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May 24, 2016 - Synergistic Effects between Biogenic Ligands and a Reductant in Fe. Acquisition from Calcareous Soil. Walter D. C. Schenkeveld,*,†...
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Synergistic Effects between Biogenic Ligands and a Reductant in Fe Acquisition from Calcareous Soil Walter D. C. Schenkeveld,*,† Zimeng Wang,‡ Daniel E. Giammar,§ and Stephan M. Kraemer*,† †

Department of Environmental Geosciences and Environmental Science Research Network, University of Vienna, Althanstraße 14 (UZA II), 1090 Vienna, Austria ‡ Department of Civil and Environmental Engineering, Stanford University, 473 Via Ortega Stanford, California 94305, United States § Department of Energy, Environmental & Chemical Engineering, Washington University in St. Louis, 1 Brookings Drive, CB 1180, St. Louis, Missouri 63130, United States S Supporting Information *

ABSTRACT: Organisms have developed different strategies to cope with environmental conditions of low Fe availability based on the exudation of reducing, ligating, and acidifying compounds. In the context of Fe acquisition from soil, the effects of these reactive compounds have generally been considered independent and additive. However, highly efficient Fe acquisition strategies may rely on synergistic effects between reactive exudates. In the present study, we demonstrate that synergistic effects between biogenic ligands and a reductant (ascorbate) can occur in Fe mobilization from soil. Synergistic Fe mobilization was found for all ligands examined (desferrioxamine B (DFOB), 2′-deoxymugineic acid (DMA), esculetin, and citrate). The size and duration of the synergistic effect on Fe mobilization varied with ligand: larger effects were observed for the sideorphores compared to esculetin and citrate. For DFOB, the synergistic effect lasted for the 168 h duration of the experiment; for DMA, an initial synergistic effect turned into an antagonistic effect after 4 h because of enhanced mobilization of competing metals; and for esculetin and citrate, the synergistic effect was temporary (less than 24 h). Our results demonstrate that synergistic effects greatly enhance the reactivity of mixtures of compounds known to be exuded in response to Fe limitation. These synergistic effects could be decisive for the survival of plants and microorganisms under conditions of low Fe availability.

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INTRODUCTION Iron (Fe) is an essential micronutrient to plants and most micro-organisms.1,2 Despite its relatively high abundance in the earth’s crust and most soils, its bioavailability is often limited as a result of the poor solubility of Fe(hydr)oxide minerals in oxic systems, particularly at circumneutral pH.3 To cope with conditions of low Fe availability, organisms have developed biogeochemical Fe acquisition strategies. Most of these strategies have, in part, a basis in classical mineral-dissolution mechanisms: proton-promoted dissolution, ligand-promoted dissolution, and reductive dissolution.4 In other words, organisms may enhance the rate and extent of Fe mobilization into solution, and hence increase the bioavailability of Fe, through the exudation of protons, ligands, and reductants. Among plants, two Fe acquisition strategies are distinguished as Strategy I and Strategy II Fe acquisition.5 Strategy I is employed by all plants except for grasses and involves the exudation of protons, the up-regulation of a plasma membranebound reductase, and the exudation of low-molecular-weight organic ligands and reductants,2 including organic acids (e.g., citric acid) and phenolic compounds. Known exuded phenolic compounds are flavonoids6 and coumarins.7 Recently, it was © XXXX American Chemical Society

discovered that certain coumarin compounds (e.g., esculetin and fraxetin) have catecholate functional groups, which can form chelate complexes with Fe3+;7,8 in fact, some of the most stable Fe(III) chelates known are the Fe complexes of tricatecholate siderophores like protochelin and enterobactin.9 Strategy II Fe acquisition is based on chelating agents called phytosiderophores.5,10 Phytosiderophores are nonproteinaceous amino acids, which are exuded by graminaceous plants (grasses including wheat, corn, and rice). Upon exudation into the rhizosphere, phytosiderophores can complex and mobilize soil or apoplastic Fe, and the resulting Fe−phytosiderophore complex can be taken up at the root surface through facilitated transport.11 For Fe efficient grass species like wheat and barley, phytosiderophores are exuded in a diurnal pulse release that sets in shortly after the onset of daylight and lasts for a few hours.12,13 Recently, it has been shown that the effectiveness of Strategy II Fe acquisition is compromised by rhizosphere Received: April 1, 2016 Revised: May 23, 2016 Accepted: May 24, 2016

