Oxidation Kinetics in Seawater - American Chemical Society

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Effect of Dunaliella tertiolecta Organic Exudates on the Fe(II) Oxidation Kinetics in Seawater A. G. González, J. M. Santana-Casiano,* M. González-Dávila, N. Pérez-Almeida, and M. Suárez de Tangil Departamento de Química, Facultad de Ciencias del Mar, Universidad de Las Palmas de Gran Canaria, Campus de Tafira, 35017 Las Palmas, Spain S Supporting Information *

ABSTRACT: The role played by the natural organic ligands excreted by the green algae Dunaliella tertiolecta on the Fe(II) oxidation rate constants was studied at different stages of growth. The concentration of dissolved organic carbon increased from 2.1 to 7.1 mg L−1 over time of culture. The oxidation kinetics of Fe(II) was studied at nanomolar levels and under different physicochemical conditions of pH (7.2−8.2), temperature (5−35 °C), salinity (10−37), and dissolved organic carbon produced by cells (2.1−7.1 mg L−1). The experimental rate always decreased in the presence of organic exudates with respect to that in the control seawater. The Fe(II) oxidation rate constant was also studied in the context of Marcus theory, where ΔG° was 39.31−51.48 kJ mol−1. A kinetic modeling approach was applied for computing the equilibrium and rate constants for Fe(II) and exudates present in solution, the Fe(II) speciation, and the contribution of each Fe(II) species to the overall oxidation rate constant. The best fit model took into account two acidity equilibrium constants for the Fe(II) complexing ligands with pKa,1 = 9.45 and pKa,2 = 4.9. The Fe(II) complexing constants were KFe(II)‑LH = 3 × 1010 and KFe(II)‑L = 107, and the corresponding computed oxidation rates were 68 ± 2 and 36 ± 8 M−1 min−1, respectively.

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rich substances, and transparent exopolymeric particles.12,18−22 Recently, Catrouillet et al.23 studied the strong complexation of Fe(II) with organic matter in anoxic conditions suggesting the importance of the organic ligands in keeping Fe(II) in soluble forms. Laboratory studies with individual and axenic cultures are needed in order to improve our knowledge about the role played by the natural exudates on the Fe(II) chemistry. Currently, few studies have addressed this research line. Steigenberger et al.24 and González et al.25 have studied the effect of exudates from Phaeodactylum tricornutum on the Fe(II) oxidation rate, one of the most common diatoms used in laboratory studies. But there are other species that can be used, such as Dunaliella tertiolecta. This species is a biflagellated unicellular marine green alga, and it is one of the marine eukaryotic algae that possess a CO2 concentration mechanism in their cells.26 The special interest of this algae is related to its physiological, biochemical, ecological, and commercial applications.27−29 Other important factors include (I) the ease of culture, (II) the ability of several species to grow over wide-ranging degrees of salinity, (III) the accumulation of extremely high levels of β-carotene, glycerol, proteins, and minerals,29 and (IV) its broad tolerance to heavy metals and pesticides by the production of high amounts of

INTRODUCTION Fe(II) oxidation kinetics has been the goal for a number of studies during recent years1−7 because iron is an essential element for life and it participates in important metabolic processes.8,9 In addition, iron may react with reactive oxygen intermediates (O2•− and H2O2)7,10,11 and plays an important role as a detoxifying agent for microorganisms.10 In the ocean, iron can be found as Fe(II) and Fe(III), with Fe(III) being the more stable form. The oxidation of Fe(II) by different reactive oxygen species has been previously described including the presence of organic ligands.12 Dissolved Fe(II) is the species most easily incorporated by microorganisms, and they have to improve their mechanisms to maintain Fe(II) in solution longer.13 Thus, unusual Fe(II) concentrations have been measured in surface seawater for many places in the ocean (from 0.5 to >1 nM)14−17 where the presence of Fe(II) has been explained by the role played by organic compounds in solution, photoreduction, and physical conditions such as temperature. Moreover, kinetic studies of Fe(II) with different natural organic matter and with selected organic compounds have shown Fe(II) binding differed from one organic matter to another. Therefore, the organic ligands excreted by microorganisms play a key role in the chemistry of iron in natural waters. Several substance classes have been reported to be capable of reacting with trace metals: the carboxylic and polyphenolic groups, the amino acid groups (glutathione-like), humic substances, microbiologically modified organic matter, sulfur© 2014 American Chemical Society

Received: Revised: Accepted: Published: 7933

March 17, 2014 June 16, 2014 June 18, 2014 June 18, 2014 dx.doi.org/10.1021/es5013092 | Environ. Sci. Technol. 2014, 48, 7933−7941

Environmental Science & Technology

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peptides and phytochelatins.30,31 D. tertiolecta has also been shown to release extracellular ligands that bind copper, cadmium, and lead.32,33 The genus Dunaliella is one of the most abundant types of marine phytoplankton34 and is responsible for most of the primary production in hypersaline environments worldwide such as saline lakes and brine or in closed seas such as the Dead Sea35. The aim of this research is to study the effect of organic exudates excreted by D. tertiolecta on the Fe(II) oxidation rate as a function of the concentration of natural ligands, pH, temperature, and salinity. The Marcus theory has been applied to the Fe(II) oxidation rate in the presence of the natural ligands excreted by D. tertiolecta over growth to improve the developed kinetic model and in order to compute the speciation and contribution of the individual rates on the overall Fe(II) oxidation process.

