Nitrogen, phosphorus and silicon uptake kinetics by marine diatom Chaetoceros calcitrans under high nutrient concentrations

Journal of Experimental Marine Biology and Ecology 446 (2013) 67–75

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Nitrogen, phosphorus and silicon uptake kinetics by marine diatom Chaetoceros calcitrans under high nutrient concentrations Chayarat Tantanasarit a, Andrew J. Englande b, Sandhya Babel a,⁎ a Environmental Technology Program, School of Biochemical Engineering and Technology, Sirindhorn International Institute of Technology (SIIT), Thammasat University, P.O. Box 22, Pathum Thani 12121, Thailand b Department of Environmental Health Sciences, School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA 70112-2704, USA

a r t i c l e

i n f o

Article history: Received 11 February 2013 Received in revised form 2 May 2013 Accepted 8 May 2013 Available online 31 May 2013 Keywords: Chaetoceros calcitrans Kinetics Nitrogen Phosphorus Silicon

a b s t r a c t Nitrogen, phosphorus, and silicon uptake rates under very high nutrient concentration were investigated by culturing Chaetoceros calcitrans in varying nitrite and nitrate–nitrogen (NO2− + NO3−-N), phosphate– phosphorus (PO43−-P), and silicate–silicon (Si(OH)4-Si) concentrations to understand nutrient uptake kinetics. Uptake rates were evaluated by using the classic Michaelis–Menten equation. Maximum uptake rates (ρmax) of 0.0529, 0.0088, and 0.0150 pmol/cell/h, and half-saturation constants (Ks) of 623, 133, and 71 μm were determined for NO2− + NO3−-N, PO43−-P and Si(OH)4-Si, respectively. Results of this study indicate that under very high nutrient concentrations, C. calcitrans can effectively reduce nutrients from the surrounding waters. These nutrients are accumulated within intracellular vacuoles as nutrient pools and assimilated by the cell later. Assimilation efficiencies for nitrogen, silicon, and phosphorus were 55.29%, 19.23, and 15.87%, respectively. Uptake rates of NO2− + NO3−-N and Si(OH)4-Si may be applied for other marine phytoplankton under similar conditions of high nutrient conditions such as in shrimp farms or areas contaminated by wastewater. Results using the kinetic model can be applied to estimate nutrient uptake by phytoplankton in estuarine and coastal environments. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Loadings of inorganic nitrogen, phosphorus, and silicon into estuarine and coastal environments originate from land erosion and from the input of untreated municipal wastewater (Lui et al., 2010), sewage effluents (Hydes and Wright, 1999), diffuse pollution, and aquaculture, such as shrimp farming (Boopathy et al., 2007; Jackson et al., 2003). These high levels of nutrient loadings result in eutrophication and harmful algal blooms, including “red tide” (Trainer et al., 2007). Eutrophication can be defined as the process of excessive growth of algae when high concentrations of nutrients, especially phosphates and nitrates, enrich into the water body. Generally, “harmful algal bloom” means aggressive abundance of algae, which produces environmental hypoxia and releases different toxins from a variety of algal species. The term “red tide” is the harmful algal blooms usually caused by specific species of dinoflagellates. Inorganic nutrients represent limiting factors for living organisms, phytoplankton, and bacteria (Lerner, 2004; WEF and ASCE/EWRI, 2006). Phytoplankton blooms can act as a large sink for inorganic nutrients during productive periods (Humborg, 1997; Torres-Valdes and Purdie, 2006). High chlorophyll a concentrations in coastal waters (>15 μg/l) and the mid estuaries (up to 70 μg/l) demonstrate that ⁎ Corresponding author. Tel.: +66 29869009x2307; fax: +66 29869112. E-mail address: [email protected] (S. Babel). 0022-0981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jembe.2013.05.004

phytoplankton can play an important role in removing nutrients, especially nitrogen, from the water column (Howard et al., 1995; Iriarte and Purdie, 1994; Kifle and Purdie, 1993; Leakey et al., 1992). Nutrient uptake rates by phytoplankton have been evaluated by many researchers. In most cases, the Michaelis–Menten equation was employed to describe the maximum nutrient uptake rate (ρmax) (MacIsaac and Dugdale, 1969). Findings indicated that uptake rates depend on many factors. These include cell size (Nishikawa et al., 2010), temperature of solution (Nishikawa et al., 2009), light (Sinclair et al., 2009), fraction of nutrients (Lomas and Glibert, 2000), and most importantly available nutrient concentrations (Baines et al., 2011). Results indicate that when nutrients are spiked into solution, a rapid higher nutrient uptake is observed (Quarmby et al., 1982). Thomas et al. (2010) found that at very high nutrient concentrations in a shrimp farm, enhanced phytoplankton growth was observed. However, there is a lack of research on quantification of nutrient uptake by phytoplankton under very high nutrient concentrations. It is thus important to understand nutrient uptake kinetics under these conditions. Results may assist in understanding phytoplankton growth occurring during shrimp farming operations, as well as that observed in estuarine systems exposed to high nutrient concentrations. Generally, many researchers have documented that nitrogen, phosphorus and silicon are required for phytoplankton growth (Baines et al., 2011; Fuhs, 1969; Ho et al., 2003). These nutrients are assimilated into algal cells as biochemical components, such as protein, nucleic acids,