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DOI: 10.1021/acs.est.6b01623 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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to limited selectivity of exudates for the target element (in this case, Fe). Exuded reductants may react with other reducible soil constituents like natural organic matter,26 and ligands may bind other metals like Cu, Ni, and Co.14 Recently, synergistic Mn mobilization from soil by plant-exuded ligands and reductants was found.27 The aim of the present study is to explore synergistic effects in Fe acquisition between biogenic ligands and reductants in calcareous soil environment. In this context, we hypothesize that such synergistic effects can occur but that the nature of the ligand will strongly influence the effect size. For testing this hypothesis, a series of batch interaction experiments were done in which a calcareous soil interacted with a reductant and a ligand, either separately or in combination. Various biogenic ligands were included to represent Strategy I and II Fe acquisition in plants as well as microbial Fe acquisition.

processes like adsorption, biodegradation of the phytosiderophore ligands, and competitive complexation of metals other than Fe and that the success and failure of the Fe acquisition strategy can be interpreted with a conceptual “Window of Fe acquisition” model.14 This model defines a time window during which plants can benefit from phytosiderophore-facilitated elevated Fe concentrations in soil solution. Many microorganisms also exude siderophores for acquiring Fe.15 Microbial siderophores are more diverse than phytosiderophores in terms of structure and chemical affinity for Fe complexation. Over 500 different ones have been identified with hydroxamate, catecholate, and α-hydroxycarboxylate functional groups and molecular masses of 500−1500 Da.16 Stability constants for Fe complexes roughly range from 1020 to 1050.17 In addition to siderophore exudation, certain microbes were also shown to exude reductants (e.g., pyocyanin and phenazine) in relation to Fe acquisition, particularly under Fe deficiency stress.1,18,19 In the context of Fe acquisition strategies, dissolution mechanisms have so far mainly been considered separately and per compound. However, organisms often exude mixtures of compounds, the composition of which can strongly depend on the Fe nutritional status of the organism.20,21 Furthermore, in natural and agricultural ecosystems, microorganisms and plant species often live in close association, each contributing their exudates to the mixture. The compounds that comprise such mixtures may participate in different dissolution mechanisms.1 Synergistic effects between compounds in exudate mixtures may arise. In the context of this work, we define Fe mobilization as synergistic if Fe mobilization by a mixture of compounds is larger than the sum of Fe mobilization by the same compounds at the same concentration in separate treatments. Such synergistic effects in Fe mobilization have been observed in model systems consisting of goethite suspensions and multiple ligands, e.g., organic acids including oxalate, and siderophores or phytosiderophores.22,23 Synergistic effects have also been observed in model systems containing a ligand and a reductant: Banwart et al.24 demonstrated that the combined addition of ascorbate (reductant) and oxalate (ligand) could strongly enhance Fe mobilization in goethite suspensions at low pH (pH < 4). Recently, synergistic Fe mobilization was also demonstrated in goethite suspensions under oxic conditions at circumneutral pH (pH 6) with ascorbate and the bacterial siderophore desferrioxamine B (DFOB).25 In natural systems, such synergistic effects may dramatically affect the rate at which organisms acquire Fe from the surrounding environment. Hence, identifying these synergisms and determining their mechanisms is important for understanding how organisms cope with conditions of limited Fe availability. Synergisms may occur between exudates from a single plant or microorganism species and also between exudates from different species that live in close association, such that the exudates can mix (e.g., bacteria living in the plant rhizosphere or plants growing together). Hence, understanding these synergisms may generate insight into the functioning of ecosystems under Fe limitation and aid crop species and variety selection for agricultural practice, e.g., in intercropping systems. So far, studies examining potential synergistic effects in Fe acquisition between exudates have exclusively focused on model systems. In more complex environmental systems like soils, synergistic effects could easily become obscured, e.g., due

