Clay-organic matter interactions drive microbial necromass preservation in soils

Domain: chemistry
Published: 2026-03-03
Editorial outcome: minor_revision

Abstract
Microbial necromass is increasingly recognized as a major source of stable soil organic matter (SOM), and its persistence is often attributed to interactions with clay-sized minerals. However, the mechanisms underlying this mineral-mediated stabilization remain poorly understood. Here, we conducted an in situ dual-labelled (13C and 15N) microbial necromass experiment across a clay gradient to quantify how clay content and necromass origin (bacterial vs. fungal) regulate necromass persistence. We find that higher clay content markedly enhances necromass retention by strengthening mineral protection, suppressing microbial activity and diversity, and limiting leaching losses. NanoSIMS imaging shows that new necromass preferentially associates with organic matter coatings on the rough mineral surfaces, highlighting organo-organic interfaces as important stabilization pathways. Necromass origin exerts little effect on retention despite marked differences in C:N ratios and bulk chemical composition, indicating that finer-scale molecular features, rather than broad compositional differences, govern necromass stabilization in soil.

Introduction
Soil organic matter (SOM) represents the largest actively cycling carbon pool in terrestrial ecosystems1, and its persistence is vital for maintaining soil fertility and mitigating climate change2,3. Along with plant inputs, microbial necromass has emerged as an important source of SOM4-7. Microbial necromass consists of the dead microbial biomass, including intact cells, cell fragments, and intracellular components released upon lysis8,9. Compared with plant-derived SOM, necromass is enriched in nitrogen-containing and polar functional groups (e.g., amide, carboxyl, and amino groups) that confer greater affinity for soil mineral surfaces10,11. Thus, the long-term accumulation and persistence of necromass in soil is widely attributed to its associations with secondary minerals, which counteracts its inherent chemical lability and limits microbial mineralization12,13. Consequently, soils with higher clay content are expected to store more necromass14,15. However, this expectation does not account for how variation in clay content shapes the multiple soil environment in ways that could increase necromass decomposition and thus constrain the magnitude of necromass accumulation.

Soil clay content is thought to influence microbial necromass persistence through multiple, sometimes competing, mechanisms16,17. On one hand, clay particles possess high surface charge and specific surface area, promoting strong adsorption of low-molecular-weight necromass and forming abundant mineral-necromass associations that limit microbial access10,18. The fine pores and microaggregates characteristic of clay-rich soils can further occlude necromass and physically restrict its contact with microbes and extracellular enzymes19-22, while high water retention reduces leaching losses23,24. Additionally, reduced oxygen diffusion in moisture-rich, high-clay soil can enhance reducing conditions and promote partial Fe(III) reduction, weakening mineral sorption and altering necromass mobility25. On the other hand, these same clay-rich environments can stimulate microbial activity that accelerates necromass mineralization26. Abundant fine pores create microhabitats that support microbial growth and protect microbes from predation22,27,28, while elevated nutrient and moisture availability alleviate resource and water limitations23,29-31, thereby enhancing microbial activity and diversity. Clay content can also influence microbial community composition32,33, which in turn shapes both necromass production and decomposition. Overall, clay can simultaneously promote necromass protection and stimulate necromass loss, underscoring the need to quantify necromass fate under in situ conditions where these protective and degradative pathways interact.

