the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 03 Nov 2025
Differences in organic carbon fractions and stability explain limited accumulation in loam and sandy loam under greenhouse conditions
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.
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Status: open (until 28 Dec 2025)
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CC1: 'Comment on egusphere-2025-5094', Yi Cheng, 09 Nov 2025
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AC1: 'Reply on CC1', Wei Han, 15 Nov 2025
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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
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AC1: 'Reply on CC1', Wei Han, 15 Nov 2025
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CC2: 'Comment on egusphere-2025-5094', Yi Cheng, 15 Nov 2025
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I recommend acceptance.
Citation: https://doi.org/10.5194/egusphere-2025-5094-CC2
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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: