although results were sufficient to fit a Michaelis-Menten model for NH 4 + uptake, NH 4 + uptake by NO 3 2 -deplete P. donghaiense was not saturated even at the highest concentrations used in this experiment (Fig. 2b). Maximum specific uptake rates (V m ) of (66.966.0)610 23 h 21 were calculated for NH 4 + (  3 2 , NH 4 + , urea, and algal amino acids, respectively (Table 2) suggesting NO 3 2 -deplete P. donghaiense had the highest affinity for urea and the lowest for amino acids.
In NO 3 2 -replete cultures of P. donghaiense, maximum specific uptake rates (V m ) of NH 4 + were greatest and not significantly different than those observed in NO 3 2 -deplete cultures (p.0.05; Table 2) (Table 2). Similarly, V m for urea and algal amino acids were lower than those observed for NH 4 + but comparable to those observed in NO 3 2 -deplete cultures (  3 2 -deplete cultures. Similarly, the derived a values, a measure of nutrient affinity, were also similar for NO 3 2 -replete and -deplete cultures of P. donghaiense even though there were significant differences (p,0.05) in a between NO 3 2 -replete and -deplete cultures for algal amino acids ( Table 2).

Discussion
Nutrient enrichment has been implicated as a causal factor in the occurrence of HAB events worldwide [5,14,33]. Both the form of dissolved N and its concentration in coastal and marine environments are thought to be important to the formation and development of HABs and phytoplankton blooms in general [13,15,34,35]. However, different phytoplankton species and groups have different uptake capacities and affinities for specific inorganic and organic nitrogen compounds [19,20,36]. Many phytoplankton can also mobilize N from complex organic compounds such as peptides using extracellular enzymes thereby making N from these compounds available for uptake [32,37]. In coastal areas, the concentration and composition of the dissolved N pool and the relative concentrations of various N compounds vary temporally and spatially, likely impacting the phytoplankton community composition and abundance on both short and longer timescales [15,38,39].
P. donghaiense is a mixotrophic species capable of both autotrophic and heterotrophic metabolisms. Previous research demonstrated that P. donghaiense can utilize inorganic and organic N compounds including urea, amino acids, small peptides, and cyanate [16,19] as N sources. In addition, this organism can also ingest cyanobacteria, cryptophytes, and other dinoflagellates [40]. To better understand the N nutrition and capabilities of P. donghaiense, we conducted growth and N uptake kinetic experiments in cultured isolates to determine their affinity, preference, and capacity for growth on different N compounds and found that P. donghaiense had high maximum growth rates and exhibited high uptake capacities and affinities for a diverse suite of N compounds under both NO 3 2 -deplete and -replete conditions.

Growth kinetics
The Monod equation [41] has been used to describe saturation kinetics for nutrient-limited phytoplankton growth in many field and laboratory studies [42][43][44]. Maximum specific growth rates (m m ) and half saturation constants (K m ) are two important kinetic parameters derived from this model that quantify organismal growth responses to environmental nutrient concentrations. The initial slope of the Monod function (m m /K m ) is thought to be a competitive index and used to assess the affinity of phytoplankton for particular nutrient substrates [45]. This model provides several useful tools for evaluating fitness of co-occurring phytoplankton with respect to the external nutrient availability in the environment [46]. Theoretical studies of resource competition suggest that the functional relationship between growth rate and the concentration of nutrient elements in the environment determines a species' ability to compete for that nutrient in the environment [46].
In our culture experiments, we found P. donghaiense grew well on DIN (NO 3 2 and NH 4 + ) and DON (urea, glu, diala and cyanate) compounds when they were supplied as the sole source of N (Fig. 1). P. donghaiense had comparable maximum specific growth rates when growing on NO 3 2 , NH 4 + , cyanate, or diala (0.71-0.83 d 21 ); but maximum specific growth rates were a factor of 2  Table 1). In contrast, half saturation constants for growth (K m ) were more variable (0.2860.08 to 5.2560.40 mmol N L 21 ); K m values were lower (0.28-0.51 mmol N L 21 ) for cultures growing on NO 3 2 , NH 4 + , or cyanate, and were an order of magnitude higher (2.05-5.25 mmol N L 21 ) in cultures grown on urea, glu, or diala (Table 1), suggesting a higher affinity for the inorganic N compounds. So, while P. donghaiense had higher maximum growth rates when supplied urea or glu as a sole source of N, cells had a higher affinity for cyanate, NO 3 2 , and NH 4 + relative to urea, glu, and diala (Table 1) suggesting that the former compounds might be important sources N when nutrient concentrations are submicromolar.
