Certain transition metals, such as iron (Fe), molybdenum (Mo), zinc (Zn), copper (Cu), nickel (Ni), and cobalt (Co), are micronutrients for organisms as they serve as metal cofactors for one-third of the enzymes involved in cellular processes. 1,2 These metalloenzymes catalyze many biogeochemical processes such as nitrogen fixation, 3,4 sulfur oxidation and reduction, 5,6 and methane generation and oxidation. 7,8 However, transition metals are commonly entrained in mineral structures, rendering them inaccessible for direct biological uptake. 9 Consequently, organisms have developed complex strategies to overcome metal limitation, including secretion of low-molecular weight organic acids 10 and siderophores, 11,12 to facilitate dissolution of minerals and cellular uptake of released transition metals. [12][13][14][15][16] Traditionally, siderophores are considered as small ironchelating molecules that effectively bind Fe to form Fesiderophore complexes, which can be transported into cells to sustain cellular Fe needs. 12,17,18 Currently, over 500 different types of siderophores have been identified. 17 Microbial siderophores are generally classified into three main groups: 19,20 hydroxamate, catecholate, and α-hydroxycarboxylate. Siderophore-promoted mineral dissolution is typically achieved through the formation of a stable complex between the siderophore and the metal ion center in mineral structures, followed by detachment and/or potential redox state changes of the metal. [21][22][23] For instance, the hydroxamate siderophore desferrioxamine B (DFOB), 24 which shows a high binding affinity for Fe(III) (logK = 32), 12,24 promotes dissolution of iron hydroxides (e.g., goethite and ferrihydrite) and silicates (e.g., clay minerals, olivine, and hornblende). [21][22][23][25][26][27][28][29][30][31][32][33][34][35] DFOB is also able to dissolve solid minerals MnOOH and CuOOH. 22,36 The dissolution of minerals by DFOB, in consequence, promotes microbial growth by supplying cells with essential elements released from minerals. 37 Other than Fe, Mo is an important transition metal for various metalloenzymes, specifically crucial for nitrogen-fixing, denitrifying, and sulfur-oxidizing microorganisms to synthesize nitrogenase, 3,38,39 nitrate reductase, 40 and sulfur-oxidizing complex, 6 respectively. Mo speciation is governed by redox transformations between its different oxidation states, e.g., Mo(VI) and Mo(IV), which influence its mobility and bioavailability. 41,42 Under oxic conditions, Mo is released from Mo-minerals and predominantly exists as the soluble molybdate (MoO 4 2-
), which readily adsorbs to metal oxides or complexes with organic matter. 43 Under anoxic and/or euxinic conditions, the formation of insoluble minerals makes Mo less available for microbial uptake. 44 Such a Mo scarcity exists in various environments. For instance, serpentine-rich soils contain less Mo than other soil types; 45,46 thus, plants have to form a symbiosis with nitrogen-fixing bacteria in root nodules to assimilate Mo. 40 Temperate and tropical soils are also challenged with limited Mo availability due to Mo adsorption to organic particles. 45,47,48 For marine environments, dissolved Mo in modern seawater is abundant (∼105 nM), 49 despite the presence of certain oxygen minimal zones and euxinic conditions where dissolved Mo quickly precipitates. 50,51 However, Mo was scarce (<2-3 nM) in the Archaean Ocean before oxidative weathering occurred (∼2.3-2.5 Ga) 44,52 because it was "locked" in minerals and rocks. Many modern sedimentary, porewater, and freshwater environments are also Mo-limited (<20 nM), 45 where Mo may undergo reduction or conversion to thiomolybdate and ultimately associate with sulfide minerals. 53 Despite Mo scarcity, the biological reactions catalyzed by Mo-containing enzymes are prevalent across various modern environments and throughout geological time. 54,55 Microbes living in Mo-depleted environments have evolved strategies to overcome Mo scarcity, such as developing highaffinity ModABC molybdate transporters, building Mo storage systems inside cells, and/or exudating organic acids or siderophores capable of binding Mo (also named molybdophores) to enhance Mo bioavailability. 45,47 To date, catecholate has been identified as the only microbial siderophore that can bind with Mo. 22,56 The production of catecholate siderophores (e.g., aminochelin, azotochelin, and protochelin) by the a model aerobic diazotroph Azotobacter vinelandii was observed in response to Mo and Fe scarcity. [57][58][59][60][61][62] Our previous study demonstrated that A. vinelandii accumulated both catecholate and hydroxamate siderophores to support its growth when molybdenite (MoS 2 ) was the sole source of Mo under oxic conditions. 43 However, the potential partial oxidative weathering of the molybdenite surface may complicate its dissolution process. The specific roles of these catecholate and hydroxamate siderophores in the weathering of Mo-minerals under varying redox conditions, as well as the underlying mechanisms, remain unknown.
