Unraveling iron oxides as abiotic catalysts of organic phosphorus recycling in soil and sediment matrices

Domain: earth_science
Published: 2024-07-18
Editorial outcome: major_revision

Abstract
In biogeochemical phosphorus cycling, iron oxide minerals are acknowledged as strong adsorbents of inorganic and organic phosphorus. Dephosphorylation of organic phosphorus is attributed only to biological processes, but iron oxides could also catalyze this reaction. Evidence of this abiotic catalysis has relied on monitoring products in solution, thereby ignoring iron oxides as both catalysts and adsorbents. Here we apply high-resolution mass spectrometry and X-ray absorption spectroscopy to characterize dissolved and particulate phosphorus species, respectively. In soil and sediment samples reacted with ribonucleotides, we uncover the abiotic production of particulate inorganic phosphate associated specifically with iron oxides. Reactions of various organic phosphorus compounds with the different minerals identified in the environmental samples reveal up to twenty-fold greater catalytic reactivities with iron oxides than with silicate and aluminosilicate minerals. Importantly, accounting for inorganic phosphate both in solution and mineral-bound, the dephosphorylation rates of iron oxides were within reported enzymatic rates in soils. Our findings thus imply a missing abiotic axiom for organic phosphorus mineralization in phosphorus cycling.

Introduction
Phosphorus (P), an essential element for all organisms, has a unique yet incompletely understood biogeochemical cycle. The predominant P form in the critical zone is the oxyanion, phosphate (PO43-), which cycles between precipitated minerals (e.g. apatite), adsorbed, and dissolved inorganic and organic species in soils1, sediments2, freshwater and marine3,4 environments, and atmospheric dust5. A significant fraction of the total P in environmental matrices is found as organic phosphate (Porg), accounting for 20–70% of total P in soils6 and 26–64% in lake sediments7. Since orthophosphate or inorganic phosphate (Pi) is the primary bioavailable form for biological P uptake and assimilation, plants and microbes employ phosphatases and related enzymes to catalyze the hydrolysis of Porg to produce Pi through nucleophilic attack8–11. This enzymatic reaction has been the focus of studies on P transformation in environmental matrices. However, there is growing research implicating also mineral surfaces, in particular iron (Fe) oxides and oxyhydroxides, in mediating Porg dephosphorylation12–16, the mechanism of which may resemble the metal active sites of phosphate-cleaving enzymes17 such as alkaline phosphatase.

The Fe oxide and Fe oxyhydroxide minerals, hereafter referred to collectively as Fe oxides, play a critical role in the regulation of biogeochemical cycles such as P through species adsorption and subsequent release following desorption or mineral dissolution3,18,19. Consequently, Fe oxide adsorbents are considered to serve as both a source and a sink of Pi in natural environments20,21. The adsorption of Pi and Porg species onto Fe oxides has been extensively studied22–27. The Fe oxide content in soils and sediments, which can be up to 166 g Fe kg-1, has been reported to sequester up to half of the total soil P18. However, the difficulty of determining which specific P species (Pi or Porg) are associated with Fe oxides has prevented a comprehensive understanding of the role of Fe oxides in mediating the fate of Porg.

Prior studies of mineral-mediated hydrolytic cleavage of Porg have focused on monitoring products in solution. Using ultraviolet-visible (UV-vis) absorption spectroscopy for solution analysis of para-nitrophenyl phosphate (p-NPP)12, a synthetic model Porg, Fe oxides (goethite, hematite, and an amorphous phase), manganese (Mn) oxides (akhtenskite, pyrolusite, and an amorphous-type phase), and titanium oxides (anatase and rutile) were all shown to be effective at cleaving the phosphoester bond in this synthetic Porg. With respect to environmentally relevant Porg, Fe oxide nanoparticles were reported to mediate the dephosphorylation of adenosine-5’-triphosphate (ATP, a triphosphorylated ribonucleotide) and glucose-6-phosphate (G6P, a monophosphorylated sugar) based on colorimetric measurements of Pi in solution using molybdate and UV-vis absorbance13. Combining this colorimetric technique with 31P nuclear magnetic resonance for solution Pi analyses, a crystalline Fe oxide (hematite), a hydrated Mn oxide (birnessite), and, to a lesser degree, an aluminum (Al) oxide (boehmite) were all reported to generate Pi in solution from the phosphomonoesters G6P, glycerophosphate, and adenosine-5’-monophosphate (AMP), as well as the triphosphorylated ATP16. High-resolution liquid chromatography-mass spectrometry (LC-MS) has been used for solution analysis of organic products to confirm the dephosphorylation of ATP, adenosine-5’-diphosphate (ADP), and AMP by ferrihydrite, a short range-ordered Fe oxide14 and birnessite28. Furthermore, hydrolysis of RNA by goethite and hematite was reported by using high-performance LC and UV-vis absorbance to detect nucleobases in solution29. Therefore, the traditional view that adsorbed Porg on Fe oxides is protected from transformation due to a presumed low catalytic reactivity of Fe oxides needs to be revisited, particularly within the context of P cycling.

Yet, the aforementioned solution-based data captured an incomplete picture of the mineral-catalyzed Porg dephosphorylation, because adsorbed (or particulate) species associated with the mineral surface could include the produced Pi from Porg. Advances in mineral surface characterization by synchrotron-based P K-edge X-ray absorption near-edge structure (XANES) spectroscopy have made it possible to distinguish between Pi and Porg bound to Fe in minerals30 or Fe oxides in a soil matrix31. In terms of monitoring mineral-catalyzed Porg dephosphorylation, one application of the XANES technique with ferrihydrite revealed the generation of particulate Pi from adsorbed ribonucleotides, while Pi was notably absent from solution14. This latter finding, which was confirmed by quantifying the dephosphorylated organic products in solution by LC-MS14, highlights the need for quantitative analysis of particulate P, in addition to dissolved Pi, especially for minerals such as Fe oxides with strong adsorption affinity for Pi species. For instance, soil P content was found to be associated predominantly with semi-crystalline Fe oxides18, but it remains unknown whether these minerals contribute to the particulate Pi fraction in soil by catalyzing the dephosphorylation of mineral-bound Porg. Furthermore, Fe oxide-associated P fractions have been implicated in enhancing plant-bioavailable P, but an underlying abiotic catalytic process has not been explored32.

Here we tested the hypothesis that Fe oxides would catalyze the recycling of P through the hydrolytic cleavage of Porg bound to Fe oxides in heterogeneous environmental matrices. To this end, we coupled the surface-sensitive XANES analysis with high-resolution LC-MS to investigate the role of Fe oxides as simultaneous adsorbents and catalysts for ribonucleotides reacted with natural sediment and soil samples. As remnants of nucleic acids, ribonucleotides represent a ubiquitous and abundant class of naturally occurring Porg compounds. A major fraction of Porg is stored in RNA and DNA, which have been found in soils at 56 µg g-1 and 435 µg g-1, respectively33,34. Through biotic and abiotic reactions, monophosphorylated ribonucleotides are generated from the nucleic acid polymers; moreover, diphosphorylated and triphosphorylated ribonucleotides are essential metabolites widely involved in carbon and energy metabolism in plants and microorganisms3, and may account for >25% of P content in certain microbial populations4,35. In this study, we focused on performing reactions with two ribonucleotides with different phosphate bonding: ATP (containing a phosphoester and two phosphoanhydride, P-O-P, bonds) and AMP (containing only a phosphoester, C-O-P, bond)9. Because phosphomonoesters are common Porg types in sediment and soil systems36–38, we performed experiments with two other relevant naturally-occurring phosphomonoesters: G6P, a sugar phosphate involved in sugar metabolism; inositol hexakisphosphate or phytate, the primary P storage in terrestrial plants. To investigate the reactivity of the different mineral types in the natural samples, we reacted the four different Porg compounds with pure forms of Fe oxides (goethite, hematite, and ferrihydrite) and various silicate-bearing minerals (quartz, clays, mica). Taken collectively, the findings from this research challenge the lack of consideration in the P cycle, beyond enzymatic processes, of an abiotic contribution of Fe oxide minerals in the generation of Pi from particulate Porg sources.

Results
Characterization of the sediment and soil samples
For our experiments with natural samples, we chose lake sediment and forest soil samples with similar endogenous Fe content (~50 mg Fe kg-1 dry sample), excavated from Missisquoi Bay in the U.S. state of Vermont and an Ultisol from the Calhoun Experimental Forest in the U.S. state of South Carolina, respectively (Fig. 1a). Missisquoi Bay is a eutrophic section of Lake Champlain that undergoes diel and seasonal oxic-anoxic cycles39 (Fig. 1a, b). These redox cycles promote dynamic formation of Fe oxides, which are implicated in the mobility and fate of the bioavailable sediment P, albeit the mechanism is not well understood. In this lake environment, P transformation is attributed to enzymatic and biological activity, but little is known about the potential conversion of Porg to Pi by Fe oxides (Fig. 1b). In our complimentary forest setting, the soils at the Calhoun Experimental Forest contain redoximorphic features and undergo redox fluctuations in response to rainfall and organic carbon pulses, leading to periodic dissolution and reformation of Fe minerals40 (Fig. 1b).

We combined multiple characterization techniques to determine the specific minerals in the sediment and soil samples that would be responsible for adsorption and catalytic reactivities towards Porg compounds: X-ray fluorescence (XRF) to determine the major elements present [Supplemental Information (SI), Fig. S1]; X-ray diffraction (XRD) to identify the major crystalline mineral phases (quartz, micas, feldspars, clays, or Fe oxides) in accordance with the elemental composition from the XRF analysis (SI, Fig. S2); and, Fe K-edge XANES spectroscopy to resolve different forms of Fe phases such as Fe oxides of low crystallinity (i.e., ferrihydrite) and high crystallinity (i.e., hematite, goethite), an Fe phosphate mineral (i.e., vivianite), and an Fe-rich mica (i.e., biotite) (SI, Fig. S3-Fig. S5, and Table S1). While our Fe XANES data accounted for low-crystallinity Fe-mineral phases, our XRD data do not account for the possible presence of low-crystallinity silicate or aluminosilicate phases in the natural samples. With respect to the possible presence of low-crystallinity aluminum (Al)-containing minerals, we obtained 27Al nuclear magnetic resonance (NMR) spectra of the sediment and soil samples to probe for the expected five-coordinated Al in the reactive sites of these minerals41–43. Our NMR data only detected tetrahedral and octahedral Al as would be found in clays and micas, which can be identified by XRD; no five-coordinated Al was detected (SI, Fig. S6). Furthermore, as will be discussed in the next section, there was minimal to no catalytic reactivity of silicate or aluminosilicates toward the different Porg compounds (Fig. 2). No Mn-bearing minerals were included in the mineral composition analysis because neither environmental sample exhibited a quantifiable amount of Mn (SI, Fig. S1).

Taken collectively, our analysis revealed that the sediment sample contained primarily glauconite (a mica, 26%), followed by quartz (21%), illite (a 2:1 clay, 16%), albite (a feldspar, 16%), and biotite (a mica, 8%); about 12% of the sediment contained Fe oxides identified as ferrihydrite (~10%) and goethite (2%), with a minor fraction (<1%) present as vivianite (Fig. 1c). The soil sample contained primarily kaolinite (a 1:1 clay, 69%) and quartz (12%), with the remaining fraction constituted of Fe oxides as ferrihydrite (10%), hematite (4%), and goethite (4%); vivianite was also present as a minor fraction (<1%) (Fig. 1c). Our sediment characterization was in agreement with a previously reported mineral composition for the sediment sample44, albeit the Fe content was greater than a previously reported value39. For the soil, the ratios of Fe oxide minerals in the soil mineral composition were in close agreement with the reported values from extracted Fe content, but the absolute values were not directly comparable to previous 57Fe Mössbauer spectroscopy data45. These discrepancies with previous data were likely due to differences in sample preparation or characterization techniques, such as the different scales of ordered minerals probed by Fe XANES versus Mössbauer spectroscopies and characterization of separate 5 µm soil sections versus bulk sample. In sum, based on our analysis, we found that about 80% (or more) on a per-mass basis of both sediment and soil samples was comprised of silicate minerals of different types (quartz, micas, feldspars, clays), and <20% constituted the Fe oxide fraction (Fig. 1c). Next, we explored the reactivities of these different mineral types in the environmental samples towards Porg compounds.

