Iron-mediated organic matter decomposition in humid soils can counteract protection

Synopsis

Consistent with a protective role, under static oxic conditions we found that Fe^II oxidation in the presence of added ¹³C-DOM resulted in SRO Fe–C associations that not only inhibited the mineralization of ¹³C-DOM by 35% relative to controls, but also suppressed the priming of native SOM mineralization by 47%, which consequently decreased overall CO₂ production by 22% (Fig. 1d). However, when ¹³C-DOM was not added, Fe^II oxidation and the production of reactive oxygen species stimulated mineralization of native SOM by 8% relative to the controls (Fig. 1d). Thus, the formation of additional SRO–Fe phases did not provide net protection to SOM unless there was additional DOM present. As might be expected, the protective role of Fe was reversible under anoxic conditions. Although CO₂ production from non-Fe amended treatments during the anoxic period was 68–70% lower than in the static oxic treatment (Fig. 1d), the de novo SRO Fe–MAOM formed via Fe^II oxidation was disproportionately vulnerable to subsequent reduction. This consequently stimulated the mineralization of both added ¹³C-DOM and the native SOM by 74% and 32–41%, respectively, and thus increased overall CO₂ production by 41–49% relative to both non-Fe amended treatments (with or without added DOM, Fig. 1d). As a result of Fe-stimulated C mineralization, the anaerobic ¹³C-DOM mineralization was 81% greater than the oxic control. Below we provide details on the production of the Fe–MAOM, discuss the data supporting Fe protection of C along with the data supporting Fe stimulation of C loss, and then provide a synthesis of the work.

Generation of FeIII-(oxyhydr)oxides

The oxidation of ⁵⁷Fe^II after a 1-d equilibration with the soil under anoxic conditions generated SRO Fe^III (oxyhydr)oxides that impacted C cycling. Exposure to O₂ (day 1–6) led to the oxidation of Fe^II, with aqueous Fe^II completely oxidized within 6 h. The sorbed Fe^II substantially decreased by 91% over the first day and slowly declined thereafter (Fig. 2). The treatment with both ⁵⁷Fe and ¹³C-DOM added had 10% more adsorbed ⁵⁷Fe^II than the ⁵⁷Fe^II-only treatment before oxidation (Fig. 2a and b), likely due to co-sorption of the Fe²⁺–DOM complex, as observed previously⁴⁴. The variable-temperature Mössbauer spectroscopy technique that we use to track the mineral composition of the ⁵⁷Fe additions, gives excellent information on the crystallinity of the Fe phases, with high crystallinity phases ordering at higher temperatures. Both ⁵⁷Fe addition treatments led to the formation of de novo SRO ⁵⁷Fe^III phases of lower crystallinity (lower Mössbauer ordering temperature) than the bulk soil Fe (Table 1; Supplementary Fig. 2 and Fig. 3), resulting in a 26–31 mmol kg⁻¹ increase in lepidocrocite and 3–14 mmol kg⁻¹ increase in nanogoethite and very-disordered Fe^III (oxyhydr)oxides that preclude assignment (Fig. 3; Supplementary Table 1). The addition of ⁵⁷Fe and ¹³C-DOM together resulted in the formation of even lower crystallinity SRO Fe^III (oxyhydr)oxides than the ⁵⁷Fe addition-only treatment as illustrated by the lower 35K/5K and 12K/5K crystallinity ratios (Table 1; Supplementary Fig. 3). Suppression of Fe^III crystallinity by co-precipitation with dissolved fulvic acids has been shown previously in synthetic pure systems⁴⁴, and here we extended this finding to a complex soil system containing a mixture of aluminosilicates, Fe^III (oxyhydr)oxides, and a variety of organic compounds. In general, lower crystallinity Fe (oxyhydr)oxides (often measured by oxalate-extraction) have higher surface area, sorb more OM, and are thought to be associated with persistent OM in soils⁴⁵.

Fig. 2: Fe^II concentration and ⁵⁷Fe to total Fe ratio.

Time-dependent a aqueous Fe^II, b sorbed (HCl-extractable) Fe^II, and c dissolved ⁵⁷Fe to total Fe ratio in the aqueous phase in the redox-fluctuating treatment. Fe^II and ⁵⁷Fe were undetectable during day 1.5–6 and 17.5–22. The gray shaded region represents the anoxic phase of the redox-fluctuating treatment. Error bars indicate s.e.m. (n = 3).

