Nitrate uptake of the red tide dinoflagellate Prorocentrum micans measured using a nutrient repletion method: effect of light intensity

Preparation of experimental organisms

P. micans used in this study was originally isolated from a plankton water sample collected from Shiwha Bay, Korea, in October 2009. Phototrophic cells were grown in enriched f/2-Si seawater media (Guillard and Ryther 1962, Guillard 1975) at 20°C under an illumination of 20 μE m⁻² s⁻¹ of cool white fluorescent light on a 14 : 10 h light-dark cycle.

Nitrate uptake as a function of nitrate concentrations under different light intensities using the N repletion method

Experiment 1 was designed to investigate the nitrate uptake of P. micans as a function of nitrate concentration at each of the 6 different light intensities when cells were originally maintained under nitrate (NO₃) repletion conditions (Table 1, Fig. 1).

Table 1

Experimental design and comparison of the 3 different methods of measuring the nitrate (NO₃) uptake of Prorocentrum micans

Fig. 1

Diagram of the 3 different methods of measuring nitrate (NO₃) uptake rates. The NO₃ conditions of cells at the beginning of the experiments, acclimation period, and incubation time were compared. Closed arrows and open arrow indicate this study and Qi and Zhu (1994), respecetively.

Dense cultures of P. micans growing photosynthetically in f/2-Si medium were transferred to 1-L polycarbonate (PC) bottles and the bottles were placed on a shelf in a culture room at 20°C under a 14 : 10 h light : dark cycle and acclimated at the target light intensity for 4 d. Three 1-mL aliquots were subsampled and then cells were enumerated to determine the cell concentration.

P. micans cells were added to triplicate 800 mL culture flasks by transferring predetermined volumes of cultures (final cell concentration = ~1,000 cells mL⁻¹, but ~500 cells mL⁻¹ for 200 μE m⁻² s⁻¹ and medium initial nutrient concentrations) (Table 1). A stock solution of nitrate made based on the f/2-Si medium concentration was added to the 2 different final concentrations (NO₃ = 20–30 μM for medium initial concentrations and 100–130 μM for high initial concentrations) (Table 1). A sufficient amount of stock solution of phosphate (PO₄) also prepared based on the f/2-Si medium concentration was added so that it would not be limiting before NO₃ was limiting. Trace metals and vitamins were also added plentifully with consideration of the ratio of nitrate to each chemical in an f/2-Si medium.

The flasks were placed on shelves in 3 temperature-controlled culture rooms. By adjusting distances between light source and flasks, target light intensities of 0 (complete darkness), 10, 20, 50, 100, and 200 μE m⁻² s⁻¹ were established (Table 1). Triplicate culture flasks for target light intensity (14 : 10 h light : dark cycle) were incubated at 20°C. The duration of the incubation was ca. 1 week for medium nitrate concentrations (20–30 μM), but 2–3 weeks for higher concentrations (100–130 μM) (Table 1).

Twenty-mL aliquots were subsampled from each flask every day, and 6-mL aliquots were used for the determination of cell concentration and 14-mL aliquots for the determination of nutrient concentrations.

Cell concentration was determined by enumerating cells on three 1-mL Sedgwick-Rafter counting chambers. Fourteen-mL aliquots were filtered through GF/F filters and then concentrations of nitrate (actually nitrate + nitrite in the Cd-coil reduction method) and phosphate were measured using a nutrient analyzer (QuAAtro; Seal Analytical, Norderstedt, Germany).

Nitrate uptake using the N depletion method

Experiment 2 was designed to investigate the nitrate uptake of P. micans at a single light intensity by cells originally maintained under nitrate depletion conditions (Table 1, Fig. 1).

Cells in a dense culture of P. micans growing photosynthetically in f/2-Si medium were concentrated using a 20 μm mesh filter after nitrate was depleted and immediately transferred to 1-L PC bottles containing oligotrophic oceanic waters (NO₃ concentration = 0.67 μM, PO₄ = 0.12 μM). The bottles were placed on a shelf in a culture room at 20°C under a 14 : 10 h light : dark cycle and then maintained for 9 d. The final concentration of nitrate was 0.35 μM. Three 1-mL aliquots were subsampled and then cells were enumerated to determine the cell concentration.

P. micans cells were added to triplicate 250-mL culture flasks by transferring predetermined volumes of cultures (final concentration = ~1,000 cells mL⁻¹). A stock solution of NO₃ based on f/2-Si medium concentrations was added to the 7 different final concentrations (1, 2.5, 5, 10, 25, 50, and 100 μM) (Table 1). Stock solutions of PO₄, trace metals, and vitamins based on the f/2-Si medium were added sufficiently so as to not be limiting.