Exceptional adsorption and catalysis by Fe oxides
We performed reactions of various Porg compounds with the different mineral types identified in the natural samples (at 1 g L-1 or 4 g L-1): biotite (a mica), quartz, kaolinite (a 1:1 clay), illite (a 2:1 clay), ferrihydrite (a low-crystallinity Fe oxide), hematite (a crystalline Fe oxide), and goethite (a crystalline Fe oxide) (Fig. 2; SI, Fig. S6–Fig. S10). In our first set of experiments, we chose ATP (ATP-P, 150 µM or 4.6 mg L-1) as our representative Porg that contains both phosphoanhydride (P-O-P) and phosphoester (C-O-P) bonds that are ubiquitous in the reservoir of Porg compounds derived from metabolism and biomass of plants and microbes. We used high-resolution LC-MS to quantify particulate and aqueous species of Pi and Porg for the ribonucleotide reactants and products in solution (i.e., ATP, ADP, AMP, adenosine), a visible light absorption spectroscopy method for solution Pi, and synchrotron-based P K-edge XANES spectroscopy for the relative fractions of particulate Pi and particulate Porg associated with Fe-bearing minerals; particulate Pi on non-Fe minerals was determined via mass balance based on the concentrations of organic and inorganic products in solution after reactions with a Porg reactant. We note that the XANES analysis can determine the relative organic fraction versus inorganic fraction of the total particulate P, but does not discriminate between possible different Porg compounds in the particulate Porg fraction14.

Following the reaction with ATP, quartz and the aluminosilicates (mica, kaolinite, illite) exhibited minimal to no catalytic reactivity whereby aqueous Pi or particulate Pi accounted for <4% of the reacted Porg; in terms of adsorption reactivity, less than 20% of the reacted Porg was found as particulate Porg with these minerals (Fig. 2a). In contrast, the reactions with the different Fe oxides (ferrihydrite, hematite, or goethite) yielded substantial dephosphorylation or adsorption of the Porg reactant, characterized by up to 31% as aqueous Pi, 12–36% as particulate Pi, and up to 46% as particulate Porg (Fig. 2a). In regard to the fate of the reacted Porg (i.e., sum of transformed and adsorbed Porg-derived P) across the Fe oxide minerals, ferrihydrite exhibited the highest adsorption with greater than 75% of reacted P as particulate Porg, while goethite was the most catalytically active, with 53–79% of the reacted P as collectively adsorbed Pi and aqueous Pi (Fig. 2a). Despite a greater than 10-fold difference in the specific surface area of ferrihydrite (230 m2 g-1) compared to goethite (16 m2 g-1), there was a near 3-fold higher fraction of particulate Pi species on goethite than on ferrihydrite (p<0.05) (Fig. 2a), suggesting that mineral surface chemistry rather than specific surface area governs the extent of catalytic reactivity, a worthwhile research avenue to pursue in future investigations.

Catalytic reactivity of Fe oxides in mineral mixtures
Since Fe oxides are present in heterogeneous mineral mixtures in natural samples, we also investigated how the presence of other minerals (ferrihydrite, quartz, or kaolinite) would influence the high catalytic reactivity of goethite towards ATP (Fig. 2b). We prepared mineral mixtures based on the composition of the natural samples (Fig. 1c). First, with a 4:1 ferrihydrite:goethite mixture, we found that the amount of particulate Pi generated in the mixture was equivalent to that of goethite alone (p=0.21) while there were an 8-fold increase in particulate Porg (p<0.001) and no Pi in solution, indicating that the higher adsorption reactivity of ferrihydrite relative to goethite overwhelmed the reactivity in the ferrihydrite:goethite mixture (Fig. 2b). Second, when comparing the goethite-only condition to 4:1 quartz:goethite and kaolinite:goethite mixtures, the same amount of dissolved Pi was produced from the Porg reactant (p≥0.75) but only 20% of the particulate Pi fraction remained (p<0.01), and this was accompanied by a 6-fold increase in particulate Porg (p<0.01) (Fig. 2b). Our data thus implied that, in terms of the production of solution Pi or particulate Pi, Fe oxides would likely retain their catalytic reactivity in mixed-mineral matrices in environmental samples.

Catalytic reactivity of Fe oxides with different Porg types
Amongst the chemical diversity of Porg types found in biomolecules, phosphomonesters are widely found in soils36–38,46,47. To probe Fe oxide reactivity with these other types of Porg, we performed reactions involving each of the three Fe oxides (ferrihydrite, hematite, and goethite) with three phosphomonoesters: AMP (AMP-P, 50 µM or 1.5 mg P L-1), G6P (G6P-P, 50 µM or 1.5 mg P L-1); and phytate (phytate-P, 300 µM or 9.0 mg P L-1) (Fig. 2c–e). To compare the Fe oxide reactivity with the other mineral types in the environmental samples, we also performed experiments of the phosphomonoester compounds reacted with quartz and the aluminosilicates (mica, kaolinite, illite) (Fig. 2c–e).

In contrast to the ATP reactions, all the Pi derived from the reacted AMP with goethite and ferrihydrite was retained as particulate Pi while aqueous Pi was absent, a significant finding that was made possible here due to the application of the XANES technique (Fig. 2c). On the one hand, the catalytic reactivity of ferrihydrite was higher for AMP than for ATP, as reflected by the 20-fold increase in the particulate Pi fraction (p<0.001) (Fig. 2a, c). On the other hand, the catalytic reactivity of goethite was less for AMP than for ATP as characterized by a 3-fold decrease in particulate Pi fraction (p<0.01) accompanied by no change in the particulate Porg fraction (p=0.29) and no measured solution Pi (Fig. 2a, c). Hematite did not display any adsorption or catalytic reactivity towards AMP (Fig. 2a and c). These findings here are consistent with a previous report15 of 2-fold to 6-fold greater reactivity of goethite than hematite for p-NPP dephosphorylation, albeit direct comparison between p-NPP (a synthetic Porg) and AMP (a natural biomolecule) is not appropriate due to the smaller molecular weight of p-NPP versus AMP (263.1 g mol-1 versus 324.23 g mol-1) and the difference in their chemical structures such as the presence of a sugar base and two heterocyclic nitrogenous rings in AMP whereas p-NPP has one benzyl ring. Interestingly, after all the ATP reacted with goethite was transformed or adsorbed in our aforementioned experiments with ATP and goethite, 20–24% of the initial ATP-P remained as AMP-P in solution and no ADP was detected (SI, Fig. S11). The results with the AMP-goethite experiment implied that this accumulation of AMP in the ATP-goethite experiment was due to the lower reactivity of goethite for AMP relative to ATP (Fig. 2c; SI, Fig. S11). Notably, the silicate and aluminosilicate minerals did not exhibit any adsorption or catalytic reactivity towards AMP (Fig. 2c).

With respect to phytate, the silicate and aluminosilicate minerals were all found to adsorb phytate (from 5% to 50% of the total reacted phytate-P) and, rather than an Fe oxide, the clay illite adsorbed the most phytate (Fig. 2d). However, similar to the results with AMP and ATP, catalytic reactivity towards phytate was only obtained with Fe oxides (Fig. 2d). Relative to controls, phytate-derived particulate Pi was higher by 8–14% (p<0.01) with hematite and by 24–32% with goethite (p<0.001); the particulate Pi with ferrihydrite, however, corresponded to the adsorption of solution Pi from the control experiment (Fig. 2d). As with AMP, the most significant adsorption for G6P was with ferrihydrite, with 47–78% of the reacted G6P found only as particulate Porg (Fig. 2e). Some G6P adsorption (4–12%) was observed with two of the aluminosilicates (mica and kaolinite) and one of the other Fe oxides (goethite) (Fig. 2e). None of the investigated minerals catalyzed G6P dephosphorylation (Fig. 2e). Only data with ferrihydrite and goethite were consistent with reports by Wan et al.16 of higher aqueous Pi production from AMP than from G6P and phytate. Even accounting for particulate Pi, we obtained lower reactivity of AMP and G6P with hematite than reported by Wan et al.16. Compared to our experiments, these previous experiments16 were performed with 60% less hematite (on a per-mass basis), 20-fold higher concentration for G6P and AMP, and 3-fold higher concentration for phytate (Fig. 2c–e). Therefore, the discrepancy could be due to possible lower reactive sites in hematite compared to ferrihydrite and goethite, thus necessitating higher Porg concentration to observe reactivity.

In sum, our data revealed that the silicates and aluminosilicates either had minimal to no catalytic reactivity or exhibited variable extent of adsorption reactivity towards the different compounds. Only the Fe oxides were found to catalyze appreciable to significant dephosphorylation of Porg compounds containing phosphoester and phosphoanhydride bonds. Importantly, the Fe oxide-catalyzed reactions seemed to be dependent on both the mineral surface chemistry and the type of Porg species. As proposed previously14,28, we posit that the differences in catalytic reactivity may stem from the binding conformations of different Porg compounds on the surface of the Fe oxide minerals.

Evolution of Particulate Pi from reacted Porg in natural samples
Our findings with the pure minerals stressed the exceptional adsorption and catalytic reactivities of the Fe oxides relative to the other minerals identified in the natural samples. Notably, we found that particulate Pi, which was largely ignored in previous investigations, was an important product of the reaction of Fe oxides with Porg species bearing a phosphomonoester bond or a phosphoanhydride bond. Furthermore, our data with mineral mixtures implied that the Fe oxide-mediated catalysis would likely remain prominent even in the heterogeneous mineral matrix of environmental samples. To evaluate this, we performed Porg reactions with natural sediment and soil samples.

First, we determined the starting P content in the natural samples: the sediment sample contained, on average, 1334 mg P per kg, with 49% as particulate Porg and 51% as particulate Pi; the soil sample only had 0.43 mg P per kg, with 56% as particulate Porg and 44% as particulate Pi (Fig. 3a). Relative to the starting P content in the natural samples, reacting ATP-P (300 µM or 9.3 mg L-1) with the sediment and soil samples (at 1 g L-1) generated excess particulate Pi of, on average, 2.7 mg P per g of the sediment sample and 4.1 mg P per g of the soil sample (Fig. 3a, b). Importantly, of this ribonucleotide-sourced particulate P, 20–50% was particulate Pi and 52–79% was particulate Porg (Fig. 3a, b). Therefore, following reactions with ATP, the sediment sample had a near 3-fold increase in the amount of both Porg and Pi in the particulate fraction and the soil sample had 3 orders of magnitude higher of particulate Porg and Pi (Fig. 3a). Interestingly, of the total P evolved from the Porg reactant, only about one-third was solution Pi while nearly two-thirds remained as particulate Pi fraction, thus highlighting a significant underestimation of the produced Pi would result in the absence of particulate Pi speciation (SI, Fig. S12). Biologically mediated P dephosphorylation was not expected to be significant in our natural samples due to long-term storage (~4 years) of both samples and low carbon loading (<0.2% g C g-1 soil) particularly for the soil sample. Nevertheless, we tested the possibility of residual microbial or enzymatic reactions in the natural samples by performing experiments with an antimicrobial agent or an enzyme denaturing agent, respectively (SI, Fig. S13). We determined that these biotic reactions accounted for only 0–5% and 23–26% of the total reactivity in the sediment and soil samples, respectively (Fig. 3a, b). Taken collectively, our findings bring attention to the occurrence of a pool of abiotically generated particulate Pi from mineral-mediated P transformation that has been hitherto unaccounted for in environmental matrices. Next, we probed which of the minerals within the heterogeneous matrix of the natural samples may be responsible for the P reactivity.

