Emerging investigator series: Coprecipitation with glucuronic acid limits reductive dissolution and transformation of ferrihydrite in an anoxic soil

Ferrihydrite, a poorly crystalline Fe(iii)-oxyhydroxide, is abundant in soils and is often found associated with organic matter. Model studies consistently show that in the presence of aqueous Fe(ii), organic carbon (OC)-associated ferrihydrite undergoes less transformation than OC-free ferrihydrite. Yet, these findings contrast microbial reductive dissolution studies in which the OC promotes the reductive dissolution of Fe(iii) in ferrihydrite and leads to the release of associated OC. To shed light on these complex processes, we quantified the extent of reductive dissolution and transformation of native Fe minerals and added ferrihydrite in anoxic soil incubations where pure 57Fe-ferrihydrite (57Fh), pure 57Fe-ferrihydrite plus dissolved glucuronic acid (57Fh + GluCaq), a 57Fe-ferrihydrite-13C-glucuronic acid coprecipitate (57Fh13GluC), or only dissolved glucuronic acid (13GluCaq) were added. By tracking the transformation of the 57Fe-ferrihydrite in the solid phase with Mössbauer spectroscopy together with analysis of the iron isotope composition of the aqueous phase and chemical extractions with inductively coupled plasma-mass spectrometry, we show that the pure 57Fe-ferrihydrite underwent more reductive dissolution and transformation than the coprecipitated 57Fe-ferrihydrite when identical amounts of glucuronic acid were provided (57Fh + GluCaqversus57Fh13GluC treatments). In the absence of glucuronic acid, the pure 57Fe-ferrihydrite underwent the least reductive dissolution and transformation (57Fh). Comparing all treatments, the overall extent of Fe(iii) reduction, including the added and native Fe minerals, determined with X-ray absorption spectroscopy, was highest in the 57Fh + GluCaq treatment. Collectively, our results suggest that the limited bioavailability of the coprecipitated OC restricts not only the reductive dissolution of the coprecipitated mineral, but it also limits the enhanced reduction of native soil Fe(iii) minerals.


Soil profile location, description, and mineralogy
Soils for characterization and the incubation study were collected in July 2020.The soil profile was described following FAO guidelines. 2 Individual horizons were manually homogenized and packaged in their field-moist state into plastic bags which were then stored at 4°C in the dark.Subsets of each soil horizon were air dried (30°C) and sieved (<2 mm, nylon) for characterization.Soil pH was determined after re-suspending the dried soil in UPW at a solid:solution ratio of 1:5 for 1 hr.Total element contents of each soil horizon were measured with energy-dispersive X-ray fluorescence (XRF) spectrometry (Spectro X-Lab 2000) and total C and N contents with an elemental analyzer (Vario MAX Cube, Elementar).
Mineral composition of the soil horizon was determined by powder X-ray diffraction (XRD, D8 Advance, Bruker).For these analysis, 30 °C dried and sieved soil was milled to ~50 µm using a disk swing mill.Milled soil material was analyzed as powder XRD in Bragg−Brentano geometry using Cu Kα1,2 radiation (λ = 1.5418Å, 40 kV, and 40 mA) and a high-resolution energydispersive 1-D detector (LYNXEYE).Diffractograms were recorded from 10° to 70°2θ with a step size of 0.02°2θ and 6 s acquisition time per step.The relative contributions of the crystalline mineral phases in the diffraction patterns were determined by Rietveld Quantitative Phase Analysis (QPA) using the TOPAS software (Version 5, Bruker AXS) in combination with published crystallographic structure files.Additionally, the amount of amorphous material was estimated by the internal standard method in the TOPAS software using aluminum oxide (Al2O3, Fluka) as the internal standard mixed into the soil at a mass ratio of 1:2 (Al2O3:soil).
For the synthesis of 57 Fh, the 57 Fe(III) stock solution was titrated with 1 M NaOH (Titrisol®) under vigorous stirring (1200/min) until a pH of 7.1 ± 0.1 was reached.For the synthesis of 57 Fh 13 GluC, 13 C-labelled glucuronic acid ( 13 GluC, 99.99% 13 C, D-[UL-13 C6]glucuronic acid sodium salt monohydrate, Omicron Biochemicals) was equilibrated overnight in darkness in 1 L UPW water adjusted to pH 7.0 with 1 M NaOH under vigorous stirring (1200/min).The 13 C-glucuronic acidcontaining solution was then acidified to pH 4.0 with 1 M HNO3 (Normatron®, VWR) and purged with N2(g) for 15 min.After adding an aliquot of the 57 Fe(III) stock solution, the solution was titrated with 1 M NaOH as described in the synthesis of ferrihydrite.The mineral suspensions were then centrifuged at 3600 g for 15 minutes, decanted, and re-suspended in UPW three times until the conductivity of the supernatant was <350 µS/cm.Afterwards, the suspensions were shock frozen by dropwise injection into liquid N2 and freeze dried, homogenized with a mortar and pestle, and stored in brown glass bottles in a desiccator until use.Detailed characterization of the resulting solid phases, including total element content, the fraction of easily-desorbed C in the coprecipitate, and confirmation of the mineral phases present using powder XRD, has been previously published. 1Briefly, the C:Fe molar ratio of the ferrihydrite-glucuronic acid coprecipitate 57 Fh 13 GluC was 0.42 and ~10 mg g -1 C was easily-desorbed, accounting for ~22% of total C in the coprecipitate.For both 57 Fh and 57 Fh 13 GluC, XRD patterns confirmed the presence of 2-line ferrihydrite, visible as broad maxima around 2.54 and 1.49 Å.   13 GluC, or (13-) GluC.