flask containing ferrozine (50 μL, 0.01 M), acetate buffer (2 mL, pH 5.5) and NaF (50 μL, 7.1 × 10−4 M). The absorbance reading was stable for over 30 min. The addition of Fe(II) (25 nM) to the sample corresponded with the zero time of reaction. The system used to measure the Fe(II) at nanomolar levels consisted of a 5 m long waveguide capillary flow cell (World Precision Instruments), connected to the UV−vis detector USB4000 (Ocean Optics) and a halogen light source (HL-2000FHSA from Mikropack). The system was connected via optical fiber. The spectra were registered using OOIBase 32 software (Ocean Optics). The samples were injected through the column using a peristaltic pump (EXPECTEC Perimax 12) with a flux of 1 mL/min. The apparent rate constant kapp (M−1 min−1) was computed considering

EXPERIMENTAL SECTION Chemicals. The Fe(II) oxidation rates were studied in seawater collected off the coast of Gran Canaria (The Canary Islands, Spain). The experiments were carried out using a stock Fe(II) solution (4 × 10−4 M) prepared using ammonium iron(II) sulfate hexahydrate (Sigma) acidified at a pH of 2 with Suprapur HCl (Sigma) in NaCl (0.7 M). The initial concentration of Fe(II) was kept constant at 25 nM in all of the studies. All solutions were prepared with Milli-Q (18 MΩ) and filtered through 0.1 μm to avoid the collapse of the analytical system. All chemicals used for the Fe(II) determination were trace analytical grade. Oxidation Experiments. The Fe(II) oxidation experiments were carried out in a glass thermostatic vessel (250 mL) as a function of the pH, temperature, and salinity both for the seawater control (seawater with f/2 nutrients) and for the seawater enriched with natural organic exudates from D. tertiolecta. The experiments were performed under air-saturated conditions, aerating the solution with pure air for 1 h before and during the experiments. The samples were stirred with a Teflon-coated magnetic stirrer. After bubbling, the pH was adjusted to the desired value (±0.01 pH units) with small additions of suprapure HCl 0.1 M using an automatic titrate system (Titrino 719S, Methrom). The pH was measured on the free hydrogen ion scale, pHF = −log[H+] with an Orion pHmeter and calibrating the combination electrode (Ross Combination, glass body) with tris(hydroximethyl)aminomethane (Tris)−artificial seawater buffers.36 The buffers were prepared in 0.005 mol kg−1 Tris and Tris−HCl in artificial seawater. The effects of temperature and salinity on the pK* of the Tris-buffers were also taken into account in this study.36 The temperature was controlled via an AG-2 bath over the ranges used in the experiments (5−35 ± 0.02 °C). The seawater salinity was determined by a salinometer (Portasal, 8410A) with error of determination ±0.001. The salinity effect was produced by diluting the samples with Milli-Q water, and the bicarbonate effect was corrected as in Santana-Casiano et al.6 Fe(II) Analysis. The total Fe(II) concentration was measured spectrophotometrically using the ferrozine method.37 In order to work at nanomolar levels of Fe(II), a modified version of the ferrozine method was used.5 The oxidation experiments were carried out following this technique because it allows us to follow the full-spectra over time and if any intermediate or interaction is formed, it can be taken into account during the experiment. At each selected time, 10 mL of sample was added to a 25 mL glass

where, under air-saturated conditions, kapp = k′/[O2]. The values of [O2] were determined from the solubility equation.38 The pseudo-first-order kinetic rate constant, k′, was determined for times over half-lifetime (t1/2) and always for R2 ≥ 0.98. The enrichment with organic ligands is properly explained in the Supporting Information (S1 and Figure S1).

d[Fe(II)]/dt = −kapp[Fe(II)][O2 ] = −k′[Fe(II)]

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(1)

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RESULTS AND DISCUSSION The total natural organic ligands excreted during the growth of D. tertiolecta were measured as dissolved organic carbon (DOC) and are plotted in Figure S2 (Supporting Information) together with the growth curve. The natural organic exudates increased with the number of cells in the culture from 2.1 ± 0.1 to 7.1 ± 0.1 mg L−1. This result indicated the natural organic ligand production increases with the cell density during their growth, in accordance with previous results.39−43 Once it was demonstrated that the culture of D. tertiolecta was a satisfactory source of natural organic ligands, the first series of experiments were carried out in order to determine the effect of natural organic exudates produced during the alga growth on the Fe(II) oxidation rate. It was carried out as a function of the DOC concentration produced at constant temperature (T = 25 °C) and pH = 8.0. The kinetic studies were performed in seawater and seawater enriched with natural exudates from the culture. Cells were never present in the oxidation experiments and they were always removed by filtration. Figure 1 shows that the log kapp decreased with cell concentrations reaching minimum values at the maximum DOC produced by cells, 7.1 ± 0.1 mg L−1. At this concentration, the Δlog kapp was 0.41 (M−1 min−1). This is equivalent to a decrease of 61% in kapp. The experimental data were fitted to eq 2, with R2 = 0.992 and a standard error of estimation of 0.01 in log kapp. log kapp,cell = 3.02( ± 0.01) + 0.019( ±0.001)[DOC] − 0.011( ±0.001)[DOC]2

(2)

