Differences in organic carbon fractions and stability explain limited accumulation in loam and sandy loam under greenhouse conditions

Tan, Boyuan; Yang, Lu; Xiao, Xun; Li, ChunJi; Tan, Jing; An, Ning; Meng, Lingxuan; Yi, Yanli; Qian, Fengkui; Li, Na; Liu, Xue; Li, Song; Han, Wei

doi:10.5194/egusphere-2025-5094

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https://doi.org/10.5194/egusphere-2025-5094

Preprints

03 Nov 2025

| 03 Nov 2025

Differences in organic carbon fractions and stability explain limited accumulation in loam and sandy loam under greenhouse conditions

Boyuan Tan, Lu Yang, Xun Xiao, ChunJi Li, Jing Tan, Ning An, Lingxuan Meng, Yanli Yi, Fengkui Qian, Na Li, Xue Liu, Song Li, and Wei Han

Abstract. Manure is widely applied in greenhouses to enhance soil organic carbon (SOC) and improve fertility. However, how SOC fractions and their chemical stability change under different soil textures and long-term manure inputs remains unclear. We investigated greenhouse soils with 2–50 years of manure application in loam and sandy loam. SOC, easily oxidizable carbon (EOC), microbial biomass carbon (MBC), dissolved organic carbon (DOC), particulate organic carbon (POC), and mineral-associated organic carbon (MAOC) were quantified. The molecular structures of SOC were analyzed via ¹³C NMR spectroscopy. Results showed that SOC in loam stabilized after about 20 years of manure application, whereas sandy loam reached equilibrium within 2 years. In loam, aromatic C and carbonyl C in SOC increased, raising the aromaticity index (ARM); in sandy loam, alkyl C increased, elevating A/OA and the hydrophobicity index (HI). Loam contained higher SOC, EOC, POC, and MAOC contents than sandy loam, with SOC positively correlated with EOC, POC, and MAOC, whereas in sandy loam SOC was positively correlated only with EOC and MAOC, and negatively with fPOC. In loam, ARM and HI promoted SOC accumulation by stimulating EOC and POC, which enhanced MAOC formation. In sandy loam, HI mainly promoted SOC through increasing EOC, which enhanced MAOC formation. In loam, MAOC formation was mainly associated with POC, whereas in sandy loam it was driven by EOC. Overall, in greenhouses, long-term stability of SOC depends on the transformation of labile carbon into stable fractions, with fine-textured soils exhibiting greater sequestration efficiency due to higher structural stability and greater MAOC accumulation.

Received: 15 Oct 2025 – Discussion started: 03 Nov 2025

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Boyuan Tan, Lu Yang, Xun Xiao, ChunJi Li, Jing Tan, Ning An, Lingxuan Meng, Yanli Yi, Fengkui Qian, Na Li, Xue Liu, Song Li, and Wei Han

Status: final response (author comments only)

CC1:
'Comment on egusphere-2025-5094', Yi Cheng, 09 Nov 2025
Greenhouses are characterized by enclosed spaces, high nutrient inputs, and frequent irrigation, which likely result in mechanisms of SOC accumulation and stabilization that differ from those in open-field systems. Therefore, the long-term evolution and dominant mechanisms of SOC under greenhouse conditions remain to be further explored. This manuscript addresses this issue by analyzing the molecular structure of SOC, the distribution of different organic carbon fractions, and their relative contributions to carbon pool stability across varying durations of manure application in greenhouse soils. Overall, the research question is well defined, the experimental design is generally sound, and the data provide solid support. The study offers valuable insights into understanding long-term SOC dynamics and optimizing organic fertilizer management in intensive greenhouse systems, and the study generally meets the scientific standards of SOIL. However, improvements are still needed in the rigor of argumentation and the precision of academic writing. Minor revision is recommended. Specific comments are as follows:
Line 82: The manuscript points out that organic manure inputs are high under greenhouse conditions, but SOC accumulation remains below expectations. It is suggested to briefly explain the possible reasons why high inputs have not resulted in corresponding SOC increases, or to clearly state that the underlying mechanisms remain unclear, so as to better introduce the research gap and objectives of this study, with particular attention to the importance of different SOC fractions.

Lines 86–89: The statement that “most greenhouse studies last only 1–10 years, and short-term stability does not represent the final stage” requires supporting references.

Section 3.1: This section provides numerous numerical values describing SOC content and stock changes in loam and sandy loam. While detailed, it appears somewhat lengthy and may obscure the main findings. Consider summarizing or emphasizing key patterns.

Line 362: The study did not include microbial community sequencing or functional analysis. Therefore, this discussion point is only indirectly supported and may weaken the focus of the argument. It is suggested that the authors narrow the discussion scope or explicitly clarify that the explanation is speculative and based on previous studies, to avoid logical inconsistency with the present data.

Lines 401–402: The sentence “lower content of clay and silt in the sandy loam… reduces the physical protection of soil particles on SOC” is grammatically and logically awkward. The current phrasing does not clearly convey the relationship between particle-size composition and SOC protection. Please revise for smoother and more precise expression, and check for similar issues throughout the manuscript.

Section 2.2 : The sampling description lacks clarity. The authors mention 6–8 samples per group (totaling 90), but later indicate that only 42 samples were retained after screening. Although selection criteria are mentioned, the numerical transition appears abrupt, requiring careful reading to understand. Consider improving logical continuity.

Lines 487–497: The manuscript appropriately acknowledges several limitations (e.g., lack of data on clay mineral types and microbial communities) and proposes directions for future research. In addition, it is recommended to note another limitation: the study used a space-for-time substitution approach (different greenhouse ages representing long-term effects). Although variables were controlled as much as possible, initial soil differences among greenhouses may still affect the SOC baseline. This “chronosequence” assumption should be recognized.