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Fig. 3: Solid-phase speciation of added ⁵⁷Fe.

⁵⁷Fe partition was calculated from respective Mössbauer spectra (corrected to exclude the signal from the native soil Fe) for the (a)  ⁵⁷Fe-only and (b) ¹³C-DOM-⁵⁷Fe addition treatments, prior to the oxic phase (day 1) and at the end of the 1st oxic (day 6), anoxic (day 17) and 2nd oxic (day 22) phases. Error bars represent standard errors associated with Mössbauer data modeling (see Supplementary Information).

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Iron protection of organic matter

In the static oxic treatment, addition of ⁵⁷Fe suppressed the mineralization of ¹³C-DOM by 35% (p < 0.01, Figs. 1d, 4 and Table 2): cumulative CO₂ production was 25.6 ± 0.8 and 39.5 ± 1.1 mmol C kg⁻¹ with and without ⁵⁷Fe, respectively, equivalent to 17.1% and 26.3% of the added ¹³C-DOM (Fig. 4c and Table 2). Although ⁵⁷Fe addition also inhibited net ¹³C-DOM mineralization in the fluctuating redox treatments (p < 0.01, Fig. 4c and Table 2), this inhibition was confined to the oxic portions (days 1–6 and days 17–22) of the incubation and was partly offset by a 74% enhanced ¹³C-DOM mineralization during the anoxic phase (days 6–17) relative to the treatment without added Fe (Figs. 1d, 4b, and Table 2, see below).

Fig. 4: Mineralization of the amended ¹³C-labeled DOM.

a The percent contribution of ¹³C-labeled DOM to total CO₂ production, b CO₂ production rates from ¹³C-labeled DOM, and c cumulative CO₂ production from ¹³C-labeled DOM. The gray shaded region represents the anoxic phase of the redox-fluctuating treatment. Error bars indicate s.e.m. (n = 3).

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The generation of low crystallinity SRO–Fe^III (oxyhydr)oxides from the oxidation of ⁵⁷Fe^II in the presence of ¹³C-DOM resulted in a lower DOC concentration than in the ¹³C-DOM-only treatment and a concurrent increase in solid-phase ¹³C content (Fig. 5). This likely reflects the formation of SRO Fe–C complexes with ¹³C-DOM adsorbing or co-precipitating with the newly-formed SRO lepidocrocite and nanogoethite phases (Fig. 3). It is generally assumed that SRO Fe phases contribute to soil C persistence by protecting it against microbial mineralization⁵, but few studies have directly measured the bioavailability of Fe-associated OM³⁹. Our study provides evidence that de novo formation of SRO Fe–C complexes inhibit the mineralization of fresh DOM inputs to soil. Others have also observed a large decrease in OM decomposition when glucose or fulvic acid sorbed to synthetic Fe minerals (ferrihydrite/goethite) was added to soils, as compared to additions of the free organic compounds^40,41. The bioavailability of mineral-associated OM is generally thought to be linked to C loadings (e.g., C/Fe ratios), with a maximum adsorption capacity occuring at a C/Fe molar ratio of about one⁴⁶. Co-precipitation could result in Fe–OM associations with much higher C/Fe ratios^11,12,19. In our study, the initial C/Fe molar ratio of the added ¹³C-DOM and ⁵⁷Fe was 2.1. If we assume that all DOM that was removed from the solution during the Fe^II oxidation event sorbed to the newly-formed Fe^III (oxyhydr)oxides, the C/Fe ratio of those OM–Fe^III (oxyhydr)oxide complexes would be ~1.7. Thus, there was likely ¹³C-DOM with a low affinity for Fe^III (oxyhydr)oxides that remained as unprotected ¹³C-DOM in the aqueous phase and this likely led to our observation of significant ¹³C-DOM mineralization even in the presence of de novo Fe^III (oxyhydr)oxides (Fig. 4).