Aqueous-and solid-phase sampling procedure
To prevent accumulation of CO2 in the headspace of the septum bottles between samplings, the headspace was purged with humidified N2 gas at a flow rate of 750 mL min −1 for 10 minutes every 2-4 days during the entire experiment.During the purging, the bottles were placed on an orbital shaker (150 rpm) at room temperature.After 72 h and 1, 2, 4, 5, and 6 weeks, following the purging of the headspace, the septum bottles were moved into the glovebox, where they were opened for anoxic sampling.First, pH and Eh (reported as Eh7; the redox potential converted to pH 7) were measured directly in the soil slurry.Then, the bottles were then manually agitated to ensure resuspension of all soil particles and ~5 mL of the soil slurry was poured into 15 mL Falcon tubes which were then capped, wrapped in Parafilm, and removed from the glovebox for centrifugation (3000 g for 15 minutes).The centrifuged tubes were returned to the glovebox, the supernatant pipetted off and additionally filtered (<0.45 µm, nylon) and acidified for further aqueous analyses (described below).To ensure the removal of all aqueous Fe(II), the residual solid-phase was then resuspended by adding 5 mL of anoxic UPW to the Falcon tube and manually shaking it.The Falcon tubes were then again capped, wrapped in Parafilm, and removed from the glovebox for centrifugation (3000 g, 15 minutes), then returned to the glovebox.The supernatant was pipetted off and the residual solid phase allowed to dry in the glovebox atmosphere in the dark (<24 h).
Solid-phase samples were then manually homogenized with a mortar and pestle and stored in the dark in the glovebox until further analyses.After sampling, the septum bottles were then re-capped and removed from the glovebox and returned to the orbital shaker (150 rpm) at 25°C.Supporting Information to ThomasArrigo et al.

