Coprecipitation with Ferrihydrite Inhibits Mineralization of Glucuronic Acid in an Anoxic Soil

It is known that the association of soil organic matter (SOM) with iron minerals limits carbon mobilization and degradation in aerobic soils and sediments. However, the efficacy of iron mineral protection mechanisms under reducing soil conditions, where Fe(III)-bearing minerals may be used as terminal electron acceptors, is poorly understood. Here, we quantified the extent to which iron mineral protection inhibits mineralization of organic carbon in reduced soils by adding dissolved 13C-glucuronic acid, a 57Fe-ferrihydrite-13C-glucuronic acid coprecipitate, or pure 57Fe-ferrihydrite to anoxic soil slurries. In tracking the re-partitioning and transformation of 13C-glucuronic acid and native SOM, we find that coprecipitation suppresses mineralization of 13C-glucuronic acid by 56% after 2 weeks (at 25 °C) and decreases to 27% after 6 weeks, owing to ongoing reductive dissolution of the coprecipitated 57Fe-ferrihydrite. Addition of both dissolved and coprecipitated 13C-glucuronic acid resulted in increased native SOM mineralization, but the reduced bioavailability of the coprecipitated versus dissolved 13C-glucuronic acid decreased the priming effect by 35%. In contrast, the addition of pure 57Fe-ferrihydrite resulted in negligible changes in native SOM mineralization. Our results show that iron mineral protection mechanisms are relevant for understanding the mobilization and degradation of SOM under reducing soil conditions.


Soil profile location, description, and mineralogy
In the Borgarfjörður catchment in western Iceland, basalts are primarily Tertiary (older than 3.1 Ma) 1 and the region receives a low influx of aeolian deposition of volcanic ash (25-100 g m -2 yr -1 ). 2 The mean annual temperature (MAT) is 4.6 °C with average annual precipitation of 988 mm yr -1 (station Hvanneyri, 2002-2020; Iceland Meteorological Office, IMO). The Hestur_GA site represents an example of the drainage-impacted low lying (<200 m elevation) wetlands typical across north and western Iceland. 3 Soils for characterization and the incubation study were collected in July 2020. The soil profile was described following FAO guidelines. 4 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 = 1.5418 Å, 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).
Compared to the same soil profile sampled in 2019, (Hestur_GA in ref. 5), the depth of soil horizons in the 2020 soil profile varied slightly, reflecting the heterogeneity of soils affected by cryoturbation. Still, physical, elemental, and mineralogic characteristics of the soil horizon selected for this incubation study, positioned at 60-72 cm depth, were most similar to the soil horizon Hestur_GA_45-60 used in ref. 5 (compare to values reported in Table S1). Soil pH was 4.56 and total Fe and C contents were 73.1 mg g -1 and 21.6 wt.%, respectively. X-ray diffraction patterns indicated the presence of plagioclase feldspars, pyroxenes, and small contributions from quartz and contained a significant amorphous fraction ( Figure S2 and Table S2).   Table S2. Black lines show the measured data, red lines the QPA fit, the dark gray line directly under the pattern shows the background, and the lower light gray line indicates the model misfit. Additional colored lines (purple = plagioclase, gold = augite, teal = quartz) represent the fitted contribution from each respective mineral phase.

Mineral synthesis and characterizations
All solutions used in this experiment were prepared from ultra pure water (UPW, Milli-Q®, Millipore, 18.2 MΩ·cm). Synthesis of isotope-labelled ferrihydrite ( 57 Fh) and the ferrihydriteglucuronic acid coprecipitate ( 57 Fh 13 GluC) followed previously published methods 14 Figure S3). Being a derivative of glucose, a high energy substrate that can be rapidly utilized by soil microorganisms, 17 mineralization of glucuronic acid is expected to be similarly rapid. For the synthesis of 57 Fh 13 GluC, 13 C-labelled glucuronic acid ( 13 GluC, 99% x( 13 C), D-[UL-

13
C6]glucuronic acid sodium salt monohydrate, Omicron Biochemicals) was equilibrated overnight in darkness in UPW water adjusted to pH 7.0 with 1 M NaOH under vigorous stirring (1200/min). The 13 C-glucuronic acid-containing 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 to pH 7.1 ± 0.1 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.
Total Fe content of the (co)precipitates was determined after acid dissolution and subsequent analysis with inductively coupled plasma-optical emission spectrometry (ICP-OES, Agilent 5100). Total C content of 57 Fh 13 GluC was measured with an elemental analyzer (CHNS-932, LECO; n = 4). The Fe:C molar ratio of the ferrihydrite-glucuronic acid coprecipitate 57 Fh 13 GluC was 0.42. The fraction of easily-desorbed C in the coprecipitate was determined after re-suspending ~4 mg 57 Fh 13 GluC in 1 mL of UPW and setting on an orbital shaker (150 rmp) for 4 hours followed by centrifugation (18620 rcf for 10 min) and measuring of the supernatant for dissolved organic carbon (DOC) as described below. Results showed that ~10 mg g -1 C was extracted by H2O, accounting for ~22% of total C in the coprecipitate. The mineral composition of the (co)precipitates was confirmed by powder XRD. For these analyses, dried sample material (∼10 mg) was resuspended in ethanol (∼30 μL, Merck) and pipetted onto a polished silicon wafer (Sil'tronix Silicon Technologies, France). Diffractograms were recorded as described above however using a step size of 0.02°2θ and 10 s acquisition time per step. 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 ( Figure S4).

