Article pubs.acs.org/est

Effect of Natural Organic Matter on Iron Uptake by the Freshwater Cyanobacterium Microcystis aeruginosa M. Fujii,†,‡ T. C. Dang,‡ M. W. Bligh,‡ A. L. Rose,§,‡ and T. D. Waite*,‡ †

Department of Civil Engineering, Tokyo Institute of Technology, 2-12-1-M1-4 Ookayama, Tokyo, Japan School of Civil and Environmental Engineering, The University of New South Wales, Sydney, NSW 2052, Australia § Southern Cross GeoScience, Southern Cross University, Lismore 2480, Australia ‡

S Supporting Information *

ABSTRACT: The mode of Fe uptake by the cyanobacterium Microcystis aeruginosa cultured in Fraquil* (pH 8) containing Suwannee River fulvic acid (SRFA) was examined using short-term radiolabeled 55Fe uptake assays and a kinetic model that describes extracellular Fe transformations. Both Fe(II) and Fe(III) uptake rates decreased substantially with increasing SRFA concentration as the availability of unchelated Fe decreased due to complexation by SRFA. Fe uptake rates under illuminated conditions were comparable to or slightly higher than those observed in the dark at the same Fe:SRFA concentration ratio, in contrast to results for systems containing ethylenediaminetetraacetic acid where Fe uptake rates were much greater under illumination than in the dark. The limited effect of light principally resulted from the relatively high rates of thermal dissociation and dark reduction of Fe(III) bound to SRFA and complexation of photogenerated Fe(II) by SRFA. Our findings imply that Fe uptake by M. aeruginosa at a fixed total Fe concentration of 200 nM is close to saturation when fulvic acid is present at concentrations near those typically found in natural waters (< ∼5 mg·L−1), with cellular growth likely to be limited by Fe availability only when natural organic matter is present at very high concentrations (>25 mg·L−1).

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INTRODUCTION

The mode of Fe uptake by phytoplankton has been investigated by numerous researchers in both freshwater and seawater media. Studies over the last few decades have found that organically complexed Fe species, including complexes with metal buffering ligands used in algal culturing media, are too large or hydrophilic to directly permeate the outer plasma membrane of eukaryotic and prokaryotic phytoplankta. Thus, Fe availability for uptake by phytoplankton is typically described as a function of unchelated Fe concentration rather than total or chelated Fe concentration in current models.19−21 Although Fe bound to some specific biogenic molecules such as siderophores and citrate may be recognized by bacterial outermembrane receptors and actively transported to intracellular compartments via energy-dependent processes,22,23 recent studies have indicated that, even under Fe-limited conditions, siderophore-independent uptake (i.e., uptake of unchelated Fe) is dominant for freshwater cyanobacteria such as Microcystis24 and Anabaena sp.25 The lack of siderophore-associated genes in a number of prokaryotic phytoplankton26 confirms the conclusion that siderophore-independent Fe uptake is important in many cases. There is also increasing evidence

Iron (Fe) is an essential micronutrient for the growth of almost all organisms. Cyanobacteria have a particularly high requirement for Fe compared to other microorganisms due to the critical role of Fe in metabolic processes including photosynthesis, respiration, nitrogen fixation, and regulation of intracellular reactive oxygen species.1 Because of the low solubility of inorganic ferric iron (Fe[III]) at circumneutral pH (∼10−11 M at pH 7.5−92), the majority of dissolved Fe(III) in natural waters is present in the form of complexes with natural organic matter (NOM) including humic substances,3−5 siderophores,6 and potentially, other ligands such as polysaccharides.7 In airsaturated surface waters at circumneutral pH, ferrous iron (Fe[II]) is oxidized to more thermodynamically stable Fe(III) relatively rapidly with a half-life of no more than a few minutes. The oxidation process is mediated in many instances by dissolved oxygen8,9 and possibly by secondary produced radicals including reactive oxygen species.10−12 Due to intensive investigations of Fe redox transformations over the past two decades, however, it is now recognized that Fe(II) can be generated at appreciable rates in euphotic waters via photochemical, thermal, and biological reduction of Fe(III) species, mediated by light-induced ligand-to-metal charge transfer (LMCT),13,14 superoxide,15−17 humic substances,18 and cell membrane reductases.19,20 © 2013 American Chemical Society

Received: Revised: Accepted: Published: 365

September 13, 2013 November 17, 2013 November 21, 2013 November 21, 2013 dx.doi.org/10.1021/es404090h | Environ. Sci. Technol. 2014, 48, 365−374

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Table 1. Kinetic Model and Rate Constants Used in This Studya

a Boldface reactions with a light gray band represent important reactions that directly affect calculation of steady-state substrate concentrations, as determined by sensitivity analysis. Fe binding capacity (CFe) of 260 μmol·g−1 (ref 31) was used for SRFA in the calculation of steady-state unchelated Fe concentration. Reactions for Fe uptake are also highlighted. bPhotoreduction rate constant determined in the presence of fluorescent light as

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Table 1. continued described in Supporting Information. cPhotoreduction rate constant determined in the presence of simulated sunlight as described in Supporting Information. dDissolved oxygen concentration in air-saturated water at 27 °C (∼0.26 mM) was used in model calculations. eB is a redox-active organic moiety that results in oxidation of superoxide. Total concentration for B ([BT]) was assumed to be 20 nmol (mg-SRFA)−1.34 fUptake parameters were determined for M. aeruginosa PCC7806 grown in Fraquil* under conditions identical to those employed in this work (i.e., exponential growth phase, light conditions, and nutrient composition, including Fe availability).