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MATERIALS AND METHODS Materials. Calcareous clay soil was collected from the top layer (0−20 cm) of a site in Santomera (Murcia, Spain). Santomera soil is a good example of an Fe-poor calcareous soil and has been used in multiple studies on Fe deficiency and Fe acquisition.12,28−30 The soil has a pH(CaCl2) (ISO/DIS 10390 soil quality determination of pH) of 7.8, a calcium carbonate content of 500 g kg−1, and a clay content of 300 g kg−1. It is low in organic matter (15 g kg−1), reactive Fe (ammonium oxalate extractable Fe: 0.5 g kg−1), and bioavailable Fe (diethylenetriaminepentaacetic acid (DTPA) extractable: 4.9 mg kg−1). The soil was air-dried and sieved over 2 mm. Selected soil parameters are presented in Table 1. Table 1. Selected Soil Properties of Santomera Soil (Data Previously Reported by Schenkeveld et al.)14 extraction origin (name) region country soil classification pH CaCl2 EC (mS cm−1) SOC (g kg−1) clay (g kg−1) CaCO3 (g kg−1)

Santomera Murcia Spain entisol 7.8 0.11 7.3 300 500

CDB AmOx DTPA

Fe (g kg−1) Fe (g kg−1) Fe (mg kg−1) Cu (mg kg−1) Ni (mg kg−1) Zn (mg kg−1) Co (mg kg−1) Mn (mg kg−1)

10.2 0.5 4.9 1.6 0.3 0.5 0.0 3.1

The ammonium salt of 2′-deoxymugineic acid (DMA) was synthesized in accordance with Namba et al.31 Purity was determined to be higher than 95% by H NMR. Esculetin (6,7dihydroxycoumarin; purity: >98%) was purchased from Alfa Aesar, sodium citrate (purity: >99) and ascorbate from Merck, and the mesylate salt of desferrioxamine B (DFOB, commercial name Desferral; >97% pure (personal communication with Novartis)) was purchased from the local pharmacy. Except for esculetin, all ligands readily dissolved in water. Esculetin was dissolved by adding sufficient 1 M NaOH solution to prepare the stock solution. For the preparation of experimental solutions analytical grade chemicals and ultrapure water were used. Ligand and reductant stock solutions had pH values between 5 and 9. Batch-Interaction Experiments. To explore potential synergistic effects between biogenic ligands and reductants in Fe mobilization from soil, we investigated how a single reductant and four biogenic ligands interacted with a calcareous B

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DFOB and Ascorbate. Fe mobilization by DFOB-only increased over time from 0.9 μM (0.25h) to 5.2 μM (168h) (Figure 1a). A lasting synergistic effect on Fe mobilization was

soil from Santomera (Spain), either separately or as a mixture of a ligand and the reductant. Ascorbate (1 mM) was used as a model biogenic reductant. Citrate and esculetin (1 mM) were selected as representative ligands for strategy I plant Fe acquisition, DMA (100 μM) as a representative ligand for strategy II plant Fe acquisition, and DFOB (500 μM) as a representative ligand for bacterial Fe acquisition. Experiments were carried out in 50 mL polypropylene tubes (Greiner bio one) in a soil−solution ratio (SSR) of 1 kg l−1. To exclude the influence of microbial activity on metal mobilization, we added sterilant (0.2 g l−1 bronopol) to all treatments. CaCl2 (10 mM) was used as background electrolyte. Blank treatments without addition of ligand or reductant were included. Soil samples were pre-equilibrated with electrolyte solution and sterilant for 2 days at 90% of the final solution volume. Reductant and chelating agents were then added to make up the remaining 10% of the volume, and samples were placed in an end-overend shaker and rotated at 18 rpm in the dark at 20 ± 1 °C. The amount of reductant added was insufficient to deplete the oxygen in the headspace of the tubes; less than 5% of the oxygen could be consumed at most. Samples were taken after 0.25, 0.5, 1, 2, 4, 8, 24, 48, 96, and 168 h for treatments involving chelating ligands and after 0.25, 1, 8, 24, and 168 h for treatments only involving ascorbate. The samples were centrifuged for 3 min at 4500 rpm and filtered with 0.45 μm cellulose acetate filters (Whatman Aqua 30/0.45 CA; experiments with DMA and DFOB) or with 0.1 μm PVDF filters (Merck Millipore, catalog no. SLVV033RS; experiments with citrate and esculetin). The pH of the filtrate was measured, and the filtrate was further analyzed. The high calcium carbonate content of Santomera soil strongly buffered the pH of the samples, which ranged from 7.0 to 7.7. Analysis. Metal concentrations (Al, Ca, Co, Cu, Fe, Mn, Mo, Na, Ni, and Zn) were determined by ICP-OES (Optima 5300 DV, PerkinElmer). Samples were acidified with nitric acid prior to analysis. Metal mobilization by the ligands and the reductant has been calculated as the difference in metal concentration between the treatment involving the ligand or reductant (or the ligand and the reductant) and the blank.