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and for cell wall formation. However, there is still a lack of information on nutrient assimilation efficiency. Comparison between nutrient uptake by phytoplankton and their assimilation into cells can help in understanding how phytoplankton remove nutrients from aquatic systems. Therefore, the objective of this study is to investigate the kinetics of nutrient uptake by phytoplankton at very high concentrations of nitrogen, phosphate, and silicon. Nutrient assimilation efficiency and physiological response of phytoplankton were observed and analyzed. Pure culture of Chaetoceros calcitrans (C. calcitrans) was chosen as a representative of marine phytoplankton because it is widely present as a dominant marine diatom species found in estuarine and coastal areas and is commercially employed in marine culture (Gorcharoenwat, 2007). High nutrient concentrations were set, corresponding to those observed for wastewaters from municipal discharges and shrimp farming operations (approximately: nitrite and nitrate 200–2,000 μm, and phosphate 50–80 μm), as examples of nutrient excessive loadings which may occur in estuarine and coastal areas (Boopathy et al., 2007; Jackson et al., 2003; Liu et al., 2010). 2. Material and methods 2.1. Culture condition An axenic culture of C. calcitrans was collected from Sriracha Fisheries Research Station, Chonburi, Thailand. Cultures were incubated in a 1 l culture flask using Conway medium (Walne, 1974) and an axenic method. One liter of stock solution was prepared containing: NaNO3 100 g, Na2HPO4 · H2O 20 g, Na2SiO3 30 g, EDTA 45 g, H3BO3 33.6 g, FeCl3 · 6H2O 1.3 g, MnCl2 · 4H2O 0.36 g, thymine (vitamin B1) 0.2 g, cyanocobalamin (vitamin B12) 10 g, and biotin 10 g. The culture medium was prepared using 1 ml of stock solution per liter of seawater. Seawater was filtered through glass microfiber paper (GF/C). Filtered seawater, glassware and equipment were sterilized using an autoclave prior to use. The temperature was maintained in the range 27–30 °C. Salinity and light were set at 30 psu (practical salinity unit) and 300 μmol m −2 s −1 using cool-white fluorescent lamps, respectively. Aeration was carried out in order to keep cells in suspension and provide sufficient CO2 stripping to prevent excessive pH changes. 2.2. Experimental design C. calcitrans from the stock solution (initially subcultured for 3 days), was cultured in 200 ml of sterile seawater with appropriate aeration to yield initial algal cell densities of 0.21 ± 0.03 106 cells/ml. Nutrient concentrations were varied by adding Conway medium 0.1, 0.2, and 0.3 ml to yield concentrations of nitrite and nitrate–nitrogen (NO2− + NO3−-N), phosphate–phosphorus (PO43−-P), and silicate–silicon (Si(OH)4-Si) in the range of 346–2110, 15–203, and 36–405 μm, respectively. A control was employed without nutrient addition to the seawater. Each treatment was done in duplicate, with culturing for seven days. C. calcitrans cells were counted daily using a hemocytometer. Concurrently, water samples were taken to determine dissolved inorganic N, P, and Si depletion. Samples were filtered through filter paper GF/F (pore size 0.7 μm) to remove all suspended particles and bacteria, and were kept frozen at − 20 °C. Nitrite and nitrate–nitrogen (NO2− + NO3−-N), phosphate–phosphorus (PO43−-P), and silicate– silicon (Si(OH)4-Si) concentrations were determined using a SKALAR Nutrient Auto Analyzer (San plusSystem) (sensitivity ± 0.01 μm). At days 0, 3, and 6, C. calcitrans cells from the experimental flasks were filtered through Whatman glass microfiber filter paper (GF/F) using a low pressure vacuum pump. The samples were next dehydrated by freeze-drying. Samples were then fumed with concentrated hydrochloric acid (HCl) for 24 h at room temperature to remove extracellular calcium carbonate or carbohydrate before using

a CHN analyzer (2400 Series II, Perkin Elmer) for carbon and nitrogen analysis. The persulfate digestion method was employed for phosphorus and silicon determination (APHA et al., 2009). 2.3. Data analysis C. calcitrans growth rates were computed using the following equation (Fogg and Thake, 1987): μ¼

lnNt  lnN0 : t

Where: μ is the specific growth rate (day −1), Nt is cell concentration at time t (cells/ml), N0 is initial cell concentration at time 0 (cells/ml), and t is time (days). Doubling times were also calculated from the above equation using Nt = 2N0. Two methods were used to calculate nutrient uptake rate (ρ, pmol (picomol)/cell/h). The first method was based on the reduction of dissolved inorganic nutrient concentrations from solution. Hence, the nutrient uptake rate, ρs (pmol/cell/h), was calculated by measuring nutrient concentration at day 0 (initial) and day 1 of the experiment as shown in the equation below (time units changed to hours). ρs ¼