Therefore, the primary objective of this research was to investigate the interactions between metallophores and natural Mo-bearing minerals. The tricatecholate protochelin and the trihydroxamate DFOB were selected as model siderophores to assess their respective functions in the dissolution of molybdenite, the largest solid Mo reservoir on Earth. The extent of mineral dissolution was monitored by changes in soluble Mo concentrations before and after reactions with metallophores under both oxic and anoxic conditions. Siderophore contents, metal-siderophore complexes, and changes in mineral surface morphology and chemistry were probed to unravel the dissolution mechanism. This research expands our understanding of the ability of metallophores to dissolve Mo-bearing minerals and offers valuable insights into biological metal acquisition strategies.
Chemical and Mineral Preparations. Protochelin, a tricatecholate siderophore with the ability to bind Mo (also called "molybdophore"), 63,64 was synthesized by the Duke University Small Molecules Custom Synthesis Facility. DFOB, a trihydroxamate siderophore, was purchased in the form of a mesylate salt from Sigma-Aldrich and used without any treatment. Siderophore characteristics are summarized in the Supporting Information (SI) Table S1. Due to its low solubility and high instability in water, 63,65 protochelin was initially dissolved in 100% methanol to obtain a 10 mM stock solution and diluted in deionized (DI) water to obtain working solution. DFOB was readily dissolved in DI water (stock concentration of 10 mM). Stocks were prepared immediately before the start of the experiments.
Molybdenite, a molybdenum disulfide mineral (Table S2), was provided by the Miami University Limper Geology Museum (Oxford, OH). This mineral was specifically selected as it represents the largest solid reservoir of Mo on earth, making it an ideal model mineral. 44,66 Molybdenite was free of impurities as indicated by X-ray diffraction (XRD) patterns (Figure S1, XRD details in Supporting Information Section 1). The molybdenite was ground and sieved to acquire a specific size fraction (63-250 μm) and prepared as 20 g/L stock suspension in DI water (resistivity >18 MΩ). Prior to use, all glassware was EDTAsoaked (30 mM) and acid-soaked (5% HCl) overnight to remove any potential metal contamination.
Batch Dissolution Experiments. Dissolution of molybdenite was investigated in the absence and presence of the siderophores at near-neutral pH (7.5-7.9) (Figure S2). Experiments were initiated by mixing a siderophore at different concentrations with 2 g/L mineral in Balch tubes. Siderophore concentrations ranged from 0.0001 mM (100 nM) to 0.5 mM for protochelin and 0.01 to 1 mM for DFOB, based on previous studies on siderophore-promoted Fe-mineral dissolution 22,25,[28][29][30] and measured siderophore concentrations in natural environments (>10 nM). 67 All experiments were conducted for 7 days, except for those with lower protochelin concentrations (0.0001-0.01 mM), which were extended to 30 days. In the DFOB experiments, bicarbonate buffer (2.5 g/L NaHCO 3 with 0.1 g/L KCl electrolyte solution) was utilized to maintain the pH at 7.5-7.9. DI water was used as the working solution for the protochelin experiment because of the potential chemical reaction between bicarbonate buffer and protochelin (data not shown). In this case, 0.1 M NaOH and 0.1 M HCl were used to maintain a pH of 7.5-7.9. To allow an even comparison between two sets of siderophore experiments using different working solutions, additional DFOB experiments were also performed using DI water with the same manual adjustment of pH. Controls containing only minerals were set up under identical conditions. All tubes were incubated at 25 °C in a 150 rpm shaker in the dark to avoid light interference and were established with triplicates.
Considering that both molybdenite and protochelin solutions could be partially oxidized under oxic aqueous conditions, 63,64,68 which could affect their interaction, dissolution experiments were conducted under both oxic and anoxic conditions. Four sets of molybdenite dissolution experiments were set up:
Environmental Science & Technology 1. Siderophores and molybdenite under oxic conditions (Figure S2A).
2. Siderophores and molybdenite under anoxic conditions (Figure S2B).
3. Siderophores and preoxidized molybdenite under anoxic conditions (Figure S2C). Molybdenite suspension was preoxidized in air for 7 days and then mixed with an anoxically prepared siderophore solution under anoxic conditions.
4. Preoxidized protochelin and molybdenite under anoxic conditions (Figure S2D). Protochelin suspension was preoxidized in air for 7 days and then mixed with an anoxically prepared molybdenite solution under anoxic conditions.