Calculated growth kinetic parameters for P. donghaiense indicate that maximum specific growth rates and half saturation constants (K m ) for NO 3 2 , NH 4 + , and urea, were comparable to each other and those measured for other bloom-forming dinoflagellate species, Prorocentrum minimum, Cochlodinium polykrikoides and for Scrippsiella trochoidea growing on urea (Table 3). Maximum growth rates for P. donghaiense growing with NO 3 2 , NH 4 + , urea or glu as the sole source of N were higher than those previously measured for P. minimum, C. polykrikoides, and S. trochoidea growing on the same N compounds (Table 3). S. trochoidea, P. minimum and C. polykrikoides had comparable maximum specific growth rates when going on urea (0.44-0.47 d 21 ; Table 3), but these were all lower than the maximum specific growth rate observed for P. donghaiense in this study(1.50 d 21 ) (Table 1). While P. donghaiense (this study) and C. polykrikoides [44] exhibited comparable maximum specific growth rates on NO 3 2 , NH 4 + , urea and glu, P. minimum exhibited higher specific growth rates in cultures growing on urea or NO 3 2 (0.45 and 0.4360.01 d 21 , respectively) relative to cultures growing on NH 4 + or glu (0.2760.05 and 0.2360.02 d 21 , respectively) [43]. But unlike P. donghaiense, P. minimum had the lowest half saturation constants for growth on urea, followed by NO 3 2 and NH 4 + ; both had the highest half saturation constants for glu (Table 3) [43]. In contrast, C. polykrikoides had higher half saturation constants for urea and NH 4 + than for NO 3 2 and glu [44]. The half saturation constant for S. trochoidea growing on urea was much higher than for the other three dinoflagellates suggesting a low affinity for growth on this compound [47].
In this study, P. donghaiense also exhibited maximum growth rates and half saturation constants comparable to those for growth on NH 4 + and NO 3 2 , when growing with cyanate and dialanine as the sole source of N ( Fig. 1; Table 1). Because peptides and cyanate are degradation products of decaying cells [32,48], these compounds were likely available to P. donghaiense during bloom maintenance when NO 3 2 concentrations are exhausted and recycling processes are important for maintaining cell biomass and turnover. The concentration and turnover of these organic compounds have not been assessed in natural systems where blooms of P. donghaiense occur. Comparisons of P. donghaiense growth kinetics with those of co-occurring species relative to the bioavailable DIN and DON compounds in the environment is warranted to elucidate how competitive interactions among phytoplankton populations contributes to bloom initiation and maintenance.