Fig. 5: Dissolved organic carbon concentration and solid-phase ¹³C enrichment.

a Dissolved organic carbon concentration (MES buffer concentration was subtracted) and b solid-phase ¹³C atom fraction from ¹³C-DOM addition treatments. The gray shaded region represents the anoxic phase of the redox-fluctuating treatment. Error bars represent s.e.m. (n = 3).

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Labile C inputs are often observed to alter the decomposition of extant SOM, defined as priming^47,48. During the oxic periods of the experiment, ⁵⁷Fe^II oxidation in the presence of added ¹³C-DOM not only suppressed the mineralization of the amended ¹³C-DOM, but also partially inhibited the priming of native SOM decomposition compared to the DOM-only treatment (Fig. 6; Table 2; Supplementary Table 2). In the static oxic treatment, addition of ¹³C-DOM alone or together with ⁵⁷Fe increased native SOM-derived CO₂ production compared to the soil-only control (priming effect) (p < 0.01, Fig. 6a, c and e; Table 2; Supplementary Table 2). However, adding ¹³C-DOM and ⁵⁷Fe together resulted in a significantly smaller priming effect on native SOM mineralization than adding ¹³C-DOM alone under the static oxic treatment (p < 0.01, Fig. 6e, Table 2 and Supplementary Table 2). With the addition of ¹³C-DOM, cumulative primed CO₂ from native SOM under the static oxic treatment measured 10.2 ± 1.2 and 19.1 ± 0.9 mmol C kg⁻¹ with and without ⁵⁷Fe addition, respectively (Fig. 6e and Supplementary Table 2). Cumulatively, adding ¹³C-DOM together with ⁵⁷Fe suppressed aerobic priming of native SOM by 47% relative to adding ¹³C-DOM alone (Fig. 1d, Table 2 and Supplementary Table 2). Collectively, ⁵⁷Fe oxidation in the presence of ¹³C-DOM resulted in 22% less overall C mineralization, compared to addition of ¹³C-DOM alone (Fig. 1d and Table 2).

Fig. 6: Native soil C mineralization.

CO₂ production rates from soil C under a static-oxic and b redox-fluctuating conditions, cumulative CO₂ production from soil C under c static-oxic and d redox-fluctuating conditions, and priming of soil C under e static-oxic and f redox-fluctuating conditions. The gray shaded region represents the anoxic phase of the redox-fluctuating treatment. Error bars indicate s.e.m. (n = 3).

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We propose that de novo SRO Fe^III minerals protected the added ¹³C-DOM under static oxic conditions, decreasing DOM availability for microbial growth, and suppressing the priming of native SOM. An alternative explanation, that the sorption of native SOM onto the de novo SRO Fe^III (oxyhydr)oxides inhibited priming, is unlikely because when we added ⁵⁷Fe^II alone it actually increased the mineralization of native SOM (due to reactive oxygen species production, as discussed below) (Fig. 6a, e and Table 2). Similarly, prior studies have shown that the addition of new SRO Fe^III phases to soils has little to no impact on the mineralization of native SOM^41,49. Rather, it is likely that DOM–Fe^III interactions create physico-chemical barriers that limit priming by decreasing microbial access to the new DOM. Thus, when reduced soils receive oxygenated water due to rainfall, snowmelt, or irrigation, the oxidation of Fe^II in the presence of DOM and formation of OM–Fe^III complexes may contribute to C protection both directly, as previously known, and indirectly, by suppressing priming.

Iron stimulation of DOM and SOM mineralization

Only in the treatment where ¹³C-DOM and ⁵⁷Fe were added together were we able to confirm that Fe had an overall protective effect on OM, and that protection was limited to the oxic portions of the experiment. Below, we quantified the impact of Fe on OM mineralization via Fe-stimulated Fenton chemistry during the first few days of oxic exposure and via Fe reduction-mediated reactions during the anoxic periods.