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MATERIALS AND METHODS Detailed procedures for the preparation and storage of stock solutions, pH measurement, and cleaning of containers are described in the Supporting Information. Culturing Media. The detailed preparation procedure and nutrient composition of the modified Fraquil medium (Fraquil*) used in this study are described in Supporting Information. Briefly, for long-term cellular incubations, Fraquil* buffered by EDTA was prepared using at least reagent grade salts inside a trace metal clean room supplied with HEPAfiltered air with final concentrations of 0.1 μM Fe and 26 μM EDTA at pH 8. Under these conditions, growth of M. aeruginosa is moderately limited and the specific growth rate decreases by ∼2-fold compared to that with Fe-sufficient Fraquil* medium (∼0.8 d−1) due to the relatively low Fe availability.24,37 For the assay of short-term 55Fe uptake, Fe- and ligand-free Fraquil* was prepared using procedures identical to those described for the long-term culturing medium, except that Fe and ligand were omitted. As described below, the Fe uptake assay was initiated by adding the solution of radiolabeled 55Fe complexed by SRFA or model ligands to the Fe- and ligand-free Fraquil* containing resuspended algal cells. Long-Term Culturing Conditions. A batch culture of the unicellular cyanobacterium Microcystis aeruginosa PCC7806 was incubated under sterile conditions in a temperature- and lightcontrolled incubator (Thermoline Scientific) at 27 °C. The light was horizontally supplied by three cool-white fluorescent tubes (36 W, 28 mm diameter, 1.2 m length, Philips) with a 14:10 light:dark cycle. Fe-limited cells were regularly subcultured into fresh media at an initial cell concentration of ∼104 cells·mL−1. The subculturing was performed approximately 2 weeks after the commencement of incubation when cultures reached stationary growth phase. The specific growth rate was determined to be ∼0.7 d−1. Cell numbers in the cultures were obtained using a Neubauer hemocytometer (0.1 mm depth) under an optical microscope (Nikon, Japan). Light Conditions. All experiments in the light were performed using either the incubator fluorescent lighting or a simulated sunlight source (a ThermoOriel 150 W Xe lamp equipped with AM1 filter). While the former light source provides only visible radiation, the latter is able to simulate the solar spectrum at the Earth’s surface providing UV light. Cell cultures and other abiotic samples were consistently incubated 10 and 40 cm (horizontally) from each light source with total radiation intensities at this distance from the fluorescent light and solar simulator determined to be 178 and 2,393 μmolquanta·m−2·s−1, respectively (Supplementary Figure S1 and Table S1). During dark incubations, the vessels were covered with aluminum foil to prevent light penetration into the solution. A full description of the light conditions used in these studies is provided in Supporting Information. Short-Term 55Fe Uptake Experiments. Prior to the Fe uptake assay, Microcystis cells were harvested by filtering the long-term culture onto a 25 mm diameter, 0.65 μm PVDF

that the reduction of organically complexed Fe(III) to Fe(II) via photochemical21,27 and nonphotochemical19,28−30 processes is critical in increasing Fe bioavailability in Fe-limited environments. Such previous studies undoubtedly provide significant insight into understanding the mode of Fe uptake by phytoplankton. However, the underlying experimental and theoretical findings on biological Fe uptake have almost exclusively been obtained in laboratory incubation assays using model Fe-binding ligands such as ethylenediaminetetraacetate (EDTA),19−21,24,28,29 desferrioxamine B (DFB),19,20 and citrate,24,29 as transformation kinetics for extracellular Fe bound to these ligands are reasonably well-defined. Therefore, an important issue remaining to be addressed is whether the current consensus based on studies using model ligands is consistent with the mode of Fe uptake occurring in natural systems where Fe is generally buffered by structurally and chemically heterogeneous NOM. Transformation kinetics of Fe bound to NOM have been extensively studied over the past decade, particularly for the widely used standard humic substance Suwannee River fulvic acid (SRFA). By using Fe complexed with this heterogeneous material, chemical reactions potentially occurring in natural surface waters including complexation,31,32 dissociation,31−33 and nonphotochemical8,9,11,16,18 and photochemical34,35 redox reactions have been carefully examined. Consequently, a comprehensive set of published rate constants is now available, enabling the prediction of Fe transformations in the presence of SRFA for particular solution conditions (e.g., at pH ∼8, Table 1). In this work, we investigate the mechanism of Fe uptake by the freshwater cyanobacterium Microcystis aeruginosa PCC7806 in the presence of SRFA with particular attention given to examination of the effects of the Fe:SRFA ratio and nonphotochemical and photochemical Fe redox transformations on cellular Fe uptake at pH 8. Microcystis aeruginosa PCC7806 is used in studies here both because of its prevalence as a troublesome organism in freshwaters worldwide and because Fe uptake kinetics have been well-defined for this organism in previous studies using the model Fe-binding ligands citrate and EDTA.21,24 Although there are only a few reports36 of growth limitation of freshwater phytoplankton due to low Fe availability, Fe nutritional status is recognized to affect the synthesis of primary and secondary metabolites including cyanotoxins even under conditions where optimal growth rates are observed.37 As such, investigation of the mode of Fe uptake by this organism is of importance in understanding the environmental and nutritional factors influencing growth and toxin production in cyanobacteria. Proper understanding of ecological function and adaptation of cyanobacteria is required in managing the occurrence of toxic phytoplankton blooms in many water bodies and particularly reservoirs used for drinking water supplies. 367