S0  S1 : N

Where: S0 is nutrient concentration at day 0 (μm), S1 is nutrient concentration at day 1 (μm), and N is the number of C. calcitrans cells at day 1. The second method of calculation considered nutrient accumulation in each phytoplankton cell. Thus, the nutrient uptake rates, ρc (pmol/cell/h), were calculated based on nutrient content in the cells at day 0 (initial) and day 3 using the following equation: ρc ¼

C3 N3  C0 N0 : t

Where: C0 is the nutrient content of C. calcitrans cells at day 0 (pmol/cell), N0 is the number of C. calcitrans cells at day 0, C3 is the nutrient content of C. calcitrans cells at day 3 (pmol/cell), N3 is the number of C. calcitrans cells at day 3, and t is time (hr). All uptake rate results were fitted to the Michealis–Menten equation (MacIsaac and Dugdale, 1969) as follows: ρ ¼ ρmax

S : ðKS þ SÞ

Where: ρmax is the maximum nutrient uptake rate (pmol/cell/h) (calculated from Michaelis–Menten equation), S is the nutrient concentration (μm), and KS is the half-saturation constant (μm) which is the nutrient concentration at ρ = ρmax/2. The maximum specific nutrient uptake rate (Vmax , hr −1) was calculated using the following equation: Vmax ¼

ρmax : Q0

Where: Q0 is the minimum cell quota (pmol of N, P and Si content/ cell). As defined by Droop (1968), cell quota is the weight of internal nutrients per unit biomass and the minimum cell quota (Q0) is the minimum nutrient content inside the cell that is enough for survival and binary fission. This parameter was used to compare nutrient uptake rates for phytoplankton of different sizes (Nakamura and Watanabe, 1983).

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3. Results As shown in Fig. 1, the growth rates of C. calcitrans were found to be initially similar at different nutrient concentrations. However after three days, changes in the growth rates were observed due to variation in nutrient concentration. To better understand the actual C. calcitrans growth rate under favorable nutrient conditions, the specific growth rate (μ) and doubling time (D) were calculated from results obtained over the first three days of growth before deviation in trends occurred. These were determined to be 0.94 ± 0.05 day −1 and 0.74 ± 0.04 day, respectively. Fig. 2 shows that nutrient concentrations were reduced over time in seawater, corresponding with a simultaneous increase in the density of C. calcitrans cells, as reflected in Fig. 1. Thus, nutrient uptake by C. calcitrans resulted in a decreasing nutrient concentration in seawater, as would be expected. Due to cell diffusion, the maximum nutrient uptake rates were observed during the first day and then decreased significantly with time (Fig. 2). At higher initial nutrient concentrations, higher nutrient uptake rates by C. calcitrans were observed. As shown in Fig. 3, the nutrient uptake rate, ρs, calculated from soluble nutrient reduction fits the Michaelis–Menten kinetic model. Nitrogen uptake in Fig. 3 is presented in terms of nitrite and nitrate– nitrogen (NO2− + NO3−-N). The maximum calculated uptake rate (ρs,max) was 0.0529 pmol/cell/h, and the half saturation constant (Ks), was 623 μm. The ρs, max for phosphate–phosphorus (PO43−-P) and silicate–silicon (Si(OH)4-Si) were 0.0088 and 0.0150 pmol/cell/h, respectively. Ks was 133 and 71 μm, respectively, for PO43 −-P and Si(OH)4-Si. In this study the nutrient uptake rate, ρc, indicates nutrient assimilation in C. calcitrans cells. By multiplication of nutrient content in C. calcitrans cells with the total number of C. calcitrans cells, nutrient assimilation can be determined. This calculation is based on changes in C. calcitrans cell densities and nutrient content per cell over time. As indicated by Fig. 1, the growth trends of C. calcitrans were similar only during the first three days. The number of cells then declined in the control and 0.1 ml Conway medium condition due to a lack of available nutrients (Fig. 2). Therefore, the ρc values were calculated based on the difference between nutrient assimilation in C. calcitrans cells between day 0 and day 3. Results are presented in Fig. 4. As shown, the ρc values increased to a plateau at high soluble nutrient concentrations. The uptake rates of nitrogen (N), phosphorus (P), and silicon (Si) fitted Michaelis–Menten kinetics with coefficients of determination (R 2) 0.99, 0.97, and 0.99, respectively. The elemental composition and stoichiometry of C. calcitrans are summarized in Table 1. The initial values of the carbon (C), nitrogen (N), phosphorus (P), and silicon (Si) contents are 3.02 ± 0.3, 0.34 ± 0.03, 0.041 ± 0.010, and 0.85 ± 0.23 pmol/cell, respectively. Consequently, the ratio of C:N:Si:P is 75.83:8.44:21.42:1.

Fig. 2. Changes in nutrient concentrations in seawater, with and without Conway medium.

As indicated in Table 1, the nutrient cell content was found to decrease on day 3 and slightly increase on day 6. Considering day 6 for the control, cell nutrient contents increased greatly in cells with less remaining nutrients in the surrounding seawater (Fig. 2). The concentration of C. calcitrans cells in the control also decreased (Fig. 1). This indicates that nutrients in seawater were not sufficient for C. calcitrans cell binary fission. Thus, nutrients were accumulated within C. calcitrans cells and assimilated later when needed for growth. 4. Discussion

Fig. 1. Changes in cell densities of C. calcitrans (106 cells/ml) under different nutrient concentrations with and without Conway medium.