Adding ⁵⁷Fe alone strongly stimulated CO₂ production from native SOM during the first 3 days of the static oxic treatment (and the oxic portions of the fluctuating redox treatment) relative to the soil-only control (p < 0.01), with no stimulatory impact afterwards (Fig. 6a and e). Cumulatively, Fe^II oxidation stimulated CO₂ production by 8% (Fig. 1d and Table 2). To confirm the role of Fenton chemistry, we performed a parallel experiment with added terephthalate—an effective hydroxyl radical scavenger—and found similar CO₂ production between Fe^II-added treatments and soil-only controls (Supplementary Fig. 4). Recent studies have similarly shown that Fe^II oxidation is linked to increases in soil CO₂ production via the generation of radical oxygen species^50,51, which facilitate the breakdown of complex biopolymers to produce labile substrates for microbial respiration^34,35. Others have also attributed increased CO₂ production following Fe^II oxidation to an increase in acidity that can promote DOC release³⁶. Given that we conducted these experiments in a strong buffer at a constant pH, the increased CO₂ production following Fe^II oxidation was most likely derived from the production of reactive oxygen species such as the hydroxyl radical.

Soil C mineralization rates typically decrease as O₂ becomes limiting^22,52,53. In our soil-only control, CO₂ production from native SOM during the anoxic period was 70% lower than in the static oxic treatment (Figs. 1d, 6 and Table 2). However, during the anoxic portions of the experiment, Fe addition stimulated native SOM mineralization relative to the no-Fe treatment (Fig. 6b and f; Table 2). In the ⁵⁷Fe-addition treatment, the degree of anoxic suppression of CO₂ production decreased from 70 to 58% of that under oxic conditions (p < 0.05; Table 2), as a result of a 41% higher anoxic native SOM-derived CO₂ production in the Fe addition treatment than in the no-addition control (p < 0.05, Table 2; Figs. 1d, 6b, d and f). This likely resulted from enhanced microbial use of Fe^III as an electron acceptor in the ⁵⁷Fe addition treatments. Following the transition from oxic to anoxic conditions in the fluctuating redox treatments, substantial Fe^III reduction occurred (day 6–17, Fig. 2a and b) and adding ⁵⁷Fe increased the total Fe^II production rates (2.9 mmol kg⁻¹ d⁻¹) compared to the soil-only control (1.6 mmol kg⁻¹ d⁻¹). This was most likely due to the facile reduction of de novo ⁵⁷Fe SRO lepidocrocite and nanogoethite, which had a much lower crystallinity (and thus a higher reactivity) than native soil Fe^III (oxyhydr)oxides (Table 1 and Fig. 3; Supplementary Table 1 and Fig. 3). The availability of native SRO Fe^III phases likely limits Fe reduction in this subtropical agricultural soil, and the de novo SRO ⁵⁷Fe^III phases were preferentially utilized as electron acceptors for microbial respiration as evidenced by the preferential release of ⁵⁷Fe^II in the aqueous phase (Fig. 2c) and the measured decrease of these ⁵⁷Fe mineral phases following reduction (Fig. 3a and Table 1; Supplementary Table 1).