The presence of natural organic exudates of D. tertiolecta affected the oxidation process and the corresponding apparent oxidation rate constants. The half-lifetime (t1/2) increased from 3.0 min (seawater control) to 8.0 min (7.1 ± 0.1 mg L−1) (Table S1, Supporting Information), confirming that the natural organic ligands produced by microorganisms affected the Fe(II) oxidation.12,24,25 These natural ligands can complex Fe(III) and Fe(II), can oxidize both free and complexed Fe(II), and also favor the reduction of free and organically complexed Fe(III) in the solution, which could increase, decrease, or not affect the iron 7934

dx.doi.org/10.1021/es5013092 | Environ. Sci. Technol. 2014, 48, 7933−7941


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Table 1. Fe(II) Oxidation Rate As a Function of the pH for the Seawater and the Seawater Enriched with Natural Organic Exudates Excreted by the D. tertiolectaa media seawater (control)

seawater enriched with exudates from 5.52 × 107 cell L−1 [DOC] = 3.1 ± 0.3 mg L−1


Figure 1. Fe(II) oxidation rate constant in the seawater enriched with the natural exudates produced by D. tertiolecta at pH = 8.0 and T = 25 °C. The line represents the fitting obtained from eq 2.


redox chemistry. Under the experimental conditions of this study with D. tertiolecta, Fe(II) production from the reduction of Fe(III) was found to be lower than 1 nM during the reaction time at pH 8 (Figure S4, Supporting Information). Santana-Casiano et al.12,18 reported the reduction of Fe(III) in the presence of 1 μM of polyphenols (catechin and sinapic acid) and catechol. Catechin and sinapic acid was identified from exudates of P. tricornutum. They showed that the Fe(II) regeneration, at 50 min of reaction, was 0.9−0.2% (pH = 7.5) and 0.2−0% (pH = 8) for catechin and sinapic acid, respectively. At the pH range from 7.5 to 8.0, the reduction of Fe(III) to Fe(II) by the presence of the ligands excreted by D. tertiolecta was less than 5%, which could be due to either a major role played by the seawater ions Ca and Mg that block the redox process or the presence of organic ligands with lower reductive strength.12,18 The results obtained in the present study suggested the decrease in the Fe(II) oxidation rate has to be directly linked to the effect of the natural ligands on the Fe(II) chemistry. As the Fe(II) oxidation rates decreased as a function of DOC concentration, three different concentrations were selected in order to study the effect of pH, temperature, and salinity on the oxidation process. They were 3.1 ± 0.3, 5.3 ± 0.3, and 7.1 ± 0.3 mg L−1, which were produced by 5.52 × 107, 2.17 × 108, and 5.04 × 108 cell L−1, respectively (days 2, 4, and 8). These concentrations of natural exudates were also considered because they were produced for three different growth stages. pH Dependence. The dependence of the Fe(II) apparent oxidation rate constant, log kapp, as a function of pH was studied in the pH range 7.2 to 8.2 (Table 1, Table S1 and Figure S3, Supporting Information). As has previously been demonstrated,1−3,5−7,44,45 the log kapp decreased as a function of the pH. The log kapp also decreased in the presence of natural exudates at any pH value with greater effect at [DOC] = 7.1 ± 0.1 mg L−1. The log kapp changed from 3.21 ± 0.03 M−1 min−1 (seawater control) to 2.96 ± 0.03 M−1 min−1 (7.1 ± 0.1 mg L−1) at pH 8.2. At pH 7.2, the log kapp changed from 2.22 ± 0.01 M−1 min−1 (seawater control) to 2.06 ± 0.03 M−1 min−1 (3.1 ± 0.3 mg L−1), 2.01 ± 0.02 M−1 min−1 (5.3 ± 0.2 mg L−1) and 1.85 ± 0.01 M−1 min−1 (7.1 ± 0.1 mg L−1). The kapp decreased by 57% and 44% at pH 7.2 and 8.2, respectively, in seawater enriched with natural organic exudates from D. tertiolecta (7.1 ± 0.1 mg L−1).

a

pH

log kapp (M−1 min−1)

t1/2 (min)

7.2 7.5 7.8 8.0 8.2 7.2 7.5 7.8 8.0 8.2 7.2 7.5 7.8 8.0 8.2 7.2 7.5 7.8 8.0 8.2

2.22 2.40 2.76 3.02 3.21 2.06 2.34 2.71 2.97 3.17 2.02 2.30 2.63 2.84 3.10 1.85 2.16 2.44 2.62 2.96

19.9 13.2 5.9 3.0 2.0 28.3 14.9 6.4 3.5 2.2 31.8 16.3 7.6 4.7 2.6 46.5 22.5 11.8 8.0 3.6

The temperature was kept constant (25°C).