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Figure 1. Model for kinetics of Fe transformation and uptake by M. aeruginosa at pH 8. Unchelated Fe(II) (i.e., Fe(II)′) is formed due to the photochemical and nonphotochemical reduction of the FeIIISRFA complexes. Unchelated Fe(III) (i.e., Fe(III)′) is also generated from thermal dissociation of the parent complex. Unchelated Fe is subsequently taken up by M. aeruginosa. However, cellular Fe uptake competes with (i) Fe complexation by extracellular Fe-binding ligands such as Fe-binding sites in SRFA and ferrozine (FZ) if present at appropriate concentrations and (ii) Fe(II)′ oxidation by dissolved oxygen. Solid arrows represent major processes under the conditions examined in the short term 55Fe uptake assays, whereas dotted arrows show relatively minor reactions. Rate constants depicted near the arrows correspond to those listed in Table 1.

was determined to be far less chemically reactive than the added 55Fe as discussed in Supporting Information. Thus, the bioavailability of Fe originally present in the SRFA solution is most likely negligible in this work. In addition, adsorption of SRFA to cells and its effect on 55Fe uptake is likely unimportant under the conditions examined in this work, as discussed in Supporting Information. 55 Fe uptake assays were initiated by adding the preequilibrated 55FeIIISRFA at final concentrations of 200 nM Fe and 1−100 mg·L−1 SRFA, respectively (except for the experiments using the solar simulator where 5−100 mg·L−1 SRFA was employed). In all cases except for the experiments where the time course of 55Fe uptake was examined, cells were incubated at 27−28 °C for 2 h (based on the linearity of 55Fe uptake) in the absence and presence of light. For comparison, 55 Fe uptake experiments using model ligands were also undertaken in a manner identical to that described above except that EDTA or citrate was used as the Fe-binding ligand instead of SRFA. After incubation, cells were vacuum-filtered onto 0.65 μm PVDF membrane filters and then rinsed three times with 1 mL of EDTA/oxalate and twice with 1 mL of 2 mM NaHCO3 (total rinsing time was ∼10 min). The filtered cells were then placed in glass scintillation vials with 5 mL of scintillation cocktail (Beckman ReadyScint). The activity was measured in a Packard TriCarb Liquid Scintillation Counter, with scintillation counts (disintegrations per minute) of the samples converted to moles of Fe using concurrent counts of 5−50 μL of 55Feligand stock in 5 mL of scintillation cocktail. Process blanks

membrane (Millipore) during daytime of late exponential growth phase (at densities ∼3 × 106 cells·mL−1). Cells on the filter were washed gently with a chelate solution (50 mM Na2EDTA and 100 mM Na2oxalate at pH 7, hereafter referred to as “EDTA/oxalate”) and subsequently rinsed with 2 mM NaHCO3 in order to remove iron hydroxides and iron adsorbed on cell surfaces via a ligand-promoted process.38 The washing treatment was achieved by passing ∼10 mL of the solutions through the filter for ∼10 min. The washed cells were resuspended into the Fe- and ligand-free Fraquil* medium at cell densities of ∼2−4 × 106 cells·mL−1. In experiments where the effect of chemical treatments on 55Fe uptake were examined, the Fe- and ligand-free culture was prepared in the additional presence of 1 mM ferrozine (FZ) by supplementing chemical stock solutions as described in Supporting Information. Solutions of Fe(III) complexed by organic ligands were made by mixing radiolabeled 55Fe stock with organic ligand stocks as described in Supporting Information. Briefly, in the case of the SRFA complexes, the 55Fe solution (prepared by dilution of 23 mM 55FeIIICl3 in 0.5 M HCl, 185 MBq, Perkin-Elmer, Australia, with 1 mM nonradiolabeled FeIIICl3 in 0.01 M HCl at total Fe concentration of 1.9 mM), was mixed with an appropriate volume of 10 g·L−1 SRFA (International Humic Substance Society) in the bottom of a polypropylene microtube. Two millimolar NaHCO3 was then added to the mixture to maintain circumneutral pH followed by adjustment to pH 8. The 55 III Fe SRFA solution was equilibrated for 24 h in the dark at 25 °C before use. Although SRFA inherently contains 1.5 nmolFe·(mg-SRFA)−1, most of the Fe originally present in SRFA 368

