Response to Comment 1:

Thank you for this valuable suggestion. We agree that introducing state-of-the-art methods for characterizing soil organic carbon (SOC) will provide a stronger foundation and better justify our methodological choices.

We have revised the introduction to incorporate a discussion of state-of-the-art techniques for assessing SOC quantity and quality. Furthermore, we have explicitly outlined the rationale for employing a combined NMR and TG approach in our study, balancing the need for molecular-level characterization with practical considerations of analytical efficiency and minimal sample pretreatment.

Lines 86-91: “Despite the emergence of advanced techniques such as nano-scale secondary ion mass spectrometry (NanoSIMS) for spatial visualization of organo-mineral associations and high-resolution mass spectrometry for molecular characterization, these approaches often require complex instrumentation and are less suited for the comparative analysis of multiple samples. Therefore, to efficiently evaluate the chemical nature and stability of SOC across contrasting management conditions, this study employed a combined NMR and TG approach.”

Response to Comment 2:

Thank you for raising this critical point regarding the potential impact of HF pretreatment on organo-mineral complexes. We appreciate the opportunity to clarify the necessity of this step and to address its implications in our study.

We fully acknowledge that HF pretreatment can dissolve short-range-order minerals and potentially disrupt certain organo-mineral associations, which may alter the native state of the SOC. ​However, the application of HF is a well-established and often necessary procedure in solid-state ¹³C NMR analysis of soils.​​ This is primarily because the paramagnetic iron (Fe³⁺) prevalent in soils, particularly in our red soil samples, causes severe signal broadening and a drastic loss of spectral resolution and sensitivity. Without HF treatment to remove these interfering minerals, the NMR spectra would be of insufficient quality to allow for any meaningful interpretation of SOC functional groups. We have revised the Materials and Methods section to explicitly state the potential effect of HF and our rationale for its use.

Lines 174-177: “While HF pretreatment can dissolve short-range-order minerals and potentially disrupt certain organo-mineral associations, it is an essential step to improve the signal-to-noise ratio and spectral resolution required for the reliable quantification of carbon functional groups.”

Response to Comment 3:

Thank you for this helpful suggestion. We have carefully revised Figure 3 to improve its clarity and informational value. We believe these modifications have significantly enhanced the readability and quantitative information presented in Figure 3. The updated figure is included in the revised manuscript (Lines 705-711). 

Response to Comment 4:

(1) Dominance of the MAOM-C pool: We fully agree with your point. In the revised section 4.2, we have now explicitly emphasized that despite the substantial relative increase in POM-C, the mineral-associated organic carbon (MAOM-C) pool remained the overwhelmingly dominant reservoir, accounting for more than 65% of the total SOC across all treatments. This underscores the fundamental role of organo-mineral interactions in C sequestration in these soils, even under significant organic amendment. We have emphasized the dominance of the MAOM-C pool in the Discussion.

Lines 354-357: “MAOM C remained the dominant SOC reservoir, consistently comprising over 65% of the total SOC across all treatments. This underscores that organo-mineral complexation, rather than physical protection within aggregates, is the primary stabilization pathway in these red soils, even under significant organic input.”

(2) Mechanistic discussion beyond correlation: We sincerely thank you for directing us to the critical need to discuss specific binding mechanisms. We have thoroughly revised this part of the discussion. While our correlation between Feₒ and MAOM-C implies an interaction, we now clearly state that this does not constitute proof of chemical protection. As you suggested, we have incorporated a discussion on the potential molecular mechanisms, specifically citing recent synchrotron-based evidence (e.g., Ruiz et al., 2024) that demonstrates mechanisms such as ​ligand exchange​ and ​coprecipitation​ are likely responsible for the strong Fe-C associations observed in similar systems. This addition moves our discussion from a simple correlation to a more sophisticated, mechanism-based interpretation.

Lines 376-385: “The nature of this interaction likely involves specific molecular-scale binding mechanisms. Recent advances in synchrotron-based spectroscopy (e.g., STXM-NEXAFS) have provided direct evidence that poorly crystalline iron oxides (e.g., ferrihydrite) stabilize organic carbon primarily through ligand exchange, where carboxyl (-COOH) and hydroxyl (-C-OH) functional groups in organic molecules replace the surface hydroxyl groups on Fe oxides (Keiluweit et al., 2012; Ruiz et al., 2024). Furthermore, coprecipitation, where organic matter is encapsulated within the matrix of forming Fe (oxyhydr)oxides, represents another potent stabilization mechanism that can generate long-term sinks for organic carbon (Chen et al., 2014). While our bulk data cannot unequivocally distinguish between these mechanisms, the correlation suggests that similar processes are likely operative in our system, contributing to the stability of the large MAOM-C pool.”