Samples were centrifugated at 7000g for 5 min and filtered through 0.22 μm polyethersulfone (PES) filters. Filtrates were then collected and diluted with 2% nitric acid to measure soluble Mo concentrations by using high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS, Nu instruments AttoM ES). The Mo detection limit was 0.1 ppb with an uncertainty of 0.05 ppb.
Measurement of Siderophore Concentration and Speciation. Siderophore concentration in mineral experiments was monitored over time. Protochelin concentration was measured by using the Arnow Assay 69 (Supporting Information
Section S2). Liquid chromatography-mass spectroscopy (LC-MS) analysis was performed to investigate protochelin oxidation (Supporting Information Section S3). The DFOB concentration was determined by measuring the absorbance of filtrate at 430 nm after mixing with 2% FeCl 3 solution in a 1:1 molar ratio. 70 At circumneutral pH (∼7), Mo(VI) complexes with protochelin to form an orange-colored complex. 64,71 Therefore, suspension colors in mineral-protochelin experiments were visually monitored, with the standard Mo(VI)-protochelin complex prepared in a 1:1 molar ratio under identical conditions (Supporting Information Section S4). However, Mo(VI) does not complex with DFOB at this pH condition. 72 To determine the pH range of Mo(VI)-DFOB complexation, a titration experiment was conducted over a pH range of 2.79 to 8.93 using an autotitrator (Metrohm, Switzerland) (Supporting Information Section S5).
To semiquantitatively measure time-course change in metalsiderophore complexes, UV-Vis spectra were recorded for both protochelin and DFOB-treated mineral solutions using a UV-Vis spectrophotometer (Thermal scientific Genesys 150). UV-Vis spectra of siderophores alone were monitored overtime as controls. To further quantify the Mo-siderophore/organic complexes in solutions, filtrates were extracted by using solid phase extraction (SPE) cartridges (Supporting Information Section S6). Any Mo associated with hydrophobic organic compounds (e.g., protochelin and its oxidation products) should be adsorbed onto the cartridges. The adsorbed Mosiderophore/organic complexes were eluted from the cartridges and acidified with 2% nitric acid, followed by measurements using HR-ICP-MS (Mo detection limit of ∼0.1 ppb). Filtrates from "mineral only" and "protochelin only" groups were processed the same way.
Mineral Characterization after the Interaction with Siderophores. At the end of dissolution experiments, minerals from both experimental (minerals + siderophores) and control groups (minerals only) were collected by centrifugation at 7000g for 10 min. To study the possible mineralogical changes, XRD patterns were obtained (Supporting Information Section S1). Scanning electron microscopy (SEM) observations were made to investigate any morphological changes of minerals after their interaction with siderophores (Supporting Information Section S7). Additionally, the BET specific surface area (SSA) and particle size of minerals before and after interaction with protochelin were determined by using a surface area and porosity analyzer (micromeritics, TriStar II) (Supporting Information Section S8).
To investigate any compositional changes of metals and carbon on the surface of molybdenite, X-ray photoelectron spectrometry (XPS) analysis was conducted (<10 nm as the probing depth) before and after interaction with protochelin and DFOB using a Thermo Scientific Nexsa XPS equipped with a hemispherical analyzer and a monochromatic Al Kα source (Supporting Information Section S9).
To further investigate the changes in surface chemistry of molybdenite before and after interaction with protochelin, timeof-flight secondary ion mass spectroscopy (ToF-SIMS) analysis was performed using a TOF.SIMS5 instrument (IONTOF GmbH, Munster, Germany) (Supporting Information Section S10 and Table S3). ToF-SIMS provides information to the topmost atomic layers and allows for accurate detections of sensitive chemicals (i.e., C, O, and N-containing compounds) with minimal damage artifacts. 73 To better interpret complex ToF-SIMS spectra, principal component analysis (PCA) was conducted with MATLAB.
Molybdenite dissolution exhibited a contrasting behavior in the presence of protochelin and DFOB (Figure 1 and Table S4). Under oxic conditions for 7 days, molybdenite dissolution was significantly promoted by protochelin in the concentration range of 0.1 to 0.5 mM (t tests, Table S5), as evidenced by significantly higher Δsoluble Mo concentration (i.e., the difference in Mo concentration in the solution between day 7 and day 0) in the presence of protochelin compared to its absence. Although molybdenite dissolution was not significant at low concentrations of protochelin (0.0001-0.01 mM) over 7 days, the dissolution was significantly enhanced over extended periods (over 15 and 30 days) (Figure S3). This promoting effect was directly proportional to protochelin concentration (R 2 = 0.97, p < 0.001) (Figure S4 and Table S6). At a concentration of 0.1 mM protochelin, the total Mo release rate over 7 days was 2.17 × 10 -12 mol s -1 (Table S7).