As shown in Tables 2, 3, and 4, the previous researchers conducted the experiment within a small timeframe to eliminate the different nutrient uptake rates which relate to differences in algal size. However, under realistic conditions, phytoplankton change size over time due to accumulation and storage of internal nutrients in their cells. Once binary fission occurs, the algal size reduces and starts to accumulate nutrients again. This is a typical daily cycle of the changing

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Fig. 3. Nitrite and nitrate (NO2− + NO3−-N), phosphate (PO43−-P), and silicate (Si(OH)4-Si) uptake rates of C. calcitrans, ρs calculated from nutrient depletion in solution, as a function of nutrient concentrations (S). The curves were fitted to the Michaelis–Menten equation.

size of phytoplankton (Yamaguchi, 1992). Thus, this study suggests that to find more practical nutrient uptake rates, the daily data (24 h) is more useful than shorter time periods. Similar experiments designed by Spilling et al. (2010) also support this conceptual idea. They observed silicon uptake rate by using daily changes of silicon concentration in seawater in order to simulate the realistic conditions in the Baltic Sea. 4.1. Nutrient reduction from solution Nutrient uptake by C. calcitrans under excessive nutrient concentrations in seawater was investigated. Results indicate that at increasing nutrient concentrations, phytoplankton exhibit higher nutrient uptake rates. This finding is in agreement with previous researchers on nutrient uptake by phytoplankton under low nutrient concentrations (Baines et al., 2011; Collos et al., 1992; Kudo et al., 1996). Results from this study also show that under very high nutrient concentrations, the uptake rates of NO2− + NO3−-N and Si(OH)4-Si were similar

Fig. 4. Nitrogen (N), phosphorus (P), and silicon (Si) uptake rate of C. calcitrans, and ρc calculated from nutrient assimilation in cells, as a function of nutrient concentrations (S). The curves were fitted to the Michaelis–Menten equation.

to those obtained under lower nutrient concentrations (Lomas and Gilbert, 2000; Spilling et al., 2010; Tarutani, 1999). However, PO43−-P uptake was much lower in this study. Thus, results clearly indicate that when enrichment of nutrients in seawater occurs, phytoplankton may effectively remove nitrogen and silicon at higher rates than phosphorus. Nutrient uptake rates by phytoplankton determined from the reduction of nutrients from solution (ρmax) in C. calcitrans were different when compared to other marine diatom species (Tables 2, 3, and 4). However, the maximum specific nutrient uptake rates (Vmax) in many cases were similar, especially for uptake of nitrate and nitrite nitrogen. Nakamura and Watanabe (1983) observed that different sizes of phytoplankton exhibit different nutrient uptakes. Hence, in this study, Vmax was used for comparison of the nutrient uptake rate as related to phytoplankton size. Results indicate that although there is variability in parameters affecting kinetics such as differences in species, nutrient concentration, and time of observation, the Vmax of

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Table 1 Comparison of nutrient elements in C. calcitrans cultured in varying Conway medium. Sample

Nutrient content (pmol/cell) C

Ratio (mol/mol)

N

P

Si

C:N

N:P

C:P

Si:P

C:Si

N:Si

Day 0 Control Conway 0.1 ml Conway 0.2 ml Conway 0.3 ml Average SD

2.90 3.45 2.99 2.75 3.02 0.30

0.31 0.38 0.31 0.35 0.34 0.03

0.035 0.055 0.037 0.036 0.041 0.010

0.55 0.88 0.83 1.12 0.85 0.23

9.34 9.16 9.70 7.93 9.03 0.77

8.81 6.79 8.38 9.77 8.44 1.24

82.32 62.27 81.31 77.42 75.83 9.29

15.65 15.85 22.54 31.64 21.42 7.52

5.26 3.93 3.61 2.45 3.81 1.16

0.56 0.43 0.37 0.31 0.42 0.11

Day 3 Control Conway 0.1 ml Conway 0.2 ml Conway 0.3 ml Average⁎ SD⁎

1.08 0.79 0.73 0.75 0.76 0.03

0.17 0.13 0.12 0.12 0.12 0.003

0.004 0.006 0.006 0.007 0.006 0.001

0.10 0.06 0.04 0.04 0.04 0.01

6.42 6.20 5.95 6.07 6.07 0.13

39.12 21.90 21.61 17.16 20.22 2.66

251.35 135.89 128.58 104.07 122.85 16.67

22.74 9.42 6.46 5.84 7.24 1.91

11.05 14.43 19.91 17.81 17.39 2.77

1.72 2.33 3.35 2.94 2.87 0.51

Day 6 Control Conway 0.1 ml Conway 0.2 ml Conway 0.3 ml Average⁎ SD⁎

43.32 2.79 1.06 0.95 1.60 1.03

7.01 0.48 0.18 0.14 0.27 0.18

0.597 0.007 0.006 0.007 0.007 0.0003

10.14 0.08 0.04 0.03 0.05 0.03

6.18 5.80 5.82 6.59 6.07 0.45

11.73 71.06 28.52 20.55 40.04 27.16

72.51 412.36 166.03 135.54 237.97 151.79

16.97 11.36 5.93 3.69 6.99 3.95

4.27 36.29 27.99 36.77 33.68 4.93

0.69 6.25 4.81 5.57 5.55 0.72

⁎ Exclude control.