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using an incubation time of 2 h. Iron uptake rates determined from these assays for a range of SRFA concentrations (while maintaining total Fe concentration constant) revealed that uptake rates under dark and light conditions decreased with increasing SRFA concentration (Figure 2A). Iron uptake rates determined under fluorescent light increased by only 1.1−1.4-fold compared to dark uptake for [SRFA] ≥ 5 mg·L−1 even though these 55Fe uptake rates were substantially below the maximum uptake rate by more than 2fold (Figure 2A and B). In simulated sunlight, 55Fe uptake rates increased by 2.0- and 2.3-fold compared to dark uptake at SRFA concentrations of 5 mg·L−1 and 25 mg·L−1, respectively, while a slight decrease in Fe uptake was seen at a SRFA concentration of 100 mg·L−1. The uptake in the presence of simulated sunlight for 5−25 mg·L−1 SRFA was enhanced to a greater degree compared to the enhancement observed with fluorescent light. As discussed further below, this result can most likely be attributed to the higher rate of photoreduction of FeIII present in the FeIIISRFA complexes due to the greater photon flux and proportion of UV light in the simulated sunlight compared to the fluorescent light (Supplementary Figure S1). Photo-oxidation of SRFA (into lower molecular weight DOM of different Fe complexation capacity to the original SRFA) followed by dissociation of unchelated Fe may also be important in photomediated Fe transformations under some conditions,41 although this effect was not considered in our kinetic model. These results in the SRFA system contrast with the behavior observed in assays with EDTA and citrate where substantial increases in 55Fe uptake rates were observed in the light (64− 720-fold in the presence of EDTA and 1.8−6.2-fold in the presence of citrate, Figure 2B) even though pFe(III)′ values calculated in the dark were comparable to those in the synthetic ligand systems (9.9−12.5, 12.7−13.2, and 10.0−10.8 for 1−100 mg·L−1 SRFA, 26−100 μM EDTA, and 26−100 μM citrate systems, respectively). Since all cells used were preconditioned in an identical manner, the different effects of light on 55Fe uptake for the synthetic ligands and SRFA are presumably associated with differences in Fe availability resulting from differences in abiotic processes (e.g., photochemical Fe transformation) occurring in these systems, rather than differences in cellular activity (e.g., Fe uptake affinities). Effect of Fe Redox Transformation on 55Fe Uptake. To examine the contribution of Fe redox transformation to cellular Fe uptake, uptake assays were undertaken in the presence of the strong Fe(II) chelator FZ21,24,28,29 (Figure 2C). The presence of this membrane-impermeable molecule resulted in statistically significant decreases in Fe uptake rate (p < 0.05) by 22−63% and 39−79% in the dark and under fluorescent light, respectively, except for 25 mg·L−1 SRFA in the dark. Although there is a concern that use of high concentrations of FZ (≥400 μM) might actively reduce Fe(III)′ and result in artificial inhibition of Fe uptake by simply lowering [Fe(III)′],42 our previous work24 confirmed that use of 1 mM FZ negligibly inhibits Fe uptake by Microcystis in citrate-buffered Fraquil* where Fe(III)′ uptake dominates. Therefore, the decrease in 55 Fe uptake observed here in the FZ-treated system suggests that Fe(III) reduction to Fe(II) played a substantial role in Fe uptake in both the dark and light conditions. Mode of Fe Uptake in the Dark. To investigate the mechanism of dark Fe uptake in further detail, we examined the chemical speciation of Fe for the Fe:SRFA ratios used in this work. The incomplete inhibition in cellular 55Fe accumulation

were determined by performing the procedure in the absence of cells. Kinetic Model for Fe Transformation and Uptake. The kinetic model used for the calculation of Fe chemical speciation and Fe uptake by M. aeruginosa is shown in Table 1 and illustrated in Figure 1. In this work, we have assumed that unchelated Fe (Fe′) is the only form of Fe directly available for uptake by M. aeruginosa. Steady-state concentrations of unchelated Fe ([Fe(III)′]SS, [Fe(II)′]SS, and [Fe′]SS) were calculated over the range of 55Fe uptake assay conditions listed in Supporting Information. While the complete set of reactions is shown, important reactions that directly influence the steadystate concentration of substrate were extracted as shown in boldface in Table 1 via sensitivity analysis described in Supporting Information. Michaelis−Menten-type saturation theory with published uptake parameters was then used to calculate the rate of cellular Fe uptake (ρs, amol·cell−1·h−1), as follows:

ρS =

ρSmax [S] KS + [S]

(1)

where [S] indicates the steady-state concentration of the biologically available portion of Fe in the extracellular environment (i.e., unchelated Fe), and KS and ρmax represent S the half saturation constant and the maximum uptake rate under conditions examined, respectively. Total uptake rate was calculated by summing Fe(II) and Fe(III) uptake (i.e., ρFe′ = ρFe(II)′ + ρFe(III)′). Although the cellular nutritional status during the preconditioning stage influences short-term Fe uptake kinetics,39 the previously published Fe uptake parameters for M. aeruginosa PCC780624 in Table 1 were determined using cells acclimated under conditions identical to those used in this work.

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RESULTS AND DISCUSSION Effect of SRFA Concentration and Light on 55Fe Uptake. 55Fe accumulated over several hours in cells incubated both in the absence and presence of fluorescent light (Supplementary Figure S3). The nonlinear accumulation evident over several hours of incubation suggests that (i) the concentration of Fe available for uptake varied in a timedependent manner, possibly due to the heterogeneity of Febinding strength or redox properties of SRFA, and/or (ii) cell growth or activity changed over the course of the Fe uptake study. Given that linearity of 55Fe uptake in the synthetic ligand systems is typically maintained for more than a few hours depending on the affinity of the ligand for Fe and the Fe:ligand molar ratio (e.g., 3 h for citrate24 and 4 h for EDTA39), the first factor is likely to contribute substantially to the observed nonlinear 55Fe uptake after 2 h. However, down-regulation of Fe uptake systems in the SRFA medium may also account for the nonlinear uptake, since algal uptake systems for trace metals such as Fe, Mn, and Zn are usually under negative feedback regulation.40 As discussed below, Fe availability is higher in the SRFA medium used in the 55Fe uptake assay than in the EDTA growth medium; as such, the cellular uptake systems may down-regulate over the course of the 55Fe uptake assay as intracellular Fe pools increase. Factors accounting for the nonlinear uptake were not examined further in this work. However, the initial Fe uptake rate was determined by linear regression analysis of data collected during the initial 2 h of incubations, and further 55Fe uptake assays were undertaken 369