The Fe(II) oxidation rate constant as a function of pH and cell concentration was fitted to a second order equation (eq 3), where R2 was 0.993 and the standard error of estimation in log kapp was under 0.05. log kapp,pH = 9.1(± 0.7) − 2.7(± 0.2)pH + 0.24(± 0.01)pH2 − 0.041( ±0.001)[DOC] (3)

The effect of pH on the Fe(II) oxidation rate solely as a result of the exudates was computed (eq 4) by subtracting the log kapp values in the seawater control and those in eq 3 for the three levels of DOC considered in the present study. R2 was 0.981, and the standard error in the estimation in log kapp was 0.14. Δlog kapp,pH = −0.40( ±0.01)pH + 0.076(± 0.002)pH2 + 0.138( ±0.003)[DOC]

(4)

The observed dependence over the whole pH range, determined in the absence and in the presence of natural exudates, showed that changes in the Fe(II) speciation could represent the major process of control as it is reflected in eq 4, which considered only the effect of natural ligands. The t1/2 (Table 1) increased when the exudate concentration increased in solution at the same pH values, indicating that Fe(II) can persist in surface seawater for longer periods of time as a result of the Fe(II)−organic exudate interactions over a wide range of pH values. In this study, the exudates were always produced at natural seawater pH and were then used under the different physicochemical conditions applied in this research. No attempt was made to study the effect of the pH on the amount of exudates produced. Further work on this aspect should be considered in order to understand the effect of a decreasing pH in the ocean.46 7935



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Temperature Dependence. The effect of temperature on the Fe(II) oxidation process in the range 5−35 °C was studied in seawater enriched with exudates of D. tertiolecta, produced at 25 °C and equilibrated at the selected temperatures (Figure 2). The

The energy of activation (Ea) can be computed through the Arrhenius equation using the data from Figure 2.47 In this study, the energy of activation slightly decreased from 89.9 ± 1.8 kJ mol−1 in the seawater control to 84.2 ± 2.6 kJ mol−1 in the seawater enriched with natural organic exudates from D. tertiolecta (7.1 ± 0.1 mg L−1). This Ea is comparable with 95.8 kJ mol−1 found in natural Pacific Subartic seawater.19 These similar values of Ea suggested that the mechanism of reaction is not strongly affected in seawater and in seawater enriched with natural exudates from D. tertiolecta. The slight decrease in Ea when the concentration of ligands increased can be related to a change in the proportion of free and complexed Fe(II) species in solution. Salinity Dependence. The dependence of the salinity on the oxidation rate of Fe(II) was studied for the range of salinities from 10 to 37 (Figure 3). The Fe(II) oxidation rate showed a greater level of dependence due to salinity in the control seawater than in the presence of natural organic exudates from D. tertiolecta.

Figure 2. Fe(II) oxidation rate constant as a function of temperature for the seawater and the seawater enriched with the organic exudates excreted by the D. tertiolecta ([DOC] = 3.1 ± 0.3, 5.3 ± 0.2, and 7.1 ± 0.1 mg L−1) according to Arrhenius plot. The pH was kept constant (8.0). The lines represent the fitting results obtained for eq 5

experimental data showed a linear dependence of log kapp and temperature. The log kapp values were the lowest in seawater enriched with the higher natural exudate concentration (7.1 ± 0.1 mg L−1). The log kapp was 1.90 ± 0.02 (M−1 min−1) at 5 °C, in the seawater control, and its value decreased to 1.82 ± 0.01, 1.77 ± 0.01, and 1.56 ± 0.03 M−1 min−1 for 3.1 ± 0.3, 5.3 ± 0.2, and 7.1 ± 0.1 mg L−1, respectively. The apparent Fe(II) oxidation rate constant decreased by 54% at 5 °C when [DOC] = 7.1 ± 0.1 mg L−1. Fe(II) remained longer in seawater in the presence of organic exudates of D. tertiolecta at the different temperatures considered. At 5 °C, where the Fe(II) oxidation rate is the lowest, the t1/2 was 27.6 min in the control seawater and became 33.5, 37.5, and 57.3 min in the seawater enriched with 3.1 ± 0.3, 5.3 ± 0.2, and 7.1 ± 0.1 mg L−1 of DOC, respectively (Table S1, Supporting Information). In the presence of high exudate concentrations, Fe(II) can remain in cold waters up to 30 min longer compared to the seawater controls. The experimental results were fitted to a linear equation (eq 5), where R2 was 0.993 and the standard error of estimation was 0.08 in log kapp terms. log kapp,T = 18.2( ± 0.5) −

Figure 3. Fe(II) oxidation rate constant as a function of salinity for the seawater and the seawater enriched with the organic exudates excreted by the D. tertiolecta at the three different ligand concentrations ([DOC] = 3.1 ± 0.3, 5.3 ± 0.2, and 7.1 ± 0.1 mg L−1). The pH and temperature were kept constant at 8.0 and 25 °C, respectively. The lines represent the fitting results obtained from eq 7

The experimental data were fitted to a second order polynomial function (eq 7), with R2= 0.980 and a standard error of estimation of 0.08 in log kapp.

4512( ±15) T

− 0.043( ±0.008)[DOC]

log kapp,S = 3.5(± 0.1) − 0.018( ± 9 × 10−3)S + 3.0( ±0.2) × 10−4S2 − 0.07( ±0.01)[DOC]

(5)

(7)

The change in the Fe(II) oxidation rate constant due to the presence of organic ligands was also calculated (eq 6), where R2 was 0.986 and with a standard estimate error of 0.4. Δlog kapp,T = 13( ±2) −

The effect of the organic ligands excreted by D. tertiolecta on the Fe(II) oxidation rate constant compared to the values for the control seawater as a function of salinity was estimated (eq 8) with R2 = 0.970 and a standard error of estimation of 0.08 in log kapp.

3579( ±83) T

+ 0.23( ±0.07)[DOC]

Δlog kapp,S = 2.7( ±0.2) − (1.0( ±0.1) × 10−2)S

(6)

− (2.0( ±0.3) × 10−4)S2 + 0.09( ± 0.01)

The changes in oxidation rate under different temperatures are highly affected by the presence of natural organic ligands.