The discussion alternates between the mechanisms of physical protection and chemical recalcitrance, but it does not clearly identify which mechanism predominates under greenhouse conditions.

The manuscript frequently cites general conclusions from open-field studies; however, greenhouse soils exhibit distinct hydrothermal characteristics. It is recommended to further emphasize the mechanistic differences revealed in this study compared with open-field.
Citation: https://doi.org/10.5194/egusphere-2025-5094-CC1
- AC1: 'Reply on CC1', Wei Han, 15 Nov 2025
  
  We sincerely thank Reviewer Yi Cheng for the time and effort spent reviewing our manuscript, which has greatly improved the overall quality of our work. We have carefully considered all comments and made the necessary revisions accordingly. Detailed responses to each comment are provided below.
  Comment 1:
  The manuscript points out that organic manure inputs are high under greenhouse conditions, but SOC accumulation remains below expectations. It is suggested to briefly explain the possible reasons why high inputs have not resulted in corresponding SOC increases, or to clearly state that the underlying mechanisms remain unclear, so as to better introduce the research gap and objectives of this study.
  Response:
  We sincerely appreciate this valuable suggestion. In the revised manuscript (Lines 83–89), we have added a clear explanation to address why high manure inputs in greenhouse soils do not necessarily lead to proportional SOC accumulation. Specifically, we now emphasize that elevated temperature and moisture, frequent irrigation, and intensive tillage under greenhouse conditions collectively accelerate organic matter decomposition and carbon mineralization. Consequently, the mechanisms governing SOC accumulation and stabilization under greenhouse conditions remain poorly understood, limiting our ability to accurately predict carbon sequestration potential and to develop optimized manure management strategies. The revised text now reads as follows:
  “This discrepancy may result from the unique environmental and management conditions of greenhouse systems, such as elevated temperature and moisture, frequent irrigation, and intensive tillage, which collectively accelerate organic matter decomposition and carbon mineralization. Consequently, the mechanisms governing SOC accumulation and stabilization under greenhouse conditions remain poorly understood, limiting our ability to accurately predict carbon sequestration potential and to develop optimized manure management strategies (Zhang et al., 2022; Tan et al., 2025).”
  Comment 2:
  The statement that ‘most greenhouse studies last only 1–10 years, and short-term stability does not represent the final stage’ requires supporting references.
  Response:
  A supporting reference has been added. Das et al. (2023b) has been cited after Line 95. This long-term manure experiment demonstrates that SOC stabilization occurs over much longer timescales than the typical 1–10-year greenhouse trials, supporting our statement that short-term SOC stability does not represent the final stage.
  Comment 3 (Section 3.1):
  This section provides numerous numerical values describing SOC content and stock changes in loam and sandy loam. While detailed, it appears somewhat lengthy and may obscure the main findings. Consider summarizing or emphasizing key patterns.
  Response:
  Section 3.1 has been revised to streamline numerical descriptions and to emphasize the major patterns. The revised text now highlights: (i) the stabilization of SOC content and stock in the loam after approximately 20 years, (ii) the substantially lower SOC accumulation in the sandy loam despite similar manure inputs, and (iii) the consistent decline in SOC sequestration efficiency in both soils as application years increase. These revisions improve readability while retaining the essential quantitative information.
  Comment 4 (Line 362):
  The study did not include microbial community sequencing or functional analysis. Therefore, this discussion point is only indirectly supported and may weaken the focus of the argument. It is suggested that the authors narrow the discussion scope or explicitly clarify that the explanation is speculative and based on previous studies, to avoid logical inconsistency with the present data.
  Response:
  The discussion has been narrowed to avoid overinterpreting microbial processes. The previous explanatory content has been removed, and only a brief statement is retained to indicate that the variables may indirectly reflect microbial metabolism and carbon transformation, without suggesting direct evidence from this study. The revised sentence appears in Lines 362–364 as follows:
  “Although their contributions are limited, they could serve as indicators of microbial metabolism and carbon transformation processes (Francioli et al., 2016; Li et al., 2021; Yan et al., 2023).”
  Comment 5:
  The sentence describing how lower clay and silt contents in the sandy loam reduce SOC protection is grammatically and logically awkward. The relationship between particle-size composition and SOC protection is not clearly conveyed. Please revise for smoother and more precise expression.
  Response:
  The sentence has been revised to improve clarity and better convey the mechanism linking particle-size composition to SOC protection. The updated version reads: “This is likely due to the lower proportions of clay and silt in the sandy loam, which reduce the availability of fine mineral surfaces and thereby weaken the physical protection of SOC (An et al., 2021; Yao et al., 2022).”. Line 408–410.
  Comment 6 (Section 2.2):
  The sampling description lacks clarity. The authors mention 6–8 samples per group (totaling 90), but later indicate that only 42 samples were retained after screening. Although selection criteria are mentioned, the numerical transition appears abrupt, requiring careful reading to understand. Consider improving logical continuity.
  Response:
  The sampling description in Section 2.2 has been revised to improve logical continuity and clarity. The updated text now explicitly explains the initial collection of 90 samples and the subsequent screening process based on soil texture consistency within each region and pH constraints. This clarification makes the numerical transition from 90 to 42 samples more transparent. The revised version reads:
  Comment 7:
  The manuscript acknowledges several limitations but should additionally recognize that the space-for-time substitution (chronosequence) approach may introduce uncertainty, as initial soil differences among greenhouses can affect the SOC baseline. This assumption should be explicitly acknowledged.
  Response:
  This section has been revised to explicitly acknowledge the uncertainty associated with the chronosequence (space-for-time substitution) approach. We now note that, although unified management conditions and sample-screening procedures were applied, initial soil differences and microenvironmental variability among greenhouses may still influence the SOC baseline and confound the interpretation of long-term patterns. In our study region, all greenhouses belong to the same village collective and were gradually expanded over time into a coherent production system. This developmental pattern provides a higher degree of comparability across sites and helps reduce confounding effects related to disparate management histories, while our screening procedures further excluded individual outlier greenhouses with inconsistent practices. In addition, the limitations related to the absence of controlled experiments on manure type or dosage, the lack of submicron-scale structural characterization of mineral–organic complexes, and the absence of microbial community or functional data have been integrated into a unified and clearer paragraph. The revised text has been added to Lines 502–519 of the manuscript and is provided below.
  “The results of this study provide guidance for the management of manure application in greenhouse soils with different textures. However, several limitations should be noted. First, the study relied on a chronosequence (space-for-time substitution) approach based on long-term greenhouse plots of different ages. Although samples were collected under unified management conditions, residual differences in initial soil properties and microenvironmental heterogeneity among plots may still influence SOC baselines, making it difficult to fully disentangle fertilization effects from environmental factors. Second, the study did not include structural characterization of submicron-scale mineral–organic complexes (e.g., using X-ray absorption fine structure spectroscopy), preventing us from distinguishing the roles of different clay mineral types (e.g., montmorillonite vs. kaolinite) in the formation and stabilization of MAOC. Third, although the relationships between SOC fractions and stabilization were analyzed, direct evidence on microbial communities and their functional traits was lacking. Future work should integrate metagenomic or functional gene sequencing to identify the key microbial groups driving carbon transformation under different soil textures. Overall, future studies combining long-term field experiments with controlled factor-based additions will be needed to disentangle the coupled effects of manure characteristics, mineral composition, and microbial processes, thereby advancing a multi-scale understanding of SOC stabilization in greenhouse soils.”
  Comment 8:
  The discussion alternates between the mechanisms of physical protection and chemical recalcitrance, but it does not clearly identify which mechanism predominates under greenhouse conditions.
  Response:
  This section has been revised to explicitly acknowledge the limitation associated with the chronosequence (space-for-time substitution) approach. We now clarify that, despite consistent management practices and sample screening, initial soil differences among greenhouse plots may still influence SOC baselines and cannot be fully eliminated without controlled long-term experiments. In addition, the revised text further integrates this limitation with other constraints of the study (e.g., lack of submicron-scale mineral–organic complex characterization and absence of microbial community data). The revised paragraph appears in Lines 490–501 as follows:
  “In summary, our findings show that long-term SOC stabilization in greenhouse soils is maintained through a coupled process in which labile carbon fractions are progressively transformed and incorporated into more stable pools, rather than through the dominance of any single carbon pathway. The operation of this process differs substantially between the two soil types examined. In the loam, the higher proportions of clay and silt provide abundant mineral surfaces that promote the microbial reworking and mineral association of particulate carbon, making mineral-associated physical protection the primary mechanism supporting long-term stabilization. In contrast, the sandy loam—characterized by limited mineral binding sites and weaker aggregation—relies more heavily on the persistence of hydrophobic and alkyl-rich organic structures, rendering chemical recalcitrance a comparatively more important stabilization pathway. These texture-related mechanistic differences explain why the two soils exhibit distinct SOC accumulation and stabilization patterns despite receiving similar manure inputs.”
  Comment 9:
  The manuscript frequently cites general conclusions from open-field studies; however, greenhouse soils exhibit distinct hydrothermal characteristics. It is recommended to further emphasize the mechanistic differences identified in this study compared with open-field systems.
  Response:
  This section has been revised to explicitly highlight the mechanistic differences between greenhouse and open-field soils. We now clarify that, although both systems receive organic inputs, their contrasting hydrothermal conditions fundamentally alter SOC decomposition dynamics and stabilization pathways. The revised text emphasizes that open-field soils experience lower temperature and moisture, which slow organic matter decomposition and promote MAOC as the dominant long-term stabilization mechanism. In contrast, greenhouse soils are exposed to elevated hydrothermal conditions that enhance microbial activity, accelerate the turnover of labile carbon, and shorten the residence time of POC, thereby reshaping both the sequence and efficiency of precursor–product transformations. These revisions strengthen the conceptual distinction between the two systems and better articulate the novel mechanistic insights provided by our study. The revised paragraph appears in Lines 365–372 as follows:
  “Moreover, the SOC stabilization mechanisms revealed in this study differ markedly from those in open-field soils of the same region. Open-field soils generally experience lower temperature and moisture, which slow organic matter decomposition and promote MAOC as the primary pathway of SOC stabilization (Heckman et al., 2023; Saljnikov et al., 2025; Zhou et al.). In contrast, the elevated hydrothermal conditions in greenhouses enhance microbial activity, accelerate the turnover of labile carbon, and shorten the residence time of POC, thereby reshaping both the sequence and efficiency of transformations from carbon precursors to more stabilized pools (Niu et al., 2024; Zhang et al., 2022).”
  
  Citation: https://doi.org/10.5194/egusphere-2025-5094-AC1
CC2: 'Comment on egusphere-2025-5094', Yi Cheng, 15 Nov 2025

I recommend acceptance.