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formation of FeIIISRFA complexes. Therefore, to describe the processes involved in dark Fe(III) uptake in a simplified manner, the steady-state concentration of Fe(III)′ present in the culture medium ([Fe(III)′]SS) was calculated solely from the balance of dissociation and formation of FeIIISRFA complexes. Given the chemical heterogeneity of fulvic acid, the various Fe-binding sites present in fulvic acid must be taken into account for the rigorous calculation of [Fe(III)′]SS. These binding sites are classically characterized by continuous or discrete ligand models.4 While recognized to be a simplification, the dissociation and complexation reactions (reactions 7 and 8 in Table 1) were described in this work by a model assuming a single ligand class, since this simplified reaction set together with associated rate constants has been shown to successfully describe the kinetics of FeIIISRFA complex formation at pH ∼8 in previous studies:32 kdFe(III)

Fe IIIL ⎯⎯⎯⎯⎯→ Fe(III)′ + L

(2)

k fFe(III)

Fe(III)′ + L ⎯⎯⎯⎯⎯→ Fe IIIL

(3) III

Because of the predominance of Fe SRFA relative to other Fe species, the FeIIISRFA concentration was approximated to be equal to the total Fe concentration in the system (i.e., [FeT] ≈ [FeIIIL]). Thus, [Fe(III)′]SS was calculated by the following equation: [Fe(III)′]SS =

kdFe(III)[Fe IIIL] k fFe(III)[L]

≈

kdFe(III)[Fe T] k fFe(III)([L T] − [Fe T]) (4)

where [L] and [LT] indicate concentrations of free Fe-binding ligand and total ligand, respectively, with [LT] calculated from the product of SRFA concentration (mg·L−1) and Fe-binding capacity of SRFA (CFe = 260 μmol·g−1).31 Rates of Fe(III) uptake, calculated (by substituting [Fe(III)′]SS in eq 1) using independently determined kinetic and uptake parameters, agreed relatively well with measured 55Fe uptake rates in the presence of FZ where a majority of 55Fe is most likely assimilated from the Fe(III)′ pool. Comparison of measured and calculated uptake rates is illustrated in Supplementary Figures S4-A and S4-D with root-mean-squared errors listed in Supplementary Table S3. When the data for 1 mg·L−1 SRFA were excluded, correlation between the calculated and measured Fe(III) uptake rates was improved, suggesting that the contribution of insoluble Fe species such as ferric oxyhydroxides may be substantial for the 1 mg·L−1 SRFA case (resulting in overestimation of Fe(III)′ concentration or Fe(III) uptake) as discussed in further detail below. The reasonable fit for [SRFA] ≥ 5 mg·L−1 supports the notion that the concentration of Fe(III) available for uptake is governed simply by formation and dissociation of FeIIISRFA complexes, consistent with previous work using model ligands EDTA and citrate.24 In this context, the decrease in 55Fe uptake rate with increasing concentration of SRFA (Figure 2A) can be explained by the lower availability of unchelated Fe due to the increase in formation rate for FeIIISRFA complexes. Fe(II)′ generation via reductive dissociation of chelated Fe(III) followed by back oxidation of Fe(II)′ to Fe(III)′ was determined to negligibly contribute to the steady-state Fe(III)′ concentration in the dark SRFA system due primarily to faster production of Fe(III)′ via thermal dissociation of FeIIISRFA, as described in Supporting Information. In contrast this reductive

Figure 2. Effect of light and chemical treatments on 55Fe uptake by M. aeruginosa in the SRFA-, citrate-, and EDTA-Fraquil* systems. 55Fe uptake as a function of (A) SRFA and (B) model ligand concentrations in the dark (□, black bars), fluorescent light (×, white bars), and simulated sunlight (◇, shaded bars). The 55Fe uptake assays were performed at concentrations of 200 nM Fe, 1−100 mg·L−1 SRFA, and 26−100 μM citrate or EDTA. Solid and dashed lines indicate model fits under dark and simulated sunlight conditions, respectively. (C) Effect of ferrozine (FZ) on 55Fe uptake. In the control treatment, Fe uptake assays were undertaken under dark (black bar) or fluorescent light (white bars) conditions at concentrations of 200 nM Fe and 1−25 mg·L−1 SRFA. In the FZ treatments, 55Fe uptake assays were performed under identical conditions except for the additional presence of FZ under dark (gray bar) or fluorescent light (shaded bar) conditions. All short-term Fe uptake assays were performed in Fraquil* for 2 h at a cell density of ∼2 × 106 cells·mL−1. Symbols and error bars represent the mean ± standard deviation from triplicate experiments. In panel C, asterisks indicate that the 55Fe uptake rate in the presence of FZ treatment was significantly different from that of the control at confidence levels of p < 0.01 for ** and p < 0.05 for * using a single-tailed heteroscedastic t test. The term “N.D” indicates that no data were collected for this treatment.