In contrast, molybdenite dissolution was inhibited by DFOB, as evidenced by significantly lower Δsoluble Mo concentration in the presence of DFOB compared to its absence (Figure 1B, t tests in Table S8). The release rate of Mo with 0.1 mM DFOB over 7 days was 1.28 × 10 -12 mol s -1 (Table S7). This inhibition was unaffected by the bicarbonate buffer, as no difference was observed between experiments using bicarbonate buffer or DI water as the working solution (Figure S5). However, the extent of inhibition did not increase with increasing DFOB concentration (Figure 1B and Table S9).
Moreover, to study whether the presence of oxygen affected the dissolution process, a molybdenite dissolution experiment was conducted under anoxic conditions for comparison. In the absence of oxygen, neither protochelin nor DFOB significantly influenced molybdenite dissolution (Figure 1C,D and Tables S4, S10). However, when either molybdenite or protochelin was preoxidized in air for 7 days, molybdenite dissolution was promoted under anoxic conditions (Figure 1C). Notably, the extent of dissolution when molybdenite and protochelin reacted under oxic conditions (d = 5.18) was equivalent to the sum of those when molybdenite reacted with preoxidized protochelin (d = 3.28) and when preoxidized molybdenite reacted with protochelin (d = 2.31) under anoxic conditions (5.18≈2.31 + 3.28 within errors, Figure 1C and Table S4). These results suggested that the air-oxidation of both molybdenite and protochelin contributed to the dissolution process under oxic conditions. DFOB did not significantly affect the dissolution of the preoxidized molybdenite under anoxic conditions (Figure 1D).
Siderophore Degradation and Adsorption to Molybdenite Surface. To explain the contrasting behavior of molybdenite dissolution caused by siderophores, adsorption experiments were performed because adsorption should be the first step for the subsequent dissolution. 21,22 Under oxic conditions, aqueous concentrations of protochelin and DFOB, along with their respective metal complexes, were monitored for 7 days (Figure 2). In the absence of molybdenite, protochelin concentration decreased over time at the experimental pH of 7.5-7.9, by as much as ∼52% after 7 days (Figure 2A). This observed degradation of protochelin was consistent with its recognized instability in water at pH exceeding 7.5 under oxic conditions, 63 which may affect its metal complexation capacity Environmental Science & Technology to dissolve molybdenite. In the LC-MS chromatogram, protochelin eluted at 5.25 min (Figure S6A), primarily consisting of protochelin-2H (m/z = 623.3) and protochelin-H (624.3) (Figure S6B). After air-oxidation, the intensity of protochelin peaks largely reduced, with new peaks appearing at 4.87 min (Figure S6A), likely assigned to degradation products (e.g., m/z = 621.3 and 622.3, Figure S6C). These new peaks were potentially attributed to the oxidation of the catechol group into quinones (Figure S6D). 74,75 Moreover, a large group of small fragments (e.g., m/z < 200) were observed after airoxidation (Figure S6C), which may also affect molybdenum dissolution. Relative to oxic conditions, protochelin was less degradable under anoxic conditions. In contrast, without molybdenite, DFOB concentration did not vary (<5%) over 7 days (Figure 2B).
The presence of molybdenite significantly lowered the aqueous concentrations of both siderophores, indicating their adsorptions onto the mineral surface. Adsorption of both siderophores to molybdenite was lower under anoxic conditions compared to oxic conditions (Figure 2A,B).
Characterization of Metal-Siderophore Complexation. Upon mineral dissolution, Mo released into the solution may form colored Mo-siderophore complexes due to the metalligand charge transfer bands. 64,71,76 A light yellow color developed after the reaction of molybdenite with protochelin (Figure S7A), corresponding to the standard color for the Mo(VI)-protochelin complex (Figure S7A). In contrast, after the reaction of molybdate or molybdenite with DFOB, no color was observed in the solutions (Figure S7B), indicating no Mo(VI)-DFOB complexation at this pH condition (7.5-7.9). However, this result did not necessarily suggest a lack of Mo(VI)-DFOB complexation under other pH conditions. Thus, a titration experiment was conducted to investigate Mo(VI)-DFOB complexation from an acidic pH (2.79) to an alkaline pH (8.93) (Figure S8). Indeed, Mo(VI) formed a complex with DFOB as the pH decreased from 9.4 to 2.1 (indicated by an arrow in the 280-300 nm wavelength region, Figure S8A). A yellow color emerged as the pH decreased to 2.79 (Figure S8B). This observation suggested that DFOB bound with Mo(VI) only under acidic conditions, not under the experimental pH condition (7.5-7.9) used in the current study, which explained the lack of any color in the DFOB + Mo(VI) experimental groups (Figure S7B).