[DOC] 7936

(8)



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The range of variability in log kapp values in seawater control at the salinities studied was 0.31 ± 0.02 M−1 min−1 whereas in the seawater enriched with exudates, the range was reduced to values of 0.14 ± 0.04 M−1 min −1 for all the natural ligand concentrations. This behavior indicates that the organic ligands play a key role in the Fe(II) oxidation rate stabilization compared to seawater without ligands, and that there is a greater control over the oxidation process exerted by the interaction with the major ionic species. This is also suggested by eq 8 where the variability in the oxidation rate constant is mostly explained by the salinity dependence (eq 7). In the seawater control, the t1/2 increased from 1.3 min at S = 10 to 3.0 min at S=37. In the presence of exudates, this effect was scarce and the difference in the t1/2 between S = 10 and S = 37 amounted to 1.2 min (3.1 ± 0.3 mg L−1), 2.1 min (5.3 ± 0.2 mg L−1), and 2.7 min (7.1 ± 0.1 mg L−1) (Table S1, Supporting Information). The experimental results for the oxidation of the Fe(II) apparent rate constant (log kapp, M−1 min−1) at DOC concentrations (mg L−1), pH (free ion scale), temperature (K), and salinity were fitted to the polynomial function (eq 9)

kapp = 1+

0 ΔG° = −F[EO0 2 → O−2 − E Fe − L]

+ (2.0( ±0.2 )) × 10 )S − 0.050( ± 0.003)

(13)

Fe(III) − L + e− → Fe(II) − L E 0Fe − L

KFe − L =

[Fe(II) − L] [Fe(III) − L][e−]

(14)

(15)

E0Fe−L

Therefore, from eq 16, = 0.32 V. From the Nernst equation and using this E0Fe−L value, the equilibrium constant ratio can be accounted (KFe(III)−L/KFe(II)−L) for the possible complexes present in solution with organic Fe(III) and Fe(II) complexes.

2

log kapp,exudates = 67(± 4) − 14(± 1)pH + 0.98(± 0.07)pH 3962( ±57) + 0.030( ±0.4 × 10−2)S T

− (1.3( ±0.1) × 10−3)S2 + 0.15( ±0.03)[DOC]

−1

where F is the Faraday constant, EO0 2 → O−2 = −0.16 V, and E0Fe−L is the half potential for reduction of the Fe−L complex.

(9)

with R2 = 0.990 and a calculated error of 0.07 in log kapp. In addition, the effect solely due to the presence of cell exudates excreted by D. tertiolecta (log kapp,exudates) is shown in eq 10, where R2 was 0.920 and the standard error of estimation was 0.3 in log kapp.

−

−1

where kd is the diffusion-controlled limit (10 M sec ), kd/ KdZ has a constant value of 0.1,49 and λ is a fitting parameter related with the necessary energy to reorganize the transition state and has been accounted as 103 kJ mol−1 and 132 kJ mol−1 for inorganic49 and organic compounds,48 respectively. R is the universal gas constant and T is the temperature in Kelvin units. The Marcus equation was applied to the experimental data shown in Figure 1, where the Fe(II) oxidation rate was measured as a function of DOC produced by cells in the culture. The free energy was between 39.6 ± 0.4 and 42.91 ± 0.04 kJ mol−1 for λ = 103 kJ mol−1. When λ = 132 kJ mol−1, ΔG0 ranged from 48.5 ± 0.3 to 51.50 ± 0.03 kJ mol−1. These values are in the same order as those measured in natural seawater, 46 kJ mol−1 and 49.7 kJ mol−148,50,51 and for different types of organic ligands in the range of 25.1− 44.5 kJ mol−1.50 The free energy (ΔG0) computed for the oxidation of one Fe− L complex by oxygen is defined as

2

[DOC]

(12) 10

4531( ±12) − 0.016( ±0.9 × 10−3)S T −4

(11)

Fe(II) + O2 → Fe(III) + O•− 2

log kapp = 25.1(± 0.7) − 2.9(± 0.2)pH + 0.26(± 0.01)pH2 −

kd K dZ

kd ⎡ λ ⎛⎜ ΔG° ⎞⎟2 ⎤ ⎢ 4 ⎝1 + λ ⎠ ⎥ exp⎢ RT ⎥ ⎢⎣ ⎥⎦

⎡ KFe(III) − L ⎤ 0 0 ⎢ ⎥ = − E Fe E 0.059 log −L Fe ⎢⎣ KFe(II) − L ⎥⎦

(10)

(16)

E0Fe

This equation allows for the calculation of changes in the apparent oxidation rate under different experimental conditions in the presence of ligands excreted by the D. tertiolecta. In addition, if the exudates excreted by the D. tertiolecta are considered to be model organic ligands in seawater, this equation may be used to compute the fractional contribution of organic exudates in the Fe(II) oxidation rate constant relative to those in natural seawater. Equation 10 indicates that the ligand effect also depends on local water conditions such as pH, temperature, and salinity, as these may also affect the properties of the organic ligands present in the solution. Rate Constants in the Context of Marcus Theory. Marcus theory can be applied in order to describe the effect of organic complexes on the Fe(II) oxidation rate in aquatic environments.48,49 The Marcus equation (eq 11) can be used in order to compute the second order rate constant (kapp in M−1 s−1) if the free energy (ΔG0) for one electron transfer is known,50 which is the case for the reaction eq 12