X-ray absorption spectroscopy
Iron K-edge (7112 eV) X-ray absorption spectroscopy (XAS) spectra were collected at the XAFS beamline of ELETTRA (Trieste, Italy) and at BM23 of ESRF (Grenoble, Italy).At ELETTRA, Xray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectra were recorded in transmission mode at ∼80 K using a N2(l) cryostat.Higher harmonics in the beam were eliminated by detuning the monochromator by 30% of its maximal intensity and two to four scans were collected and averaged.At ESRF, spectra were recorded in transmission mode at ~10 K using a He(l) cryostat and higher harmonics in the beam were eliminated by mirrors.At both beamlines, the Si(111) monochromator was calibrated to the firstderivative maximum of the K-edge absorption spectrum of a metallic Fe foil (7112 eV).The foil was continuously monitored to account for small energy shifts (<1 eV) during the sample measurements.
All spectra were energy calibrated, pre-edge subtracted, and post-edge normalized in Athena. 5Linear combination fit (LCF) analyses of Fe K-edge XANES spectra were conducted over an energy range of -20 to 30 eV (E-E0) with E0 of sample and reference compound spectra defined as the highest peak in the first XANES derivatives.Linear combination fit analyses of k 3weighted Fe K-edge EXAFS spectra were performed over a k-range of 2-12 Å -1 with the E0 of all spectra and reference compounds set to 7128 eV.No constraints were imposed during LCF analyses, and initial fit fractions (XANES: 101±1%, EXAFS: 88±5%) were recalculated to a compound sum of 100%.
Iron-containing reference compounds used for LCF analysis were selected based on previous measurements of similar soils from Iceland. 6Visual comparison of the E0 of the illite (1M1-1) reference sample suggested the sample contains both Fe(II) and Fe(III).This was confirmed with LCF, which showed that 1Mt-1 contained ~20% Fe(II) (Figure S6).S3. a Fe(III) and Fe(II) were fit by the references ferrihydrite and SWA-1_red, respectively.b Normalized sum of squared residuals (100∑i(datai-fiti) 2 /∑idata 2 ).c Fit accuracy; reduced χ 2 = (Nidp/Npts)∑i((dataifiti)/εi) 2 (Nidp-Nvar) -1 .Nidp, Npts and Nvar are, respectively, the number of independent points in the model fit, the total number of data points (249 or 38 for 57 Fh+GluCaq), and the number of variables in the fit (2).εi is the uncertainty of the i th data point.Initial fit fractions (101±1%) were recalculated to 100%.d Theoretical contributions calculated based on (co-)precipitate additions listed in Table S2.
Supporting Information to ThomasArrigo et al.

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Figure S5.k 3 -weighted Fe K-edge EXAFS spectra of references used in linear combination fits (LCF). 57Fh and 57 Fh 13 GluC are included here for comparison but were not used in LCF analyses.
The total amount of Fe in poorly-crystalline or amorphous mineral form (Feo) remained relatively stable over the incubation, with minor increases (Control, 57 Fh 13 GluC and GluCaq treatments) and decreases ( 57 Fh, 57 Fh+GluCaq treatments) noted.The lack of clear trends agrees with previous anoxic incubations of a similar soil horizon (Hestur_GA_45-60 in ref. 6 ), where only minor increases in Feo were recorded (note that in ref. 6 , extractions were not sequential).In contrast to Feo, trends in Fep; organically-bound or colloidal Fe, were more easily discernible and increased across all treatments during the incubation.For the 13 GluCaq and 57 Fh+GluCaq treatments, the increase in Fep may be linked to the newly formed fraction of organicallycomplexed Fe(II) indicated by LCF analysis of EXAFS spectra (Table 1).It is possible that organically-complexed Fe(II) also formed in the other treatments, however contributions were less than our accepted detection limit for LCF analysis of EXAFS spectra (5 %).Increases in Fep in all treatments may also be linked to colloidal Fe, as previous anoxic incubations recorded the formation of iron-and organic-rich fine colloids (3 kDa to 0.45 µm).  3) a As determined by the 1,10-phenanthroline method 7,8 in ( b ) 0.5 M HCl extractions 9 as the first step of a 2-step sequential extraction method.c Acid ammonium oxalate extraction 7 as the second step of a 2-step sequential extraction method.d A sodium-pyrophosphate treatment 10 was conducted on separate samples.e Theoretical contributions calculated for 57 Fh, 57 Fh 13 GluC and 57 Fh+GluC treatments based on extractions of the (co-)precipitates, whereby Fe in the 57 Fh and the 57 Fh 13 GluC coprecipitate was completely (>98%) mobilized in the 0.5 M HCl extraction and was hardly mobilized (<4%) by the sodium-pyrophosphate treatment.Errors in parenthesis represent the standard deviation of triplicate incubation bottles for the 0.5 M HCl and acid ammonium oxalate extractions.For the sodiumpyrophosphate treatment, solid phases from the triplicate incubation bottles were combined and the treatment was conducted in duplicate (error shown in parenthesis).
Supporting Information to ThomasArrigo et al.The soil horizon selected for this incubation study (from the Hestur_GA (2020) soil profile, 60-72 cm depth) was analyzed with Mössbauer spectroscopy 77 K and 5 K (Figure S6, Table S7).The and therefore likely corresponds to a mixture of these two minerals.The second sextet, Fe(III)-S2, contributed to 32 % of the spectrum and had fitting parameters compatible with lepidocrocite: CS = 0.50 mm s -1 , ε = 0.00 mm s -1 and H = 45.3 T. 11 Alternatively, the high carbon content of this soil (21.6 wt.%) suggests that the Fe(III)-S2 sextet is ferrihydrite coprecipitated in the presence of dissolved organic matter, which has been shown to cause significant decreases in the hyperfine field. 12The collapsed phase, CF, observed in the 5 K spectra, accounted for 23 % of the spectrum and contained Fe oxyhydroxides near their ordering temperatures, thus assigning them to individual mineral phases is not possible.However, similar Fe phases have been suggested to consist of organic matter-mineral associations 12 or Fe minerals associated with Al or Si. 13 The Fe(II) doublet, contributing to 5 % of the spectrum, had fitting parameters that could be compatible with Fe(II) in clays or Fe(II) sorbed onto Fe minerals: CS = 1.20 mm s -1 and QS = 2.27 mm s -1 . 14wever, the loss in the area of the Fe(II) doublet in the 5 K spectrum compared to the 77 K spectrum, combined with the high center shift value of the collapsed feature (0.73 mm s -1 ) suggests the area assigned to the collapsed feature may additionally contain Fe(II) minerals that are ordered into an octet indistinguishable from the rest of the collapsed feature.The Fe(III) doublet, Fe(III)-D1, contributed to 13 % of the spectrum and had fitting parameters compatible with monomeric Fe(III)-OM or Fe(III) in clays: CS = 0.48 mm s -1 and QS = 0.75 mm s -1 . 15