nitrite–nitrogen (NO2− + NO3−-N) uptake was similar to those found in other species (Table 2). The Vmax determined in this research, is similar to that previously reported for the same genus Chaetoceros sp. (Lomas and Gilbert, 2000; Spilling et al., 2010) except for phosphate–phosphorus (PO43 −-P) uptake. The PO43 −-P uptake rate in this study was found to be much lower than that reported for other species (Table 3). This may be because C. calcitrans is much smaller and contains less phosphorus than other species. Moreover, Peters et al. (2006) found that under a phosphorus-limited medium, larger diatoms increase nutrient flux towards the cells by increasing alkaline phosphate activity, in an attempt to scavenge as much phosphorus as possible from the surrounding environment. In contrast, in this research, there was an abundance of external cellular phosphate. Therefore, it was not necessary for C. calcitrans to store phosphorus. Thus, PO43 −-P uptake results from this study may be used to estimate phosphorus uptake rate where PO43 −-P is excessive in marine environments.

As shown in Table 4, the Vmax of silicate–silicon (Si(OH)4-Si) observed in this study was found similar to those commonly observed for other diatoms (Spilling et al., 2010). This may be due to passive uptake of Si(OH)4-Si by phytoplankton. Wischmeyer et al. (2003) found that Si(OH)4 can be transported into the cell through a diffusive boundary layer by diffusion even at low Si concentrations. They also found that only the diffusion process is sufficient to sustain the phytoplankton growth rate. Thus, even though cell size and initial Si(OH)4-Si concentrations are different, the Vmax value determined for C. calcitrans may be used as a base line for marine diatoms in estuarine and coastal areas. 4.2. Nutrient assimilation in C. calcitrans cells For nutrient uptake rates, when calculated from nutrient reduction from seawater and nutrient assimilation in cells, a large difference was observed. As shown in Tables 2, 3, and 4, the maximum

Table 2 Comparison of kinetic parameters for nitrate and nitrite–nitrogen (NO2− + NO3−-N) uptake for C. calcitrans with other marine phytoplankton. Species

Chaetoceros calcitrans Reduction from solution (NO2− + NO3−-N) Assimilation in cell (N) Chaetoceros sp. Thalassiosira weissflogii Skeletonema costatum Eucampia zodiacus Coscinodiscus wailesii Chattonella antiqua Gymnodinium catenatum Karenia brevis Pseudo-nitzschia australis

ρmax

Q0

Vmax

Ks

(pmol/cell/h)

(pmol/cell)

(hr−1)

(μm) 623

0.0529

0.31

0.17

0.0291 0.024 0.310 0.063 0.916 95.5 0.910 6.480 0.670

0.31 0.21 1.93 0.65 1.00 440 8 31.3

0.09 0.11 0.17 0.10 0.92 0.22 0.12 0.21

Note: ρmax Q0 Vmax Ks S0

The maximum nutrient uptake rate (pmol/cell/h). Nutrient content in cell from control. The maximum specific nutrient uptake rate (hr−1). Half-saturation constant (μm). Initial nutrient concentration (μm).

0.11

35 3.10 2.80 0.40 2.92 5.08 2.81 7.59 0.66 0.201

V max Ks

S0

Duration

Reference

1 day

Present study

1 day 20 min 20 min 20 min 30 min 30 min 60 min 30 min 30 min 20 min

Present study Lomas and Gilbert (2000) Lomas and Gilbert (2000) Lomas and Gilbert (2000) Nishikawa et al. (2009) Nishikawa et al. (2010) Nakamura (1985) Yamamoto et al. (2004) Sinclair et al. (2009) Cochlan et al. (2008)

(μm) 0.0003

346–934

0.0027 0.035 0.061 0.250 0.315 0.043 0.043 0.027

346–934 0–40 0–40 0–40 1–100 1–100 11–300 2.5–50 0–50 0.007–2.571

0.522

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Table 3 Comparison of kinetic parameters for phosphate–phosphorus (PO43−-P) uptake for C. calcitrans with other marine phytoplankton. Species

Chaetoceros calcitrans Reduction from solution (PO43−-P) Assimilation in cell (P) Chaetoceros sp. Skeletonema costatum Eucampia zodiacus Coscinodiscus wailesii Chattonella antiqua Gymnodinium catenatum

V max Ks

ρmax

Q0

Vmax

Ks

(pmol/cell/h)

(pmol/cell)

(hr−1)