However, color observations qualitatively describe only metal-siderophore complexation. Therefore, subsequent semiquantitative and quantitative analyses of metal-siderophore complexation were conducted using UV-Vis spectroscopy and SPE methods coupled with HR-ICP-MS measurements, respectively. The time-course UV-Vis spectra of protochelin after interaction with molybdenite displayed a gradual increase in the intensity of the broad peak around 400-420 nm (as marked by the arrow in Figure S9A). Based on the standard Mo(VI)-protochelin complex, this peak suggested the formation of the Mo(VI)-protochelin complex (Figure S9A). However, the intensity of this broad peak also slightly increased in the presence of protochelin alone, possibly caused by protochelin degradation 63 (Figure S9B). Therefore, the observed increase in this peak was likely caused by a combination of the Mo(VI)protochelin complex formation and partial degradation of protochelin. In contrast, the UV-Vis spectrum of DFOB after interaction with molybdenite did not show any observable peaks beyond 300 nm (Figure S10), again suggesting no Mo(VI)-DFOB complexation at this experimental pH. 77 The concentrations of Mo complexed with hydrophobic compounds were quantified (Figure 2C). Over time, the concentration of Mo-siderophore complexes (typically protochelin or its oxidation products in our system) increased significantly, in contrast to the negligible levels observed in the molybdenite solution without protochelin or in the protochelin solution without molybdenite. This rise in complexation overtime further underscored the interaction between protochelin-related organics and Mo from molybdenite, emphasizing protochelin's role in facilitating the dissolution process.
Changes in Molybdenite Structure and Surface Chemistry. Upon the addition of siderophores, the XRD peaks of molybdenite remained detectable (Figure S11), suggesting that the overall crystal structure of molybdenite remained intact. However, those characteristic peaks became less intense, consistent with a decrease in particle size and an increase in SSA observed after the reaction, suggesting partial dissolution 78 (Table S11). SEM images showed significant morphological changes in molybdenite after interaction with protochelin, where dissolution pits and holes were present (Figure 3A2), in contrast to the relatively smooth surface of pure molybdenite (Figure 3A1). Conversely, upon the addition of DFOB, no noticeable morphological change was observed (Figure S12).
XPS analysis was performed to investigate any chemical changes in the molybdenite surface after interaction with siderophores (Figures 3B,C, S13-S15). The observed Mo 3d and S 2s peak positions and relative intensities of the original Environmental Science & Technology molybdenite (Figures 3B1 and S13A1) were consistent with a previous study. 79 However, significant changes were observed after molybdenite interaction with protochelin under oxic conditions, with the emergence of two MoO 3 characteristic peaks at the binding energies of ∼232.5-232.7 eV and ∼235.7 eV (Figure 3B2). This observation suggested oxidation of the molybdenite surface, from Mo 4+ in molybdenite (MoS 2 ) to Mo 6+ in MoO 3 . Additionally, following addition of protochelin, the relative peak areas of C�O and C-O-C significantly increased (Figure 3C2) relative to those without protochelin (Figure 3C1), suggesting adsorption of protochelin/byproducts on the molybdenite surface. Expectedly, Mo speciation of molybdenite was unchanged in the presence of DFOB (Figure S13A), but C�O and C-O-C groups increased from 0 to 6.3% and from 8.5 to 11.2%, respectively (Figure S13B), because of adsorption of DFOB on molybdenite surface. In contrast to oxic conditions, no significant modifications of Mo 3d, S 2s, or C peaks were observed for molybdenite with protochelin under anoxic conditions (Figure S14), consistent with little molybdenite dissolution without air (Figure 1C). The Mo 3d and S 2s peaks also remained unchanged after molybdenite reaction with preoxidized protochelin under anoxic conditions (Figure S15A), while C�O, C-O-C, and OH-C�O groups increased (Figure S15B), suggesting adsorption of protochelin degradation products on the molybdenite surface.
Other than XPS, ToF-SIMS analysis was performed for a more detailed investigation of molybdenite surface chemistry (Figure 4). In PCA of ToF-SIMS negative spectra, PC1 and PC2 loadings accounted for 69.78 and 22.97% of the total variation, respectively (Figure 4A). Samples were divided into two main groups (Figure 4A): (1) protochelin and oxidized protochelin, characterized by high PC1 scores, and (2) untreated molybdenite, oxidized molybdenite, and protochelin/oxidized protochelin treated molybdenite under either oxic or anoxic conditions, with relatively low PC1 scores.
