Iron (Fe) is abundant in soils but often exhibits low bioavailability due to its occurrence in insoluble (oxyhydr)oxide minerals. To overcome Fe limitation, microorganisms and plants produce siderophores, high-affinity organic ligands that solubilize and transport Fe(III). However, the environmental fate of siderophores in soils remains poorly understood because of the chemical complexity of soil matrices and the difficulty of distinguishing siderophore-associated Fe from large background Fe pools. In this study, gallium (Ga(III)) was employed as an analog for Fe(III) to investigate the behavior of structurally distinct siderophores in soils. Synchrotron-based micro-X-ray fluorescence (μ-XRF) mapping and extended X-ray absorption fine structure (EXAFS) spectroscopy were used to examine the distribution and speciation of Ga(III) complexes with the hydroxamate siderophore desferrioxamine-B (DFOB), the catecholate siderophore protochelin, and the carboxylate phytosiderophore proline-2′-deoxymugineic acid (PDMA) in three contrasting soils: a Cassville clay, a Belhaven muck, and a Gruver series soil. Siderophore fate varied strongly with both ligand structure and soil composition. In the Cassville clay, interactions were dominated by retention of Ga(III)-siderophore complexes and sorption to Fe- and Mn-oxide-coated clay domains, indicating that clay-rich soils can act as strong sinks for siderophores. In the Belhaven muck, retention of DFOB and protochelin complexes was driven by interactions between hydrophobic ligand backbones and nonpolar organic matter domains. In the Gruver soil, all three siderophores were associated with poorly crystalline Fe phases, while electrostatic repulsion between negatively charged mineral surfaces and negatively charged siderophore complexes promoted complex dissociation. Across all soils, the lower metal-binding affinity of PDMA relative to DFOB and protochelin resulted in extensive dissociation of Ga(III)-PDMA complexes and redistribution of Ga among diverse soil components. These findings demonstrate that both siderophore structure and soil physicochemical properties exert strong controls on the retention, transport, and stability of siderophore-metal complexes. This work provides new insight into the mechanisms governing Fe acquisition and micronutrient cycling in soils and highlights the importance of soil composition in regulating siderophore-mediated nutrient availability.

2.1. Materials. All solutions were prepared using ASTM Type 1 (>18.2 MΩ∙cm) deionized water (DI). All chemicals used were of reagent grade from Fisher Scientific (Hampton, NH) or Sigma-Aldrich (St. Louis, MO). The three siderophores examined in this study were chosen for their structural diversity. The mesylate salt of the trishydroxamate siderophore DFOB was purchased from Sigma Aldrich (>92.5%) and used without any further purification. Protochelin (a triscatecholamide siderophore) were synthesized by the Duke University Small Molecules Custom Synthesis Facility (Harrington et al. 2012). Proline-2′-deoxymugineic acid PDMA was provided as PDMA∙2HCl by Aichi Steel Corporation (Japan) (Suzuki et al. 2021).

2.2. Soils Characterization. Soils chosen for this study include a Cassville clay, Gruver series, and a Belhaven muck. All soils were air dried and passed through a 2 mm sieve. The Cassville series was sourced from Cassville County, NC and with a sandy clay loam texture (51.5% sand, 17.6% silt, 30.9% clay) and a pH of 6.5 ± 0.2. The Gruver series was sourced from Goodwell, Oklahoma, and is a no-till agricultural soil (irrigated corn into alfalfa) with a pH of 8.0 ± 0.4 and a loam texture (38.1% sand, 45.0% silt, 16.8% clay). The Belhaven series was sourced from a field in Plymouth, NC and is a poorly drained organic soil with a pH of 6.3 ± 0.3. Total Fe and Mn concentrations in the soils was determined by using a XRF analyzer (Niton 5XL+) in soils mode.

2.3. Ga K-edge extended X-ray absorption fine structure spectroscopy.Bulk Ga K-edge XAS spectra were collected at the Stanford Synchrotron Radiation Lightsource (SSRL) under normal operating conditions on beamline 11−2 on samples consisting of Ga(III)-siderophore adsorbed to soils. Spectra were energy calibrated by adjusting the E₀ to an elemental Ga sample. Spectra were collected in fluorescence mode using a 100-element Ge detector or in transmission mode. The energy was selected by using a variable-exit Si(220) double-crystal monochromator and harmonics were rejected with a rhodium-coated mirror. Spectra were collected using Soller slits and a Zn X-ray filter improved the signal-to-noise ratio. Three spectra were collected for each sample, with no evidence of beam damage in successive scans, and averaged to improve the signal-to-noise ratio. For two samples, transmission data was used due to poor fluorescence data quality.

Ga K-edge spectra were energy calibrated, averaged, background-subtracted, splined, and linear combination fit (LCF) (Kelly, Hesterberg, and Ravel 2008) using the SIXPACK (Webb 2005) interface which makes use of the IFEFFIT code (Newville 2001). LCF analyses were conducted with previously published Ga(III)-siderophore mineral standards (Evers in prep), which are summarized in SI Table 1. All spectral components used in fits were prepared as described by Evers at al and were extensively characterized in Evers et al. Because the previous work found that Ga(III)-DFOB, Ga(III)-PDMA, and Ga(III)-protochelin adsorbed as intact complexes to a variety of minerals, the presence of free Ga(III)-DFOB, Ga(III)-PDMA, and Ga(III)-protochelin in LCF fits can be interpreted as  mineral associated, inner sphere, outer sphere, ternary complexes, or intercalated species. In the fits, the presence of standards composed of only Ga(III) sorbed to minerals, with no siderophore present, were interpreted as dissociation of the complex followed by sorption of Ga(III) to the surface. Components found to make up less than 5% of the spectral reconstruction were removed, and fits were recalculated with the remaining standards.