Citation: https://doi.org/10.5194/egusphere-2025-5094-CC2
RC1:
'Comment on egusphere-2025-5094', Anonymous Referee #1, 16 Dec 2025

The manuscript of Tan et al. investigates how soil organic carbon fractions and chemical forms vary with manure application duration in greenhouse soils with diverging textures. The conditions in greenhouses are warmer, and management is intensive, which may result in carbon accumulation patterns that differ from those in the field. The data presented are comprehensive, and the interpretation considers different pathways of C stabilization. In addition, combining physical and chemical measures of soil carbon is a valuable approach. There is still some room for improvement regarding clarity. In particular, many abbreviations are used; some are commonly known, but some of them should be introduced more clearly in the introduction (full name and meaning), and the meaning could again be more directly connected in the discussion (e.g., for the stability indexes, for them also Figure 5cd was not entirely clear) to help the reader. In general, the result and discussion could be slightly shortened by checking for repetition of result descriptions in the discussion and in the argumentation. If needed, some of the figures could also be moved to the SI (e.g. correlation figures).

Specific comments
L.29: A/OA description is missing
L.63: First occurrence of the abbreviations (A/OA, ARM) in the main manuscript, please add the description/meaning.
L.66: How does this example (tropics) relate to the study? I suggest removing examples that do not directly support the arguments.
L.74: How does this example (rice cultivation) relate to the study? I suggest removing examples that do not directly support the arguments.
L.117-120: This is a repetition, please remove.
L142: Word “aged” is unspecific, state more directly that you refer to the duration for which manure was applied in these greenhouses
L.194: Acquisition time?
L.198: With which NMR regions were the indices calculated?
L. 226-229: The use of SOCs as an abbreviation for sequestration here is confusing. For the other variables (SOC content and SOC stock), you use the full term, which is easier to understand. So, I advise also using the full term for SOC sequestration and dropping this additional abbreviation from the manuscript. Also, the sequestration is shown in Figure 1c, currently missing after the first mention of SOCs.
L.278-286: Can you add literature references for the NMR functional group description?
Figure 5 c and d: The visualization of the different ratios as stacked boxes is unclear. The ratios don’t add up to a total, and this visualization makes it difficult to get the actual information about the ratios. Also, the additional proportional change is adding complexity. Maybe you can visualize the different ratios as different points (plus standard error) per treatment (no boxes)? Then, the change should be visible without additionally plotting it.
L.308: This paragraph is now discussing correlations between physical fractions and chemical composition. Could it be a new section itself?
L.348: Literature references?
L.382-384: Literature references?
L.395: This last sentence of the paragraph is stating the opposite of the discussion before (e.g. L.380)? Please clarify.
Figure 6: Is the correlation of the fractions based on the fraction concentrations or proportions? Please clarify the units in the Figure description (also in the other correlation Figure 4).

Citation: https://doi.org/10.5194/egusphere-2025-5094-RC1
- AC2: 'Reply on RC1', Wei Han, 02 Jan 2026
  
  The manuscript of Tan et al. investigates how soil organic carbon fractions and chemical forms vary with manure application duration in greenhouse soils with diverging textures. The conditions in greenhouses are warmer, and management is intensive, which may result in carbon accumulation patterns that differ from those in the field. The data presented are comprehensive, and the interpretation considers different pathways of C stabilization. In addition, combining physical and chemical measures of soil carbon is a valuable approach. There is still some room for improvement regarding clarity. In particular, many abbreviations are used; some are commonly known, but some of them should be introduced more clearly in the introduction (full name and meaning), and the meaning could again be more directly connected in the discussion (e.g., for the stability indexes, for them also Figure 5cd was not entirely clear) to help the reader. In general, the result and discussion could be slightly shortened by checking for repetition of result descriptions in the discussion and in the argumentation. If needed, some of the figures could also be moved to the SI (e.g. correlation figures).
  Response to General comments
  We sincerely thank the reviewer for the careful and constructive evaluation of our manuscript. The comments and suggestions have been extremely helpful in improving the clarity, rigor, and overall presentation of the study. We have carefully considered each comment and revised the manuscript accordingly. All changes have been implemented in the revised version, and detailed point-by-point responses are provided below.
  Comment 1: L.29 and L.63
  A/OA description is missing. First occurrence of the abbreviations (A/OA, ARM) in the main manuscript, please add the description/meaning.
  Response
  We have revised the manuscript accordingly. The abbreviations A/OA and ARM are now fully defined at their first occurrence in the Introduction, including their full names and meanings.
  Specifically, ARM (Aromaticity Index) was revised to AI (Aromaticity Index) to improve clarity and consistency, following the definition and usage in Stability of soil organic carbon under long-term fertilization: Results from ¹³C NMR analysis and laboratory incubation. The corresponding formula and interpretation have been clarified in the Methods section, and the terminology has been made consistent throughout the manuscript.
  Comment 2: L.66
  How does this example (tropics) relate to the study? I suggest removing examples that do not directly support the arguments.
  Response
  We agree with the reviewer that the regional example was not directly relevant to the focus of this study. Therefore, we have removed the comparison between tropical and non-tropical regions and revised the sentence to emphasize the role of hydrothermal conditions in regulating the effectiveness of long-term manure application on SOC accumulation, which is more consistent with the environmental context of greenhouse systems investigated in this study.
  Comment 3: L.74
  How does this example (rice cultivation) relate to the study? I suggest removing examples that do not directly support the arguments.
  Response
  We agree with the reviewer that the example related to rice cultivation was not sufficiently relevant to the present study. Accordingly, we have removed the rice-related example from the revised manuscript to keep the Introduction focused on mechanisms directly applicable to greenhouse soils under manure application.
  Comment 4: L.117–120
  This is a repetition, please remove.
  Response
  We have carefully re-examined the content in Lines 117–120 to ensure that the description is concise and non-redundant.
  Comment 5: L.142
  Word “aged” is unspecific, state more directly that you refer to the duration for which manure was applied in these greenhouses.
  Response
  We agree that the term “aged” was ambiguous. The sentence has been revised to explicitly indicate the duration of manure application, replacing “aged” with “years of manure application” to improve clarity.
  Comment 6: L.194
  Acquisition time?
  Response
  The acquisition parameters of the 13C CPMAS solid-state NMR measurements have been clarified and fully specified in the revised Methods section. In CPMAS experiments, the contact time refers specifically to the duration of magnetization transfer during the cross-polarization process and is therefore conceptually distinct from acquisition time. In the present study, the acquisition conditions are defined by the recycle delay and the total number of scans, which determine the effective signal accumulation and signal-to-noise ratio. Accordingly, we have added detailed information on the magic angle spinning rate (5 kHz), contact time (1 ms), recycle delay (0.8 s), and the total number of scans (140,000 scans per sample). These parameters together ensure reliable spectral quality and reproducibility for SOC chemical structure characterization.
  Comment 7: L.198:
  