Uptake kinetics
The Michaelis-Menten equation is often used to describe the relationship between external nutrient concentrations and their uptake rates [49][50][51]. As for growth kinetics, kinetic parameters for N uptake have been used to assess the relative preference (and affinity) for different N substrates in the environment [50,52]. While kinetic parameters are usually measured in nutrient-deplete cultures, we conducted N uptake kinetic experiments in both NO 3 2 -deplete and -replete batch cultures of P. donghaiense because NO 3 2 concentrations are generally high in the Changjiang River  Table 2. Mean (6SD, n = 2) uptake kinetic parameters (maximum specific uptake rate (V m ), half saturation constant (K s ) and affinity (a)) for P. donghaiense growing on NO 3 2 deplete culture media. Michaelis-Menten type uptake kinetics were observed for all N compounds tested except glu (Figs. 2d, 3d). For NH 4 + , the uptake capacity (V m ) for exponentially growing P. donghaiense was ,1.5, 1.9, and 2.1 times greater than those for urea, NO 3 2 , and the algal amino acids, respectively, in both NO 3 2 -deplete and -replete cultures. The half saturation constants, which are also used to estimate nutrient affinity under nutrient-limiting conditions, suggest similar nutrient preferences under NO 3 2 -deplete andreplete conditions; urea ...NO 3 2 .NH 4 + . algal amino acids for NO 3 2 -deplete P. donghaiense, and urea ...NH 4 + . algal amino acids for NO 3 2 -replete P. donghaiense (Table 2; Figs. 2, 3). Differences in kinetic parameters are often observed in cells growing under N-replete versus -deplete conditions [53]. Many N-starved cells are capable of enhanced N uptake when nutrients are resupplied, and N-replete cells can have higher half-saturation constants than those grown under N-depleted conditions [36]. While our kinetics parameters conformed to this norm based on our statistical analyses, it was surprising that uptake kinetics for NH 4 + , urea, and amino acids were similar in nutrient-replete anddeplete cultures of P. donghaiense (Table 2; Figs. 2&3). Ambient NO 3 2 concentrations were ,43 mmol N L 21 when we initiated our uptake experiments in the N-replete cultures and this should have been saturating for NO 3 2 uptake (Table 2). However, kinetic parameters in NO 3 2 -replete and -deplete batch cultures were similar suggesting that even when NO 3 2 concentrations are high, such as during bloom initiation, P. donghaiense has a high affinity for other N compounds that may available in the environment and this may give them a competitive edge over other phytoplankton. NO 3 2 and NH 4 + are the dominant DIN species thought to fuel the bulk of primary production in estuarine, coastal, and oceanic environments. In Chinese estuaries and coastal waters where P.  Table 3. Summary of mean (6SD) maximum specific growth rates (m max , d 21 ) and half saturation constants (k m , mmol N L 21 ) for nitrate (NO 3 2 ), ammonium (NH 4 + ), urea, and glutamic acid (glu) estimated for different dinoflagellate species.    donghaiense blooms, NO 3 2 and NH 4 + concentrations are commonly 0-20.0 and 0-3.0 mmol N L 21 , respectively [16]. In this study, we found P. donghaiense had higher maximum uptake rates for NH 4 + than NO 3 2 , urea and amino acids in both NO 3 2 -deplete andreplete batch cultures (Table 2; Fig. 2). However, because of the low half saturation constants for urea uptake (0.13 and 0.12, respectively), both N-deplete and -replete cultures of P. donghaiense had a much higher affinity for urea than for inorganic N or amino acids. This is consistent with results from a field study in the East China Sea during a P. donghaiense dominant bloom [11,16]. As we know, DON can constitute more than 80% of the TDN in surface waters in coastal systems and the open ocean [21] where it can also contribute to primary productivity [20]. In the East China Sea, DON accounted for 44.0%-88.5% of the TDN, and urea and DFAA accounted for 1.7%-36.3% of the DON, before and during a P. donghaiense bloom [16]. In our experiments, P. donghaiense had similar maximum specific uptake rates for urea and amino acids under NO 3 2 -deplete and -replete conditions ( Table 2) and half saturation constants for urea uptake were much lower than for NO 3 2 , NH 4 + and amino acids, suggesting a higher affinity for urea than for other N compounds tested. Further, during a bloom dominated by P. donghaiense in the East China Sea in 2005, maximum specific uptake rates for urea were 11-28610 23 h 21 and half saturation constants were 0. 