Figure 1. Mobilization of (a) Fe, (b) Mn, (c) Co, and (d) Al from Santomera soil as a function of time by solutions containing 500 μM DFOB and 1 mM ascorbate, either separately or combined (SSR = 1; 10 mM CaCl2). The solutions contained a sterilant (0.2 g l−1 bronopol) to prevent biodegradation. The red two-headed arrow indicates the synergistic effect in Fe mobilization. Error bars indicate standard deviations.

observed for combined application of DFOB and ascorbate (indicated by the two-headed arrow, Figure 1). The Fe concentration in the combined treatment continuously increased from 3.8 μM after 0.25 h to 9.2 μM after 168 h. The initial synergistic effect was particularly strong: after 0.25 h, it was 2.8 μM, corresponding to a factor 3.6 more Fe mobilization in the combined treatment than in the separate treatments. The synergistic effect reached a maximum of 4.5 μM after 24 h, after which it did not significantly change. The fact that 58% of the maximum synergistic effect was already reached after 0.25 h suggests a high reactivity of ascorbate directly after contact with soil. The mechanism underlying the synergistic effect is not yet understood; synergistic Fe mobilization may be caused by electron input into Fe(hydr)oxide minerals, labilizing the structure and facilitating ligandpromoted dissolution, or by direct complexation and mobilization of the short-lived, more labile Fe(II) before it becomes reoxidized by oxygen.25 Additionally, iron reduction and reoxidation may result in transient Fe(III) species that are more available for complexation (e.g., more labile Fe-bearing minerals or Fe complexes with soil organic matter (SOM)). In a previous study, we had observed a synergistic effect in Fe mobilization by ascorbate and DFOB in goethite suspensions, both under aerobic and anaerobic conditions.25 We found that Fe mobilization by DFOB from pure goethite increased linearly over time in suspensions far from equilibrium and that the synergistic effect developed more gradually and increased continuously. The more rapid development of the synergistic

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RESULTS AND DISCUSSION The term synergistic effect is used to express the positive concentration difference between the combined ligand-andreductant (ascorbate) treatment and the sum of the ligand-only and reductant-only treatments. A negative concentration difference between the combined treatment and the sum of the ligand-only and reductant-only treatments is referred to as an antagonistic effect. Ascorbate-Only. In the ascorbate-only treatment, the Fe concentration was not significantly elevated relative to the blank treatment. After Fe3+ reduction by ascorbate to the more soluble Fe2+, Fe2+ was presumably rapidly reoxidized by dissolved oxygen, preventing the Fe solution concentration from increasing. As a consequence of the negligible Fe mobilization in the ascorbate-only treatment, the synergistic effect corresponds to the difference between the ligand-andascorbate treatment and the ligand-only treatment. With exception of Mn, Al, and Mo, mobilization of other metals in the ascorbate-only treatment was also small ( 25);36 DFOB did not mobilize divalent metals like Ni2+, Zn2+, and Cu2+. For Mn and Co mobilization from soil, a temporary synergistic effect was found (Figure 1b,c). For Al, an antagonistic effect was observed; less Al was mobilized in the combined treatment than in the treatment with DFOB-only (Figure 1d). Presumably, the antagonistic effect on Al mobilization is due to increased competition resulting from the enhanced Fe, Mn, and Co mobilization in the combined treatment. A more extensive description of Al, Mn, and Co mobilization is included in the Supporting Information. The metals for which a synergistic effect was found (Fe, Mn, and Co) can occur in multiple redox states. Fe and Mn are present in soil as oxide phases that are potentially labilized by reductive processes followed by reoxidation. Co(III) is often associated with Mn oxides and may be mobilized and reimmobilized by these transient processes. DFOB may have an important effect on Co(III) mobilization due to its high complexation constant (logß11(Co(III)DFOB) = 37.4),37,38 preventing readsorption before the system reverts to its original redox state. Similarly, Mn(III) and Co(III)39 associated with oxy(hydr)oxide minerals40 may be transiently released by electron transfer and atom exchange (ETAE).41 Further research is needed to elucidate the processes involved. DMA and Ascorbate. In the DMA-only treatment, Fe mobilization increased from 3.1 μM (0.25 h) and reached a maximum of 20.0 μM (24 h), after which it declined to 10.9 μM (168 h) (Figure 2a). In the combined treatment, Fe mobilization increased from 9.9 μM (0.25 h) to a maximum after of 15.6 μM (1 h) and then declined to 1.8 μM (168 h). Hence, in the presence of DMA and ascorbate, initial Fe mobilization was faster than in the DMA-only treatment, the maximum was reached earlier and at a lower concentration level, and the final concentration was lower. In other words, combined application of ascorbate and DMA initially had a synergistic effect on Fe mobilization reaching a maximum of 9.7 μM after 1 h, but the synergic effect wore off, and ultimately antagonistic effects of combined DMA and ascorbate application were observed after 4 h (Figure 2a). Recently, Schenkeveld et al.14 introduced a “Window of Fe acquisition” conceptual model that explained the eventual decline in FeDMA concentration in treatments in which biodegradation was prevented as a result of the depletion of free DMA ligand and the subsequent competitive displacement of Fe from the FeDMA complex by other metals, particularly Cu, Ni, and Co. In the context of this model, enhanced mobilization of competing metals as a result of ascorbate addition explains the decline in FeDMA concentration setting in earlier, leading to a dramatic decrease in size of the Fe