  With which NMR regions were the indices calculated?
  Response
  
  We have revised the Methods section to explicitly state the 13C NMR regions used for each index and to provide the calculation formulas directly. Specifically, the aromaticity index (AI) was calculated as:
  
  AI = Aromatic C / (Alkyl C + O-alkyl C + Aromatic C).
  
  The hydrophobicity index (HI) was calculated as:
  
  HI = (Alkyl C + Aromatic C) / (O-alkyl C + Carbonyl C).
  
  The ratio of alkyl C to O-alkyl C (A/OA) was calculated as:
  
  A/OA = Alkyl C / O-alkyl C.
  Comment 8: L. 226-229:
  The use of SOCs as an abbreviation for sequestration here is confusing. For the other variables (SOC content and SOC stock), you use the full term, which is easier to understand. So, I advise also using the full term for SOC sequestration and dropping this additional abbreviation from the manuscript. Also, the sequestration is shown in Figure 1c, currently missing after the first mention of SOCs.
  Response
  The reviewer is correct, and we appreciate this helpful suggestion. We have carefully revised the manuscript to improve clarity and consistency in terminology. Specifically, the abbreviation “SOCs” referring to SOC sequestration has been removed throughout the entire manuscript, and the full term “SOC sequestration” is now used consistently, in line with the expressions for SOC content and SOC stock. In addition, the reference to Figure 1c has been added at the first mention of SOC sequestration to ensure consistency between the text and the figure presentation.
  Comment 9: L. 278–286
  Could you add relevant references to support the description of NMR functional groups?
  Response
  We agree with the reviewer’s suggestion. Relevant references have now been added to support the interpretation and description of the ¹³C NMR functional groups in this section (L. 278–286).
  Comment 10: Fig. 5c and 5d
  The visualization of the different ratios as stacked boxes is unclear. The ratios don’t add up to a total, and this visualization makes it difficult to get the actual information about the ratios. Also, the additional proportional change is adding complexity. Maybe you can visualize the different ratios as different points (plus standard error) per treatment (no boxes)? Then, the change should be visible without additionally plotting it.
  Response
  We thank the reviewer for this constructive suggestion and agree that the previous visualization (stacked boxplots) could be confusing because these ratios are not additive quantities. We have therefore revised Fig. 5c and 5d to improve interpretability. Specifically, we now present each ratio index (e.g., A/OA, HI, and AI) as one value per treatment (individual data points), rather than as stacked boxplots.
  Comment 11: L. 278-286
  Can you add literature references for the NMR functional group description?
  Response
  Following the reviewer’s suggestion, the visualization of the SOC stability indicators in Figure 5c and 5d has been revised to improve clarity. In the revised figures, the aromaticity index (AI), hydrophobicity index (HI), and the ratio of alkyl C to O-alkyl C (A/OA) are now presented separately as individual bar charts for each treatment. This modification reduces visual complexity and allows the absolute values and treatment-related differences of each index to be more clearly and directly interpreted.
  Comment 12: L. 308
  This paragraph is now discussing correlations between physical fractions and chemical composition. Could it be a new section itself?
  Response
  We appreciate the reviewer’s suggestion. In response, we have separated this paragraph into an independent subsection in the Discussion. This subsection specifically addresses the relationships between SOC fractions and SOC chemical composition, which improves the structural clarity of the Discussion.
  Comment 13: L. 348
  Literature references?
  Response
  Relevant literature references have been added to support this statement (Mustafa et al., 2022; Zhou et al., 2024).
  Comment 14: L. 382-384
  Literature references?
  Response
  Relevant references have been added to support this description (Panettieri et al., 2014; Kubar et al., 2018).
  Comment 15: L. 395
  This last sentence of the paragraph is stating the opposite of the discussion before (e.g. L.380)? Please clarify.
  Response
  The apparent inconsistency resulted from an overly generalized statement. We have revised the sentence to clarify that SOC stabilization followed texture-dependent pathways: in the loam, stability increased mainly with higher proportions of aromatic C and carbonyl C, whereas in the sandy loam, alkyl C played a more important role. This revision resolves the inconsistency and is consistent with the results presented in Fig. 5 and Table 2.
  Comment 16:
  Is the correlation of the fractions based on the fraction concentrations or proportions? Please clarify the units in the Figure description (also in the other correlation Figure 4).
  Response
  In Fig. 4, SOC fractions shown as EOC, DOC, MBC, cPOC, fPOC, and MAOC refer to their concentrations (g kg⁻¹ soil), whereas variables expressed as EOC/SOC, DOC/SOC, MBC/SOC, cPOC/SOC, fPOC/SOC, and MAOC/SOC represent the relative proportions of each fraction to total SOC. This distinction has now been explicitly clarified in the figure caption to avoid any confusion.
  