Iron’s stimulation of C mineralization during anoxic periods was greatly enhanced when ⁵⁷Fe and ¹³C-DOM were added together, yielding increases in mineralization of native SOM (anoxic priming) and ¹³C-DOM by 32 ± 3% and 74 ± 7%, respectively, relative to adding ¹³C-DOM alone (p < 0.05; Table 2; Figs. 1d, 4b, 4c, 6b, d and f). In fact, when¹³C-DOM and ⁵⁷Fe were added together, anaerobic ¹³C-DOM mineralization in the fluctuating redox treatment was even 81% greater than the aerobic ¹³C-DOM mineralization in the static oxic treatment at the same point in time (p < 0.01; Fig. 4b; Table 2). The stimulation of ¹³C-DOM mineralization under anoxic conditions was linked in part to its molecular composition, given that thermodynamic constraints on Fe reduction limit metabolism to relatively oxidized C substrates^22,42,43. During the anoxic periods, the mineralization of ¹³C-DOM over the native SOM (in both the ¹³C-DOM only and DOM–Fe addition treatments) was 2–3 times higher than that in the static oxic treatment at the same point (Fig. 4a). Characterization of the molecular composition of ¹³C-DOM and water-extractable native SOM using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) revealed that the ¹³C-DOM had significantly less lignin-derived materials and much more aliphatic formulae than the water-extractable native SOM (Supplementary Table 3, Figs. 5 and 6), which represents the most bioavailable fraction of native SOM⁵⁴. The preferential anaerobic mineralization of ¹³C-DOM over SOM may be due to a lower abundance of lignin-derived compounds, which are not readily depolymerized under anoxic conditions⁵⁵. In addition, compared to water-extractable native SOM, ¹³C-DOM contains compounds with higher nominal oxidation state of C (NOSC values > 0.5, Supplementary Fig. 6), which are associated with a higher likelihood of thermodynamic favorability (−ΔG_r) when coupled to Fe^III reduction than the bioavailable fraction of native SOM^22,43. Fe reduction was also stimulated by the addition of ¹³C-DOM alone (producing 2.3 mmol kg⁻¹ d⁻¹ of Fe^II compared to the soil-only control rate of 1.6 mmol kg⁻¹ d⁻¹) (Fig. 2a and b), consistent with prior work²⁶. However, when Fe and DOM were added together, Fe reduction was greatly increased to 7.4 mmol kg⁻¹ d⁻¹, which was even greater than the additive effect of separate ¹³C-DOM (2.3 mmol kg⁻¹ d⁻¹) and ⁵⁷Fe additions (2.9 mmol kg⁻¹ d⁻¹) (Fig. 2a and b). This was because compared to oxidation of ⁵⁷Fe^II alone, oxidizing ⁵⁷Fe^II in the presence of ¹³C-DOM led to formation of even less-crystalline SRO lepidocrocite and nanogoethite phases (all ordering at <35 K in the Mössbauer spectra, Table 1 and Supplementary Fig. 3). These SRO ⁵⁷Fe-¹³C-OM phases exhibited high rates of Fe reduction, releasing significant ⁵⁷Fe²⁺(aq) and ¹³C-DOM (Figs. 2 and 5) when exposed to anoxic conditions, leaving the solid phase depleted in its lowest crystallinity Fe phases (Fig. 3 and Table 1; Supplementary Table 1 and Fig. 7), and preferentially stimulating anaerobic mineralization of the added ¹³C-DOM (Fig. 4a).

Fe reduction can solubilize significant amounts of OM adsorbed or coprecipitated with Fe^III (oxyhydr)oxides directly, as shown in our experiment, or indirectly because of an increase in pH^30,32,56. This re-mobilized ¹³C-DOM often includes biochemically labile C^32,57, and may potentially offset the kinetic/thermodynamic constraints often limiting anaerobic decomposition^22,43,58. We find that collectively, the reduction of SRO Fe^III phases offset O₂ limitations on C mineralization by 24 ± 3% relative to the non-Fe amended treatment (Table 2).

Synthesis

A recent survey of over 5500 soil profiles spanning continental scale environmental gradients found that SRO Fe and Al (oxyhydr)oxide abundance was the best predictor of C content in humid soils, among the geochemical and climate variables that were available⁴⁵. This is consistent with other work showing that SRO Fe^III phases are broadly implicated in the persistence of OM in soil^1,3,59. However, the nature of the relationship between Fe and C in humid soils—and redox dynamic soils in general, which would include floodplain and perennial wetland soils from all climatic regions—is far from straightforward. Humid soils are replete with microsites that undergo dynamic anoxia in response to high labile C loads during periods of high moisture and experience appreciable Fe^III reduction rates^23,25,60,61. Oxidation of the Fe^II generated from Fe^III reduction is a common mechanism for MAOM formation in humid and redox-dynamic soils, yet Fe is also responsible for OM loss and our work here illustrates two principal refinements in this regard.

First, the production of SRO Fe–MAOM via Fe^II oxidation will likely increase CO₂ production in the short-term. Only when we formed MAOM in the presence of DOM and maintained strict oxic conditions was there a net decrease in C mineralization (both in the added ¹³C-DOM and the native SOM, i.e. via decreased priming). When we simply generated MAOM via Fe^II oxidation without added DOM, Fenton chemistry caused an 8% increase in C mineralization (Fig. 1d). Upon the inevitable return to periodic anoxia in humid soils, our work suggests that C mineralization would be accelerated by 41–49% by Fe reduction (Fig. 1d), thus counteracting the stabilization effect on OM of SRO Fe phases. The magnitude of these counteracting mechanisms may also be influenced by soil structure, which we largely eliminated in our study by conducting experiments in soil slurries. Hence, direct application of our results to in situ soil environments is tentative. However, the general principles of our work are also likely to be applicable to structurally complex soil systems. For example, Fe mineral-associated C is often released in natural soils under in-situ flow conditions as a consequence of dissimilatory Fe reduction (e.g.,⁶²) and thus becomes more vulnerable to microbial decomposition. In our study, we even found that the added DOM was preferentially degraded under anoxic conditions relative to the oxic control (Fig. 4), which highlights how the thermodynamic constraints of anaerobic metabolism and the molecular composition of C sources can influence the fate of fresh DOM inputs^22,42,43. Consequently, the net effect of Fe–C interactions in dynamic redox environments likely hinges in part on the composition of DOM inputs, a worthy topic for further research.