The flasks were placed on shelves in a temperature-controlled culture room at 20°C at 20 μE m⁻² s⁻¹ with a 14 : 10 h light : dark cycle (Table 1). Triplicate culture flasks were also set up. The duration of the incubation was 6 d.

Fifteen-mL aliquots were subsampled from each flask every day and 5-mL aliquots were used for the determination of cell concentration and 10-mL aliquots for the determination of nutrient concentrations. Cell and nitrate concentrations were determined as described above.

Calculation of nitrate uptake rates and half saturation constant in the N repletion method

In experiment 1, the nitrate uptake rate of a P. micans cell was determined by dividing the reduction in nitrate concentration (N) by the mean cell concentration at 1 or 2 d intervals;

where t₂ – t₁ = 1 d, but 2 d for the high nitrate concentration at 10 μE m⁻² s⁻¹.

where t₂ – t₁ = 1 d, but 2 d for the high nitrate concentration at 10 μE m⁻² s⁻¹.

Day 0 to 1 or day 2 were treated as the acclimation period, and thus data from these days were not used in calculation. Data on daily nitrate uptake rates from day 2 or 3 to the day before the growth rate of P. micans exceeded half the maximum growth rate in each experiment were plotted by the Michaelis-Menten equation;

where V_max = maximum uptake rate (pM cell⁻¹ d⁻¹), N* = mean nitrate concentration (μM), and K_s-NO3 = half saturation constant for nitrate uptake (μM).

where N = nitrate concentration at a single day, t₂ – t₁ = 1 d, but 2 d for the high nitrate concentration at 10 μE m⁻² s⁻¹.

The specific growth rate of P. micans (μ, d⁻¹) was calculated as:

The maximum growth rate (μ_max) of P. micans was obtained after data were fitted to a Michaelis-Menten equation:

where N* = mean nitrate concentration (μM), K_GR = the nutrient concentration sustaining 1/2μ_max.

Data were iteratively fitted to the model using DeltaGraph (SPSS Inc., Chicago, IL, USA), and statistical analyses were conducted using IBM SPSS Statistics version 21 (IBM Corp., Armonk, NY, USA).

Calculation of nitrate uptake rates and half saturation constant in the N depletion method

In experiment 2, the nitrate uptake rate of a P. micans cell was also determined by dividing the reduction in nitrate concentration (N) by the mean cell concentration at 1 d intervals; the reduction in the nitrate concentration, mean nitrate concentration, and mean cell concentration were obtained using Eqs. (1), (2), and (3), respectively.

Day 0 to 1 was treated as an acclimation period, and thus data obtained in this period were not used. The data on daily nitrate uptake rates from day 1 or 3 were plotted to Eq. (3) and (4) as the repletion method and iteratively fitted to the model using DeltaGraph (SPSS Inc.).

Daily variations in nitrate and cell concentrations and growth rate in the N repletion method

In experiment 1, with increasing elapsed incubation time, the P. micans concentration increased and then became saturated, while the nitrate concentration rapidly decreased and then became depleted (Figs 2 & 3).

Fig. 2

Change in the concentration of Prorocentrum micans (cells mL⁻¹) as a function of elapsed time (d) at 0, 10, 20, 50, 100, and 200 μE m⁻² s⁻¹ when high (A–F) and medium (G–K) initial NO₃ concentrations were provided. Data points indicated by closed circles were used for calculating NO₃ uptake and growth rates, while those indicated by the open circles were not used.

Fig. 3

Change in the concentration of nitrate plus nitrite (NO₃, μM) as a function of elapsed time (d) at 0, 10, 20, 50, 100, and 200 μE m⁻² s⁻¹ when high (A–F) and medium (G–K) initial NO₃ concentrations were provided. Closed circles were used for calculating NO₃ uptake rates, while those indicated by the open circles were not used.