  Citation: https://doi.org/10.5194/egusphere-2025-5094-AC2
RC2:
'Comment on egusphere-2025-5094', Anonymous Referee #2, 28 Dec 2025

General comments
This paper investigates the characteristics of SOC fractions (contents, chemistry and stability) under greenhouse conditions with various durations of manure addition (2 to 50 years) in two contrasted soil textures. This article is interesting as such long timespans are quite rare; it uses many sites and combines both physical fractions (POC and MAOC) and stability-defined fractions.
The differences between the two soils are rather marked, as the new equilibrium in loam reaches high SOC levels and takes at least 20 years to establish, versus only 2 years in the sandy loam soil with smaller SOC accumulation. The chemical composition of the SOC also varies between the soils.
Overall, the article is nice and well written, although indices could be made more accessible to the reader for better understanding; physical vs other fractions should also be separated more carefully. Please find below a few suggestions.

Specific comments
L.29: what is A/OA?
L.32: you introduced POC but not fPOC: fine? free?
L.33: Aromatic molecules are usually less labile, I am surprised it ‘stimulates’ EOC. Could you clarify? Also, I understand you mean that POC will later adsorb on the mineral phase and form MAOC so it ‘enhances’ MAOC, but you can state it more clearly.
L.63: again, define A/OA here.
L.121: ‘22–36 °C’ is not an average temperature, except if you are presenting the result under the form ‘Wujianfang–Yangshibao’. Same for nighttime.
L.134: you can add the durations here, so we know how many groups were studied (although it is in the Table 1).
L.141: if I get it well, more than half of the samples do not match your criteria of soil texture and pH. While having these criteria is interesting to keep homogeneity in the soil properties, how do you ensure the results you present after can be applied to greenhouse soils if half of them are not covered by the conditions of your experiments?
L.159: I think it would be useful to clarify which fractions are complementary (POC and MAOC), and which are just corresponding to properties (EOC, DOC, MBC). Having them all presented together can be confusing.
L.160: from this, I guess cPOC and fPOC stand for coarse and fine, but you should add it.
L.164: you could add what the fumigation agent is.
L.198: you can add more information about these indices: in particular, formula, extreme values and their interpretation.
L.230: while this is correct, it feels a bit strange to compare the result at 20 years in loam vs at 2 years in sandy loam – even though the value doesn’t change much after 2 years. Either compare similar durations, or state clearly that you do it this way because the equilibrium has been reached.
L.250: I don’t understand well Fig.2 a and b. It is the ‘content and variations of SLOCF and POC, MAOC’ for loam and sandy loam soils: content at which duration? Also, you talk about ‘reductions’ in sandy loam ‘relative to loam’: to me, reduction means it was higher and decreased. Here, it seems you mean it is smaller, does it? Apologies if I am misunderstanding this.
L.253: I am not comfortable with comparing fractions that are not complementary. POC and MAOC belong together, as complementary physical fractions, but not the others as they are not extracted and measured the same way.
Discussion: the whole part is a bit too long, the reader ends quite lost after some time, especially when discussing the chemical composition of SOC. I think this can be made clearer, maybe shorter.

Technical corrections
L.97: ‘durations’ instead of ‘years’?
L.99: same

Citation: https://doi.org/10.5194/egusphere-2025-5094-RC2
- AC3: 'Reply on RC2', Wei Han, 02 Jan 2026
  