Association of evolved Pi with Fe oxides in natural samples
Through µ-XRF mapping, we found that Fe was distributed throughout the sediment and soil samples and that P appeared to be co-localized with Fe in distinct regions (Fig. 3c and d); we confirmed the lack of strong correlation of P with calcium (Ca) (SI, Fig. S14). Subsequently, we obtained extensive bulk P K-edge XANES data to determine the mineral types associated with the particulate Pi generated from the reacted ribonucleotide by conducting linear combination fitting (LCF) using XANES spectra obtained from separate experiments of Porg or Pi with the different mineral components identified in the natural samples (Fig. 4; SI, Table S6 and Table S7). It was not possible to use the XANES spectra to distinguish the specific P species associated with the different aluminosilicates nor the specific Fe oxide minerals associated with either particulate Pi or particulate Porg species (SI, Fig. S15). Specifically, we were able to employ the LCFs of the XANES spectra to distinguish Pi associated with Ca using apatite as a reference, Pi (without discriminating between Porg or Pi) associated with silicates and aluminosilicates, Pi associated with Fe in Fe oxides, Porg species associated with Fe in Fe oxides, and Pi in Fe-P clusters using vivianite as reference (Fig. 4a–e). We determined the fate of newly generated Pi and Porg following the ATP reaction with the natural samples by accounting for the total particulate P in excess of the background amount in the reference samples and characterizing the differences in the fractions of Pi and Porg associated with specific minerals in natural samples before and after reactions with ATP (Fig. 4f, g). The following were characteristic features in the XANES spectra that enabled the LCFs to capture distinct P speciation in the natural samples reacted with Porg: differences in the rising edge of the white line for Porg versus Pi (Fig. 4c, d), the shift in the peak of the white line with different fractions of Pi versus Porg bound to Fe oxides (Fig. 4d), and the pre-edge region of silicate-bound Porg (4e).

In accordance with the observed higher reactivity of the Fe oxides relative to the other minerals in the reactions with pure minerals (Fig. 2a, b), we found that both particulate Porg and particulate Pi generated after reactions of the sediment and soil samples with the ribonucleotide reactant were associated primarily with Fe and Fe-oxide fractions in the sample matrix (Fig. 4f, g). On a dry sediment basis, about 72% of the ribonucleotide-derived particulate P was found in clusters of Fe-complexed Pi (1066±118 µg g-1) and particulate Pi associated with the Fe oxide fraction (912±502 µg g-1); the remaining was P associated with aluminosilicate minerals (651±97 µg g-1), and Pi in Ca-phosphate mineral (110±22 µg g-1) (Fig. 4f). Based on our experiments with pure minerals (Fig. 2a), the accumulation of Fe-P clusters in the sediment sample matrix was attributed to strong Fe binding of Pi catalytically generated from the high abundance of particulate Pi associated with Fe oxides (Fig. 4f). In contrast to the lake sediment, the particulate P generated from the reacted ribonucleotide with the soil sample was nearly completely associated with the Fe oxide fraction, whereby ~28% was Fe oxide-bound Pi (1124±279 µg g-1) and the remaining 72% was Fe oxide-bound Porg (2758±103 µg g-1); there was a relatively minor amount of P associated with silicates (181±127 µg g-1) (Fig. 4g). Based on the different mineral compositions of sediment versus soil samples and informed by our experiments with pure minerals (Fig. 2a), we posit that the observed differences between the natural samples in the mineral associations of the ribonucleotide-sourced P was due to the different forms of Fe oxides (hematite-free versus hematite-rich Fe-oxide fractions), silicate types (quartz-enriched versus clay-enriched), and available Ca (nearly 12-fold higher Ca content in the sediment sample) (Table S8).

Rates of Fe oxide-mediated catalysis versus soil enzymes
Our data thus far point to the production of both aqueous Pi and particulate Pi during Fe oxide-mediated Porg dephosphorylation, the environmental relevance of which need to be considered in relation to reported enzymatic rates (Fig. 5; SI, S11 and S12). To this end, with the three different Fe oxides (goethite, hematite, ferrihydrite) identified in the natural samples, we performed kinetic experiments to obtain the production rates of aqueous Pi and particulate Pi species as a function of reacted ATP concentrations (25–400 µM), and subsequently determined two apparent maximal dephosphorylation rates (Vmax): one Vmax for the production rate of dissolved Pi and one Vmax for the production rate of particulate Pi (Fig. 5a, b; SI, Fig. S16). While the goethite-catalyzed reaction generated both dissolved and particulate Pi, only particulate Pi was found in appreciable quantities with both hematite and ferrihydrite (Fig. 5b). The Vmax for particulate Pi with ferrihydrite (2.59–4.24 µmol P h-1 g mineral-1) was up to 4-fold higher than with goethite (0.455–1.70 µmol P h-1 g mineral-1, p<0.05) and up to 20-fold higher than with hematite (0.164–0.576 µmol P h-1 g mineral-1, p<0.05) (Fig. 5b). This marked difference in the production rate of particulate Pi is consistent with the well-known higher adsorption reactivity of low-crystallinity Fe oxides relative to crystalline Fe oxides48–50. With goethite, the Vmax for Pi in solution (2.70–4.65 µmol P h-1 g mineral-1) was nearly 5-fold greater than the corresponding Vmax for the particulate Pi measured on the goethite surface (p<0.05), indicating that goethite exhibited more of an enzyme-like behavior compared to the other Fe oxides (Fig. 5b).

As a way of normalizing the Vmax value for each mineral, we performed Pi adsorption experiments to determine the site density for Pi binding on each mineral surface (SI, Fig. S17). While goethite exhibited nearly 3-fold higher capacity for Pi binding than hematite (26.0±1.2 µmol P g-1 versus 9.1±0.9 µmol P g-1, p<0.001), ferrihydrite had the highest capacity for Pi binding (184.1±6.0 µmol P g-1) at 7 and 20 times higher than for goethite (p<0.001) and hematite (p<0.001), respectively (SI, Fig. S17). By using the mineral-dependent Pi binding site density to normalize the combined Vmax for particulate Pi and dissolved Pi, we determined the total turnover number for Porg dephosphorylation (kcat) by each mineral (Fig. 5c). Further highlighting the higher catalytic efficiency of goethite compared to the other Fe oxides, we found that the total kcat for goethite was 2-fold to 4-fold higher than the kcat for hematite (p<0.01) and up to 9-fold greater than the kcat for ferrihydrite (p<0.001) (Fig. 5c). As we have already pointed out, mineral-dependent reactivity did not appear to be due to differences in surface area. Future research on the surface mechanisms underlying this abiotic catalysis will need to address the dependence of the observed difference in catalytic turnover on both the surface chemistry and mineral structure of each mineral.

Here we evaluated the environmental relevance of our findings by comparing reported global values for phosphatase enzyme activity in soils51 to our Fe oxide-mediated dephosphorylation activity as a function of soil Fe oxide content estimated from our total Vmax values (Fig. 5d). Even at 2% fraction of Fe oxide content in soil, we found that the estimated rate of Fe oxide-catalyzed dephosphorylation (0.04–0.06 µmol P h-1 g soil-1) was above the minimum rate reported for enzymes in soils (0.01 µmol P h-1 g soil-1)51 (Fig. 5d). We further estimated that a soil Fe oxide content of ~20% or greater to be of significance for obtaining abiotic rates that would be approximately within one order of magnitude or less of the averaged value of soil phosphatase rates (11.6±0.8 µmol P h-1 g soil-1)51 reported in soils globally (Fig. 5d). In accordance with this estimation, we did observe the association of ribonucleotide-sourced Pi primarily with Fe oxides in our heterogeneous soil sample, which had adequate Fe oxide content (~18%) (Figs. 1d and 4g).

Methods
Chemicals
The ribonucleotide compounds [ATP (≥93% purity as determined by our independent LC-MS analysis), ADP (≥95%), and AMP (≥97%)], G6P (≥98%), glucose (≥99.5%) and phytate (≥82% purity as determined by our independent LC-MS analysis) were purchased from Millipore Sigma (St. Louis, MO); adenosine (100%) was obtained from Chem-Impex International (Wood Dale, IL). Phytate dephosphorylation products [inositol pentaphosphate (>98%), inositol tetraphosphate (>98%), inositol triphosphate (>98%), inositol biphosphate (>95%), and inositol monophosphate (>8%)] were purchased from Cayman Chemical (Ann Arbor, MI). Minerals were either purchased or synthesized. Goethite was purchased from Alfa Aesar (Ward Hill, MA), magnetite from Millipore Sigma (St. Louis, MO), hematite from Strem Chemicals (Newburyport, MA), quartz from Honeywell (St. Louis, MO), kaolinite from Fluka (Muskegon, MI), and mica (muscovite) from Spectrum Chemical (Gardena, CA). The illite source clay ISCz-1, obtained from the Clay Minerals Society’s repository of Purdue University, was ground in a mortar and pestle before use. Ferrihydrite was synthesized by the method detailed by Schwertmann and Cornell53. We obtained XRD spectra using a Bruker D8 Advance powder X-ray diffractometer to confirm the identity of all mineral phases synthesized and purchased matched their reference spectra in the Crystallography Open Database. All other chemicals used were analytical grade and purchased from Millipore Sigma (St. Louis, MO).

Location and characterization of the natural samples
For natural samples, we obtained samples from a lake sediment and a forest soil. Lake sediment samples were taken from a platform sampling buoy in Missisquoi Bay off Lake Champlain (N44°59’33” W73°8’20”) located in Vermont, United States of America. The site, which undergoes seasonal oxic-anoxic cycles, is notable for its high P loading (1.39 mg-P g-1 sediment) and presence of redox-sensitive Fe in the sediment (39.8 mg-Fe g-1 sediment)37. Soil samples were sourced from the Calhoun Critical Zone Observatory (N34°36’33.012” W81°43’40.62”), located in the Sumter National Forest in South Carolina, United States of America. A sample core was taken from the subsoil at a depth of 58–86 cm. The soil is characterized by high Fe oxide content (59.3 mg Fe g-1 soil) and minimal amounts of P (0.43 µg P g-1 soil)66. Both natural samples were sieved to a particle size of 250 μm and ground in a mortar and pestle before use. The pH of the pore water at the Calhoun Critical Zone Observatory soil was determined to be 5.8 at the time of excavation, however the pH has been reported previously to range between and 4.5–6.267,68. The mean annual temperature at the soil sampling site is 289 K (or 16°C), ranging between 278 K (5°C) and 298 K (25°C)67. For the Missisquoi Bay sediment sample, the pore water pH was determined to be circumneutral with a temperature of 297 K (24°C)69,70. The natural samples and pure minerals used in this study were characterized by XRF, XRD, and Fe K-edge µ-XRF and µ-XANES. The XRD and XRF data were used in tandem to confirm the presence of specific elements and crystalline phases. The Fe K-edge µ-XRF and µ-XANES data determined the Fe speciation in the natural samples. Location and topography maps with the sample site locations were created using QGIS version 3.2471 and topography data72 from the U.S. Geological Survey.

Elemental XRF analysis of the natural samples
We performed XRF spectroscopy on a Xenemetrix Ex-Calibur EX-2600 spectrometer equipped with a rhodium (Rh) X-ray tube and a silicon (Si) energy-dispersive detector. The samples were placed in a special plastic cup and powder was supported on a 6 µm Mylar film. Standards and samples were used in the form of a pellet and diluted with urea. Data collection was performed at room temperature under vacuum using the nEXt version 1.9 software. The X-ray tube was operated at 20 keV and 10 µA, and fluorescence spectra were collected for 10 min. Both natural samples exhibited significant amounts of Fe, silicon (Si), and Al; the lake sediment also contained potassium (K) (SI, Fig. S1). The XRF analysis did not determine any appreciable amount of manganese in both the lake sediment and forest soil (SI, Fig. S1). Therefore, the presence of manganese oxides was not considered in later analysis.

Characterization of crystalline minerals in the natural samples via XRD
The XRD diffraction patterns were recorded using a Bruker D8 Advance powder X-ray diffractometer fitted with a copper anode. The diffractometer was operated at 40 kV and 40 mA, with a Göebel Mirror, a Soller 0.2° optic, and a SSD160_2 detector used in 0D mode. Throughout data collection, a temperature of 25°C was maintained in the sample chamber. The XRD profile was collected in continuous mode with a 2θ step size of 0.005°, a 12 s counting time per step, and from 2θ = 5–72°. Bruker’s DIFFRAC.suite V7.5.0 software was used for data collection and subsequent analysis. Peak fitting was performed with DIFFRAC.EVA V6.0.0.7 using candidate peaks in the Crystallography Open Database to determine the percent contribution of each crystalline phase in each natural sample (SI, Fig. S2).