Figure 2. Mobilization of (a) Fe, (b) Cu, (c) Ni, (d) Co, (e) Mn, and (f) Zn from Santomera soil as a function of time by solutions containing 100 μM DMA and 1 mM ascorbate, either separately or combined (SSR = 1; 10 mM CaCl2). A sterilant (0.2 g l−1 bronopol) was added to prevent biodegradation. Error bars indicate standard deviations.

acquisition window (note the logarithmic scale for the x-axis in Figure 2a). Although synergistic Fe mobilization only lasted up to 4 h, the effect may nonetheless be of great significance to Strategy II plants for acquiring Fe from the environment. In the rhizosphere, phytosiderophore ligands are subject to biodegradation, limiting their residence time. Therefore, it is particularly the period directly after release into the soil when an increase in Fe concentration is most beneficial to plants. Because of the diurnal pulse in which phytosiderophores are exuded, the beneficial effect from reductants in the rhizosphere would be recurring on a daily basis. In a previous study, Cu was identified as the quantitatively most important competing metal in Santomera soil, particularly at low DMA concentrations.14 However, the difference in Cu concentration between the DMA-only treatment and the combined treatment was marginal (Figure 2b); CuDMA concentrations increased from 2.2−2.6 μM (0.25 h) to 16.6− 18.7 μM (168 h). This demonstrates that ascorbate did not have a strong effect on Cu mobilization by DMA. Toward the D

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to occur in soils under anaerobic conditions.45 Possibly, it can also transiently occur under aerobic conditions when there is input of electrons from ascorbate. The relatively strong increase in Mn solution concentration, also in the absence of DMA (Figure 2e), suggests reductive dissolution of Mn(hydr)oxides, to which Ni and Co tend to be strongly associated in environmental systems.39,46,47 The lack of Ni and Co mobilization in the ascorbate-only treatment shows that a ligand is required as a scavenger because otherwise, the liberated metals appear to bind to other solid phases. Additionally, introduction of electrons into oxy(hydr)oxide minerals may cause the labilization of bonds between Fe and adsorbed Ni and Co facilitating ligand-promoted dissolution. Also, the reduction of Co(III), the common redox state when associated with Fe(hydr)oxides, to Co(II) increases the ionic radius, which makes it less favorable for reincorporation into certain Fe(hydr)oxide minerals40 and more prone to complexation and mobilization by DMA. Esculetin and Ascorbate. Synergistic Fe mobilization was also observed for ascorbate and esculetin (Figure 3). No