  This paper investigates the characteristics of SOC fractions (contents, chemistry and stability) under greenhouse conditions with various durations of manure addition (2 to 50 years) in two contrasted soil textures. This article is interesting as such long timespans are quite rare; it uses many sites and combines both physical fractions (POC and MAOC) and stability-defined fractions.
  The differences between the two soils are rather marked, as the new equilibrium in loam reaches high SOC levels and takes at least 20 years to establish, versus only 2 years in the sandy loam soil with smaller SOC accumulation. The chemical composition of the SOC also varies between the soils.
  Overall, the article is nice and well written, although indices could be made more accessible to the reader for better understanding; physical vs other fractions should also be separated more carefully. Please find below a few suggestions.
  Response to General comments
  We thank the reviewer for the positive and constructive assessment of our manuscript. We appreciate the helpful suggestions regarding the clarity of SOC indices and the presentation of different SOC fractions. In response, we have revised the manuscript to improve the definition and interpretation of the indices used and to more clearly distinguish physical SOC fractions from other property-based carbon pools. These changes were made to enhance clarity and readability. The detailed responses to the specific comments are provided below.
  Comment 1:
  L.29: what is A/OA?
  Response:
  We have revised the text at L.29 to explicitly define A/OA as the ratio of alkyl C to O-alkyl C. The sentence now reads: in sandy loam, alkyl C increased, elevating alkyl C to O-alkyl C (A/OA) and the hydrophobicity index (HI).
  Comment 2:
  L.32: you introduced POC but not fPOC: fine? free?
  Response:
  In this study, fPOC is used to denote fine particulate organic carbon. To avoid potential confusion, we have revised the text at L.32 by spelling out fPOC at its first occurrence.
  Comment 3:
  L.33: Aromatic molecules are usually less labile, I am surprised it ‘stimulates’ EOC. Could you clarify? Also, I understand you mean that POC will later adsorb on the mineral phase and form MAOC so it ‘enhances’ MAOC, but you can state it more clearly.
  Response:
  We agree with the reviewer’s concern and have revised the sentence to avoid implying that aromatic molecules directly stimulate labile carbon pools. In the revised text, increases in SOC aromaticity (AI) and hydrophobicity (HI) are described as occurring concurrently with changes in DOC, EOC, and POC, rather than as direct causal effects. This wording is consistent with the structural equation model, which indicates that these carbon pools collectively contribute to MAOC formation in loam through integrated pathways, while in sandy loam MAOC formation is mainly associated with EOC and cPOC contributes to SOC independently without a significant pathway to MAOC. The sentence has been revised accordingly (L.33).
  Comment 4:
  L.63: again, define A/OA here.
  Response:
  The abbreviations A/OA and AI have now been defined at their first occurrence in the main text by adding their full names (alkyl C to O-alkyl C ratio and aromaticity index, respectively) at Line 63. The text has been revised accordingly.
  Comment 5:
  L.121: ‘22–36 ℃’ is not an average temperature, except if you are presenting the result under the form ‘Wujianfang–Yangshibao’. Same for nighttime.
  Response:
  The temperature description has been revised to avoid using ‘average’ for a temperature range. The text now states that daytime and nighttime temperatures ranged from 22 to 36 ℃ and from 15 to 18 ℃, respectively (Line 121). The text has been revised accordingly.
  Comment 6:
  L.134: you can add the durations here, so we know how many groups were studied (although it is in the Table 1).
  Response:
  The manure application durations and the number of experimental groups at each site have now been explicitly added to the main text at Line 134 to clarify the study design. Although this information was previously provided in Table 1, it is now stated in the text to improve clarity and readability. The text has been revised accordingly.
  Comment 7:
  L.141: if I get it well, more than half of the samples do not match your criteria of soil texture and pH. While having these criteria is interesting to keep homogeneity in the soil properties, how do you ensure the results you present after can be applied to greenhouse soils if half of them are not covered by the conditions of your experiments?
  Response:
  We apologize for this oversight. Upon re-examination of the particle-size data, we found that the silt and clay fractions were inadvertently interchanged in the original manuscript, which resulted in an apparent inconsistency between the predefined soil texture criteria and part of the dataset. This was a reporting error, and the particle-size measurements themselves are correct.
  The soil texture classification has now been recalculated using the correct silt and clay fractions according to the USDA soil texture triangle. After correction, all samples consistently fall within the defined loam and sandy loam texture classes used in this study. The relevant tables and text have been revised accordingly. Importantly, this correction does not affect the subsequent analyses or the main conclusions of the manuscript.
  In addition, although fertilization and broad management practices (e.g., fertilizer formulation and annual application schedules) were consistent across greenhouse plots, minor local variability in soil properties may still occur due to subtle differences in management execution and historical operations, such as irrigation practices, localized soil amendment history, or micro-site effects within greenhouses. Such variability is commonly observed in operational greenhouse systems and does not indicate systematic differences in primary management regimes. The screening criteria were therefore applied to ensure comparability of soils under broadly similar texture and pH conditions.
  Comment 8:
  L.159: I think it would be useful to clarify which fractions are complementary (POC and MAOC), and which are just corresponding to properties (EOC, DOC, MBC). Having them all presented together can be confusing.
  Response:
  We agree that POC and MAOC represent complementary physical SOC pools, whereas EOC, DOC, and MBC correspond to carbon lability and microbial activity rather than discrete or mutually exclusive SOC pools.
  To avoid potential conceptual ambiguity, we have revised the Methods section to explicitly distinguish between these two categories and to clarify their different analytical roles. Specifically, the Methods section now states:
  ‘We quantified (i) complementary physical SOC pools obtained by physical fractionation—POC (coarse particulate organic carbon, cPOC, and fine particulate organic carbon, fPOC) and MAOC—which together represent SOC stored in particulate versus mineral-associated forms, and (ii) labile C indicators—EOC, DOC, and MBC—which reflect short-term bioavailable and microbially mediated C dynamics rather than mutually exclusive SOC pools. These two categories were jointly analyzed to evaluate how long-term manure application influences both SOC accumulation and turnover in greenhouse soils.’
  This clarification ensures that physically defined SOC pools and labile carbon indicators are conceptually separated, while also explaining why they are presented together in the analysis..
  Comment 9:
  L.160: from this, I guess cPOC and fPOC stand for coarse and fine, but you should add it.
  Response:
  We agree with the reviewer. The abbreviations cPOC and fPOC have now been explicitly defined as coarse particulate organic carbon and fine particulate organic carbon, respectively, at their first occurrence in the Methods section
  Comment 10:
  L.164: you could add what the fumigation agent is.
  Response:
  The Methods section has been revised to specify that ethanol-free chloroform vapor was used as the fumigation agent in the fumigation–extraction procedure (Vance et al., 1987).
  Comment 11:
  L.198: you can add more information about these indices: in particular, formula, extreme values and their interpretation.
  Response:
  We agree with the reviewer’s comment. The Methods section has been revised to clarify that the relative contribution of each SOC functional group was determined by integrating the corresponding ^13C NMR spectral regions. Based on these relative intensities, three SOC stability indicators were explicitly defined and calculated as follows:
  The aromaticity index (AI) was calculated as:
  AI = Aromatic C / (Alkyl C + O-alkyl C + Aromatic C)
  The hydrophobicity index (HI) was calculated as:
  HI = (Alkyl C + Aromatic C) / (O-alkyl C + Carbonyl C)
  The ratio of alkyl C to O-alkyl C (A/OA) was calculated as:
  A/OA = Alkyl C / O-alkyl C
  These formulas have now been explicitly provided in the Methods section as Eqs. (5)–(7) to improve clarity and avoid ambiguity.
  Comment 12:
  L.230: while this is correct, it feels a bit strange to compare the result at 20 years in loam vs at 2 years in sandy loam – even though the value doesn’t change much after 2 years. Either compare similar durations, or state clearly that you do it this way because the equilibrium has been reached.
  Response:
  We agree that directly comparing SOC values at different application durations may be confusing. In the revised manuscript, we have therefore removed the explicit comparison between sandy loam and loam at mismatched durations. The Results section now describes SOC content and stock in the sandy loam independently, focusing on their temporal pattern within this soil type. This approach is explicitly justified in the main text by stating that SOC in the sandy loam reached an apparent equilibrium after approximately 2 years of manure application, whereas SOC in the loam continued to increase and approached equilibrium only after around 20 years. The text has been revised accordingly.
  Comment 13:
  L.250: I don’t understand well Fig.2 a and b. It is the ‘content and variations of SLOCF and POC, MAOC’ for loam and sandy loam soils: content at which duration? Also, you talk about ‘reductions’ in sandy loam ‘relative to loam’: to me, reduction means it was higher and decreased. Here, it seems you mean it is smaller, does it? Apologies if I am misunderstanding this.
  Response:
  Thank you for the comment. In Fig. 2a and b, the values do not represent a specific manure application year. Instead, they show the overall distributions of SOC fractions using all available application durations for each soil type. We have revised the Results text to state this more clearly.
  In addition, we replaced the term ‘reduction’ with ‘lower than’ to avoid confusion, because the intention was to indicate smaller contents in sandy loam than in loam, rather than a temporal decrease. These revisions were made to improve clarity and consistency with Fig. 2.
  Comment 14:
  L.253: I am not comfortable with comparing fractions that are not complementary. POC and MAOC belong together, as complementary physical fractions, but not the others as they are not extracted and measured the same way.
  Response:
  The sentence that ranked SOC fractions across different fractionation schemes has been removed to avoid inappropriate comparisons between non-complementary fractions. The paragraph has been revised accordingly, and the results are now described within their respective fractionation frameworks.
  Comment 15:
  Discussion: the whole part is a bit too long, the reader ends quite lost after some time, especially when discussing the chemical composition of SOC. I think this can be made clearer, maybe shorter.
  Response:
  We agree with the reviewer that the original Discussion section was overly long and that the interpretation of SOC chemical composition could be difficult to follow. To address this concern, we have substantially revised and streamlined the Discussion to improve clarity and logical structure.
  In the revised version, the discussion of SOC chemical composition (Section 4.2) is more concise and mechanism-focused, emphasizing only the key transformations relevant to SOC stabilization. Interpretation of texture-related differences is now mainly integrated into the correlation analysis (Section 4.3) and the pathway synthesis (Section 4.4), rather than being repeatedly stated across sections.
  Specifically, we reorganized the Discussion into four clearly defined subsections (Sections 4.1–4.4).
  Section 4.1: Effects of manure application on SOC fractions in greenhouse
  The former Section 4.1, which combined SOC fractions and chemical structure, was refocused exclusively on SOC fractions (POC, MAOC, EOC, DOC, and MBC). The key observation that SOC reached an apparent equilibrium after approximately 20 years in loam but after about 2 years in sandy loam was retained and explicitly framed as a texture-dependent equilibration pattern, thereby avoiding confusing comparisons across mismatched durations. Examples not directly supported by the present dataset (e.g., microbial functional gene case studies) were removed, while DOC and MBC were retained as process-related indicators with limited contribution to total SOC.
  Section 4.2: Effects of manure application on SOC chemical composition in greenhouse
  The discussion of SOC chemical composition based on ¹³C NMR was separated from the former Section 4.1 and consolidated into an independent subsection. This section was shortened and streamlined by removing repetitive descriptions and merging overlapping explanations of functional-group changes. The revised text focuses on the main texture-dependent trends in SOC functional groups, thereby improving readability and reducing potential confusion.
  Section 4.3: Relationships between SOC fractions and stability-related indices
  The former Section 4.2 was refined to focus on the relationships between SOC fractions and stability-related indices (AI, HI, and A/OA). Repetitive mechanistic descriptions were shortened, and the discussion was refocused on how mineral protection modulates the contribution of different SOC fractions to stability in loam versus sandy loam. Interpretations related to priming effects were condensed and presented cautiously, without extending beyond the observational scope of the data.
  Section 4.4: Sequestration pathways of SOC in greenhouse
  The synthesis of SOC sequestration pathways, previously embedded in correlation analyses, was extracted into a separate subsection to provide a clearer integrative summary.
  Overall, these revisions shorten the Discussion and improve its readability by removing redundancy and clarifying the organization, particularly for the section on SOC chemical composition.
  Comment 16 and 17:
  L.97 and L.99: ‘durations’ instead of ‘years’?
  Response:
  The term ‘years’ has been replaced with ‘durations’ at L97 and L99 to more accurately describe the length of manure application history rather than calendar time.
  
  Citation: https://doi.org/10.5194/egusphere-2025-5094-AC3

Boyuan Tan, Lu Yang, Xun Xiao, ChunJi Li, Jing Tan, Ning An, Lingxuan Meng, Yanli Yi, Fengkui Qian, Na Li, Xue Liu, Song Li, and Wei Han

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https://doi.org/10.5194/egusphere-2025-5094-supplement

Boyuan Tan, Lu Yang, Xun Xiao, ChunJi Li, Jing Tan, Ning An, Lingxuan Meng, Yanli Yi, Fengkui Qian, Na Li, Xue Liu, Song Li, and Wei Han

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Short summary

In greenhouses, long-term manure application does not always lead to lasting soil carbon. We found that loam and sandy loam store carbon in very different ways. Loam continuously converts particulate carbon into stable forms, while sandy loam relies on short-lived, easily lost carbon. These results indicate that long-term carbon storage depends on soil composition and mineral properties, providing guidance for smarter manure use in sustainable farming.


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