the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 25 Jun 2025
Long-term pig manure application increases soil organic carbon through aggregate protection and Fe-carbon associations in a subtropical Red soil (Udic Ferralsols)
Abstract. Manure is known to improve soil organic carbon (SOC) in Fe-rich red soils, while the underlying stabilization mechanisms remain poorly understood. In this study, four treatments were selected: (1) no amendment (Control), (2) low manure (LM, 150 kg N ha-1 yr-1), (3) high manure (HM, 600 kg N ha-1 yr-1), (4) high manure with lime (HML, 600 kg N ha-1 yr-1 plus 3000 kg Ca (OH)2 ha-1 3yr-1). The quantity and quality of topsoil (0–20 cm) organic carbon were investigated by physical fractionation, 13C-nuclear magnetic resonance (NMR) spectroscopy and thermogravimetry (TG) analysis. Manure application increased total SOC by 65.1 %–126.7 % (primarily in the particulate organic matter (POM) fraction), while the mineral-associated organic matter fraction (MAOM), despite its higher C content (4.18–7.09 g C kg⁻¹), contributed less (65.4 %–71.0 %) compared to the control (82.4 %). POM C was stabilized via hierarchical aggregation: fresh manure inputs acted as binding nuclei, increasing macroaggregates (>0.25 mm) while reducing microaggregates (0.05–0.25 mm), physically isolating labile C from microbial decomposition. Concurrently, manure amendments triggered Fe-mediated chemical stabilization. Elevated pH (4.8 to 5.4–7.1) enhanced non-crystalline Fe oxide (Feo) content (+25.4 %), which positively correlated with MAOM C (R² = 0.56, P < 0.05). Despite a chemical composition shift toward aliphaticity and reduced aromaticity, thermally stable organic matters increased by 8 %–12 %, revealing critical role of Feo (aggregates were destroyed before TG analysis) in offsetting inherent molecular lability. Overall, this study establishes a dual SOC stabilization framework for subtropical red soils, highlighting physical protection through aggregation processes and chemical protection via Fe-carbon associations.
Competing interests: Hu Zhou is a member of the editorial board of SOIL.
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims made in the text, published maps, institutional affiliations, or any other geographical representation in this paper. While Copernicus Publications makes every effort to include appropriate place names, the final responsibility lies with the authors. Views expressed in the text are those of the authors and do not necessarily reflect the views of the publisher.- Preprint
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Status: final response (author comments only)
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RC1: 'Comment on egusphere-2025-2405', Anonymous Referee #1, 17 Jul 2025
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AC2: 'Reply on RC1', Hui Rong, 03 Sep 2025
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: Fed defined at first use (Table 1 footnote: "Fed: crystalline Fe oxides; Feo: 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
Citation: https://doi.org/10.5194/egusphere-2025-2405-AC2
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AC2: 'Reply on RC1', Hui Rong, 03 Sep 2025
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RC2: 'Comment on egusphere-2025-2405', Anonymous Referee #2, 30 Jul 2025
The manuscript investigates the impact of organic fertilization using pig manure on soil physicochemical properties and the stabilization mechanisms of organic C in amended soils. Through soil fractionation, NMR spectroscopy, and thermogravimetric analysis, the authors examine how manure-derived C integrates into different soil pools (POM and MAOM), and how changes in pH influence mineral-organic associations involving Fe. These are relevant and timely topics in the context of soil organic matter stabilization and sustainable fertilization strategies. While I believe the data are suitable for publication, two key aspects of the manuscript require clarification and revision.
Major Comments
1-Clarification of knowledge gap and novelty. The introduction should be revised to more explicitly identify the specific knowledge gap addressed by the study. Numerous studies have already demonstrated that organic amendments increase soil pH, which can promote mineral-organic associations (e.g., via Fe precipitation), see :Huang et al., 2017; Wang et al., 2019; Chen et al., 2022. The authors must clearly differentiate their contribution from existing literature and emphasize what aspect of their approach or findings is novel. The discussion would benefit from being framed around these novel findings.