Characterization of Fe minerals by Fe K-edge µ-XANES and P hotspots by P K-edge µ-XANES in natural samples
Control and ATP-reacted natural samples were both analyzed for Fe species identification by synchrotron analysis. Samples for XANES analysis of ATP-reacted natural samples were prepared in a 500 mL polypropylene bottle by the addition of 400 mg of either the lake sediment and forest soil 400 mL and a 100 µM ATP solution comprised of 0.1 M NaNO3, 0.01 M NaHCO3 (adjusted to pH=7.0 using 2 M HNO3). Natural samples were reacted for 7 days and stopped by vacuum filtration, then rinsed with a small volume of GenPure water (Thermo Scientific; 18.2 MΩ cm) to remove excess supernatant. The collected solid was freeze-dried then ground with an agate mortar and pestle. Control dry sediment and soil samples were directly ground with a mortar and pestle. The ground samples were embedded in epoxy (Epotek-301) and thin sectioned on a Buehler PetroThin thin sectioning system to produce 100 mm thick sections mounted on quartz slides.

We performed Fe K-edge and P K-edge µ-XRF mapping and µ-XANES spectroscopy at the Stanford Synchrotron Radiation Lightsource (SSRL) on beamline 2–3. Data was collected using SSRL’s MicroEXAFS Data Collector 2.0 software. The energy was selected using a water-cooled Si(111) monochromator. Calibration was performed by setting the maximum of the first derivative of the XANES spectrum of an Fe foil to 7112.0 eV. The beam was focused to a 5 µm spot using a Sigray axially symmetric mirror and fluorescence detection was achieved using a one-element Vortex detector. µ-XRF maps with a step size of 10 microns of the samples were collected at four different energies across the Fe absorption edge: 7122, 7124, 7128 and 7130 eV. Principal component analysis (PCA) was performed on the multi-energy maps to distinguish chemically distinct Fe hot spots within the images, which were processed in Sam’s Microprobe Analysis ToolKit (SMAK)41 version 2.03. These locations were selected for collection of Fe K-edge µ-XANES spectra for the lake sediment and soil sample (SI, Fig. S3; Fig. S4). We also collected µ-XRF maps on this beamline using 3 different energies across the P absorption edge: 2148, 2152.3, and 2152.5 eV. Because the 2–3 beamline is not as sensitive as the 14-3 beamline, the P mapping was solely used to identify P hot spots in the samples and confirm P localization with Fe. Calibration for P measurements was performed by setting the maximum of the first derivative of the XANES spectrum of GaP to 2152.0 eV.

The Fe K-edge spectra collected at each spot was fit to a set of standards informed by XRF and XRD speciation. For the lake sediment samples: illite (Clay Mineral Society source clay IMt1), and Fe-rich biotite; for the soil samples: hematite; and for both samples: vivianite, ferrihydrite, and goethite. Reference spectra for Fe-rich biotite and vivianite reference XANES spectra were from Sutherland et al. 202073 and Hansel et al. 200374. All spectra used for fitting were subjected to the same normalization procedure. The normalization procedure involved fitting a line to the pre-edge region of the spectrum (from -50– -30 eV relative to E0) the maximum of the first derivative of the spectra, and a second-order polynomial to the post-edge region (from 80–250 eV for lake sediment and from 30–250 eV for soil). The pre-edge line was subtracted from the spectrum to remove the background signal and the absorption jump at the first inflection point was set to an intensity of 1.0 based on the difference between the pre- and post-edge fits. Linear combination fitting was performed using the Athena module of the Demeter 0.9.26 IFEFFIT software package75. The spectra of different Fe reference spectra were combined to generate a spectrum that fit the spectra of the natural samples. Fits were considered acceptable when visually, the rising edge, white line peak shape, and post-edge region shape matched; the sum of all components was between 0.90–1.10; and if the R-factor was <0.02. A total of 8 spots were used to generate an average lake sediment Fe speciation and 5 spots used to generate an average soil sample Fe speciation (SI, Fig. S5). Both ferrihydrite and vivianite were identified in the sediment and soil samples (SI, Fig. S5). After determination of Fe speciation, the contribution of each non-crystalline phase (ferrihydrite and vivianite) to the total mineral makeup was calculated from the XRD quantitative analysis of a crystalline Fe-containing mineral, resulting in a total speciation of each natural sample (shown in main text, Fig. 1c).

Characterization of Al coordination in natural samples by 27Al NMR
Solid state 27Al magic-angle spinning NMR data were collected at room temperature on a Bruker Avance III 400 MHz spectrometer equipped with a 4 mm HX probe using the TopSpin version 3.6.5 software. A zirconia blind-borer rotor with 4 mm diameter was spun at 10 kHz. The resonance frequency of 27Al was 399.73 MHz. 27Al NMR spectra of the lake sediment and soil sample were measured using a recycle delay of 5 s. To achieve an adequate signal-to-noise ratio, a total of 64 scans were collected for the sediment sample and 16 scans were collected for the soil sample. The 27Al chemical shifts were referenced to AlCl3·6H2O set to 0.0 ppm. In accordance to previous studies41,42, we sought to identify different coordination environments of Al based on the following chemical shifts: Al(VI) ~0 ppm, Al(V) ~35 ppm, and Al(IV) ~60 ppm.

Dephosphorylation reactions of various Porg compounds with natural samples and minerals
Triplicate experiments were conducted for 7 days reactions of ribonucleotides (ATP-P; 150 or 300 µM) with natural samples (lake sediment or forest soil; 1 g L-1). In 50 mL polypropylene tubes, 40 mL reaction solution was mixed with the adsorbent. The reaction solution comprised of 0.1 M NaNO3, 0.01 M NaHCO3 (adjusted to pH=7.0 using 2 M HNO3) and the specified concentrations of ribonucleotides. Here, to compare directly the reactivities of the different mineral present across the two different natural samples, we chose to use the standardized conditions of a reaction solution at pH 7.0, ionic strength set by 0.1 M NaNO3 and buffered by 0.01 M NaHCO3 at 25°C. We acknowledge that the actual pore water conditions in the natural samples, as highlighted above under detailed description of sample characteristics, would be different from the standardized experimental conditions. Reactions were shaken in the absence of light, in an Eppendorf Innova S44i incubator shaker (25°C, 150 rpm) for 7 days. Reactions were stopped by filtration through a 0.2 µm filter or were centrifuged at 2325 g for 20 min prior to filtration. An aliquot of the filtrate was analyzed for solution Pi immediately and the remaining filtered sample was frozen at -20°C until analysis by high-resolution LC-MS.

Using the same procedure as above, triplicate experiments were also conducted for 75 min and 7 days reactions of Porg compounds (ATP, AMP, phytate, or G6P; 50 µM) with the different minerals (1 g L-1) identified in significant fractions in the natural sample composition: quartz, clays (kaolinite, illite), micas (biotite, glauconite), and Fe oxides (ferrihydrite, goethite, magnetite, and hematite). In addition to these single mineral reactions, we performed reactions with two-mineral mixtures containing goethite (1 g L-1) and quartz (4 g L-1), kaolinite (4 g L-1), or ferrihydrite (1 or 4 g L-1). Reactions with clays (illite, kaolinite, and mica) were centrifuged at 2325 g for 20 min prior to filtration and analysis of the aqueous phase components. Samples for P K-edge XANES analysis were prepared as described above.

Kinetic experiments were performed in triplicate with goethite and ferrihydrite over a total of 5 time points: (4 h, 8 h, 12 h, 24 h, and 48 h) and five concentrations (25 µM, 50 µM, 125 µM, 200 µM, and 400 µM). The initial rate of velocity for Pi generation (solution-based and mineral-bound) at each concentration was calculated and applied to the Michaelis-Menten model for enzymatic kinetics using the GraphPad Prism version 9.4.0 software. The maximum velocity of Fe oxide-mediated Pi generation was extracted from model fits and subsequently normalized by the Pi adsorption site density for each mineral to calculate the turnover number of kcat.

Identification and quantification of aqueous dephosphorylation products using
Solution Pi concentration was quantified by a phosphomolybdate spectrophotometric method using a Cary 60 UV-Visible spectrophotometer76 using the Cary WinUV Simple Reads version 5.1.3.1042 software. We ascertained that the colorimetric method measured Pi and not the Porg products over the course of these experiments. Solution Porg compounds (ATP, ADP, AMP; referred group as AXP moving forward) and adenosine were quantified using a previously established LC-MS method14. The LC-MS system used comprised of a Dionex Ultimate 3000 LC coupled to a Q-Exactive mass spectrometer. Measurements and analysis were performed using the Xcalibur version 4.1 software (Thermo Scientific). Compounds were separated using a Waters Acquity UPLC® BEH C18 column (2.1 mm × 100 mm x 1.7 µm) and an 11-min gradient of Solvent A (3% v/v methanol/15 mM acetic acid/10 mM tributylamine) and Solvent B (100% methanol). The mass spectrometer was run in negative ion mode at full MS range (m/z 100 – 600) with a resolution of 70,000. Standards ranging from 0.5 to 15 µM AXP compounds and adenosine were used for calibration. Samples with compound concentrations above 15 µM were diluted to ensure measurements were within the calibration range. Quality controls were run every 6–9 samples and remained within 30% error. Compounds were quantified using peak area integration from the ion chromatograms.

Mineral surface-localized P speciation
We used P K-edge bulk XANES spectroscopy to determine the characterize and quantify the particulate Porg and Pi following the aforementioned Porg compound reactions with the natural samples or the representative minerals. The P K-edge XANES spectroscopy for ribonucleotide reactions was conducted at the Stanford Synchrotron Radiation Lightsource (SSRL) on beamline 14–3. Powdered samples were applied to Mylar tape in a thin layer. The spectra were collected in fluorescence mode with a Vortex detector at room temperature in a helium atmosphere. The vertical slits were set to 1 mm and the horizontal slits to 2.5 mm. A Si(111) monochromator was used to select the energy and calibrated to the energy of the first pre-edge feature of PPh4-Br (2146.96 eV) and there was no noticeable shift 12 and 24 h after the initial calibration. Due to shutdown of the SSRL for maintenance, the P K-edge XANES spectroscopy for non-ribonucleotide reactions (phytate and G6P) was conducted at the MEX2 Beamline of the Australian Synchrotron. Powdered samples were applied to carbon tape in a thin layer. The spectra were collected in fluorescence mode with a Vortex-ME4 detector at room temperature under vacuum. The vertical slits were set to 3 mm and the horizontal slits to 5 mm. A Si(111) monochromator was used to select the energy and calibrated to the energy of the first pre-edge feature of PPh4Br (2146.96 eV). The resulting spectra were analyzed by LCF with the Athena module75 of the Demeter version 0.9.26 IFEFFIT software package. The mineral associated-P reference spectra were combined to generate a spectrum that fit the spectra of the natural samples. Fits were considered acceptable when visually, the rising edge, white line peak shape, and post-edge region shape matched; the sum of all components was between 0.90 and 1.10; and if the R-factor was <0.02. To achieve the LCF, we obtained an extensive set of XANES reference spectra for the different minerals (goethite, hematite, ferrihydrite, quartz, kaolinite, illite) by reacting these minerals with Pi or with Porg compounds (ATP or AMP). The reference XANES spectra used for the phosphate minerals, vivianite and fluoroapatite, were from Gustafsson et al. 202077.

Statistical analysis
Experimental data points from LC-MS are presented as bar graphs and error bars are calculated as one standard deviation by assuming a standard normal distribution. Values determined from multiple post-processing of experimental data are shown as Box plots generated using GraphPad Prism 9.4.0. Error bars on fractions obtained from LCF modeling of XANES data represent error of model fits. All statistical significance was calculated using the unpaired t-test, except for comparisons of Vmax, kcat, and Pi site density values which utilized the F-test. Model fitting parameters and associated statistical analysis were performed in GraphPad Prism 9.4.0 and are listed in SI Table S9 and in Table S10.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "NCOMMS-23-32052: Iron Oxides as Catalyfic Traps in Organic Phosphorus Mineralizafion\nBasinski et al., showed and quanfified a new and crucial role of iron oxides as an abiofic mineral-enzyme in organic P remineralizafion. Some crifical and interesfing roles of environmental Fe and Mn oxides are found in recent research, such as iron- and manganese as catalysis to cause the transformafion of simple organic molecules into complex macromolecules (Moore et al., Nature, 2023). This manuscript is also an interesfing paper to include the quanfitafive analyses of solufion and surface P species transformafion into soil and sediment incubafion. The author then found that the Fe oxides exhibited up to great high catalyfic reacfivity (or dephosphorylafion acfivity) for ribonucleofide phosphate mineralizafion in soils and sediments. Finally, the wrifings and figure plots are well organized to show the findings and results. However, there sfill are many concerns that have to be processed before the acceptance of this manuscript. Thus, the current version cannot be accepted by Nature Communicafions and I suggested a major revision.\nMain comments:\n(1) Is ATP (has both C-O-P and P-O-P bonds) a typical organic phosphate in natural environments? Many literatures suggested that, in nature, the main organic P species include phosphate monoester, phosphate diester, and phosphonate (Baldwin, Environ Chem, 2013; George et al., Plant Soil, 2018). Inositol phosphates are the main organic P storage in terrestrial plants and the most abundant organic P in soils (Turner et al., 2002). How about the catalyfic acfivity of Fe oxides toward the dephosphorylafion of phosphate monoester in your soils and sediment incubafion? Since they are more abundant Ps in the environment, we cannot neglect their transformafion caused by Fe oxides. Wan et al., showed that Fe oxides have low reacfivity for C-O-P degradafion and high reacfivity for P-O-P degradafion (Wan et al., Geochim Cosmochim Ac 2021; Sci Total Environ, 2022). If the catalyzed degradafion of ATP by Fe oxides might be specified from the cleavage of P-O-P bond, instead of C-O-P bonds, which will weaken your conclusions about organic P (C-O-P bond-including P) mineralizafion. You may need to consider adding a C-O-P’s organic P to see how Fe oxides catalyze purely organic P mineralizafion and to strengthen your conclusions.\n(2) How about manganese oxides in organic P mineralizafion in soils and sediments since Mn oxides also show high reacfivity to P transformafion (Baldwin et al., Environ Sci Tech, 2001; Wan et al. ACS Earth Space Chem, 2018).\n(3) You should include the XRF method of P measurement (Figures 2 c-d) in the method secfion. Did you XRF in the SSRL 2-3 (Energy: 4.9-23 keV)? Could SSRL 2-3 measure P mapping? Why not use SSRL 14-3?\n(4) Line 235-236 Autoclave sterilizafion? Generally, increasing temperature will significantly change the mineralogy of soils and sediments and might increase the contents of crystalline hemafite and goethite? Could you please add the new Fe XANES to compare the mineralogical change of Fe oxides in your soil and sediment samples due to autoclave treatment? Maybe, you also need to consider how this sterilizafion way affects your findings and conclusions.\n(5) What are the parameters in your soil and sediment samples’ porewater? Local temperature? You set up pH 7 and 0.1 M NaNO3 + 0.01 M NaHCO3 to perform experiments. Why not use the porewater condifions to do experiments? Which might be closer to realisfic reacfion condifions and show the true catalyfic reacfivity/rates of Fe oxides in organic P mineralizafion.\n(6) You used XRD to do mineralogical idenfificafion. How do you know amorphous phase contents and how do they affect your conclusions?\n(7) Figure S6 showed the similar XANES spectra for both mineral-Porgnic and mineral-Pi? What is the confidenfiality of using P K-edge XANES to analyze surface organic and inorganic P?\nOverall, the manuscript is of high quality. I assume that considering these concerns/comments might further improve your manuscript quality and consolidate your findings about this new and interesfing role of Fe oxides in P biogeochemical cycles in the sub-surface Earth.", "reviewer_2": "I have concluded that the research described is sufficient quality and potenfial impact to be accepted for publicafion in Nature Communicafions, with some minor revision (see below). The manuscript is very well wriften and presented, and the findings of this study clearly demonstrated that significant mineralizafion of model organic phosphorus compounds occurred on the surface of iron oxide, which represents a potenfially important process that contributes to phosphorus transformafions and bioavailability in sediments and soils.\nI only have one significant comment/request to make on the manuscript. I am a soil scienfist with very liftle experience or experfise in sediment phosphorus dynamics. With specific regard to the dynamics of organic phosphorus in soil, I was surprised to note the complete absence of any menfion or considerafion of the role and funcfion of “iron oxide surface mineralzafion” in the cycling and bioavailability of inositol phosphates. Inositol phosphates (principally inositol hexaphosphate [commonly referred to as “phytate”]) are widely acknowledged to be important consfituenst of soil organic phosphorus, and have been shown to be strongly adsorbed on soil colloid surfaces. I would request that the authors consider including some discussion of the potenfial role of iron oxide surface mineralizafion in the fate and bioavailability of inositol phosphates in soil."}, "author_response": "Response to Reviewers\n\nReviewer #1 (Remarks to the Author):\nNCOMMS-23-32052: Iron Oxides as Catalytic Traps in Organic Phosphorus Mineralization\nBasinski et al., showed and quantified a new and crucial role of iron oxides as an abiotic\nmineral-enzyme in organic P remineralization. Some critical and interesting roles of\nenvironmental Fe and Mn oxides are found in recent research, such as iron- and\nmanganese as catalysis to cause the transformation of simple organic molecules into\ncomplex macromolecules (Moore et al., Nature, 2023). This manuscript is also an\ninteresting paper to include the quantitative analyses of solution and surface P species\ntransformation into soil and sediment incubation. The author then found that the Fe oxides\nexhibited up to great high catalytic reactivity (or dephosphorylation activity) for\nribonucleotide phosphate mineralization in soils and sediments. Finally, the writings and\nfigure plots are well organized to show the findings and results. However, there still are\nmany concerns that have to be processed before the acceptance of this manuscript. Thus,\nthe current version cannot be accepted by Nature Communications and I suggested a\nmajor revision.\nResponse: Thank you for acknowledging the new underexplored yet critical role of iron oxides\nwith enzyme-like characteristics in the phosphorus cycle. We appreciate the highlight of our\nwell-written and well-organized by the reviewers. Below we provide details on how we have\ninvested in addressing all the concerns and feedbacks by the reviewer. Notably, we were able to\nsecure additional beamtime at the Australin Synchrotron on short notice to conduct to analyze\nsamples stemming from the additional experiments the author requested with other organic P\ntypes. This would have been an impossible task for us to achieve due to the shutdown of the\nStanford synchrotron. We are grateful to our diverse team of co-authors that includes beamline\nscientists from different synchrotron facilities.\nMain comments:\nComment 1:\nIs ATP (has both C-O-P and P-O-P bonds) a typical organic phosphate in natural\nenvironments? Many literatures suggested that, in nature, the main organic P species\ninclude phosphate monoester, phosphate diester, and phosphonate (Baldwin,, Environ\nChem, 2013; George et al., Plant Soil, 2018). Inositol phosphates are the main organic P\nstorage in terrestrial plants and the most abundant organic P in soils (Turner et al., 2002).\nHow about the catalytic activity of Fe oxides toward the dephosphorylation of phosphate\nmonoester in your soils and sediment incubation? Since they are more abundant Ps in the\nenvironment, we cannot neglect their transformation caused by Fe oxides. Wan et al.,\nshowed that Fe oxides have low reactivity for C-O-P degradation and high reactivity for P-\nO-P degradation (Wan et al., Geochim Cosmochim Ac 2021; Sci Total Environ, 2022). If the\ncatalyzed degradation of ATP by Fe oxides might be specified from the cleavage of P-O-P\nbond, instead of C-O-P bonds, which will weaken your conclusions about organic P (C-O-P\nbond-including P) mineralization. You may need to consider adding a C-O-P’s organic P to\nsee how Fe oxides catalyze purely organic P mineralization and to strengthen your\nconclusions.\nResponse: We agree with the reviewer that phosphomonoesters (with C-O-P) are found to be\nthe forms of P mostly detected in environmental matrices. In our original manuscript, we did\norg\npresent data with both AMP (a phosphomonoester) and ATP reacted with the different Fe\noxides, quartz, aluminosilicates. We used the data with the two different ribonucleotides to make\na statement that the Fe oxide reactivity would be dependent on the P types because while\norg\ngoethite and hematite has more reactivity towards ATP than AMP, ferrihydrite had more catalytic\nreactivity for AMP than for ATP. However, as pointed out by the reviewer, we acknowledge there\nare different forms of P compounds with phosphomonoester bonds. To address the concern\norg\nby the reviewer that we need to consider how different phosphomonesters may react with the\nFe oxides, we conducted additional experiments with a sugar phosphate (glucose-6-phosphate)\nand phytate (the primary storage in plants), to add to our data with AMP. These new data are\nillustrated in Figures 2d and 2e. We expanded the text to discuss the new results along with the\ndiscussion of AMP (see Lines 212-251). It is important to acknowledge that P with\norg\nphosphoanhydride bonds (P-O-P) are ubiquitous in plant and microbial metabolism and\nbiomass. Therefore, the persistent detection of phosphomonoesters may be indicative of both\nlow reactivity of phosphomonoesters and the accumulation of dephosphorylation products from\nmultiphosphorylated P compounds containing phosphoanhydride bonds. In fact, in agreement\norg\nwith previous enzymatic dephosphorylation data, we found two of Fe oxides had relatively lower\nreactivity for phosphomonoesters had lower reactivity compared to ATP, but, contrary to the\nenzyme results, AMP reactivity was nearly 20-fold greater on ferrihydrite relative to ATP\nreactivity on ferrihydrite. We added to the discussion section to highlight these findings (see\nLines 407-424). This remarkable difference in mineral reactivity was only captured by surface\ncharacterization by P-XANES. We note that the publication by Wan et. al. 2022 only monitored\nthe generation of P in solution. With regards to experiments with the natural samples, we only\ni\nperformed reactions with ATP due to insufficient amounts of natural samples that precluded us\nfrom conducting experiments with both ATP and the entire set of phosphomonoesters.\nLines 212-251:\nAmongst the chemical diversity of P types found in biomolecules, phosphomonesters are widely\norg\nfound in soils34–36,41,42. To probe Fe oxide reactivity with these other types of P , we performed\norg\nreactions involving each of the three Fe oxides (ferrihydrite, hematite, and goethite) with three\nphosphomonoesters: a ribonucleotide, AMP (AMP-P, 50 µM P or 1.5 mg P L-1); a sugar phosphate,\nG6P (G6P-P, 50 µM P or 1.5 mg P L-1); and, the primary P storage in terrestrial plants36, inositol\nhexaphosphate or phytate (phytate-P, 300 µM P or 9.0 mg P L-1 (Fig. 2c, 2d, and 2e). To compare\nthe Fe oxide reactivity with the other mineral types, we also performed experiments of the\nphosphomonoester P compounds reacted with quartz and aluminosilicates (mica, kaolinite, illite)\norg\n(Fig. 2c, 2d, and 2e).\nIn contrast to the ATP reactions, all the P derived from the reacted AMP was retained as\ni\nparticulate P while aqueous P was absent, a significant finding that was made possible here due to\ni i\nthe application of the XANES technique (Fig. 2c). On the one hand, the catalytic reactivity of\nferrihydrite was higher for AMP than for ATP, as reflected by the 20-fold increase in particulate P\ni\nfraction (p < 0.001) (Fig. 2a and 2c). On the other hand, the catalytic reactivity of goethite was less\nfor AMP than for ATP as characterized by a 3-fold decrease in particulate P fraction (p < 0.01)\ni\naccompanied by no change in the particulate P fraction (p = 0.29) and no measured solution P;\norg i\n2 | Pa ge\nhematite did not display any adsorption or catalytic reactivity towards AMP (Fig. 2a and 2c).\nInterestingly, in our aforementioned experiments with ATP and goethite, 20–24% of the initial ATP-P\nremained as AMP-P in solution and no ADP was detected after nearly all the ATP reacted with\ngoethite was transformed or adsorbed (SI, Fig. S10). Thus, this accumulation of AMP in the ATP-\ngoethite experiment can be explained by the lower reactivity of goethite for AMP relative to ATP\n(Fig. 2c; SI, Fig. S10). When ATP was reacted with hematite, we measured a conversion of 17–23%\nof the initial ATP-P to ADP-P and 5–7% to AMP-P, consistent with lower reactivity for both ADP and\nAMP compared to ATP (SI, Fig. S10). Notably, the silicate and aluminosilicate minerals did not\nexhibit any adsorption or catalytic reactivity towards AMP (Fig. 2c).\nIn contrast to AMP, all the silicate and aluminosilicate minerals adsorbed phytate (from 5% to\n50% of the total reacted phytate-P) and, rather than an Fe oxide, the clay illite adsorbed the most\nphytate (Fig. 2d). However, similar to the results with AMP and ATP, catalytic reactivity towards\nphytate was only obtained with Fe oxides, specifically hematite and goethite (Fig. 2d). Relative to\ncontrols, phytate-derived particulate P was higher by 8 – 14% (p < 0.01) with hematite and by 24 –\ni\n32% with goethite (p < 0.001); the particulate P with ferrihydrite, however, corresponded to the\ni\nadsorption of solution P in the control experiment (Fig. 2d). For G6P, some G6P adsorption was\ni\nobserved with two of the aluminosilicates (mica and kaolinite) and two of the Fe oxides (ferrihydrite\nand goethite) (Fig. 2e). But, as with phytate, the most significant adsorption was with ferrihydrite,\nwith 47 – 78% of the reacted G6P found as particulate P (Fig. 2e). None of the investigated\norg\nminerals catalyzed G6P dephosphorylation (Fig. 2e).\nIn sum, our data revealed that the silicates and aluminosilicates either had minimal to no\ncatalytic reactivity or exhibited some extent of adsorption reactivity. Only the Fe oxides were found to\ncatalyze the dephosphorylation of both phosphomonoester and phosphoanhydride-bearing\ncompounds. Importantly, the Fe oxide-catalyzed reactions seemed to be dependent on both the\nmineral surface chemistry and the type of P species. As proposed previously14, we expect the\norg\ndifferences in reactivity may stem from the binding conformations of different P on the mineral\norg\nsurface.\nLines 407-424:\nWhile phosphoanhydride-containing compounds are not commonly detected in soil and\nsediment systems34,35, phosphoanhydride bonds are ubiquitous in plant and microbial metabolites,\nincluding ATP as the major P content in many microbes4,52. We posit that the persistent detection of\nphosphomonoesters in environmental matrices may be indicative of both low reactivity of enzymes\nand minerals towards phosphomonoesters and the accumulation of remnants of dephosphorylated\nP compounds containing phosphoanhydride bonds. Here we show that the Fe oxide-catalyzed\norg\nreaction for a P compound containing both phosphoanhydride and phosphoester bonds was\norg\nretained, even in heterogeneous mixtures with quartz or clays. We also found accumulation of a\nmonophosphorylated ribonucleotide after Fe oxide-catalyzed reactions with a triphosphorylated\nribonucleotide. A study on enzymatic dephosphorylation53 reported higher reactivity, by\napproximately 2 orders of magnitude, for multiphosphorylated compounds than for\nphosphomonoester compounds. Goethite and hematite, both of which represented the most reactive\nFe-oxides, had minimal reactivity towards phosphomonoester P compounds. This lack of\norg\nsignificant dephosphorylation of phytate and a sugar phosphate by the Fe oxide minerals may\ncontribute to the persistence of these phosphoester P types in environmental matrices. However,\norg\ndephosphorylation of a monophosphorylated ribonucleotide by ferrihydrite, was nearly 20-fold\ngreater to that for the triphosphorylated ribonucleotide. Therefore, both the Fe oxide type and the\nP chemistry need to be considered when predicting the extent of the catalytic fate of P in\norg org\ndifferent environmental matrices.\nComment 2:\n3 | Pa ge\nHow about manganese oxides in organic P mineralization in soils and sediments since Mn\noxides also show high reactivity to P transformation (Baldwin et al., Environ Sci Tech,\n2001; Wan et al. ACS Earth Space Chem, 2018).\nResponse: We agree with the reviewer that Mn oxides are known to be reactive towards\norganic P compounds, as we had pointed out in the Introduction of our original manuscript (see\nLines 57 and lines 63). However, the scope of our project is on Fe oxides, especially since\nthere was no quantifiable Mn amount (XRF data) or identifiable Mn-bearing minerals (XRD data)\nin our natural soil and sediment samples (See lines 136-137). We also expanded our discussion\nto point to the reason why reactivity with Mn oxides was not considered in our experiments but\nalso highlight their possible significance in marine sediments. (See lines 430-439).\nLines 136-137:\nNo manganese-bearing minerals were included in the mineral composition analysis because neither\nenvironmental sample exhibited a quantifiable amount of manganese (SI, Fig. S1).\nLines 430-439:\nCurrent models of P cycling1,3,56, which include enzymes for P dephosphorylation and minerals for\norg\nP adsorption, do not account for reported catalytic dephosphorylation of P by Fe oxides and Mn\norg org\noxides3,14,16. We only consider Fe oxides in this research due to no quantifiable Mn in our forest soil\nand lake sediment samples, but there can be approximately, on a per-mass basis, 10:1 Fe\noxides:Mn oxides in marine sediments57,58. Our quantitative findings imply an important and yet\nunaccounted dual catalytic and adsorbent role of Fe oxides which warrant consideration alongside\nbiologically mediated processes in the P cycle. Our proposed redefined role of Fe oxides as\nimportant catalytic players, if confirmed to be a widespread phenomenon with quantitative\nsignificance, would have important implications regarding the addition of an abiotic axiom at the P -\norg\nP nexus in the biogeochemical P cycling.\ni\nComment 3:\nYou should include the XRF method of P measurement (Figures 2 c-d) in the method\nsection. Did you XRF in the SSRL 2-3 (Energy: 4.9-23 keV)? Could SSRL 2-3 measure P\nmapping? Why not use SSRL 14-3?\nResponse:\nAs the reviewer pointed out, we did perform the XRF mapping on SSRL’s beamline 2-3 and\nhave now included the methodology of those P measurements (See Lines 517-521). The P\nmapping acquired at the 2-3 beamline was solely used for P and Fe correlation analysis. We\nnote that, while the 2-3 beamline at SSRL can perform P mapping, it does not have the same\nsensitivity as the 14-3 beamline. We also ran the samples with the intention of P mapping at the\n14-3 beamline, but we unfortunately do not have the data due to an issue with the memory\nduring data collection. However, because we only used the data from the 2-3 beamline for\ncorrelation analysis, the lower sensitivity at that beamline was not an issue.\nLines 517-521:\nWe also collected μ-XRF maps on this beamline using 3 different energies across the P adsorption\nedge: 2148, 2152.3, and 2152.5 eV. Because the 2-3 beamline is not as sensitive as the 14-3\nbeamline, the P mapping was solely used to identify P hotspots in the samples and confirm P\n4 | Pa ge\nlocalization with Fe. Calibration for P measurements was performed by setting the maximum of the\nfirst derivative of the XANES spectrum of GaP to 2152.0 eV.\nComment 4:\nLine 235-236 Autoclave sterilization? Generally, increasing temperature will significantly\nchange the mineralogy of soils and sediments and might increase the contents of\ncrystalline hematite and goethite? Could you please add the new Fe XANES to compare\nthe mineralogical change of Fe oxides in your soil and sediment samples due to autoclave\ntreatment? Maybe, you also need to consider how this sterilization way affects your\nfindings and conclusions.\nResponse: As the reviewer pointed out, autoclaving of samples containing Fe oxides would\nlikely change the mineralogy of these minerals. For that reason, our natural samples were not\nautoclaved and biological dephosphorylation was assumed to be minimal due to long-term\nstorage and low carbon loading. We apologize for the typo and have edited the section that\nincluded this information (see Lines 278-286).\nLines 278-286:\nBiologically-mediated P dephosphorylation was not expected to be significant in our natural\norg\nsamples due to long-term storage (~4 years) of both samples and their low carbon loading\n(<0.2% g C g-1 soil) particularly for the soil sample. Nevertheless, we tested the possibility of\nresidual microbial or enzymatic reactions in the natural samples by performing experiments with\nan antimicrobial agent or an enzyme denaturing agent, respectively (SI, Fig. S12). We\ndetermined that these biotic reactions accounted only for 0 – 5% and 23 – 26% of the total\nreactivity in the sediment and soil samples, respectively (Fig. 3a and 2c). Taken collectively, our\nfindings bring attention to the occurrence of a pool of abiotically generated particulate P from\ni\nmineral-mediated P transformation that has been hitherto unaccounted for in environmental\norg\nmatrices.\nComment 5:\nWhat are the parameters in your soil and sediment samples’ porewater? Local\ntemperature? You set up pH 7 and 0.1 M NaNO3 + 0.01 M NaHCO3 to perform\nexperiments. Why not use the porewater conditions to do experiments? Which might be\ncloser to realistic reaction conditions and show the true catalytic reactivity/rates of Fe\noxides in organic P mineralization.\nResponse: The pH of the porewater at the Calhoun Critical Zone Observatory soil was\ndetermined to be 5.8 at the time of excavation, however the reported pH at this site has ranged\nfrom 4.5 to 6.2 (Richter et. al. 1994; Chen et. al. 2020). The mean annual temperature at the soil\nsampling site is 16 °C with a range of 5 °C to 25 °C (Richter et. al. 1994). For the Missisquoi Bay\nsediment sample, the porewater was determined to be at a temperature of 24 °C with a\ncircumneutral pH (Smith et. al. 2011 and Cai et. al. 2010). We have included this information in\nLines 468-473. Rather than using reaction conditions that directly mirrored those of the\nporewater conditions of the natural systems, we chose to use the standardized conditions of pH\n5 | Pa ge\n7 and background electrolyte 0.1 M NaNO buffered with 0.01 M NaHCO at 25 °C to be able to\n3 3\ncompare directly the reactivities of the different minerals present across the two different\nsystems. Please see lines 548-553 in the main text for the inclusion of this reasoning.\nLines 468-473:\nThe pH of the porewater at the Calhoun Critical Zone Observatory soil was determined to be 5.8 at\nthe time of excavation, however the pH has been reported previously to range between and 4.5 to\n6.257,58. The mean annual temperature at the soil sampling site is 289 K (or 16 ºC), ranging\ntemperatures found to range between 278 K (5 ºC) and 298 K (25 ºC)57. For the Missisquoi Bay\nsediment sample, the porewater pH was determined to be circumneutral with a temperature of 297K\n(24.0 ºC)59,60.\nLines 548-553:\nHere, to compare directly the reactivities of the different minerals present across the two\ndifferent natural samples, we chose to use the standardized conditions of a reaction solution at\npH 7.0, ionic strength set by 0.1 M NaNO and buffered by 0.01M NaHCO at 298K (25 ºC). We\n3, 3\nacknowledge that the actual porewater conditions in the natural samples, as highlighted above\nunder detailed descriptions of sample characteristics, would be different from the standardized\nexperimental conditions.\nComment 6:\nYou used XRD to do mineralogical identification. How do you know amorphous phase\ncontents and how do they affect your conclusions?\nResponse: We acknowledge that our characterization method focused on the crystalline phases\nvia XRD and that only the amorphous phases were that of Fe phases characterized via XANES.\nThe scope of our technical analysis did not account for possible amorphous silicate and\naluminosilicate phases. However, according to our data, these silicate-bearing minerals all had\nlow catalytic and adsorption reactivities compared to the Fe oxides for our representative P .