With increasing light intensity from 10 to 200 μE m⁻² s⁻¹, the time for nitrate to be depleted (TND; <1.5 μM) and time for the growth rate of P. micans to reach a lag phase (cell concentrations >7,000 cells mL⁻¹) decreased (Figs 2 & 3). When the initial nitrate concentrations were 100–130 μM and the initial P. micans concentrations were ~1,000 cells mL⁻¹, the nitrate concentrations became depleted and the growth rate of P. micans concentrations reached a lag phase after 14 d at 10 μE m⁻² s⁻¹, but at 6–8 d at 100–200 μE m⁻² s⁻¹. However, in darkness, P. micans continuously decreased to <100 cells mL⁻¹ at 18 d and nitrate concentrations did not clearly change. In all light intensities except for darkness, P. micans cells eventually reached ca. 10,000 to 12,000 cells mL⁻¹ at the end of the experiment. With increasing light intensity, the highest growth rate of P. micans at each light intensity increased from 0.17 d⁻¹ at 10 μE m⁻² s⁻¹ to 0.36 d⁻¹ at 100 μE m⁻² s⁻¹, but decreased to 0.30 d⁻¹ at 200 μE m⁻² s⁻¹.

When the initial nitrate concentrations were ca. 20–30 μM and initial P. micans concentrations were 1,000 cells mL⁻¹ (but ~500 cells mL⁻¹ for 200 μE m⁻² s⁻¹), the TND were 7 d at 10 μE m⁻² s⁻¹, but 3–5 d at 100–200 μE m⁻² s⁻¹ (Figs 2 & 3). With increasing light intensity, the highest growth rate of P. micans at each light intensity continuously increased from 0.19 d⁻¹ at 10 μE m⁻² s⁻¹ to 0.42 d⁻¹ at 200 μE m⁻² s⁻¹.

Nitrate uptake rate measured using the N repletion method

In experiment 1, P. micans did not clearly take up nitrate in darkness (Fig. 4A). However, with increasing mean nitrate concentrations, the uptake rate of nitrate by P. micans at 10–100 μE m⁻² s⁻¹ rapidly increased at NO₃ concentrations of <20 μM, but slowed and then became saturated at higher NO₃ concentrations (Fig. 4B–E), while at 200 μE m⁻² s⁻¹ it became saturated at NO₃ concentrations of <10 μM (Fig. 4F).

Fig. 4

Maximum NO₃ uptake rates (V_max, pM cell⁻¹ d⁻¹) as a function of concentrations of NO₃ (μM). 0, 10, 20, 50, 100, and 200 μE m⁻² s⁻¹ using the NO₃ replete (A–F) and 20 μE m⁻² s⁻¹ using the NO₃ depleted (G) long incubation method. The unit of the half saturation constant for uptake rates (K_s-NO3) is μM.

When data were fitted to Eq. (3), with increasing light intensity from 10 to 100 μE m⁻² s⁻¹, the maximum uptake rate of nitrate (V_max) by P. micans also increased from 3.6 pM cell⁻¹ d⁻¹ to 10.8 pM cell⁻¹ d⁻¹, but decreased to 6.1 pM cell⁻¹ d⁻¹ at 200 μE m⁻² s⁻¹ (Table 2, Fig. 4B–F). Similarly, with increasing light intensity from 10–20 to 100 μE m⁻² s⁻¹, the half saturation constant for nitrate uptake (K_s-NO3) increased from 4.1–4.2 to 6.9 μM, but decreased to 3.5 μM at 200 μE m⁻² s⁻¹ (Table 2, Fig. 4B–F).

Table 2

The maximum uptake rate (V_max) or the volume specific maximum uptake rate (V_max-s), and half saturation constant (K_s-NO3) for the uptake rates of red tide dinoflagellates measured using cultures

Nitrate uptake rate measured using the N depletion method

In experiment 2, with increasing mean nitrate concentrations, the nitrate uptake rate of P. micans at 20 μE m⁻² s⁻¹ rapidly increased at NO₃ concentrations of <10 μM, but slowed and then became saturated at higher NO₃ concentrations (Fig. 4G). When data were fitted to Eq. (3), the V_max by P. micans was 4.4 pM cell⁻¹ d⁻¹ and the K_s-NO3 was 4.6 μM (Fig. 4G).

Maximum growth rate in the N repletion method

In experiment 1, P. micans did not grow obviously in darkness. However, when data were fitted to Eq. (6), with increasing light intensity from 10 to 100 μE m⁻² s⁻¹, the maximum growth rate of P. micans also increased from 0.15 to 0.35 d⁻¹, but decreased to 0.27 d⁻¹ at 200 μE m⁻² s⁻¹ (Table 2).

Effects of light intensity on the maximum nitrate uptake rate and maximum growth rate

In experiment 1, the nitrate uptake rates of P. micans at a mean NO₃ concentration of 80–100 μM were significantly affected by a light intensity of 10 to 200 μE m⁻² s⁻¹ (p < 0.01, ANOVA) (Fig. 5A). Similarly, the maximum growth rates of P. micans were also significantly affected by light intensity (p < 0.01, ANOVA) (Fig. 5B).