If the standard half reaction potential for = 0.77 V is used, a KFe(III)−L/KFe(II)−L ratio of 106−108 is computed. KFe(III)−L values in the bibliography ranged from 1012 to 1023;52−55 therefore, a huge range of possible KFe(II)−L values (104−1017) exist. A kinetic model approach has been developed in order to compute the best fit for KFe(II)−L. The Kinetic Modeling Approach. The kinetic model developed from Santana-Casiano et al.6 (Table S2, Supporting Information) was applied with the introduction of new species and contributions and solved by using the Gepasi software.56 The kinetic model took into consideration all the experimental conditions previously discussed in this paper (Table S2, Supporting Information). According with the literature,4,18,23,48 organic ligands can form complexes with Fe(II) as a function of pH affecting the Fe(II) oxidation rate. In order to explain the pH effect on the oxidation kinetics in the presence of ligands, two acidity groups were considered. The best fit for the experimental data gave a ligand speciation described by eqs 17 and 18, even when the proton interexchange 7937



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The kinetic modeling approach used in this work allowed us to compute the Fe(II) speciation and the contribution of the individual species to the overall rate constant. The Fe(II) speciation is shown in Figure 4 for the seawater enriched with

may occur in two different functional groups; that is, LH in eq 17 and 18 can differ due to the heterogeneity of the ligand produced by the algae. LH 2 ↔ LH + H LH ↔ L + H

K a = 10−9.45 −4.9

K a = 10

(this work)

(this work)

(17) (18)

The computed acidity constants that produce the best agreement between the experimental distribution and the model output presented values of pKa,1 = 9.45 ± 0.05 and pKa,2 = 4.89 ± 0.02, respectively. These acidity constants are in close alignment with the carboxyl and polyphenol groups for the surface cells of D. tertiolecta32 and quinone-type compounds.57 These compounds have also been identified in natural seawater and laboratory cultures of macro and micro algae.58,59 In addition, Rico et al.59 identified polyphenols in solid cells as in seawater enriched with exudates, which allow us to suggest that the functional groups identified in surface cells can be found also in solution. Recently, Lopez et al.60 measured the production of polyphenols by D. tertiolecta in seawater that was 9.4 nmol L−1. Moreover, the carboxylic and hydroxo- groups have been described as the most active groups, binding cationic metals, such as copper and iron, in aqueous solutions in numerous aquatic microorganisms.61,62 These groups are two strong bases that complex Fe(II) decreasing the Fe(II) oxidation rate. The model included the Fe(II) complexes for both types of ligands, LH and L (eqs 19 and 20), acting as two different ligands, and the corresponding oxidation processes (eqs 21 and 22). Fe(II) + LH ↔ Fe(II) − LH KFe(II) − LH = (2 ± 1 × 1010)

(this work)

(19)

Fe(II) + L ↔ Fe(II) − L KFe(II) − L = 1.1 ± 0.2 × 107

(this work)

(20)

Fe(II) − LH + O2 → Fe(III) − LH kFe(II) − LH = 68 ± 2 M−1 min−1

(this work)

(21)

(22)

Figure 4. Fe(II) speciation from the kinetic modeling approach: (A) seawater enriched with natural organic ligands extracted from cultures of D. tertiolecta as [DOC] = 3.1 ± 0.3 mg L−1; (B) [DOC] = 7.1 ± 0.1 mg L−1.

The equilibrium (eqs 19 and 20) and rate constants (eqs 21 and 22) indicated above were computed using the Gepasi software and included all the experimental conditions discussed for the three DOC concentrations. These equations reflect the complexation of Fe(II) and natural ligands and the oxidation of the complexed Fe(II) with oxygen. These values of KFe(II)−L are in the range of values accounted by the application of Marcus theory. Although the concentration of natural ligands (DOC) was measured, not all of these ligands are able to react with Fe(II) in solution. In this sense, the total ligand concentrations excreted by many phytoplankton cultures, capable to react with cationic metals, are in the 10−200 nM range. T. weissf logii cultures presented 114 nM, S. costatum produced 106 nM, and P. tricornutum between 11 and 228 nM, over the different phases of culture.25,40,41,59,63,64 The best fitting was found for specific ligand concentrations of 27 ± 3, 72 ± 2, and 185 ± 5 nM corresponding with total DOC concentrations of 3.1 ± 0.3, 5.3 ± 0.2, and 7.1 ± 0.1 mg L−1, respectively. The best complexing constant and rate constants are those presented by eqs 19−22.

natural organic exudates (expressed as DOC) with 3.1 ± 0.3 mg L−1 (Figure 4A) and 7.1 ± 0.1 mg L−1 (Figure 4B). The speciation in the seawater control and 5.3 ± 0.2 mg L−1 are showed in Figure S5 (Supporting Information). In the seawater control,45 the Fe(II) speciation was controlled from pH 6 to 8.2 for Fe2+, changing from 58% to 27%. FeCl+ and Fe(SO4) were also important species for pH values from 6 to 7.6. At pH = 8.0, Fe(CO3) and Fe(H3SiO4)+ played an important role and reached 22% and 19%, respectively. The Fe(II) speciation was radically affected when organic ligands were considered. The speciation was controlled by Fe(II)−LH, which was the most important species at pH 7.0, reaching 83% for 7.1 ± 0.1 mg L−1. The Fe(II)−LH influence decreased as the pH differed from pH 7.0. The Fe(II)−LH was the most abundant species at pH 8.0 when the DOC was over 5.3 ± 0.2 mg L−1. The second Fe(II) organic complex considered in this model (Fe(II)−L) became more important as the ligand concentration increased and when pH reached values of 8 becoming 19% at 7.1 ± 0.1 mg L−1 DOC. The