Second, our work here suggests that the initial SRO Fe–C associations are not likely to persist without protection from periodic Fe reduction events. Several researchers have identified or produced SRO–Fe^III–OM colloids that are resistant to either microbial or chemical reduction^{63,64,65,66,67}, however, the key components conferring this protection are variable and/or elusive. Some work has identified that SRO–Fe^III–OM co-precipitates with low C/Fe ratios provide resistance to microbial reduction^63,64, whereas other work has emphasized structural properties (conformation and micro-aggregation) as the mechanism that retards dissolution^65,66,67. SRO Fe–OM phases are often co-precipitated with Al and Si ions⁶⁸—which can retard recrystallization⁶⁹—and given the co-association of Al and Fe with OM in humid soils, Al is a strong candidate for protecting Fe against reduction. However, studies that have examined Al and Si co-precipitated Fe-(oxyhydr)oxides found those ions also make the co-precipitates more susceptible to reductive dissolution⁷⁰. Coward et al.⁶⁷ recently proposed several mechanisms by which SRO Fe^III–OM phases could become resistant to reductive dissolution, including acquiring reduction-resistant surface coatings, or becoming embedded in a composite aggregate structure⁶. Such a protective coating could even come from higher crystallinity Fe (oxyhydr)oxides. Hall et al. recently found that ¹⁴C-derived C residence time in humid soils was positively correlated with Fe phase crystallinity⁷¹. Consistent with that, we find here that in contrast to the initial oxidation event, the 2nd oxidation event generated more crystalline ⁵⁷Fe phases (Table 1; Supplementary Fig. 8) and did not stimulate additional C mineralization (Fig. 6 and Table 2). It may be that during repeated redox fluctuations a substantial portion of the co-precipitated OM would be lost, but a core Fe–MAOM structure would remain protected from reductive dissolution.

Perhaps most compelling is the growing evidence that various aggregation, conformation, and structural characteristics of soils confer protection for OM^5,6,7,10. Even the protective surface coatings^66,67 or conformational changes in OM at low C/Fe ratios⁶⁴ discussed above are examples of micro-aggregate structures not unlike the encasement of SRO Fe–OM phases by aluminosilicate clays or other processes that generate micro-aggregates of minerals and OM during pedogenesis^6,7,10,72. These aggregation processes can structure microaggregates with core SRO Fe phases and outer aluminosilicate or other phases that are not susceptible to reductive dissolution—as observed in Andisols by dithionite-resistant SRO–Fe phases⁶⁶. Our soil slurry approach was designed to minimize the physical constraints (macro-pore flow, spatial arrangement of microbes, minerals and OM, and the development of aggregates) on C decomposition and thereby isolate the sorbent and electron-transfer roles of Fe in C dynamics (Supplementary Fig. 1). Under these conditions, we find that Fe does not confer intrinsic protection for OM in redox-dynamic soils. In an in situ soil environment—where MAOM emerges in a dynamic three-dimensional space—structural and physical protection of MAOM is thus likely a key protective mechanism for reconciling the comparatively large proportions of SRO-OM associations in soil of very old age based on ¹⁴C-dating^1,4,5,59. Future studies should thus assess the extent that the formation and destruction of Fe-cemented microaggregates contribute to OM persistence in redox-dynamic soils. Our work demonstrates that the inherent persistence of SRO Fe-associated C cannot be guaranteed. Biological and geochemical context is critical for understanding the long-term fate of Fe^III-associated SOM under a changing climate, given the dual roles of Fe^III phases in both accelerating and inhibiting OM decomposition.

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