(3) Clarifying how mineral bonding alters thermal behavior: We fully agree that a more detailed mechanistic explanation was needed. In the revised Section 4.3, we have incorporated a discussion on the fundamental principles of how organo-mineral bonding influences thermal stability, as suggested. Specifically, we have cited Kleber et al. (2021) and other relevant literature to explain that the formation of inner-sphere complexes between organic molecules and Fe oxide surfaces via ​ligand exchange​ creates stronger chemical bonds that require more energy (higher temperature) to break during thermal decomposition, thereby shifting thermal oxidation to a higher temperature range and contributing to the Exo₂ signal.

Lines 406-409: “The observed increase in the absolute amount of thermally stable SOC (Exo2) can be attributed to the formation of stable organo-mineral complexes with newly formed Feₒ. As elucidated by Kleber et al. (2021), the thermal stability of organic matter is significantly enhanced upon binding to mineral surfaces.”

(4) Reconciling decreased TG-T₅₀ and increased absolute Exo₂:​​ This is a critical point, and we thank you for prompting us to clarify this apparent paradox. We have now explicitly stated that a decrease in TG-T₅₀ (indicating a bulk shift towards more labile SOM) and an increase in the absolute amount of thermally stable SOM (Exo₂) are ​not mutually exclusive. This can occur when the addition of fresh, labile manure-derived carbon disproportionately increases the labile pool, thereby decreasing the overall bulk stability (T₅₀), while simultaneously, the reactions driven by this manure input (e.g., pH change, Fe oxide transformation) promote the formation of new, highly stable organo-mineral complexes that increase the absolute size of the stable Exo₂ pool. This nuanced interpretation has been clearly added to the discussion.

Lines 423-431: “The observed decrease TG-T50 and the simultaneous increase in Exo2 are not contradictory. This apparent paradox can be explained by the dual effect of manure amendment: the input of a large quantity of labile, manure-derived organic compounds significantly increased the proportion of thermally labile C, thereby reducing the average stability of the entire SOM pool (as reflected by the decreased TG-T50). Concurrently, the manure-induced biogeochemical changes (e.g., increased pH and Fe oxide transformation) promoted the formation of highly stable organo-mineral complexes. While the relative proportion of this stable C might be diluted by the new labile C, its absolute amount within the soil system increased substantially, which is captured by the increase in the absolute Exo2 signal.”

Response to Comment 5:

We sincerely appreciate the reviewer's meticulous attention to technical details. All suggested improvements have been implemented as follows:

(1) Unit formatting standardization:​​ Spaces added between units and exponents.

(2) Abbreviation definition:​​ Fe_d defined at first use (Table 1 footnote: "Fe_d: crystalline Fe oxides; Fe_o: non crystalline Fe oxides"; Line 641).

(3) Comprehensive calculation workflow added to Supplementary Material:​​ Calculation details added to Supplementary Material.

(4) Literature update:​​ Three key studies integrated: Chen et al., 2022; Kleber et al., 2021; Ruiz et al., 2024

Major Comments​

Comment 1:

We sincerely thank you for this critical and constructive comment. We appreciate you highlighting these important studies, and we agree that our introduction and discussion need to more explicitly frame our work within the existing literature to better highlight its novel contributions. As you rightly point out, previous studies have established the general phenomenon that organic amendments can increase non-crystalline iron oxides and promote mineral-organic associations (e.g., Chen et al., 2022; Huang et al., 2018; Wang et al., 2019), with some highlighting the role of redox cycling in these processes.

Our study builds upon this foundation by specifically investigating the relationship between pH changes and Fe oxide speciation under manure amendment. Furthermore, the novelty of our work lies in employing a multi-method approach to elucidate the underlying mechanisms of SOC stabilization, particularly in the context of acidic red soils. We have clarified these contributions in the Introduction. This change can be found in the Lines 62-75.

Lines 62-75: “Previous studies have demonstrated that long-term application of organic fertilizers—including manure, compost, and straw—significantly increases the content of amorphous iron oxides (Fe_o) in agricultural soils (Chen et al., 2022; Huang et al., 2018; Wang et al., 2019). These studies further revealed that redox cycling promotes the reductive dissolution of crystalline iron (Fe_d), followed by its reprecipitation as amorphous Feo during oxidative periods. Soil pH was identified as a key factor dynamically influencing the Fed/Feo ratio (Liu et al., 2020; Wang et al., 2023). Although organic amendments are known to elevate pH in acidic red soils, the specific effect of this pH modulation on the formation and stabilization of Feo remains unclear. Additionally, previous studies often isolated chemical recalcitrance, physical protection (via aggregation), or organo-mineral interactions separately, thereby neglecting integrative assessments among these pathways. To address this knowledge gap, an integrative approach combining physical fractionation, molecular characterization, and thermal stability analysis is essential for elucidating the coupled effects of manure on SOC quantity and quality.”