\norg\nTherefore, we do not expect the presence of amorphous phases would affect the fate of the P\norg\nreactant. The main text has been modified (See Lines 151 – 157) to express this caveat.\nLines 151-157:\nMoreover, our XRD and Fe XANES data would not account for the possible presence of low-\ncrystallinity silicate or aluminosilicate phases in the natural samples. However, as will be discussed in\nthe next section, there was minimal to no catalytic reactivity of silicate or aluminosilicate silicates\ntowards the different P compounds (Fig. 2). Consistently, based on our analysis, we found that\norg\nabout 80% (or more) on a per-mass basis of both sediment and soil samples was comprised of\nsilicate minerals of different types (quartz, micas, feldspars, clays), and less than 20% constituted the\nFe oxide fraction (Fig. 1c).\nComment 7:\nFigure S6 showed the similar XANES spectra for both mineral-Porgnic and mineral-Pi?\nWhat is the confidentiality of using P K-edge XANES to analyze surface organic and\n6 | Pa ge\ninorganic P?\nOverall, the manuscript is of high quality. I assume that considering these\nconcerns/comments might further improve your manuscript quality and consolidate your\nfindings about this new and interesting role of Fe oxides in P biogeochemical cycles in the\nsub-surface Earth.\nResponse: Recent studies performed by Prietzel et. al., 2016, Klein et. al., 2019, and Eusterhues\net. al., 2023, have shown that P K-edge spectra LCF can be used to differentiate the difference\nbetween Fe—P and Fe—P. This is due to the analysis of the pre-edge feature, shifting of the\norg i\nwhite line, and post-edge features. As pointed out by the reviewer, it is difficult to discern\ndifferences in the P and P reference spectra and reaction spectra in the original S6 figure. We\ni org\nhave included an inset that focuses on the white line region and the post-edge region so this\ndistinction is more apparent. Please see the new Figure S6.\nReferences\nWan et al. Rethinking the biotic and abiotic remineralization of complex phosphate molecules in\nsoils and sediments. Science of The Total Environment 833, 155187 (2022).\nRichter et al. Soil chemical change during three decades in an old-field loblolly pine (Pinus taeda\nL.) ecosystem. Ecology 75, 1463 (1994).\nChen et al. Iron-mediated organic matter decomposition in humid soils can counteract\nprotection. Nature Communications 11, 2255 (2020).\nSmith et al. Relating sediment phosphorus mobility to seasonal and diel redox fluctuations at the\nsediment–water interface in a eutrophic freshwater lake. Limnology and Oceanography 56,\n2251–2264 (2011).\nCai et al. Carbon Cycling and the Coupling Between Proton and Electron Transfer Reactions in\nAquatic Sediments in Lake Champlain. Aquat Geochem 16, 421–446 (2010).\nPrietzel et al. Reference spectra of important adsorbed organic and inorganic phosphate binding\nforms for soil P speciation using synchrotron-based K-edge XANES spectroscopy. Journal of\nSynchrotron Radiation 23, 532–544 (2016).\nKlein et al. Abiotic phosphorus recycling from adsorbed ribonucleotides on a ferrihydrite-type\nmineral: Probing solution and surface species. Journal of Colloid and Interface Science 547,\n171–182 (2019).\nEusterhues et al. Importance of inner-sphere P-O-Fe bonds in natural and synthetic mineral-\norganic associations. Science of The Total Environment 905, 167232 (2023).\nReviewer #2 (Remarks to the Author):\nI have concluded that the research described is sufficient quality and potential impact to be\n7 | Pa ge\naccepted for publication in Nature Communications, with some minor revision (see below). The\nmanuscript is very well written and presented, and the findings of this study clearly\ndemonstrated that significant mineralization of model organic phosphorus compounds occurred\non the surface of iron oxide, which represents a potentially important process that contributes to\nphosphorus transformations and bioavailability in sediments and soils.\nResponse: We thank the reviewer for their kind words regarding our effort to present a well-\nwritten manuscript and acknowledging the important contribution of our research findings.\nMain comments:\nComment 1:\nI only have one significant comment/request to make on the manuscript. I am a soil\nscientist with very little experience or expertise in sediment phosphorus dynamics. With\nspecific regard to the dynamics of organic phosphorus in soil, I was surprised to note the\ncomplete absence of any mention or consideration of the role and function of “iron oxide\nsurface mineralzation” in the cycling and bioavailability of inositol phosphates. Inositol\nphosphates (principally inositol hexaphosphate [commonly referred to as “phytate”]) are\nwidely acknowledged to be important constituenst of soil organic phosphorus, and have\nbeen shown to be strongly adsorbed on soil colloid surfaces. I would request that the\nauthors consider including some discussion of the potential role of iron oxide surface\nmineralization in the fate and bioavailability of inositol phosphates in soil.\nResponse: We agree with the reviewer that inositol phosphate or phytate is an important\ncomponent of soil P reservoirs. We conducted additional experiments involving reactions of\norg\nphytate with the three Fe oxides (ferrihydrite, hematite, and goethite), quartz, and the\naluminosilicates with inositol hexaphosphate or phytate. We also performed experiments with\nthe sugar phosphate G6P, another important P in plant and microbial systems. The results can\norg\nbe found in new Figure 2d, and 2e and the accompanying discussion in see Lines 212-251.\nOur results show that the catalytic reactivity of Fe oxides (specifically goethite and hematite)\ntowards phytate was significantly lower than with ATP, but the Fe oxide was the only mineral\ndemonstrating catalytic dephosphorylation of phytate compared to the other silicate and\naluminosilicate minerals.\nLines 212-251:\nAmongst the chemical diversity of P types found in biomolecules, phosphomonesters are widely\norg\nfound in soils34–36,41,42. To probe Fe oxide reactivity with these other types of P , we performed\norg\nreactions involving each of the three Fe oxides (ferrihydrite, hematite, and goethite) with three\nphosphomonoesters: a ribonucleotide, AMP (AMP-P, 50 µM P or 1.5 mg P L-1); a sugar phosphate,\nG6P (G6P-P, 50 µM P or 1.5 mg P L-1); and, the primary P storage in terrestrial plants36, inositol\nhexaphosphate or phytate (phytate-P, 300 µM P or 9.0 mg P L-1 (Fig. 2c, 2d, and 2e). To compare\nthe Fe oxide reactivity with the other mineral types, we also performed experiments of the\nphosphomonoester P compounds reacted with quartz and aluminosilicates (mica, kaolinite, illite)\norg\n(Fig. 2c, 2d, and 2e).\nIn contrast to the ATP reactions, all the P derived from the reacted AMP was retained as\ni\nparticulate P while aqueous P was absent, a significant finding that was made possible here due to\ni i\nthe application of the XANES technique (Fig. 2c). On the one hand, the catalytic reactivity of\nferrihydrite was higher for AMP than for ATP, as reflected by the 20-fold increase in particulate P\ni\nfraction (p < 0.001) (Fig. 2a and 2c). On the other hand, the catalytic reactivity of goethite was less\n8 | Pa ge\nfor AMP than for ATP as characterized by a 3-fold decrease in particulate P fraction (p < 0.01)\ni\naccompanied by no change in the particulate P fraction (p = 0.29) and no measured solution P;\norg i\nhematite did not display any adsorption or catalytic reactivity towards AMP (Fig. 2a and 2c).\nInterestingly, in our aforementioned experiments with ATP and goethite, 20–24% of the initial ATP-P\nremained as AMP-P in solution and no ADP was detected after nearly all the ATP reacted with\ngoethite was transformed or adsorbed (SI, Fig. S10). Thus, this accumulation of AMP in the ATP-\ngoethite experiment can be explained by the lower reactivity of goethite for AMP relative to ATP\n(Fig. 2c; SI, Fig. S10). When ATP was reacted with hematite, we measured a conversion of 17–23%\nof the initial ATP-P to ADP-P and 5–7% to AMP-P, consistent with lower reactivity for both ADP and\nAMP compared to ATP (SI, Fig. S10). Notably, the silicate and aluminosilicate minerals did not\nexhibit any adsorption or catalytic reactivity towards AMP (Fig. 2c).\nIn contrast to AMP, all the silicate and aluminosilicate minerals adsorbed phytate (from 5% to\n50% of the total reacted phytate-P) and, rather than an Fe oxide, the clay illite adsorbed the most\nphytate (Fig. 2d). However, similar to the results with AMP and ATP, catalytic reactivity towards\nphytate was only obtained with Fe oxides, specifically hematite and goethite (Fig. 2d). Relative to\ncontrols, phytate-derived particulate P was higher by 8 – 14% (p < 0.01) with hematite and by 24 –\ni\n32% with goethite (p < 0.001); the particulate P with ferrihydrite, however, corresponded to the\ni\nadsorption of solution P in the control experiment (Fig. 2d). For G6P, some G6P adsorption was\ni\nobserved with two of the aluminosilicates (mica and kaolinite) and two of the Fe oxides (ferrihydrite\nand goethite) (Fig. 2e). But, as with phytate, the most significant adsorption was with ferrihydrite,\nwith 47 – 78% of the reacted G6P found as particulate P (Fig. 2e). None of the investigated\norg\nminerals catalyzed G6P dephosphorylation (Fig. 2e).\nIn sum, our data revealed that the silicates and aluminosilicates either had minimal to no\ncatalytic reactivity or exhibited some extent of adsorption reactivity. Only the Fe oxides were found to\ncatalyze the dephosphorylation of both phosphomonoester and phosphoanhydride-bearing\ncompounds. Importantly, the Fe oxide-catalyzed reactions seemed to be dependent on both the\nmineral surface chemistry and the type of P species. As proposed previously14, we expect the\norg\ndifferences in reactivity may stem from the binding conformations of different P on the mineral\norg\nsurface.\n9 | Pa ge"}, {"round": 2, "reviewer_comments": {"reviewer_1": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nI appreciate the authors had processed most of my comments and after major revision, the manuscript looks befter than the inifial version. I am sfill very concerned about use P K-edge XANES to disfinguish P species within different mineral phases and within different P species. In figure 2c and lines 225-230/423-426, the author observed the significant hydrolysis of AMP by ferrihydrite. But with comparison, ferrihydrite showed super limited hydrolysis ability of phytate (Pi in phytate may come ~4% impurity) and G6P, which three P species all contain only one P-O-C bond. Why here ferrihydrite can hydrolyze more than 70% AMP? Why other two Fe oxides show limited AMP hydrolysis ability? what mechanism/surface structure drives such a high amount of AMP’s P-O-C cleavage by ferrihydrite, but not for G6P’s P-O-C breakage? Baldwin (1995) indicated no such big difference or P-O-C cleavage between different iron oxides and Tong (2020) indicates that the main force to drive C-O-P breakage comes from the exposure facets of crystalline iron oxides. Thus, this data does not make sense, which may come from P K-edge XANES fifting (70% Pi in parficulate phase, but without any release of Pi into solufion, which does not also make sense). I suggest the author should check P K-edge XANES for these AMP-iron oxide samples to get accurate fifting results. Thus, I recommend a minor revision and the author should process this important concern, which might significantly challenge the conclusions here.\nReferences\nLi, Tong, et al. \"Enhanced hydrolysis of p-nitrophenyl phosphate by iron (hydr) oxide nanoparficles: Roles of exposed facets.\" Environmental Science & Technology 54.14 (2020): 8658-8667.\nBaldwin, Darren S., et al. \"Phosphate ester hydrolysis facilitated by mineral phases.\" Environmental science & technology 29.6 (1995): 1706-1709.", "reviewer_2": "Reviewer #2 (Remarks to the Author):\nI have considered the detailed and comprehensive responses by the authors to my original comment and the much more detailed comments made by the other reviewer, and concluded that these adequately addressed our collecfive quesfions. Accordingly, I recommend that the manuscript be accepted for publicafion without any further revision."}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nTo address clearly the comments provided by Reviewer 1, we divided up the one paragraph\nprovided by the reviewer into several comments as detailed below.\nComment 1: I appreciate the authors had processed most of my comments and after major\nrevision, the manuscript looks better than the initial version.\nResponse: We thank the reviewer for acknowledging our efforts. It was a herculean effort to secure\non very short notice additional beamtime at a synchrotron in Australia during the shutdown of the\nStanford synchrotron in California. We have done experiments with additional P biomolecules with\norg\nfive different minerals, in addition to the experimental data with two other P compounds that were\norg\nalready presented in the paper.\nComment 2: I am still very concerned about use P K-edge XANES to distinguish P species\nwithin different mineral phases and within different P species.