Initial soil + 57 Fh or 57 Fh 13 GluC
Mössbauer spectra of the unreacted soil + 57 Fh or 57 Fh 13 GluC at all temperatures were dominated by the added ferrihydrite and most the components attributed to the soil could no longer be distinguished (compare to the spectra of the Hestur_GA soil in Figure S6).At 77 K, the spectra were fit with an Fe(III) doublet (Fe(III)-D) of CS = 0.46 mm s -1 and QS = 1.06 mm s -1 (soil + 57 Fh) and CS = 0.46 mm s -1 and QS = 0.95 mm s -1 (soil + 57 Fh 13 GluC).At 5 K, the spectra were also similarly composed of a broad Fe(III) sextet corresponding to Fh 16 and magnetically ordered components originating in the soil (≥97% of 57 Fe atoms) and a small Fe(III) doublet component most likely originating from the soil (see Table S8).
Considering that the soil matrix in both treatments is identical and only minorly contributes to the overall Mössbauer signal (~10 %), differences in fitted parameters are attributed to varying characteristics of the 57 Fe-labelled ferrihydrite in the 57 Fh and the 57 Fh 13 GluC (co-)precipitates.
For example, the fitted mean hyperfine field of the Fe(III)-S1 sextet matching ferrihydrite in the soil + 57 Fh 13 GluC spectra was consistently smaller than that of the same feature in the 57 Fh + soil spectra (47.3 T vs. 48.1 T at 5 K).1][22] To further assess the crystallinity of ferrihydrite in the 57 Fh and 57 Fh 13 GluC (co- )precipitates, we additionally collected spectra at intermediate temperatures (Figure S8).Both the soil + 57 Fh and soil + 57 Fh 13 GluC spectra are mostly ordered at 45 K, suggesting that the blocking temperatures of the (co-)precipitates was between 45 K and 77 K, in agreement with values in literature for ferrihydrite. 11However, a direct comparison of the samples at lower temperatures (e.g., 35 K) revealed that the soil + 57 Fh spectra more closely resembled a sextet (as opposed to a collapsed feature or a doublet) than the soil + 57 Fh 13 GluC sample, indicating that the soil + 57 Fh was more magnetically ordered, indicative of a higher degree of crystallinity or stronger interparticle interactions.Lower ordering temperatures have been previously reported for Fe(III)(oxyhydr)oxides formed in the presence of organic carbon (e.g., refs. 12,20,214][25][26] Based on these analyses, we conclude that the 57 Fe-labelled ferrihydrite in 57 Fh was slightly more crystalline than that in the 57 Fh 13 GluC coprecipitate.S8.S9.
Supporting Information to ThomasArrigo et al.

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Table S7.Mossbauer fitting parameters of initial unreacted soil and the (co-)precipitates.