25-14.95 mmol N L 21 , which are comparable to results presented here for culture studies [16]. These results suggest that even if present at low concentrations, urea has the potential to be an important source of N fueling the growth of P. donghaiense and this may be important for outcompeting co-occurring phytoplankton during bloom initiation and/or supporting the persistence of bloom organisms in the environment once NO 3 2 has become depleted. A wide range of maximum uptake rates and half saturation constants for NO 3 2 , NH 4 + , urea, and amino acid uptake have been observed among dinoflagellates both in laboratory cultures and experiments using natural populations during blooms (Table 4). Higher maximum uptake rates have been observed for NH 4 + compared to NO 3 2 , urea or amino acids for most species including: cultures of P. minimum, Heterosigma akashiwo and Alexandrium minutum, and assemblages dominated by Dinophysis acuminate (representing ,91% of the phytoplankton biomass), P. minimum (representing .93% of the total phytoplankton cells), Alexandrium catenella (more than 99% of the phytoplankton) and Karenia mikimotoi (cell density up to 8610 26 cell?L 21 ) (Table 4). In contrast, in cultures of the diatom Pseudonitzschia australis, maximum NO 3 2 uptake rates exceeded NH 4 + uptake rates in populations growing on NO 3 2 -deplete condition [54]. The high variability in maximum uptake rates and half saturation constants between species and experiments could be due to differences in the preconditioning of cells prior to kinetic studies. Nutritional history was a significant factor affecting N uptake rates by P. minimum culture [55]. N uptake kinetics are known to vary with in response to the physiological status and their nutrient history of cells [36]. Our culture experiments demonstrated that P. donghaiense can take up NH 4 + and other N compounds at high rates even when ambient NO 3 2 concentrations are high (e.g., ,43 mmol N L 21 ). This nutritional flexibility may contribute to the initiation and long duration of P. donghaiense blooms in nature. While blooms initiate when NO 3 2 concentrations are high, NO 3 2 is rapidly depleted as blooms progress.
While there are still few studies examining N uptake during blooms of P. donghaiense, kinetic parameters for NO 3 2 , NH 4 + , urea, and glycine uptake were compared during successive dinoflagellate blooms in Changjiang River estuary and East China Sea coastal waters in 2005 [16]. In these mixed blooms, Karenia mikimotoi was the dominant species initially and then was succeeded by P. donghaiense. In most cases, when the bloom was dominated by P. donghaiense, the K s values for urea and glycine uptake (K s , 5.3 and 1.8 mmol N L 21 respectively) were higher than when the bloom was dominated by K. mikimotoi (K s , 1.4 and 1.7 mmol N L 21 respectively) ( Table. 4). The differences in the K s of the two bloom species for NO 3 2 and NH 4 + may have been a driver of species succession as concentrations of these compounds were drawn down during the K. mikimotoi bloom which preceded the P. donghaiense bloom.
Cell-normalized N uptake rates by P. donghaiense-dominated assemblages in the Chanjiang River estuary were comparable to cell-normalized N uptake rates measured in the culture studies reported here. Uptake rates of NH 4 + , urea, and glycine were ,96, ,22 and ,38 fmol N cell 21 h 21 , respectively, during blooms of P. donghaiense (cell density and Chl a were ,5.0610 6 cells L 21 and 8.66-9.68 mg L 21 , respectively) [11,16] while maximum uptake rates for NH 4 + , urea and algal amino acids observed in this study were ,119, 78 and ,53 fmol N cell 21 h 21 , respectively. This suggests that uptake may not have been saturated in the environment. The K s values of NO 3 2 and urea for P. donghaiense were much lower than the environmental concentrations, but except for NH 4 + and DFAA, which suggest that P. donghaiense have higher affinities on NO 3 2 and urea than those for NH 4 + and DFAA, but the later two also contribute on P. donghaiense bloom initiation and duration.

Summary
Results presented here demonstrate that P. donghaiense can grow on a diverse array of N compounds as their sole source of N, including inorganic N (NO 3 2 and NH 4 + ) and organic N compounds such as urea, dissolved free amino acids, small peptides, and even cyanate to support their growth. Maximum specific growth rates varied by a factor of 2 for all of the N compounds tested. In addition, uptake kinetics for regenerated N sources (e.g., NH 4 + , urea, and amino acids) were similar under NO 3 2 -replete and -deplete conditions suggesting that competition for these compounds may contribute to the success of P. donghaiense during bloom initiation when NO 3 2 concentrations are high, and maintenance, when NO 3 2 concentrations have been depleted. The nutritional flexibility exhibited by P. donghaiense likely contributes to its success in eutrophic environments where inorganic nutrient concentrations can be high, but where nutrient concentrations and the dominant form of bioavailable N rapidly change in response to stochastic events and the formation of algal blooms.