(μm) 133

0.0088

0.035

0.252

0.0014 1.40 0.04 0.55 59.10 0.14 1.42

0.035 0.470 0.004 0.160 30 1 1.8

0.040 3 10 3.44 1.97 0.23 0.78

55 2.60 0.68 4.85 6.67 1.76 3.4

S0

Duration

Reference

(μm) 0.0019

15–203

1 day

Present study

0.0007 1.154 14.706 0.709 0.295 0.131 0.228

15–203 –⁎ –⁎ 0.25–20 0.25–20 1.5–30 1–20

1 day –⁎ –⁎ 30 min 30 min 60 min 30 min

Present study Tarutani (1999) Tarutani (1999) Nishikawa et al. (2009) Nishikawa et al. (2010) Nakamura (1985) Yamamoto et al. (2004)

Note: ρmax Q0 Vmax Ks S0

The maximum nutrient uptake rate (pmol/cell/h). Nutrient content in cell from control. The maximum specific nutrient uptake rate (hr−1). Half-saturation constant (μm). Initial nutrient concentration (μm).

⁎ Data are not available.

nutrient uptake (ρmax) of nitrogen (N), phosphorus (P), and silicon (Si), based on nutrient content in C. calcitrans cells, was much lower than those calculated based on nutrient reduction from solution. These results indicate that C. calcitrans cells are able to uptake nutrients in larger amounts than needed. Similar results have been reported by previous researchers (Collos et al., 1992; Lomas and Glibert, 2000; Sinclair et al., 2009). It has also been reported that phytoplankton can effectively increase uptake rate of inorganic nutrients at higher nutrient concentrations. Phytoplankton quickly remove nutrients from solution when the external supply is suddenly increased, in order to assimilate for binary fission. They accumulate these large pools of unassimilated nutrient compounds in large internal vacuoles (Antia et al., 1963; Eppley and Coatsworth, 1968). The percentage of assimilation can be calculated by dividing the nutrient uptake rate obtained from the nutrient content in the cell (Vc, max, hr −1) by nutrient uptake rates obtained from nutrient reduction in solution (Vs, max, hr −1) (applied from Wood et al., 2005). As shown in Table 5, nitrogen assimilation efficiency was determined to be higher than for that of silicon and phosphorus (55.29% versus 19.23% and 15.87%, respectively). Many researchers (Quigg et al., 2003; Ho et al., 2003; Leonardos and Geider, 2004) report that

marine diatom cells contain a high amount of nitrogen compounds such as proteins and nucleic acids (DNA and RNA). Such storage phenomena also were reported for nitrogen as luxury consumption (Eppley and Strickland, 1968). A study on biochemical variability of green algae, Tetraselmis suecica, by Fabregas et al. (1985) found that the protein production is higher than the total nitrate uptake under very high nitrate concentrations. Evidently, Sinclair et al. (2009) also observed soluble nitrogen (urea, ammonium and nitrate) uptake by Karenia brevis in batch experiments. Their results found that assimilation efficiency varied between 35% for nitrate and 81% for urea. Thus, nitrogen is required for phytoplankton growth as a major cell component. Assimilation efficiency for phosphorus is much lower than for nitrogen (Table 5). Therefore, this nutrient can be considered as a minor component of marine phytoplankton cells. Culture experiments by Kudo et al. (1996) reported increasing nitrogen content, whereas the phosphorus content remained constant for the marine diatom Phaeodactylum tricornutum. Results indicated that the phosphorus fraction is small. Nevertheless, phosphorus plays a significant role in phytoplankton growth. Phosphorus compounds are assimilated into nucleic acids, proteins, membrane lipid (phospholipids), and energy

Table 4 Comparison of kinetic parameters for silicate–silicon (Si(OH)4-Si) uptake for C. calcitrans with other marine phytoplankton. Species

Chaetoceros calcitrans Reduction from solution (Si(OH)4-Si) Assimilation in cell (Si) Chaetoceros wighamii Skeletonema costatum Diatoma tenuis Melosira arctica Nitzschia frigid Pauliella taeniata Thalassiosira baltica T. levanderi

ρmax

Q0

Vmax

Ks

(pmol/cell/h)

(pmol/cell)

(hr−1)

(μm)

0.0150

0.552

0.026

71

0.0028

0.552

0.005 0.027 0.028 0.030 0.023 0.014 0.022 0.013 0.032

Note: ρmax Q0 Vmax Ks S0

The maximum nutrient uptake rate (pmol/cell/h). Nutrient content in cell from control. The maximum specific nutrient uptake rate (hr−1). Half-saturation constant (μm). Initial nutrient concentration (μm).

2 5.1 3.5 8.1 6.2 4.7 6.5 2.3 6.1

V max Ks

S0

Duration

Reference

(μm) 0.0004

36–405

1 day

Present study

0.0025 0.0053 0.0080 0.0037 0.0037 0.0030 0.0034 0.0057 0.0052

36–405 45 21 45 21 21 21 45 21

1 1 1 1 1 1 1 1 1

Present study Spilling et al. (2010) Spilling et al. (2010) Spilling et al. (2010) Spilling et al. (2010) Spilling et al. (2010) Spilling et al. (2010) Spilling et al. (2010) Spilling et al. (2010)

day day day day day day day day day

C. Tantanasarit et al. / Journal of Experimental Marine Biology and Ecology 446 (2013) 67–75 Table 5 Comparison of nutrient uptake rate, growth rate, and nutrient content in C. calcitrans. Nutrient Content

Reduction from solution

(pmol/cell) Vs, N P Si

0.124 0.006 0.044

max/μ

4.348 6.445 0.665

ρs,

max

0.940 0.156 0.266

Assimilation in cell ∗ D Vc,

max/μ

2.404 1.023 0.128

ρc,

max

Assimilation (%)

∗D

0.517 0.025 0.050

55.29 15.87 19.23

Note: – Nitrogen (N), phosphorus (P) and silicon (Si) contents at day 3. – The specific growth rate (μ) is 0.94 day−1, doubling time (D) is 0.74 day. – The maximum specific nutrient uptake rate calculated based on. • Nutrient reduction from solution is Vs, max (hr−1). • Nutrient content in C. calcitrans cells is Vc, max (hr−1). – The maximum uptake rate calculated based on. • Nutrient reduction from solution is ρs, max (pmol/cell/h). • Nutrient content in C. calcitrans cells is ρc, max (pmol/cell/h). – Assimilation (%) = (Vc, max/Vs, max) × 100.

transport such as ATP for photosynthesis and respiration processes (Fuhs, 1969). Thus, phosphorus is significant for phytoplankton growth even though a small amount is required. In this study, results indicated that silicon is assimilated slightly more than phosphorus. This can be explained by the fact that silicon acts as a limiting factor for diatoms (Baines et al., 2011). Diatoms require silicon for cell wall and frustules formation. Moreover, diatoms play an important role in silicon distribution in estuarine and coastal areas. Roubeix et al. (2008) found that large amounts of silicon were removed by marine diatom blooms when the silicon concentration increases in the estuary. Results, therefore, provide evidence that silicon is significant for marine diatom growth. A similar assimilation efficiency compared with phosphorus was found in this study.

4.3. Comparison of nutrient uptake rates and growth rates The accumulation of large pools of unassimilated nutrient compounds can be explained by the observed differences between the nutrient uptake rate and growth rate (Collos et al., 1992; Eppley and Thomas, 1969; Zehr et al., 1989). This comparison can be evaluated by dividing the specific maximum nutrient uptake rate Vmax (hr −1) by the specific growth rate μ (hr −1). If Vmax/μ is greater than 1, cells take up nutrients faster than they are needed for growth. If Vmax/μ is less than 1, the specific uptake rates are not sufficient to support growth (Goldman and Gilbert, 1983; McCarthy and Goldman, 1979). As shown in Table 5, nitrogen and phosphorus ratios of Vmax/μ are greater than 1, which indicates that C. calcitrans cells effectively uptake these nutrients more than what is needed for growth. Nitrogen and phosphorus are effectively transported into the phytoplankton cells by both passive (diffusion or adsorption) and active (internal or enzyme uptake) kinetics (Eppley and Rogers, 1970; Yao et al., 2011). Higher nutrient uptake rates occur and the nutrients are accumulated in large intracellular pools. It has been observed that diatoms have a high capacity and are well adapted to rapidly take up nutrients (Collos et al., 2005). Sinclair et al. (2009) found that during daylight, the marine phytoplankton (K. brevis) had a high nutrient uptake rate and stored biochemical substrates in their cells. This resulted in all values of Vmax/μ > 1. Results demonstrated that phytoplankton have the ability to uptake and store large quantities of nutrients and later assimilate these into their cells. In contrast, the ratio of Vmax/μ b 1 was observed for silicon. This indicates that C. calcitrans required more silicon than that taken up. Diatoms require silicon for building cell walls and frustules formation. Many researchers (Baines et al., 2011; Brzezinski et al., 2008; Roubeix et al., 2008; Spilling et al., 2010; Sugie et al., 2010) have reported that upwelling currents play a significant role in diatom distribution in