The untreated molybdenite and oxidized molybdenite exhibited similar characteristics, with the primary difference being an increased intensity of the O -ions, indicating molybdenite oxidation by air (Figure S16A,B). After interaction with protochelin or oxidized protochelin, organic adsorption to molybdenite surface contributed to the score increase along the PC1 axis (Figure 4A), shifting toward to the "protochelin" and "oxidized protochelin" groups. Negative loadings in PC1 were predominantly attributed to sulfur oxides (SO x ) and Mo-S compounds, whereas positive loadings mainly included carbon and nitrogen compounds (e.g., cyano-based anions CN -, C 3 N -, for molybdenite without protochelin (B1) and with protochelin (B2) addition under oxic conditions. In B2, the blue curve represents the MoS 2 phase and the brown one denotes the MoO 3 phase. High-resolution XPS profiles of C 1s for molybdenite without protochelin (C1) and with protochelin addition (C2) under oxic conditions. Red curves refer to the summary of the fitted peaks.
CNO -etc.) and benzene-containing chemicals (Figure 4B & Table S12). This result suggested that the molybdenite surface was coated by protochelin and its degradation products under either oxic or anoxic conditions (Figure S16C,D).
The oxidation of protochelin predominantly accounted for the variation along the PC2 axis with a shift from positive to negative loadings. Small organic acids and nitrogen-containing organics (i.e., acetate C 2 H 3 O 2 -, C 2 OH -, CO 2 H -, C 3 NO -etc.) were mostly found in negative loadings in PC2 (Figure 4C & Table S13), compared to the organics with larger molecular weights mostly observed in positive loadings (e.g., C
4 H 10 -, C 7 H 14 O 5 -, catechol groups C 6 H 4 O 2 -/C 6 H 5 O 2 -, protochelin C 31 H 34 N 4 O 10
-etc.). These findings aligned with the LC-MS results, confirming protochelin degradation in the presence of oxygen (Figure S17).
In contrast to negative ion spectra, positive ion spectra revealed that Na + and its associated ions significantly contributed to the differences among the samples. Compared to the spectra of the original protochelin, organic peaks (e.g., C 7 H 5 O 3 + , C 6 H 12 + , and C 5 H 10 N + ) either diminished or disappeared upon oxidation (Figure S18). All molybdenite samples showed similar positive spectral patterns (Figure S19) with only Na + showing a significant increase after the addition of protochelin. PCA of positive ion spectra showed similar results (Figure S20). The variation along the PC1 axis was mainly attributed to surface Na + and related ions (e.g., Na 2 OH + , CO 3 Na + , etc.), which were introduced during the experiments through the use of NaOH for pH adjustment (Table S14). Comparatively, the negative loadings contained primarily nitrogen-containing organics and hydrocarbons (Figure S20 & Table S15).
■ DISCUSSION Mechanism of Protochelin-Promoted Molybdenite Dissolution. Siderophore-promoted Fe mineral dissolution mechanisms were previously summarized as surface-controlled (nonreductive) and reductive mechanisms. 27,32,80,81 The surface-controlled mechanism involves: 21,81 (1) siderophore adsorption on mineral surface with the formation of the innersphere surface complex between the hydroxamate/catechol
group and metal on the mineral surface; (2) detachment of the surface metal-siderophore complex, facilitated by polarization of metal-oxygen bonds within mineral structure; (3) surface site regeneration and exposure to additional siderophore complexation. The reductive mechanism refers to electron transfer between the metal and the adsorbed siderophore, resulting in reduction of the metal and oxidation of the siderophore. 22,82 The metal reduction weakens the bond between the surface metal and the crystal lattice, promoting its detachment from the surface. In both mechanisms, the adsorption of siderophores onto minerals is the pivotal step. 21,22 Once siderophores adsorbed to Mo-minerals, similar to Fesiderophore interactions, those organics might chelate with metal Mo exposed on the surface. Among all microbial siderophores, only catecholates are known to effectively bind soluble Mo(VI) under near-neutral pH conditions. 22,56 Specifically, the tricatecholate protochelin forms a 1:1 complex with molybdate as Mo(VI)O 2 H 2 Proto 2-around pH 7.5. 61,64 However, Mo in molybdenite (MoS 2 ) is Mo(IV) rather than Mo(VI). Previous studies found that Mo(IV) can be chelated by either cyanide in the complex ion [Mo(IV)(CN) 8 ] 4-83 or dithiol groups (-SH) in dithiocatechols. 84,85 Acetate has been also shown to form acetato complexes with Mo(IV), Mo(V), and Mo(VI). 86 Stable oxomolybdenum(IV) complexes have also been observed from reduction of Mo(VI) in the presence of dithionite under neutral to alkaline pH conditions. 87,88 It has been suggested that Mo(IV)-organic matter (OM) complexes could potentially form under euxinic conditions. 51 Furthermore, the formation of monomeric Mo(IV)-catechol complexes was reported through reduction of dioxomolybdenum(VI) monomer when pH > 9. 89 However, there is a lack of direct evidence supporting the Mo(IV)-catechol complexation at a near-neutral pH.