Furthermore, the relationship between pH and Fe oxides is now depicted in Fig. 4 (Lines 722-724), with corresponding descriptions added to the Results section (Lines 288-290). Additionally, we have expanded the Discussion (Lines 368-373) to provide a more comprehensive explanation of the role of Fe oxides in SOC  stabilization within acidic red soils, emphasizing the distinctive mechanisms revealed by our study.

Lines 295-297: “Notably, Fe_o concentration was significantly and positively correlated with pH (R²=0.59, P<0.01; Fig. 4A). Conversely, the concentration of Fed was not correlated with pH (P>0.05; Fig. 4B).”

Response to Comment 2:

Thank you very much for your valuable comment. You are absolutely correct to highlight the need for normalization of the NMR spectra to allow for an accurate visual and quantitative comparison between treatments.

As you suggested, we have now normalized all ¹³C CP-MAS NMR spectra by scaling the total integral area of each spectrum to that of the control soil sample. The revised Figure 1 has been updated (Lines 701-702), and related data processing step has been added in the Material and Methods part (Lines 188-191). This normalization allows for a clear and valid comparison of the relative distribution of carbon functional groups across the different treatments.

Line188-191: " All obtained spectra were processed with phase and baseline corrections. To allow for a direct comparison of the relative distribution of carbon functional groups, the total spectral intensity of each spectrum was normalized to that of the control soil sample. The normalized spectra were then integrated, and we assigned the obtained …"

Furthermore, some statements have been clarified in the manuscript (adding “the proportion of…”):

Line 265: “…significantly increased the proportion of alkyl C by ….”

Line 266: “The proportion of O-alkyl C was significantly…”

Line 268: “Aromatic C and carbonyl C proportions…”

Line 269-271: deleted “While aromatic C was increased in the content after manure amend, its relative proportion was decreased by 12.4%-13.2% (P<0.05)” 

Specific Comments

Response to Specific Comment 1:

Thank you for this insightful comment. You are correct that the connection between red soils and carbon stabilization processes needed to be more explicitly stated. We have now revised the manuscript to clarify our rationale for choosing red soils as our focal point.

Our selection was based not only on their agricultural importance but primarily on their unique pedogenic properties that are highly relevant to organic carbon stabilization. 

We have stated the rationale in the Introduction part as follows:

Line 47-49: "Red soils cover 22% of China’s cropland and exhibit low inherent SOC but high Fe reactivity, making them ideal for studying Fe-C stabilization under organic amendments."

Response to Specific Comment 2:

Thank you for this excellent comment, which helps us to clarify an important mechanistic aspect of our study.

You are absolutely right that the phrase "transformation of Fe oxides" is too vague. Based on our data, we are specifically referring to a ​shift in speciation from more crystalline iron (oxyhydr)oxides (e.g., goethite, hematite) towards poorer crystalline phases (e.g., ferrihydrite)​. We will replace the imprecise term "transformation" with this more detailed description in the revised manuscript.

Lines 64-65: “…significantly increases the content of amorphous iron oxides (Fe_o) in agricultural soils (Chen et al., 2022; Huang et al., 2018; Wang et al., 2019) …”.

Response to Comment 3:

Thank you for this helpful suggestion. We agree that presenting the study objectives first, followed by the hypotheses, improves the logical flow and makes this section easier to follow.

We have revised the paragraph in the introduction (Lines 92-98) to clearly state our research objectives first. Subsequently, we have proposed the specific hypotheses that were derived from each objective.

Lines 92-98: “The specific objectives of this study were: 1) to evaluate the changes of Fe oxides and its effect on MAOM formation; 2) to explore how soil aggregation affected POM formation; 3) to evaluate the effect of manure application on SOC composition and stability. We hypothesized that: 1) Manure application enhance MAOM C formation by increasing non-crystalline Fe oxides (Fe_o), induced by elevated pH; 2) The physical protection was strengthened after manure application due to the soil aggregation process, which triggered labile SOC protection; 3) The application of pig manure strengthened the recalcitrance of SOC, thus improved thermal stability.” 

References:

Chen, M., Zhang, S., and Liu, L.: Organic fertilization increased soil organic carbon stability and sequestration by improving aggregate stability and iron oxide transformation in saline-alkaline soil. Plant Soil 474, 233–249. https://doi.org/10.1007/s11104-022-05326-3, 2022.

Huang, X., Feng, C., and Zhao, G.: Carbon Sequestration Potential Promoted by Oxalate Extractable Iron Oxides through Organic Fertilization. Soil Sci. Soc. Am. J. 81, 1359–1370. https://doi.org/10.2136/sssaj2017.02.0068, 2017.

Wang, P., Wang, J., and Zhang, H.: The role of iron oxides in the preservation of soil organic matter under long-term fertilization. J. Soils Sediments 19, 588–598. https://doi.org/10.1007/s11368-018-2085-1, 2019.