end the experiment, there was actually a small antagonistic effect of ascorbate on Cu mobilization by DMA (p = 0.008). A strongly enhanced mobilization of the other two principal competing metals, Ni and Co, by DMA in the presence of ascorbate (Figure 2c,d) explains the faster onset of competitive displacement of Fe from FeDMA complexes by competing metals in the combined treatment and the subsequent decline in FeDMA concentration. In the combined treatment Ni mobilization gradually increased from 4.4 to 14.7 μM, and in the DMA-only treatment, it increased from 0.2 to 6.9 μM. Co mobilization increased from 9.8 to 18.3 μM in the combined treatment and from 0.2 to 6.8 μM in the DMA-only treatment. The relative synergistic effect was largest initially, with a factor 7.5 (Ni) and 45 (Co) more mobilization in the combined treatment than in the DMA-only treatment after 0.25 h. The absolute synergistic mobilization reached a maximum of 8.5 and 12.4 μM for Ni and Co, respectively, after 48 h, after which it slightly decreased. The mobilization of the plant micronutrients Mn and Zn was also synergistically enhanced through the combined application of DMA and ascorbate. Mn mobilization by DMA-only was marginal (0.6−1.2 μM) (Figure 2e), and ascorbate-only strongly mobilized Mn up to 37.1 μM after 1 h, after which the concentration gradually declined. Upon the combined application of ascorbate and DMA, an initial synergistic effect on Mn mobilization of 9.4 μM after 0.25 h was observed that gradually diminished and was no longer significant after 24 h. In the combined treatment, Mn mobilization decreased from 39.9 μM (0.25 h) to 19.6 μM (168 h). Trends in Zn mobilization were similar to those in Fe mobilization (Figure 2f). In the combined treatment, a small, initial synergistic effect on Zn mobilization was found that transformed into an antagonistic effect after 8 h. Zn mobilization increased from 1.8 μM (0.25 h) to a maximum of 2.6 μM (4 h), after which it declined to 0.7 μM after 168 h. In the DMA-only treatment, Zn mobilization increased from 1.4 μM (0.25 h) to 2.7 μM (24 h), after which it declined to 2.3 μM (168 h). These results on metal mobilization from soil by DMA demonstrate that the effect of addition of a reductant proved strongly dependent on the metal. A synergistic effect was observed for metals that tend to be present in oxic soils as oxy(hydr)oxide minerals and can undergo redox reactions to form more soluble reduced metal species (Fe and Mn). For Mn, the addition of ascorbate-only led to increased solution concentrations (Figure 2e), but for Fe, it did not (Figure 2a). Presumably, this is related to the slow oxidation kinetics of Mn2+ compared to Fe2+,42 which is rapidly reoxidized to its insoluble trivalent form. Furthermore, results from multisurface complexation modeling of the metal speciation in Santomera soil43 suggest that a strong synergistic mobilization occurs for metals that are mainly (>85%) associated with metal− oxy(hydr)oxide phases (Ni and Co). Metals that are primarily (>90%) associated with SOM (Cu and Zn) show a small initial synergistic mobilization that eventually becomes antagonistic. This suggests that the metal speciation in the soil solid phase has a strong influence on the size of the synergistic effect. Synergistic mobilization of metals associated with metal (hydr)oxide minerals also concerns metals like Ni that are not redox reactive and, therefore, are not reduced by ascorbate. Ni and Co can be released from mineral matrixes by processes like dissolution of metal (hydr)oxide minerals and electron transfer and atom exchange40,44 and may subsequently be mobilized by DMA. Electron transfer and atom exchange was recently shown

Figure 3. Fe mobilization from Santomera soil as a function of time by solutions containing 1 mM ascorbate and 1 mM esculetin, either separately or combined (SSR = 1; 10 mM CaCl2). The solutions contained a sterilant (0.2 g l−1 bronopol) to prevent biodegradation. Error bars indicate standard deviations.

significant Fe mobilization was found throughout the experiment in the ascorbate-only and esculetin-only treatments. When applied together, a 1.1 μM Fe concentration was measured after 0.25 h. The Fe concentration gradually declined until it reached background levels after 8 h. As a fraction of the amount of ligand that was added, maximum Fe mobilization was small: 0.1%. The decline in Fe concentration does not coincide with an increase in concentration of competing metals (Figure S1); hence, there is no indication that competitive displacement of complexed Fe causes the decline. The decrease might be related to the ligand-reductant esculetin8 being oxidized and the reaction product being unable to bind and solubilize Fe. No synergistic mobilization was found for any other metal. Mn mobilization in the combined treatment proved additive (Figure S1a). Mn mobilization in the ascorbate-only treatment (21.0−35.4 μM) was much larger than in the esculetin-only treatment (2.2−2.6 μM). Because esculetin and molybdophores have catecholate functional groups in common,8,36 Mo mobilization in the esculetin treatments was examined. However, Mo mobilization by esculetin-only was negligible, E

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include the thermodynamic stability of the Fe complex, the thermodynamic and kinetic specificity of the ligand for Fe, the affinity of the Fe complex for reactive surfaces, and the susceptibility of the ligand to degradation and change that affects its ability to bind Fe.36 Among the examined ligands, DFOB forms the thermodynamically most stable Fe complexes (logß11 = 31.9)36 due to its ability to form 1:1 hexadentate complexes with Fe through hard Lewis base hydroxamate functional groups, which have a high affinity for the hard Lewis acid Fe3+. Nevertheless, on the time scales of the experiment, DFOB mobilized more Al than Fe, both in the presence and in the absence of ascorbate (Figure 1a,d). This may have either a thermodynamic or a kinetic cause. The lower affinity of DFOB for Al (log ß11 = 25.5)36 may be overcompensated by a considerably higher free Al3+ activity than free Fe3+ activity in soil solution. Also, the complexation and mobilization kinetics of Al by DFOB from Santomera soil are faster than those of Fe (Figure 1 a,d). The fact that Fe mobilization by DFOB (contrary to Al mobilization) increased until the end of the experiment implies that equilibrium was not yet reached when the experiment was terminated. The lasting nature of the synergistic effect on Fe mobilization suggests that ascorbate partly mitigated a kinetic competitive disadvantage of Fe over Al and may help to establish equilibrium more quickly. Another factor that compromises Fe mobilization from soil by DFOB is the cationic nature of both the DFOB ligand and the prevalent metal−DFOB complexes and the abundance of negatively charged reactive soil compounds (e.g., SOM and clay minerals; Table 1); sorption is likely to retain a substantial fraction of the DFOB to the soil solid phase.48,49 DMA forms 1:1 complexes with Fe that are thermodynamically less stable (logß11 = 20.3)36 than Fe complexes of DFOB. In fact, multisurface modeling of the interaction of DMA with Santomera soil suggested that no Fe should be mobilized under equilibrium conditions.43 However, it was proposed that as long as there is sufficient free ligand in soil solution, Fe is not outcompeted by other metals for complexation by DMA, keeping a time window of Fe acquisition open.14 A high kinetic specificity of DMA for mobilizing Fe combined with enhanced Fe availability related to the application of a reductant, and the formation of negatively charged Fe-DMA complexes that are repelled by negatively charged soil compounds results in a large initial synergistic Fe mobilization. The synergistic effect is temporary, however, due to the low thermodynamic specificity for Fe, leading to Fe eventually being displaced by competing metals. Because the mobilization of certain competing metals is also enhanced by the addition of a reductant, the outcompeting of Fe sets in earlier, eventually resulting in an antagonistic effect. This more complex effect from the reductant on the mobilization of Fe and other metals suggests that balancing the concentrations of the reductant and the complexing ligand is key to optimizing the window of Fe acquisition through the synergistic effect. Citrate and esculetin cannot completely fill the octahedral coordination sphere of Fe with a single ligand and may form 2:1 complexes and, in the case of esculetin, also form 3:1 complexes with Fe. In soil environments where metal ions are in abundance, it is highly unfavorable for metal mobilization if multiple ligands are required for forming soluble Fe complexes. Because of the relatively low stability of Fe citrate complex species (e.g., logß11 = 13.2),50 equilibrium with soil Fe phases at Santomera soil pH requires that the total ligand concentration in solution is in considerable excess over the Fe complex

and its contribution to the combined treatment with ascorbate was minor compared to mobilization by ascorbate-only (Figure S1b). Citrate and Ascorbate. In the presence of citrate and ascorbate, a small synergistic mobilization of Fe was observed, up to 8 h (Figure 4a). The mobilized Fe concentrations were,

Figure 4. Mobilization of (a) Fe and (b) Al from Santomera soil as a function of time by solutions containing 1 mM ascorbate and 1 mM citrate, either separately or combined (SSR = 1; 10 mM CaCl2). The solutions contained a sterilant (0.2 g l−1 bronopol) to prevent biodegradation. Error bars indicate standard deviations.