Our results emphasized the important role of oxygen in oxidizing both molybdenite and protochelin to facilitate molybdenite dissolution (Figure 1). Under oxic conditions, molybdenite undergoes oxidation in aqueous environments to form Mo(VI) on edges. 90 Previous studies have indicated that molybdenite edges are chemically reactive and prone to reaction with oxygen and water, leading to the formation of oxidized species (HMoO 4 -/MoO 4 2-/MoO 3 ) at the solid-solution interface. 91,92 The increased level of the O -signal in molybdenite ToF-SIMS spectra (Figure S16) confirmed its oxidation. Likewise, protochelin underwent significant oxidation along with adsorption onto molybdenite surface, producing various degradation products and fragments, such as quinones, aromatic compounds, cyano-based anions, N-containing organics, hydrocarbonates, and catechol compounds (Figures S6, S9B, S17, and Tables S12-S15), consistent with previously observed degradation patterns. 63,74 Therefore, it was proposed that the dissolution under oxic conditions was driven by the complexation (1) between Mo(VI) and protochelin, (2) between protochelin oxidation products and Mo(IV) within the molybdenite structure, and (3) between Mo(VI) and protochelin oxidation products.
Promoted dissolution also occurred under anoxic conditions when either molybdenite or protochelin was preoxidized in air (Figure 1C and Table S4). For the experiment with preoxidized molybdenite and protochelin, the complexation between protochelin and the resulting Mo(VI) likely facilitated anoxic dissolution. While for molybdenite and preoxidized protochelin, chelation between protochelin oxidation products and Mo(IV) likely contributed to the dissolution process. Beyond direct Mo complexation, protochelin oxidation may also contribute to molybdenite dissolution by generating intermediates with strong oxidizing potentials, such as semiquinone radicals, superoxide radicals, and hydrogen peroxide. 63,93 These reactive intermediates may oxidize surface Mo(IV), promoting the complexation of Mo(VI) with protochelin and its degradation in this system. Following adsorption, chelation, and oxidation, the complexes detach from the molybdenite surface, creating additional defects (Figures 3A and S12), edges, and active sites. This process promoted further dissolution and enhanced Mo release. 94 Taken together, the proposed protochelin-promoted molybdenite dissolution under oxic conditions involves several steps: (1) air-oxidation of molybdenite surface Mo(IV) to Mo(VI) and air-oxidation of protochelin to form degradation products (e.g., CN -, catechol, acetate, etc.); (2) adsorption of protochelin (and its oxidation products) onto molybdenite surface; (3) formation of Mo(VI)-protochelin (catechol) and Mo(IV)-organic complexes (derived from the oxidation of protochelin), along with possible Mo(IV) oxidation by oxidizing intermediates; (4) detachment of the Mo(VI)-protochelin and Mo(IV)-organic complexes from the molybdenite surface, exposing fresh surface with possible defects; (5) further oxidation of exposed Mo(IV) by air and adsorption of protochelin or its oxidation product to form additional complexes. This iterative adsorption-detachment process, facilitated by protochelin, unintentionally leads to a significant Mo release. 95 Contrasting Effects of Protochelin and DFOB on Molybdenite Dissolution. Whereas protochelin was found to promote molybdenite dissolution (Figure 1A and S3-4), DFOB inhibited it (Figures 1B and S5). At a pH range of 7 to 8, catechol groups within protochelin undergo protonation, resulting in a neutral to positive charge. 63 Similarly, DFOB is mostly protonated and carries positive charges such as H 4 DFOB + . 24 At circumneutral pH, molybdenite surface is negatively charged (Table S2). Consequently, both protochelin and DFOB should adsorb to molybdenite surface through Coulombic attraction. However, protochelin and DFOB exhibited a contrasting behavior in molybdenite dissolution. Therefore, the charge difference alone was unlikely to account for their contrasting behaviors in molybdenite dissolution.