Fe(II)L + O2 → Fe(III) − L kFe(II) − L = 36 ± 8 M−1 min−1

(this work)

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The oxidation process in the seawater control45 was controlled by the Fe2+ from pH 6.0 (96%) to 7.7 (22.5%), where the Fe(OH)2 began to be the most important contributor to the overall rate constant, with 22.5% (pH 7.7) to 55% (pH 8.5). The presence of natural organic ligands changed not only the oxidation rate but also the contribution of each individual species to the overall Fe(II) oxidation rate constant. The Fe(II)-LH complex was the most important contributor at pH values between 7.0 and 7.5 for [DOC] = 5.3 ± 0.2 mg L−1, and between 6.3 and 7.8 for [DOC] = 7.1 ± 0.1 mg L−1, which control the oxidation process. The highest contribution for the Fe(II)−LH complexes was at pH 7.0, where the value rose to 71% for the exudates produced at the highest DOC concentration (7.1 ± 0.1 mg L−1). The maximum contribution of the Fe(II)-L species was at pH 7.5 and with exudates from 7.1 ± 0.1 mg L−1 when it reached 3%. The oxidation process was controlled at pH ≥ 8.0 by the inorganic species, specifically by Fe(OH)2 and Fe(CO3)22− species. The individual contributions to the overall Fe(II) rate constant demonstrated that Fe(II)−LH played a key role in the Fe(II) oxidation rate. In addition, at pH ≥ 8.0, the oxidation process is controlled by the Fe(II) hydroxide and carbonate species.

model explained the key role played by Fe(II)−organic complexes on Fe(II) speciation in natural waters. The individual contribution of each Fe(II) species, including Fe(II)−LH and Fe(II)−L species, is shown in Figure 5 for

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ENVIRONMENTAL IMPLICATIONS The interaction between Fe(II) and natural ligands produced by phytoplankton species controls the iron biogeochemistry and algal uptake in seawater. The natural ligands excreted by D. tertiolecta retarded the Fe(II) oxidation rate in seawater. The application of Marcus theory and a kinetic model support the Fe(II) complexation with ligands produced by the algae in seawater under oxic conditions, when the direct measurements are not available. The effect of organic matters on Fe(II) chemistry oxidation has been addressed to model organic compounds but cellular exudates are one of the key organic ligands in natural waters. Thus, the effect of these ligands on iron chemistry will improve the iron chemistry that is of a great interest to environmental scientists. The role of organic exudates by D. tertiolecta under ocean acidification conditions must be factored due to the key role played by them on the Fe(II) oxidation rate at lower pH values. As they are responsible for decreasing the Fe(II) oxidation rate, these experiments indicated that the exudates of D. tertiolecta can act as an important Fe(II) supplier to other marine organisms.

Figure 5. Contribution of each Fe(II) species to the overall rate constant for (A) seawater enriched with natural organic ligands extracted from cultures of D. tertiolecta as [DOC] = 3.1 ± 0.3 mg L−1 and (B) [DOC] = 7.1 ± 0.1 mg L−1.

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seawater enriched with natural ligands (expressed as DOC): 3.1 ± 0.3 mg L−1 (Figure 5A) and 7.1 ± 0.1 mg L−1 (Figure 5B). Seawater control and 5.3 ± 0.2 mg L−1 are shown in Figure S6 (Supporting Information). The individual contribution for each Fe(II) species to the overall kinetics rate was computed from the results of the kinetic model and the speciation of the Fe(II) (eq 23), where αi = [FeXi]/[Fe(II)]T and constitutes the molar fraction of each Fe(II) species in the solution. k is the apparent overall rate constant (M−1 min−1) and ki are the individual rate constants for the Fe(II) species.

* Supporting Information Experimental details and data for Fe(II) oxidation rates under different conditions; kinetic model output. This material is available free of charge via the Internet at http://pubs.acs.org.

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*Tel: +34-928-454-448. Fax: +34-928-452-922. E-mail: [email protected]. Notes

+ k FeHCO+3 αFeHCO+3 + kFe(CO3)αFe(CO3) + kFe(CO3)22−

The authors declare no competing financial interest.

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αFe(CO3)22− + kFe(CO3)OH−αFe(CO3)OH− + k FeCl+αFeCl+ + k FeSO4αFeSO4 + k

α

FeH3SiO+4

αFe(II) − LH + kFe(II) − LαFe(II) − L

AUTHOR INFORMATION

Corresponding Author

k = k Fe2+α Fe2+ + k FeOH+αFeOH+ + kFe(OH)2αFe(OH)2

FeH3SiO+4

ASSOCIATED CONTENT

S

ACKNOWLEDGMENTS This study received financial support from the Project CTM2006-09857 and CTM2010-19517-MAR given by the Ministerio de Economiá y Competitividad in Spain. A.G.G.’s

+ kFe(II) − LH (23) 7939



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participation was financed thanks to Grant No. BES-2007-15776 of the Spanish Ministerio de Economiá y Competitividad.