however, low; the maximum concentration was 0.5 μM after 0.5 h, corresponding to 0.05% of the added ligand. In the treatments with ascorbate-only and citrate-only the maximum concentrations were 0.2 and 0.1 μM, respectively. Citrate mobilized Al relatively strongly: in the citrate-only treatment, up to 7.0 μM Al after 0.5 h, after which the concentration gradually declined to 1.4 μM after 168 h (Figure 4b). No Al was mobilized in the ascorbate-only treatment. The combined addition of ascorbate and citrate, however, had an antagonistic effect on the mobilization of Al for up to 4 h. The fact that both the synergistic Fe mobilization and the antagonistic effect on Al mobilization disappeared on the same time scale suggests that the enhanced Fe mobilization in the combined treatment was at the expense of Al mobilization. However, the synergistic effect was much smaller (max. 0.2 μM) than the antagonistic effect (max. 3.1 μM). Possibly, Fe− citrate complexes have a stronger tendency to adsorb to soil reactive compounds than Al−citrate complexes. Why the effects disappeared over time is not clear. Synergistic Mo mobilization was found for combined ascorbate and citrate application (Figure S2a). Mobilization remained 1−1.3 μM larger than in the treatment with ascorbate-only; Mo mobilization by citrate-only treatment was negligible. For Co and Ni, the combined addition of ascorbate and citrate had a small synergistic effect on mobilization of up to 0.2 μM (Figure S2b,c). The citrate-only treatment did not mobilize Co, but it did mobilize up to 0.1 μM Ni for up to 1 h. The ascorbate-only treatment mobilized up to 0.2 μM of both Ni and Co. Influence of Ligand Properties and Reactivities on Metal Mobilization and the Synergistic Effect. Our results demonstrate that the size and duration of the synergistic Fe mobilization are strongly ligand-dependent: for esculetin and citrate, a small temporary synergistic effect was found; for DMA, a large temporary synergistic effect was found that transformed into an antagonistic effect; and for DFOB, a lasting intermediate synergistic effect was observed. The ligand properties play a key role in this respect. These properties F

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ACKNOWLEDGMENTS We thank Martin Walter and Doris Gasser for assistance with the experimental work and acknowledge Christian Stanetty, Martin R. Walter, and Paul Kosma from the Division of Organic Chemistry, BOKU for synthesizing the DMA. This work was supported by the Austrian Science Fund (FWF, grant no. I1528-N19) and by the International Center for Advanced Renewable Energy and Sustainability (ICARES) at Washington University in St. Louis.

concentration. Stability constants for Fe esculetin complexes have not yet been reported. Furthermore, the specificity of citrate for Fe is low, as illustrated by the considerable Al mobilization. Finally, upon oxidation, the ability of esculetin to bind Fe may be compromised. All of these factors may contribute to limited Fe mobilization by esculetin and citrate, even though on a relative scale, Fe mobilization is considerably enhanced through the synergistic effect with ascorbate. Environmental Implications. Our results demonstrate synergistic effects between biogenic ligands and reductants in Fe mobilization from soil. The synergistic effect on Fe mobilization was observed for all examined ligands with different ligating groups, indicating that the observed effect of reductants represents an extension of the existing knowledge on ligand-promoted iron mobilization in soils. Moreover, the ligands chosen in this study are exuded by different types of organisms. Together, this indicates that synergistic Fe acquisition mechanisms may widely occur in nature and could be of great environmental significance. The biosynthesis of specific Fe-binding ligands (e.g., siderophores) is associated with metabolic costs. The exploitation of synergistic effects with low-molecular-weight reductants may increase the efficiency of Fe acquisition and concomitantly reduce these costs. Still, among the ligand− reductant systems that we investigated, we found one system (reductant−phytosiderophore) in which a synergistic effect evolved into an antagonistic effect on Fe mobilization over time. However, Strategy II iron acquisition operates in a welldefined “window of Fe acquisition” over short time-scales.14 Therefore, an initial synergism within the window or Fe acquisition may be highly beneficial, and a long-term antagonistic effect may be less relevant for plant Fe uptake. In general, it may be concluded that the exploitation of a synergistic effect needs to be regulated (e.g., addressing exudation rates and timeframes) so that the synergistic effect is maximized and a possible antagonistic effect is minimized. If these criteria are met, synergistic effects can be of great importance in the context of microbial and plant Fe acquisition and could be decisive for the survival of organisms under conditions of low Fe availability. The influence of ligand and reductant concentration on the size and duration of the synergistic effect need to be further explored and are the topic of a follow up study.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b01623. Additional information on data descriptions for soil interaction experiments, figures showing additional metal mobilization data for esculetin and citrate, and tables showing mobilization data in tables. (PDF)

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DOI: 10.1021/acs.est.6b01623 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.est.6b01623 Environ. Sci. Technol. XXXX, XXX, XXX−XXX