Details to aqueous DOC and solid-phase C isotope ratio measurements
The isotopic ratio of aqueous samples expressed as δ 13 CDOC was measured by converting the DOC into CO2 by oxidation with a heated Na2S2O8 solution followed by measurement of its isotope ratios. For data normalization and quality control, two in-house reference materials (C3 and C4 sugars) and potassium hydrogen phthalate (KHP) were measured in each run. The external reproducibility of control standards was better than 0.2‰ (±1σ).

Details to headspace gas measurements
For the 25°C incubation headspace gas sampling, ~25 mL of headspace gas was removed through a needle connected to a syringe with a 3-way stopcock valve and immediately injected into the SSIM. While measurements were running (detailed below), the headspace of the bottles was purged with humidified N2 gas at a flow rate of 750 mL min −1 for 10 minutes. During the purging, the bottles were placed on an orbital shaker (150 rpm) at room temperature. Bottles were then injected with ~30 mL of humidified N2 gas through a needle connected to a syringe with a 3way stopcock valve prior to being returned to an orbital shaker (150 rpm) in a temperaturecontrolled room (25 °C). For the 12°C incubation headspace gas sampling, an aliquot of headspace gas was removed through a needle connected to a syringe with a 3-way stopcock valve and injected into a 10 mL N2-flushed sealed septum vial for CO2 and CH4 concentration measurements with gas chromatography. An additional aliquot of headspace gas was injected into a 30 mL N2-flushed sealed septum vial for δ 13 C analysis with CRDS.
Headspace gas samples injected into the SSIM achieved a pressure of >1100 Torr (1170 ± 23, x ̅ ± σ) and measurements were conducted using N2 as a carrier gas. Samples were measured over an 8-minute measurement cycle. For all treatments, temperatures, and timepoints, production of CH4 was negligible. Concentrations of CO2 from the 57 Fh and Control treatments were quantified relative to a 5-point calibration curve of CO2 standards of known concentrations (400, 500, 700, 2600, 5000 ppm, Messer and SPECIALTY) and the δ 13 C values were corrected (value and drift) based on two certified CO2 tank standards (-3‰ and -36‰, Carbagas). Concentrations of CO2 and the 13 C atom fraction of CO2 from the 57 Fh 13 GluC and 13 GluC treatments were calibrated using 11 standards varying from 1 to 85% x( 13 C). These standards were created by mixing 98% x( 13 C) Na2CO3 (Sigma Aldrich) with natural abundance Na2CO3 (δ 13 C = 1.42‰, Fluka), digesting with an excess of 12M HCl and removing aliquots of headspace. 20 These standards were used to confirm the accuracy and precision of measuring CO2 concentrations at high delta values using CRDS. To this end, each of the 11 standards (1 to 85 % x( 13 C)) were prepared as three different concentrations ranging from ~400 ppm to ~6000 ppm CO2 and were measured both with CRDS and GC as described above. In addition, the total CO2 concentrations in selected experiment samples were additionally measured with GC. The linearity of the δ 13 C correction at varying CO2 concentrations and high δ 13 C values (R 2 = 0.9997) as well as the linear relationship between total CO2 concentrations at varying δ 13 C values measured with CRDS and GC (R 2 = 0.9985) are shown Figure S5 and confirm the viability of using CRDS for both CO2 concentration as well as δ 13 C value determination in the ranges of this study.
Dissolved CO2 in the soil slurries was estimated based on Henry's law using the dimensionless H constant. Therefore, in the Control and 57 Fh treatments, where pH remained <6 for the duration of the incubation, estimates of dissolved CO2 are likely accurate, whereas increases in pH >6 and thus a dominance of bicarbonate in solution in the 57 Fh 13 GluC and 13 GluC treatments (after 5 and 4 weeks, respectively) indicate that dissolved CO2 may be underestimated at these timepoints. Figure S5. Calculated versus measured 13 CO2 / 12 CO2 molar ratios (left panel, n = 37) and total CO2 concentrations measured by CRDS and GC (right panel, n= 42). In both panels, measured samples include synthetic standards (n = 11, 1 to 85% x( 13 C); each standard prepared at 3 different CO2 concentrations ranging from ~400 ppm to ~6000 ppm, randomly measured in duplicate). Additionally, for selected experiment samples (n = 12), total CO2 concentrations were measured with both CRDS and GC (shown in red in the right panel). Figure S6. Iron isotope composition of Feaq, shown as f n Fe, where n = 56 (blue) or 57 (green), for the 25°C, 6-week incubations. Dashed lines are: the calculated isotope composition of the total system (TS) following the addition of the isotope-labelled coprecipitates and the natural abundance (NA) isotope composition considering the iron isotopes 56 Fe and 57 Fe only. 18 Error bars show the standard deviation from triplicate incubation bottles.