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(22) Bhaskar, P. V.; Bhosle, N. B. Bacterial extracellular polymeric substance (EPS): a carrier of heavy metals in the marine food-chain. Environ. Int. 2006, 32, 191−198. (23) Catrouillet, C.; Devranche, M.; Dia, A.; Coz, M.B-L.; Marsac, R.; Pourret, O.; Gruau, G. Geochemical modeling of Fe(II) binding to humic and fulvic acids. Chem. Geol. 2014, 372, 109−118. (24) Steigenberger, S.; Statham, P. J.; Völker, C.; Passow, U. The role of polysaccharides and diatom exudates in the redox cycling of Fe and the photoproduction of hydrogen peroxide in coastal seawaters. Biogeosciences. 2010, 7 (1), 109−119. (25) González, A. G.; Santana-Casiano, J. M.; González-Dávila, M.; Pérez, N. Effect of organic exudates of Phaeodactylum tricornutum on the Fe(II) oxidation rate constant. Cienc. Mar. 2012, 38 (1B), 245−261. (26) Beardall, J.; Heraud, P.; Roberts, S.; Shelly, K.; Stojkovic, S. Effects of UV-B radiation on inorganic carbon acquisition by the marine microalga Dunaliella tertiolecta (Chlorophyceae). Phycologia 2002, 41 (3), 268−272. (27) Avron, M.; Ben-Amotz, A. Dunaliella: Physiology, Biochemistry and Biotechnology; CRC Press: Boca Raton, 1992. (28) Milledge, J. J. Commercial application of microalgae other than as biofuels: A brief review. Rev. Environ. Sci. Biotechnol. 2011, 10 (1), 31− 41. (29) Ben-Amotz, A.; Abron, M. Glycerol, β-carotene and dry algal meal production by commercial cultivation of Dunaliella. In Algae Biomass, Production and Use; Shelef, G., Soeder, C. J., Eds.; Elsevier/ North Holland Biomedical Press: Amsterdam, 1980; pp 603−661. (30) Hirata, K.; Tsujimoto, Y.; Namba, T.; Ohta, T.; Hirayanagi, N.; Miyasaka, H.; Zenk, M. H.; Miyamoto, K. Strong induction of phytochelatin synthesis by zinc in marine green alga Dunaliella tertiolecta. J. Biosci. Bioeng. 2001, 92 (1), 24−29. (31) Tsuji, N.; Hirayanagi, N.; Okada, M.; Miyasaka, H.; Hirata, K.; Zenk, M. H.; Miyamoto, K. Enhancement of tolerance to heavy metals and oxidative stress in Dunaliella tertiolecta by Zn-induced phytochelatin synthesis. Biochem. Biophys. Res. Commun.. 2002, 293 (1), 653−659. (32) González-Dávila, M.; Santana-Casiano, J. M.; Pérez-Peña, J.; Millero, F. J. Binding of Cu(II) to the surface and exudates of the alga Dunaliella tertiolecta in seawater. Environ. Sci. Technol. 1995, 29 (2), 289−301. (33) Santana-Casiano, J. M.; González-Dávila, M.; Pérez-Peña, J.; Millero, F. J. Pb2+ interactions with the marine phytoplankton Dunaliella tertiolecta. Mar. Chem. 1995, 48 (2), 115−129. (34) Foster, S.; Thomson, D.; Maher, W. Uptake and metabolism of arsenate by anexic cultures of the microalgae Dunaliella tertiolecta and Phaeodactylum tricornutum. Mar. Chem. 2008, 108, 172−183. (35) Oren, A. A hundred years of Dunaliella research: 1905−2005. Saline Systems 2005, 1, 1−14 and references cited therein. (36) Millero, F. J. The pH of estuarine waters. Limnol. Oceanogr. 1986, 31, 839−847. (37) Violler, E.; Inglett, P. W.; Hunter, K.; Roychuodhury, A. N.; Cappellen, P. The ferrozine method revisited: Fe(II)/Fe(III) determination in natural waters. Appl. Geochem. 2000, 15, 785−790. (38) Benson, B. B.; Krause, D. J. The concentration and isotopic fractionation of oxygen dissolved in fresh water and seawater in equilibrium with the atmosphere. Limnol. Oceanogr. 1984, 29, 620−632. (39) Santana-Casiano, J. M.; González-Dávila, M.; Laglera, L. M.; Pérez-Peña, J. Binding of lead to P. tricornutum and T. weissf logii cultures in seawater. Toxicol. Environ. Chem. 1999, 73 (3−4), 191−206. (40) González-Dávila, M.; Santana-Casiano, J. M.; Laglera, L. M. Copper adsorption in diatom cultures. Mar. Chem. 2000, 70 (1−3), 161−170. (41) Strmečki, S.; Plavšićc, M.; Steigenberger, S.; Passow, U. Characterization of phytoplankton exudates and carbohydrates in relation to their complexation of copper, cadmium and iron. Mar. Ecol.: Prog. Ser. 2010, 408, 33−46. (42) Sunda, W. G.; Shertzer, K. W.; Hardison, D. R. Ammonium uptake and growth models in marine diatoms: Monod and Droop revisited. Mar. Ecol.: Prog. Ser. 2009, 386, 29−41. (43) Laws, E. A.; Pei, S.; Bienfang, P.; Grant, S.; Sunda, W. G. Phosphate-limited growth of Pavlova lutheri (prymnesiophyceae) in

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