induced by FZ (Figure 2C) suggests that a certain amount of Fe was acquired from the Fe(III) pool. In this situation, the rate of Fe uptake was shown, by sensitivity analysis (Supporting Information), to be controlled by the thermal dissociation and 370

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ligand-to-metal charge transfer (LMCT), superoxide-mediated iron reduction (SMIR), and direct or indirect reduction by other organic and inorganic radicals, with the significance of each pathway likely to depend on the experimental conditions (e.g., solution pH, concentrations of Fe and SRFA and light conditions).12 Sensitivity analysis suggests that the steady-state concentration of unchelated Fe(II) in photolyzed SRFA solution can be accounted for by the balance of thermal and light-mediated III L FeIIIL reduction of FeIIISRFA (kFe red‑dark and kred‑light), oxidation of Fe(II)′ by dissolved oxygen (kFe(II) oxl ), and complexation of photogenerated Fe(II) by SRFA (kFe(II) ) (reactions 1, 2, 9, and f 12 in Table 1), as follows:

complex dissociation pathway appears to substantially contribute to the steady-state Fe(III)′ concentration in sunlit seawater containing EDTA as an iron-binding ligand.43 Dark 55Fe uptake was significantly decreased by the FZ treatment (Figure 2C), suggesting that cellular Fe uptake is accompanied by Fe(II) generation via nonphotochemical reductive process(es). Given that hydroquinone-type species typically present in humic substances are capable of reducing Fe even under dark conditions at circumneutral pH,18 the thermal reduction of Fe by such redox-active moieties in SRFA is most likely responsible for this phenomenon. The traditional FZtrapping method, where Fe(II) formed in the system is determined by measuring the kinetics of FeIIFZ3 accumulation, was employed to determine the first-order rate constant for the III L −5 −1 abiotic dark Fe reduction, yielding kFe s (as red‑dark = 1.3 × 10 described in Supporting Information). Compared to SRFAmediated reduction, the contribution of superoxide-mediated reduction to Fe(II) formation and thus Fe(II) uptake by Microcystis cells was found to be small under conditions employed in our incubations, as shown by sensitivity analysis (Supporting Information). The steady-state concentration of Fe(II)′ ([Fe(II)′]SS) was then calculated by considering SRFAmediated dark reduction and other competing reactions for Fe(II)′, selected via sensitivity analysis (i.e., oxidation to Fe(III)′ and recomplexation to FeIISRFA with second-order rate constants of kFe(II) and kFe(II) , respectively), as follows: oxl f

III

[Fe(II)′]SS =

≈

Fe L III k red ‐ dark[Fe L] Fe(II) [O2 ] + k fFe(II)[L] kox1 Fe L k red ‐ dark[Fe T] Fe(II) [O2 ] + k fFe(II)([L T] − [Fe T]) kox1

III

Fe L Fe L (k red ‐ dark + k red ‐ light)[Fe T] Fe(II) kox1 [O2 ] + k fFe(II)([L T] − [Fe T])

(6)

Steady-state concentrations of unchelated Fe(II) and Fe(III) were calculated using eq 6 and eq 4, respectively, using the published rate constants listed in Table 1. The Fe(III)′ concentration in the light was assumed to be identical to that in the dark. The Fe uptake rates calculated by substituting the unchelated Fe concentrations in eq 1 indicated reasonable agreement with measured Fe uptake under fluorescent light and simulated sunlight (Supplementary Figure S5; root-meansquared error provided in Supplementary Table S3). Incorporation of Fe(II)′ oxidation in kinetic calculations caused a slight (1.25-fold) increase in Fe(III)′ concentration under simulated solar radiation when 1 mg·L−1 SRFA concentration was employed (Supplementary Table S11). However, in the system with higher SRFA concentrations (>5 mg·L−1) where the model is particularly robust due to the negligible contribution of insoluble Fe species, the effect of Fe(II)′ oxidation on unchelated Fe concentrations was calculated to be unimportant, since the recomplexation of photogenerated Fe(II)′ by SRFA is rapid under these conditions. Thus, our kinetic model suggests that the importance of Fe(II)′ oxidation is limited to the case of low SRFA concentrations where recomplexation of Fe(II)′ by SRFA is slower than Fe(II)′ oxidation by oxygen, as described in Supporting Information. As can be seen in Supplementary Figure S6, positive relationships were obtained between lightmediated Fe uptake rates and photoreduction rates for SRFA, with the slope from linear regression decreasing as SRFA concentration increases. Model calculations also revealed that the lower response of Fe uptake to Fe(III) photoreduction at higher SRFA concentrations resulted from a higher rate of removal of unchelated Fe(II) due to complexation by SRFA. However, a relative increase in Fe(II)′ concentration due to photoreduction (by ∼14-fold relative to the dark, Supplementary Table S2) is predicted to only enhance total Fe uptake by 2.6-fold at most, since Fe(III) reduction is already substantial in the dark, and uptake rates of Fe(II) and Fe(III) are comparable in the range of SRFA concentrations examined (ratios of Fe(II) uptake relative to Fe(III) uptake are 0.15−2.0, as shown in Supplementary Table S4). A summary of all assumptions and approximations used in the kinetic model under both light and dark conditions is provided in Supporting Information.