\nResponse: Based on this comment from the reviewer, we would like to clarify that we do not apply\nthe XANES technique beyond its limitation. As pointed out in the Introduction, the P K-edge XANES\ntechnique has been demonstrated to distinguish between organic P and inorganic P bound to Fe\noxides (see Lines 72-79):\n“Advances in mineral surface characterization by synchrotron-based P K-edge X-ray absorption\nnear-edge structure (XANES) spectroscopy have made it possible to distinguish between P and P\ni org\nbound to Fe in minerals28 or Fe oxides in a soil matrix29. In terms of monitoring mineral-mediated P\norg\nrecycling, application of the XANES technique with ferrihydrite revealed the generation of particulate\nP from adsorbed ribonucleotides, while P was notably absent from solution14. This latter finding,\ni i\nwhich was confirmed by quantifying the dephosphorylated organic products in the solution by LC-\nMS14, highlights the need to perform quantitative analysis of particulate P, in addition to dissolved P,\ni i\nespecially for minerals such as Fe oxides with strong adsorption affinity for P species.”\ni\nFurthermore, we have pointed out in the original version of the manuscript both the powerful insights\nthat can be obtained from the XANES technique and for which analysis this technique can have\nlimitations (see Lines 326-332):\n“It was not possible to use the P K-edge XANES spectra to distinguish the specific P species\nassociated with the different aluminosilicates nor the specific Fe oxide minerals associated with\neither particulate P or particulate P species on Fe oxides (SI, Fig. S14). Specifically, we were able\ni org\nto employ the LCFs to distinguish P associated with Ca using apatite as a reference, P (without\ni\ndiscriminating between P or P ) associated with aluminosilicates, P associated with Fe in Fe oxides,\ni org i\nP species associated with Fe in Fe oxides, and P in Fe-P clusters using vivianite as reference (Fig.\norg i i\n4c, 4d, and 4e).”\nTherefore, in response to the statement by the reviewer, we did not apply P K-edge XANES to all P\nspecies and all minerals. This XANES technique was used to quantify the fraction of particulate P\ni\nand particulate P . This can be done for any type of P types but we can’t distinguish between\norg org\ndifferent P species present simultaneous in the particulate fraction. Importantly, we employed high-\norg\nresolution mass spectrometry to monitor different organic species (both phosphorylated and\ndephosphorylated organic species) in solution, from which preliminary quantitation of particulate P\ni\n1 | Pag e\ncan be determined from performing a mass balance on P. For example, in mineral-mediated analysis\nof ATP, the following mass balance will provide a preliminary account of particulate P: (P ) =\ni i, particuate\n(ADP ) + 2(AMP ) – (P ). Using such mass balance, we have highlighted previously that\nsolution solution i,solution\nthe absence of P in solution does not mean lack of catalyzed reaction; importantly, the presence of\ni\ndephosphorylated organics highlight missing P determined to be particulate P (Klein et. al., 2019).\ni i\nComment 3: In figure 2c and lines 225-230/423-426, the author observed the significant\nhydrolysis of AMP by ferrihydrite. But with comparison, ferrihydrite showed super limited\nhydrolysis ability of phytate (Pi in phytate may come ~4% impurity) and G6P, which three P\nspecies all contain only one P-O-C bond. Why here ferrihydrite can hydrolyze more than 70%\nAMP? Why other two Fe oxides show limited AMP hydrolysis ability? what mechanism/surface\nstructure drives such a high amount of AMP’s P-O-C cleavage by ferrihydrite, but not for G6P’s\nP-O-C breakage?\nResponse: It is reasonable to expect that different energy would be required to cleave the P-O-C\nbond in our different investigated phosphomonoesters because the different organic structures\nattached to the phosphoester bond: a ribonucleoside composed of a sugar attached to heterocycling\nnitrogenous ring in AMP, versus only a sugar moiety in G6P, versus a inositol in phytate. In fact, fact, Wan\net. al. (2022) reported, based only on monitoring aqueous P, 10-fold to 25-fold higher P production\ni i\nfrom AMP than from G6P and phytate reacted with hematite—these previous experiments were\ndone with 60% less mineral (per mass) in solution and with 20-fold higher concentration for G6P and\nAMP and 3-fold higher concentration for phytate compared to our experiments. Furthermore,\ndifferent rates of enzymatic dephosphorylation were reported for these different phosphomonoester\nP types (Solhtalab et. al., 2021). Therefore, based on these previous findings, it is not surprising\norg\nthat we found less P production with G6P compared to AMP reacted with the Fe oxides.\ni\nWith respect to mechanisms, we would like to point out that the motivation of the current study was\nto explore whether mineral-mediated catalysis of P recycling, which was proposed with pure\norg\nminerals, can actually occur with different natural P sources, with different minerals, and,\norg\nimportantly, within natural environmental samples obtained from a forest soil and a lake sediment.\nNow, it is worthwhile for the field to invest in figuring out the mechanisms of this process because we\nhave provided evidence, for the first time, that this mineral-mediated catalysis can occur within\nenvironmental samples and, importantly, we obtained abiotic rates that were comparable to\nenzymatic rates reported in certain soils. Understanding and determining the mechanisms that\ninfluence Fe oxide reactivity, which are beyond the scope of the current manuscript, is an important\nsubject for future investigation. We added discussion on exploration of these mechanisms in\nrecently published studies as well as pointing out areas that need to be investigated to gain further\ninsights. (see Lines 452-463):\n“Our findings corroborate the role of surface chemistry in dictating the extent of catalytic\nreactivity of the Fe oxide. First, P dephosphorylation by the Fe oxides was not found to be\norg\ndependent on surface area. Despite a 14-fold lower surface area for goethite compared to\nferrihydrite (16.0 m2 g-1 versus 230 m2 g-1), goethite was found to be the most reactive,\ndephosphorylating nearly 7-fold higher amount of the total P added as ATP. Second, when\norg\naccounting for the P binding site density to normalize the rate of dephosphorylation, goethite still\ni\nexhibited a turnover number up to 9-fold greater than ferrihydrite. Regarding the relevance of other\nchemical characteristics of the mineral structure, a study with hematic proposed Lewis acid sites and\nFe coordination to be determining factors for the extent of hydrolytic cleavage of a synthetic P by\norg\n2 | Pag e\nthis Fe oxise15,57. Our results with naturally-occurring P showed markedly different reactivity\norg\nbetween our three investigated Fe oxides (goethite, ferrihydrite, and hematite) for the same P\norg\ncompound. Therefore, is Fe oxide-dependent catalytic reactivity highlights the importance of further\ninvestigation into the importance of Fe oxide surface chemistry and surface acidity.”\nComment 4: Baldwin (1995) indicated no such big difference or P-O-C cleavage between\ndifferent iron oxides and Tong (2020) indicates that the main force to drive C-O-P breakage\ncomes from the exposure facets of crystalline iron oxides. Thus, this data does not make\nsense, which may come from P K-edge XANES fitting (70% Pi in parficulate phase, but without\nany release of Pi into solution, which does not also make sense).\nReferences\nLi, Tong, et al. \"Enhanced hydrolysis of p-nitrophenyl phosphate by iron (hydr) oxide\nnanoparticles: Roles of exposed facets.\" Environmental Science & Technology 54.14 (2020):\n8658-8667.\nBaldwin, Darren S., et al. \"Phosphate ester hydrolysis facilitated by mineral phases.\"\nEnvironmental science & technology 29.6 (1995): 1706-1709.\nResponse: It is not clear to us what the objection of the Reviewer is because we are reporting our\ndata that were obtained from meticulously performed experiments with independent replicates. It is\nworthwhile to note that differences in P dephosphorylation by different Fe oxides has been\norg\nreported previously by Li et. al. 2020, a reference highlighted by the reviewer (as stated above).\nSpecifically, Li et. al. (2020) reported greater dephosphorylation of pNPP, a synthetic P , by\norg\ngoethite than by hematite. In agreement with this finding, our results showed no reactivity with\nhematite but there was reactivity with goethite and ferrihydrite, albeit direct comparison between\npNPP and the ribonucleotide AMP is not appropriate (as we already discussed above).\nAs highlighted both in the abstract and the introduction of our manuscript, the motivation for using\nXANES technique is because it is critical to consider Fe oxide surfaces as both adsorbents and\ncatalysts whereby generated P from the mineral catalysis may be trapped on the surface and thus\ni\nabsent from the solution phase. As we have pointed out in the Introduction, a previous study (Klein\net al., 2019) had reported the absence of P in solution while dephosphorylated organic species were\ni\ndetected in solution by high-resolution LC-MS, which led to the quantification of missing P bound to\ni\nferrihydrite an Fe oxide surface (See our response to Comment 2 above).\nComment 6: I suggest the author should check P K-edge XANES for these AMP-iron oxide\nsamples to get accurate fitting results. Thus, I recommend a minor revision and the author\nshould process this important concern, which might significantly challenge the conclusions\nhere.\nResponse: We were transparent with our XANES data and how they were fitted (see SI Figs S7-S10,\nFig. S15, and Table S3, S5-S7) in response to comments during the first round of reviews. It is\nimportant to note that the application of the XANES technique has been validated previously, as we\nhave highlighted in the Introduction (also see our response to Comment 2). This technique has been\npreviously documented to distinguish between inorganic P and organic P either bound to pure Fe\noxides or found in the environmental matrices (Prietzel et. al. 2016, Eusterhues et. al., 2023). Here\nwe are applying this technique to monitor, for the first time the de novo generation of inorganic P\n3 | Pag e\nfrom organic P reacted with environmental samples and pure forms of Fe oxide and other minerals.\nTo add further clarification on how the XANES data were used, we added 3 additional figure panels\nto Figure 4 with close-up illustrations of different part of the XANES spectra and we expanded our\nresults section to explain in more details how these spectra were analyzed.\nReviewer #2 (Remarks to the Author):\nI have considered the detailed and comprehensive responses by the authors to my original comment\nand the much more detailed comments made by the other reviewer, and concluded that these\nadequately addressed our collective questions. Accordingly, I recommend that the manuscript be\naccepted for publication without any further revision.\nResponse: We thank the reviewer for their time in the thorough review of our manuscript in both\nrounds. Thank you for recommending our manuscript for publication.\nReferences:\nKlein, A. R., Bone, S. E., Bakker, E., Chang, Z. & Aristilde, L. Abiotic phosphorus recycling from\nadsorbed ribonucleotides on a ferrihydrite-type mineral: Probing solution and surface species.\nJournal of Colloid and Interface Science 547, 171–182 (2019).\nWan, B., Huang, R., Diaz, J. M. & Tang, Y. Rethinking the biotic and abiotic remineralization of\ncomplex phosphate molecules in soil and sediments. Science of the Total Environment 833, (2022).\nSolhtalab, M., Klein, A. R. & Aristilde, L. Hierarchical Reactivity of Enzyme-Mediated Phosphorus\nRecycling from Organic Mixtures by Aspergillus niger Phytase. J. Agric. Food Chem. 69, 2295–2305\n(2021).\nLi, T. et al. Enhanced Hydrolysis of p-Nitrophenyl Phosphate by Iron (Hydr)oxide Nanoparticles:\nRoles of Exposed Facets. Environ. Sci. Technol. 54, 8658–8667 (2020).\nBaldwin, D. S., Beattie, J. K., Coleman, L. M. & Jones, D. R. Phosphate Ester Hydrolysis Facilitated by\nMineral Phases. Environ. Sci. Technol. 29, 1706–1709 (1995).\nPrietzel, J. et al. Reference spectra of important adsorbed organic and inorganic phosphate binding\nforms for soil P speciation using synchrotron-based K-edge XANES spectroscopy. Journal of\nSynchrotron Radiation 23, 532–544 (2016).\nEusterhues, K. et al. Importance of inner-sphere P-O-Fe bonds in natural and synthetic mineral-\norganic associations. Science of The Total Environment 905, 167232 (2023).\n4 | Pag e"}, {"round": 3, "reviewer_comments": {"reviewer_1": "REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have processed my concerns well and I suggest the current version of this manuscript can be accepted for the publicafion."}, "author_response": ""}]

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