Figure S1 .
Figure S1.(L) Map of Iceland with soil profile sampling location.Orthoimage based on data from National Land Survey of Iceland.(R) The complete Hestur_GA (2020) soil profile.

Figure S2 .
Figure S2.(Left) X-ray diffraction patterns of 57 Fh and 57 Fh 13 GluC.(Co)precipitates show the broad peak features at 2.54 and 1.49 Å typical of 2-line ferrihydrite.Figure published in ref. 1 .(Right) Magnitudes of the Fourier transform k 3 -weighted Fe K-edge EXAFS spectra of 57 Fh and 57 Fh 13 GluC.Qualitative comparison suggests that the coprecipitate has lower amplitudes for features corresponding to corner-and edge-sharing Fe.

Figure S3 .
Figure S3.Aqueous geochemical data.Trends in pH (A), redox potential (Eh7; Eh calculated relative to pH 7) (B), aqueous Fe (Feaq; panel C), and dissolved organic carbon (DOC; panel D) concentrations.Error bars indicate the standard deviation calculated from triplicate incubation bottles.Parts of this data have been previously published in ref. 1.

Figure S4 .
Figure S4.(A) First derivatives of normalized Fe K-edge XANES spectra of reference spectra and incubated soil samples and (B) LCF fits of the normalized spectra.Experimental data and model fits are shown as solid lines and symbols, respectively.Fit results are reported in TableS3.

Figure S6 .
Figure S6.LCF of Fe K-edge XANES spectra of the illite (1Mt-1) reference used here.Results indicated that the reference contained ~20 % Fe(II).Experimental data and model fits are shown as solid lines and symbols, respectively.

Table S1 .
Physical and elemental characterization of the Hestur_GA (2020) soil profile.

Table S2 .
Experimental conditions.a

Table S3 .
Linear combination fit results for Fe K-edge XANES spectra after 6 weeks anoxic incubation.a

Table S4 .
Fractions of Fe(II) fit with LCF analysis of XANES and EXAFS spectra.Note that fractions of Fe(II) based on LCF of EXAFS spectra were determined assuming the Fe in clay fraction contained 20% Fe(II) and 80% Fe(III).See FigureS6and discussions above.

Table S5 .
Results from selective chemical extractions.
a 57 Fe fractions in Feaq were determined in terms of 56 Fe to57Fe only and therefore are not included in the iron isotope recovery.All other 57 Fe fractions are reported relative to the iron isotopes54Fe, 56 Fe, 57 Fe and 58 Fe.Supporting Information to ThomasArrigo et al.S147.

57 Fe Mössbauer spectroscopy 7.1 Hestur_GA 60-72 soil horizon
Center shift with respect to α-57 Fe 0 .b Quadrupole splitting, QS (for doublets) or quadrupole shift, ε (for sextets).c Mean hyperfine field.d Standard deviation of QS (doublets) or H (sextet). e Goodness of fit.Phases marked in italics are components of the previous phase.Note that the percentage of components always sums to 100% but refers to the percentage of the previous phase.*Indicates values that were fixed during the fitting process.Abbreviations: Fh = ferrihydrite.

Table S8 .
Mossbauer fitting parameters of unreacted (co-)precipitate + soil mixtures.Center shift with respect to α-57 Fe 0 .b Quadrupole splitting, QS (for doublets) or quadrupole shift, ε (for sextets).c Mean hyperfine field.d Standard deviation of QS (doublets) or H (sextet). e Goodness of fit.Phases marked in italics are components of the previous phase.Note that the percentage of components always sums to 100% but refers to the percentage of the previous phase.*Indicates values that were fixed during the fitting process.Abbreviations: Fh = ferrihydrite.

Table S9 .
Mossbauer fitting parameters of 2-and 6-week incubated samples.Mossbauer fitting parameters of 2-and 6-week incubated samples.Mossbauer fitting parameters of 2-and 6-week incubated samples.Center shift with respect to α-57 Fe 0 .b Quadrupole splitting, QS (for doublets) or quadrupole shift, ε (for sextets).c Mean hyperfine field.d Standard deviation of QS (doublets) or H (sextet). e Goodness of fit.Phases marked in italics are components of the previous phase.Note that the percentage of components always sums to 100% but refers to the percentage of the previous phase.*Indicates values that were fixed during the fitting process.Abbreviations: Fh = ferrihydrite. a