The observed contrasting behaviors between the two siderophores were likely due to the difference in their binding capacity of catechol and hydroxamate functional groups with Mo (Table S1 and Figures S7-S8). In contrast to protochelin, DFOB does not effectively chelate Mo(VI) at the experimental pH ∼ 7.5 (Figures S7 and S8), consistent with previous research showing the decomposition of Mo(VI)-DFOB complex above pH 5. 77 Indeed, no Mo-DFOB complex was observed in solution during the dissolution process (Figure S10). Therefore, the inhibitory effect of DFOB on molybdenite dissolution can be attributed to adsorption of DFOB, which would block the active sites on molybdenite surface 96 and thus inhibit molybdenite dissolution (Figure 1B). Under anoxic conditions, less DFOB was adsorbed onto the molybdenite surface (Figure 2B), likely due to fewer available reactive sites and reduced hydrogen bonding, resulting in a reduced inhibitory effect compared to that under oxic conditions (Figure 1D).
Aqueous metal is scarce across diverse ecosystems, and minerals are major metal reservoirs, 9 but they have limited bioavailability. Organic matter complexation and Mo adsorption onto Fe/Mn Environmental Science & Technology minerals lead to low levels of dissolved Mo, particularly in tropical soils, marine sediments, and ocean bottom water. 44,45 This Mo scarcity becomes more serious under euxinic conditions, as dissolved Mo precipitates as thiomolybdate or insoluble sulfide minerals, such as molybdenite (e.g., MoS 2 ). The result of the current study, the first of its kind, suggests that catecholate siderophores can effectively dissolve molybdenite to release Mo for potential microbial uptake. More importantly, the contrasting effects of the two categories of siderophores strongly suggest a need to consider the balance between the promoting and inhibitory effects of Mo-mineral dissolution in natural environments. Moreover, this work enhances our understanding of Mo release pathways in redox-fluctuating environments by showing that previously oxidized metallophores can enhance Mo-mineral dissolution when the environment becomes anoxic (Figure 1C), such as the widely observed geogenic sources of Mo in groundwater systems. 97 Our work also provides insights into agriculture by proposing the potential use of both siderophores and molybdenite as fertilizers to address Mo limitation in soils. 98,99 Compared to the modern ocean, Mo was scarce in the ancient oceans, a result of low oxidative weathering of minerals. 100 The insignificant molybdenite dissolution by protochelin under anoxic conditions implies its limited role in Mo release from minerals on early Earth. However, the enhanced dissolution of molybdenite by preoxidized protochelin under anoxic conditions hints that chemically and structurally similar compounds to the degradation products of protochelin might have promoted mineral dissolution before the full oxygenation of Earth, despite the unclear evolutionary timeline of siderophores. 101 Rising oxygen levels would have stimulated both oxidative mineral weathering and the emergence of siderophoreproducing microorganisms, further enhancing siderophoremediated mineral dissolution and contributing to the growing pool of dissolved Mo. Moreover, since biological nitrogen fixation using Mo-based nitrogenase is believed to have emerged ∼3.2 Ga ago, 102 our previous studies demonstrated that strict or facultative anaerobic nitrogen-fixing bacteria produced secondary metal-chelating metabolites for Mo uptake from molybdenite structure under strictly anoxic conditions. 44,66,68 Future research should explore the effects of other types of siderophores on the dissolution of various Mo-bearing minerals or rocks under different environmental settings. 103 Factors such as pH and Eh, which can affect the solubility and stability of both siderophores and minerals, should be considered. Furthermore, the presence of organic acids (e.g., oxalate, acetic acid, citric acid) and other minerals such as iron oxides may alter the interaction between siderophores and Mo-minerals. 21,22,27,104 ■ ASSOCIATED CONTENT * sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.4c11212.
Arnow assay, spectrophotometric titration, X-ray diffraction, solid phase extraction, liquid chromatographymass spectrometry (LC-MS) analysis, scanning electron microscopy (SEM), specific surface area (SSA) and particle size analysis, X-ray photoelectron spectrometry (XPS), and time-of-flight secondary ion mass spectroscopy (ToF-SIMS); information on siderophores and molybdenite; sample information for ToF-SIMS analysis; molybdenite dissolution; SSA with particle size; ToF-SIMS loading tables; graphs about molybdenite dissolution experiment design; XRD patterns; dissolution results; LC-MS results for protochelin degradation; solution colors; titration; spectra of Mo(VI)-siderophore complexes; SEM images; XPS profiles; and ToF-SIMS spectra (PDF)
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AUTHOR INFORMATION Corresponding Authors Yizhi Sheng -Center for Geomicrobiology and Biogeochemistry Research, State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Beijing 100083, China; orcid.org/0000-0001-7285-4695; Email: shengyz@cugb.edu.cn Hailiang Dong -Department of Geology and Environmental Earth Science, Miami University, Oxford, Ohio 45056, United States; orcid.org/0000-0002-7468-1350; Email: dongh@miamioh.edu
https://doi.org/10.1021/acs.est.4c11212 Environ. Sci. Technol. 2025, 59, 533-544