III

≈

Fe(II) kox1 [O2 ] + k fFe(II)[L] III

III

[Fe(II)′]SS =

III

Fe L Fe L III (k red ‐ dark + k red ‐ light)[Fe L]

(5)

[Fe(II)′]SS was determined assuming a single ligand class using rate constants listed in Table 1 (reactions 1, 9, and 12), as this model was found to be appropriate to describe FeIISRFA complexation kinetics in previous work.32 Fe(II) uptake rates were then calculated by substituting the substrate concentration into eq 1. Comparison of calculated values with measurements at identical Fe:SRFA ratios indicated that the proposed model can reasonably account for the measured dark Fe(II) uptake (Supplementary Figures S4-B and S4-E), supporting the notion of facilitated Fe uptake by dark Fe(III) reduction. Similar to Fe(III) uptake, the decreasing trend of Fe(II) uptake with increasing SRFA concentration is reasonably explained by a decline in the concentration of Fe(II)′ available for uptake. In addition to Fe(II) and Fe(III) uptake, total Fe uptake (ρFe′) was well predicted by the models, particularly at [SRFA] ≥ 5 mg·L−1 (Figure 2A, Supplementary Figures S4-C and S4-F). Mode of Fe Uptake in the Light. By using the traditional FZ-trapping method, the rate constant for photochemical III L III reduction (kFe red‑light) of Fe SRFA under simulated sunlight was determined to be greater by ∼6-fold (1.7 × 10−4 s−1) than the rate constant for photoreduction under fluorescent light (2.9 × 10−5 s−1; i.e., only a little higher than dark reduction, see Supporting Information for details) due to the different intensity and spectral quality of the two light sources. Although the detailed mechanism involved in the photochemical reduction of Fe(III) complexed by SRFA is not, as yet, completely understood, the photochemical reduction of FeIIISRFA potentially includes several mechanisms such as light absorption by a chromophoric complex followed by 371

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In the case of EDTA, the magnitude of the effect of Fe(II)′ oxidation on the Fe(III)′ concentration (and corresponding Fe(III)′ uptake rate) depends on the concentration of EDTA: as EDTA concentration increases, the magnitude of the influence of Fe(II)′ oxidation on the increase in Fe(III)′ concentrations decreases. At the EDTA concentrations used in this work (26−100 μM EDTA), the Fe(III)′ concentration is calculated to increase by less than 1.7-fold when the Fe(II)′ oxidation reaction is considered. Therefore, even though the rates of photoreductive dissociation of FeIIIEDTA in the light (1.2 × 10−4 s−1 for simulated sunlight and 8.6 × 10−6 s−1 for fluorescent light) are higher than (by 12-fold) or comparable to the thermal dissociation rate of FeIIIEDTA (1.0 × 10−5 s−1), the large increases in 55Fe uptake under in the light relative to the dark (722-fold for simulated sunlight and 40−64-fold for fluorescent light) cannot be explained by solely considering the increase in Fe(III)′ concentration due to Fe(II)′ oxidation following photochemical dissociation of FeIIIEDTA. In addition to Fe(III)′ concentration, the light-induced increase in Fe(II)′ concentration also needs to be considered in order to effectively explain the large increase in 55Fe uptake in the EDTA and light system. The marked differences in abiotic Fe transformation kinetics in freshwater and seawater media, particularly the slower recomplexation rate of unchelated Fe by Fe-binding ligand in seawater (due to the greater competition of alkali earth metals such as Ca and Mg for Fe complexation by the ligand), are most likely the major reason why the magnitude of the effect of Fe(II)′ oxidation on the steady-state Fe(III)′ concentration and relative importance of Fe(II)′ and Fe(III)′ in Fe uptake was different in the simulated freshwater system we have examined here and the seawater system used by Sunda and Huntsman (2003)43 (see Supporting Information). Environmental Implications of Findings. In this study, unchelated Fe concentrations have been calculated according to reactions identified, using sensitivity analysis, as important determinants of these concentrations (see Supporting Information for assumptions and approximations inherent in the model used here). Using the model, unchelated Fe(II) and Fe(III) concentrations can be calculated as a function of free ligand concentration ([L], Figure 3A). The model-predicted steady-state concentrations of Fe(II)′ are higher than Fe(III)′ concentrations at [L] ≥ ∼10−7 M (corresponding to [SRFA] ≥ 0.39 mg·L−1) by at least 1 order of magnitude under both dark and light conditions. However, the Fe(III) uptake rate was predicted to be higher than the Fe(II) uptake rate in the dark and at the Fe:SRFA ratios examined (Figure 3B), as the affinity of Microcystis for Fe(III) is higher than that for Fe(II).24 At lower ligand concentrations ([L] ≤ ∼10−7 M), [Fe(II)′] and uptake rates were calculated to be almost independent of [L] (as [Fe(II)′] is controlled by reductive dissociation of FeIIISRFA and Fe(II)′ oxidation), while [Fe(III)′] increased with decreasing [L]. [Fe(III)′] is expected to exceed the solubility limit for ferric oxyhydroxides at [SRFA] ≤ 3.1 mg·L−1 ([L] ≤ 8 × 10−7 M). Thus, both organically complexed Fe(III) and insoluble ferric oxyhydroxides are expected to be present at [SRFA] = 1 mg·L−1. Since the latter Fe species is not considered in our kinetic model, the finding that the calculated Fe uptake rates were consistently higher than the measured values at [SRFA] = 1 mg·L−1 (Supplementary Figures S4 and S5) is to be expected. According to the model calculations, Fe uptake rates at pH 8 in the presence of fulvic acid concentrations typically observed in freshwaters are expected to be relatively high (e.g., more than