Fig. 5

Nitrate uptake rate (UR, pM cell⁻¹ d⁻¹) (A) and growth rate (GR, d⁻¹) (B) of Prorocentrum micans as a function of light intensity (μE m⁻² s⁻¹) when UR and GR at each light intensity were saturated.

Comparison of the results from the 3 different methods

This study shows that the maximum nitrate uptake rates (V_max) and half saturation constants for nitrate uptake (K_s-NO3) of P. micans at 20 μE m⁻² s⁻¹ measured using the nutrient repletion method are similar to those measured using the nutrient depletion method. Thus, the new repletion method of measuring the nitrate uptake rate and K_s-NO3 using cells maintained under a nitrate repletion condition gives results similar to that using cells maintained under a nitrate depletion condition. However, the V_max of P. micans originally maintained under nitrate repletion at 50 μE m⁻² s⁻¹ obtained in this study (7.49 pM cell⁻¹ d⁻¹, equivalent to 21 × 10⁻³ h⁻¹) is much lower than that at 56–63 μE m⁻² s⁻¹ under nutrient depletion (113 pM cell⁻¹ d⁻¹, equivalent to 319 × 10⁻³ h⁻¹) as observed by Qi and Zhu (1994), while the K_s-NO3 of P. micans originally under nitrate repletion at 50 μE m⁻² s⁻¹ in this study was greater than that at 56–63 μE m⁻² s⁻¹. Qi and Zhu (1994) did not provide information on incubation time, light : dark cycle, and initial cell abundance, but only indicated that they used a modification of the method of Eppley (1973). Therefore, it is difficult to know the exact incubation time. However, the conventional and partially modified methods usually incubated cells for <3 h without a light : dark cycle in their nitrate uptake experiments (Table 3), and thus the incubation time in the study of Qi and Zhu (1994) was likely to be shorter than that in our experiment and may not have considered the light and dark cycle. Cells maintained in a nitrate depletion condition may take up nitrate rapidly and with a short incubation time regardless of the light and dark cycle and may have a relatively high maximum uptake rate. However, the physiology and behaviors of red tide dinoflagellates, such as nutrient acquisition, cell division, and vertical migration, are known to be affected by the light and dark cycle or circadian rhythm (Suzuki and Johnson 2001, Van Dolah et al. 2007). Therefore, the nutrient depletion (long and short incubation time) method may apply to the conditions under which red tide causative cells are introduced from oligotrophic offshore surface waters to eutrophic coastal waters or when highly concentrated nutrients due to heavy rains are discharged from rivers to coastal waters after nutrient depletion conditions persist (Fig. 6A). However, this new nutrient repletion method may apply to the conditions under which nutrients are added to eutrophic waters, and high nutrient conditions in surface waters persist for a long time (Fig. 6B). In addition, this new method can be applied to red tide species that inhabit deep, but partially lit, eutrophic waters below the thermocline (Fig. 6C & D). In natural environments, red tide species are exposed to nutrient repletion or depletion conditions, and both conditions alternate. Therefore, the results of both experiments obtained by using nutrient replete and depleted cells can be applied to certain waters where both nutrient replete and depleted conditions occur.

Table 3

The red tide species, taxonomical group (TG), nitrate concentration range tested (NCR), incubation time (IT), light intensity (LI), and temperature (T) in previous studies measuring NO₃ uptake in the laboratory or field and this study

Fig. 6

Diagrams of Prorocentrum micans cells under N repletion and depletion conditions and then experiencing high and low nutrient waters. (A) P. micans cells experiencing high N after being maintained in N depleted waters. (B) P. micans cells are transported from N depleted waters offshore to N replete waters near the shore. (C) P. micans cells continuously maintained in N replete waters near the shore. (D) P. micans cells migrating between well-lit oligotrophic surface waters and dim eutrophicated deep waters. NDC, nitrogen depleted cell; NRC, nitrogen replete cell; HN, high N concentration water; LN, low N concentration water; HL, high light intensity; LL, low light intensity.