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estuarine and coastal areas with a dramatic removal of dissolved silicon from the water body. Silicon uptake into phytoplankton cell is only by the diffusion process (Wischmeyer et al., 2003), which may not be sufficient for binary fission when diatom blooms occur. The amount of nutrient uptake sufficient for C. calcitrans binary fission can be determined by multiplying the maximum uptake rate (ρmax) with doubling time (D) as presented in Table 5. A comparison between this multiplication (ρmax ∗ D) and nutrient content in cells indicates that ρmax ∗ D values are much higher than the nutrient content in the cells (Table 5). Thus, it can be concluded that C. calcitrans effectively takes nutrients from the seawater and accumulates them in the cells as nutrient pools in excess of the amount that is required for growth. The nutrient assimilation process occurred later within the cell. 4.4. Uptake trend at high nutrient conditions Phytoplankton are able to take up more nutrients at higher nutrient concentrations and reach another maximum value. The nitrate uptake rate by Chaetoceros sp., as reported by Lomas and Glibert (2000), demonstrates that at nitrate concentrations between 0 and 40 μm, the nitrate–nitrogen uptake rate reached a maximum plateau of 0.024 pmol/cell/h. However, at higher nitrate concentrations, 50 to 260 μm, the uptake rate increased in a linear pattern with increasing nitrate concentration in solution. In this study the NO2− + NO3−-N uptake rate was obtained as 0.0529 pmol/cell/h for 346–934 μm concentrations. Results demonstrate that although there is a maximum nutrient uptake rate at a low concentration, phytoplankton have the capacity to uptake additional nutrients and reach a higher maximum uptake rate when the surrounding nutrient concentration is increased. Results indicate that a very high nutrient concentration resulted in a significantly lower nutrient uptake affinity in C. calcitrans. The halfsaturation constant (Ks) of the Michaelis–Menten kinetics model is an index of the affinity for nutrient uptake, and has been suggested to have ecological significance in species competitive interactions (Dugdale, 1967). A low Ks indicates that phytoplankton can effectively uptake nutrients even at very low concentrations. Results from Eppley et al. (1969) indicate that larger cell sizes can achieve higher Ks concentrations. Consequently, large phytoplankton may be at a disadvantage in nutrient uptake competition as compared to smaller phytoplankton since they require more nutrients for growth. In this study, C. calcitrans is a small marine diatom compared with other diatoms. However, Ks for all nutrient uptake rates were very high, which differed from other reported research, as shown in Tables 2, 3, and 4. The affinity index, Vmax/Ks indicates the competitive ability for nutrient uptake (Healey, 1980). Higher values of Vmax/Ks indicate a higher efficiency in nutrient uptake. In this study, results indicate very low values of Vmax/Ks as compared to other researches (Tables 2, 3, and 4). The extremely high Ks and low Vmax/Ks values demonstrate that under the conditions of this study with high nutrient concentrations, C. calcitrans is less competitive due to the presence of ample nutrients. Based on the results of nutrient uptake kinetics of C. calcitrans, nutrient uptake rate can be applied to other marine phytoplankton (Tables 2, 3 and 4). Enrichment of nutrients into estuarine and coastal areas results in a higher nutrient uptake rate until the maximum plateau (Vmax) is reached. Higher values of Ks and lower Vmax/Ks demonstrate that phytoplankton do not need to compete to store an excess of nutrients. As a result, this situation can enhance phytoplankton densities, including C. calcitrans. In contrast, at lower nutrient concentration, the nutrient uptake rate will decrease, but Ks and the affinity index (Vmax/Ks) may not change. Phytoplankton may have interspecies competition. As previously discussed, the lower Ks and higher value of Vmax/Ks demonstrates more effective nutrient uptake by phytoplankton. Hence, under the same range of S0, the comparison between these kinetic values can help in understanding nutrient

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uptake efficiency by specific algal species which may become dominant during algal blooms. These findings assist in the understanding of nutrient restoration by phytoplankton in estuarine and coastal areas. Nutrient concentrations may be used as indicators of potential phytoplankton growth. By knowing nutrient concentrations and the kinetic constants, the corresponding uptake rates can be calculated. By knowing the quantity of diatoms, an overall removal rate can be determined. Results can assist in estimating the time required for a given quantity of nutrient removal. Thus, results from this study can be used to estimate time and productivity of algal bloom including red tide as well as the duration of its prevalence. Results may also be used to assist in the improvement of seawater quality in coastal areas and to enhance commercial phytoplankton mass culture.

5. Conclusions C. calcitrans effectively removes nutrients under high nutrient concentrations. Uptake kinetic under high nutrient concentration can be described by Michaelis–Menten equation. The value of Vmax for NO2− + NO3−-N and Si(OH)4-Si in C. calcitrans was found to be similar for other marine phytoplankton, but PO43−-P was lower due to excess PO43−-P in solution. This demonstrates that Vmax obtained in this study may be used as a baseline for marine phytoplankton for NO2− + NO3−-N and Si(OH)4-Si uptake. However, the PO43−-P uptake rates determined can be applied only at high PO43−-P concentrations. It is also evident from results that C. calcitrans removes and assimilates nitrogen more than silicon and phosphorus. The ρmax values evaluated from nutrient reduction in solution were observed to be much larger than those obtained based on nutrient assimilation in cells. A comparison between uptake rate and growth rate demonstrates that C. calcitrans effectively removed nitrogen, phosphorus, and silicon from solution with consequent storage in vacuoles as nutrients pools, which can be used later, if needed, for growth. The extremely high half-saturation constants (Ks) and low affinity index (Vmax/Ks) obtained in this study indicate that at very high nutrient concentrations, the uptake affinity by phytoplankton was low due to an abundance of available nutrients. Future work may include implementation of species index at high nutrient concentration ranges to evaluate the effect of river run-off, wastewater discharges and harmful algal blooms, including red tide that results in an increase in soluble nutrients in the coastal ecosystem.

Acknowledgment The authors would like to thank the Royal Golden Jubilee Ph.D. program (RGJ) under the Thailand Research Fund (TRF) for providing the funds for the study. We also would like to acknowledge the kind help from Assoc. Prof. Dr. Shettapong Meksumpun in providing valuable comments. We are thankful to Sriracha Fisheries Research Station for providing a pure culture of Chaetoceros calcitrans and seawater, and the Department of Marine Science, Faculty of Fisheries, Kasetsart University for providing laboratory space. [RH]

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