2-NMR spectroscopy interpretation and figure representation. To my knowledge, NMR spectroscopy is not a quantitative technique, the spectra shown in Figure 1 must be normalized to allow for visual comparison between treatments. Without normalization, comparisons across amended and control soils can be misleading. Furthermore, statements such as in Lines 358–362 (“...increased the content of O-alkyl C, but declined that of aromatic C”) could be misinterpreted. The data seem to indicate an increase in O-alkyl C due to the chemical nature of pig manure, rather than an actual loss of aromatic C. A shift in relative proportions does not equate to removal of specific fractions. This distinction should be clarified.
Specific Comments
- Lines 47-48: The connection between red soils and carbon stabilization processes is unclear. Please clarify whether red soils are highlighted because they represent a major class of cultivated soils receiving compost amendments. The rationale for choosing red soils as the focal point should be more explicitly stated.
- Lines 58-60: The expression “transformation of Fe oxides” could be made more precise. The sentence “The long-term impacts of manure-induced pH shifts on Fe oxide speciation” would benefit from clarification. Are you suggesting that the changes in speciation are causing the precipitation of poorly crystalline Fe phases?
- Lines 79-85: The paragraph outlining the study goals and hypotheses would be easier to follow if the objectives were presented first, followed by the hypotheses.
References:
Chen, M., Zhang, S., Liu, L., Liu, J., Ding, X., 2022. 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
Huang, X., Feng, C., Zhao, G., Ding, M., Kang, W., Yu, G., Ran, W., Shen, Q., 2017. 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
Wang, P., Wang, J., Zhang, H., Dong, Y., Zhang, Y., 2019. 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
Citation: https://doi.org/10.5194/egusphere-2025-2405-RC2 -
AC1: 'Reply on RC2', Hui Rong, 03 Sep 2025
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 (Feo) 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 (Fed), 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, Feo 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 (Feo) 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 (Feo), 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.
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- 1
The research is entirely in the scope of the journal. SOC stabilization can improve soil fertility and mitigate climate change, thus being a concern for a long time. In this study, the authors utilized physical fractionation techniques, NMR spectroscopy and thermogravimetry (TG) analysis to study the quantity and quality of organic carbon stored in a red topsoil subject to pig manure inputs, and highlighted physical protection through aggregation processes and chemical protection via Fe-carbon associations. The experimental design is robust, data are comprehensive, and the findings hold significant implications for carbon sequestration practices.
Recommendation: Minor Revision prior to acceptance. Required improvements are outlined below.
1-In the introduction part, some state-of-art methods used for characterizing the quantity and the quality of SOC should be added.
2-In the material and methods part, the potential effect of HF pretreatment on organo-mineral complexes was missing, and please justify the necessity of this process.
3-In the results part, adjust label overlaps and add regression equations to panels in Fig.3.
4-Some discussion is not convincing.
In the Discussion 4.2, the authors should emphasize that while POM-C tripled (8.8%-26.0%), MAOM-C remains the dominant pool (>65%). In addition, in this part, correlations between Feₒ and MAOM-C implied Fe-organic carbon interactions but do not prove chemical protection. Molecular binding mechanisms (e.g., ligand exchange, coprecipitation) need to be discussed by citing synchrotron-based evidence (Ruiz et al., 2024).
In the Discussion 4.3, increased Exo2 (thermally stable SOM) was attributed to Feo offsetting molecular lability, but clarifying how mineral bonding alters thermal behavior (Kleber et al., 2021) was suggested. Additionally, explicitly state that decreased TG-T50 (more labile SOM) and increased absolute Exo2 (more stable SOM) are not mutually exclusive.
5-Some language and formatting mistakes
Units: Add spaces (e.g., "kg ha⁻¹ yr⁻¹" not "kg ha⁻¹yr⁻¹").
Abbreviations: Define all acronyms at first use (e.g., "Fed" undefined in text).
Repetition: Delete duplicated opening sentence in Section 4.3.
Another Minor Revisions
HML treatment anomaly: Explain why higher pH in HML (7.08 vs. HM’s 6.11) did not further increase Feₒ (Table 1). Cite pH-dependent Fe transformation (Vithana et al., 2015).
Carbon input calculation: Detail manure-C input calculations (dry matter basis, moisture correction) in Supplementary Material.
Literature: Update references (e.g., include 2023–2024 studies on Fe-C interactions).
In all, this manuscript presents a timely, well-executed investigation of SOC stabilization mechanisms in manure-amended red soils. The dual-protection framework advances soil carbon science, particularly for tropical/subtropical regions. Recommend acceptance after minor revisions.