Figure 3. (A) Simulated unchelated Fe concentrations and (B) simulated Fe uptake rates as a function of SRFA concentration in the dark (solid black line for total Fe, dashed black line for Fe(II), and chained black line for Fe(III)) and under simulated sunlight (solid gray line for total Fe, dashed gray line for Fe(II), and chained black line for Fe(III)). The shaded area represents the range of free ligand concentrations used in the 55Fe uptake assays of this work ([SRFA] = 1−100 mg·L−1 and [FeT] = 200 nM). The double chained line indicates the solubility limit of Fe(III) at pH 8 (> ∼10 pM). Calculation using the kinetic model suggests that Fe(III)′ concentrations at 5 mg·L−1 SRFA or greater are less than 7.4 pM, while the Fe(III)′ concentration is 135 pM in the case of 1 mg·L−1 SRFA.

1.0 amol·cell−1.h−1 at [SRFA] ≤ 5 mg·L−1 or [L] ≤ 1.3 × 10−6 M). Calculated Fe uptake rates and reported cellular Fe quotas under Fe-limited conditions (∼1.0 amol·cell−1)39 suggest that Fe uptake at [SRFA] ≤ 5 mg·L−1 would be sufficient to sustain the optimal growth rate (> ∼0.7 day−1 in Fraquil*); only when fulvic acid concentrations are very high (e.g., ≥25 mg·L−1 or [L] ≥ 6.5 × 10−6 M) would the growth rate of M. aeruginosa likely be strongly limited by low Fe availability in this laboratory culturing system where a fixed Fe concentration was employed. In some natural environments, concentrations of dissolved Fe and NOM exhibit a positive correlation.44 In such cases, therefore, Fe availability would not necessarily decrease as NOM concentration increases. The relatively minor impact of fluorescent light and slightly greater impact of simulated sunlight on 55Fe uptake in a SRFAbuffered system by M. aeruginosa contrast with the substantial effect of light in an EDTA-buffered system. Despite the faster photoreduction rate of FeIIISRFA compared to FeIIIEDTA, calculations reveal that while Fe′ concentrations in the EDTA system increase substantially in the light compared to the dark (e.g., 2,820-fold for 26 μM EDTA), much lower increases are to be expected in the SRFA system (e.g., ∼12−13-fold for 5−25 mg·L−1 SRFA, Supplementary Table S2) because thermal dissociation and nonphotochemical reduction of FeIIISRFA are already substantial in the dark, the thermal dissociation rate of FeIIISRFA is higher than the photoreductive dissociation rate, and Fe(II) is rapidly recomplexed by SRFA. Fe uptake rates in simulated sunlight are predicted to be higher than those in the dark or fluorescent light conditions by up to ∼2.6-fold at the SRFA concentrations examined, in reasonable agreement with observation at SRFA ≥ 5 mg·L−1 where precipitation of Fe(III) is predicted to be negligible (Supplementary Figure S5). 372

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Although the reason for the observed negative impact of simulated sunlight on Fe uptake at 100 mg·L−1 SRFA remains unclear, plausible explanations include a contribution of stronger Fe-binding ligand classes at higher SRFA:Fe ratios, participation of additional photo-oxidant(s) in Fe(II) oxidation in the light,35 and Fe release from photo-oxidized SRFA followed by subsequent Fe(III) precipitation,41 which were not considered in our model. Nevertheless, given the lack of a marked effect of light on Fe uptake in this work, it would seem reasonable to conclude that natural sunlight is unlikely to increase the Fe uptake by M. aeruginosa to a large extent if dissolved Fe is predominantly complexed by fulvic acid. However, while sunlight is likely to have a negligible impact under circumneutral pH and moderate temperatures, caution must be exercised in estimating Fe uptake by M. aeruginosa in the other environments, for example, at much lower temperatures (where thermal dissociation rates of Fe(III) chelate complex decreases) or higher pH (where Fe(II) oxidation rates are significantly higher). In such environments, the importance of Fe(III) chelate photolysis may differ significantly from that under more typical conditions as suggested by Sunda and Huntsman (2003).43 Despite the inherent chemical complexity of fulvic acid, we have been able to reasonably describe Fe uptake in the SRFA system using existing knowledge of underlying chemical mechanisms that influence Fe speciation under these conditions. In real natural environments, however, Fe may occur as a complex mixture of NOM complexes and Fe oxides including colloidal nanoparticles.5,45 In addition, molecular composition and characteristics of NOM including aromaticity and Fe-binding abilities may vary depending on the origin and fraction of NOM.46 Furthermore, NOM may have diverse effects on metal uptake and microbial growth, not only via buffering the activity of important trace metals but also by complexation of toxic metals (e.g., Al) and adsorption to cells.47 Further consideration of such aspects is required if we are to comprehensively understand the role of NOM in metal uptake and microbial growth.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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S Supporting Information *

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ACKNOWLEDGMENTS

This work was partially supported by the Australian Research Council and partners the Sydney Catchment Authority and Water Research Australia (WRA) through Linkage grant LP0883561. Support of a Grant-in-Aid for Young Scientists (A) (25709045) and a bilateral joint research project from the Japan Society for the Promotion of Science is also gratefully acknowledged. 373

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