Effects of light intensity on the maximum nitrate uptake rate and half saturation constant for nitrate uptake

Prior to this study, there have been no studies on the effects of light intensity on the K_s-NO3 and V_max of nitrate of red tide organisms. Eppley et al. (1969) measured the NO₃ uptake of phytoplankton with the assumption that K_s-NO3 is not influenced by irradiance. Later, most studies measured NO₃ uptake at 1 light intensity. Sinclair et al. (2006, 2009) measured NO₃ uptake at 2 different light intensities in each study and showed different rates between 2 light intensities. However, this study explored the effects of 6 different light intensities. The results of this study clearly show that the uptake rate of P. micans is affected by light intensity. The maximum swimming speed of P. micans is 380 μm s⁻¹, so it is theoretically able to descend to 14 m during 10 h of travel (Jeong et al. 2015). P. micans usually ascends at sunrise, but descends at sunset (Hasle 1950). Therefore, it is likely to experience a wide range of light intensity. Thus, it may take up nitrate at different rates at different depths when vertically migrating through the water column. In particular, the NO₃ uptake rates of P. micans at darkness were almost negligible. Therefore, it may not take up NO₃ much after sunlight completely disappears from the water column.

The light intensity at which the highest K_s-NO3 and V_max are achieved is the same as that at which the highest growth rate is achieved (i.e., 100 μE m⁻² s⁻¹ for P. micans). Thus, it is reasonable to measure the K_s-NO3 and V_max of nitrate at the light intensity after the optimal light intensity for growth is discovered because it is generally easier to measure growth rates than nutrient uptake rates.

Comparison of the maximum nitrate uptake rate and half saturation constant for the nitrate uptake of red tide dinoflagellates

The K_s-NO3 of 10 red tide dinoflagellate species measured using cultures has been reported so far, and either the V_max or volume-specific maximum uptake rates of nitrate (V_max-s) of 7 red tide dinoflagellate species have been reported (Table 2). Within the genus Prorocentrum, the V_max of the Korean strain of P. micans at 100 μE m⁻² s⁻¹ obtained in this study is 380% higher than that of P. minimum at a similar light intensity, but the volume specific maximum uptake rate of the Korean strain of P. micans was lower than that of Prorocentrum donghaiense (Table 2). The larger size of P. micans may be partially responsible for the higher V_max and lower volume-specific maximum uptake rate compared to those of P. minimum and P. donghaiense. The K_s-NO3 of the Korean strain of P. micans at 100 μE m⁻² s⁻¹ is slightly greater than that of P. minimum, but much greater than that of P. donghaiense (Table 2). Therefore, when nitrate concentrations are <4 μM, P. donghaiense may take up nitrate and grow rapidly to form red tide patches, while P. micans may not do so. Therefore, P. donghaiense may inhibit P. micans red tide outbreaks by causing nitrate depletion or by maintaining a low nitrate concentration. However, even when nitrate concentrations are >4 μM, the abundance of P. donghaiense is expected to be greater than that of P. micans because the growth rate of P. donghaiense is twice the maximum growth rate of P. micans. The maximum swimming speed of P. micans and, in turn, the depth it reaches through 10 h travel (i.e., 380 μm s⁻¹ and 14 m) are greater than those of P. donghaiense (i.e., 280 μm s⁻¹ and 10 m). Thus, P. micans is likely to outgrow P. donghaiense when the thermocline depth is deeper than ~10 m.

Implications for red tide dynamics

P. micans is a common red tide species in the waters of many countries (Allen 1941, Uchida 1981, Pybus 1990, Shumway 1990, Zheng-fang et al. 1995, Shankle et al. 2004, Peña-Manjarrez et al. 2005, Jeong et al. 2013, Kang et al. 2013, Park et al. 2013a); it is 1 of 3 major red tide dinoflagellates in the waters of southern California (i.e., Akashiwo sanguinea, Lingulodinium polyedrum, and P. micans) (Allen 1941, Cullen and Horrigan 1981, Shankle et al. 2004). It is likely to compete severally with many phototrophic species to form red tides. However, it may have difficulty in forming red tide patches in eutrophicated coastal waters due to lower growth rates, lower V_max, and higher K_s-NO3 than co-occurring red tide species. Its abilities to conduct mixotrophy and produce allelopathic materials over competitors may enable it to form red tides (Jeong et al. 2005, Ji et al. 2011); it is known to be able to feed on many algal prey species and inhibit the growth of the diatom Skeletonema costatum and the dinoflagellate Karenia mikimotoi using allelopathic materials (Jeong et al. 2005, Ji et al. 2011). Therefore, to understand the red tide dynamics of P. micans in natural environments, both NO₃ uptake from ambient waters through photosynthesis and organic nitrogen through mixotrophy should be taken into consideration.