Independent of clay content, the chemical composition of microbial necromass strongly influences its decomposition34,35. Necromass chemistry is associated with the composition of the living microbial community9,13, with different microbial taxa producing necromass with contrasting recalcitrance and affinity for mineral surfaces35,36. For instance, bacterial necromass, typically richer in nitrogen (N) and lower in chitin content than fungal necromass, may be preferentially mineralized by microorganisms37,38. Yet its higher N content can also promote stronger association with minerals, as mineral surfaces preferentially adsorb N-rich organic substrates39,40. In contrast, fungal necromass contains chitin and other complex polysaccharides that can degrade into carboxyl-rich compounds capable of forming strong inner-sphere complexes with Fe- and Al-oxide minerals41,42. Necromass chemistry may further dictate its spatial distribution, thereby influencing its vulnerability to decomposition43. For instance, N-rich necromass may preferentially accumulate on mineral surfaces, whereas cellular lipids may contribute to more microbial-accessible organo-organic interactions stacked atop existing mineral-OM associations44. Understanding whether necromass with different chemistry exhibits distinct mineralization rates is crucial for predicting its persistence45. Likewise, the balance between preferential adsorption and mineralization of N-rich necromass substrates will influence the relationship between necromass carbon (C) and N mineralization. Nevertheless, the role of necromass chemistry in SOM persistence remains poorly understood, warranting further investigation.

Here, we established an experimental clay gradient to directly assess how variation in clay content regulates in situ microbial necromass decomposition and retention (Fig. 1). We constructed artificial soils with contrasting clay contents by mixing montmorillonite clay, quartz sand, and plant litter while maintaining identical organic matter input and clay mineral. Following a pre-incubation period, model soils were placed in a natural forest site and amended with dual isotope-labelled (13C and 15N) bacterial and fungal necromass. We hypothesized that (1) necromass retention would increase with clay content, as the mineral-protective effects of clay outweigh stimulation of microbial decomposition, and (2) bacterial necromass would be more strongly retained than fungal necromass owing to its higher affinity for clay minerals. To test these hypotheses, the model soils were incubated in situ for 386 days (Fig. 1), during which the fate of the labelled necromass was traced to quantify its mineralization, retention, and redistribution among SOM pools. This experimental framework enabled us to isolate the effects of clay content and necromass chemistry on the persistence of microbial necromass under realistic environmental conditions.

Results
Microbial necromass characteristics and mineralization
Bacterial necromass contained more N and exhibited a lower C:N ratio than fungal necromass (Table S1). Fourier-transform infrared spectroscopy (FTIR) analysis further showed that bacterial necromass was enriched in aliphatic compounds but depleted in aromatics and polysaccharides relative to fungal necromass (Table S1). Upon addition to field soil, necromass was rapidly utilized by microorganisms during the initial stage of incubation (Fig. 2a, c), as reflected by the peak in 13C-CO2 flux at 3-18 days. After this peak, the mineralization rate declined markedly (Fig. S1). By the end of in situ incubation (386 days), 9.7-22.9% of the added necromass 13C was mineralized to 13C-CO2 (Fig. 2b, d). Cumulative 13C-CO2 was significantly influenced by soil clay content (P < 0.001; Table S2), with the lowest values observed in high-clay soil. In contrast, no significant differences were found between bacterial and fungal groups (Table S2).

The retention of the added necromass 13C and 15N, quantified as total recovery in the model soils, decreased rapidly during the first 10 days in low- and medium-clay soil (Fig. 3a-d). In contrast, the decline lasted for the first 30 days in high-clay soil, followed by a more gradual decrease thereafter (Fig. 3a-d). After 386 days of in situ incubation, 12.3-31.5% of the added necromass 13C remained in the model soils (Fig. 3a, b), significantly lower than the 24.6-45.4% retention of the added necromass 15N (P < 0.001, Fig. 3c, d). Accordingly, the 15N :13C retention ratios exceeded 1 across all clay contents (Fig. S2). Both 13C and 15N retention were significantly lower in low- and medium-clay soil than in high-clay soil (C: P < 0.001, N: P < 0.001; Table S3), with no significant differences between bacterial and fungal groups. Mineralization rate constants of the added necromass 13C (1.12-2.15 year−1) were higher than those of the necromass 15N (0.76-1.36 year-1; Fig. 3e, f). The mineralization rate constants of the added necromass 13C and 15N were significantly higher in low- and medium-clay soil compared to high-clay soil (C: P = 0.021, N: P = 0.032; Table S4), with no difference between microbial groups (Table S4).

Partitioning of microbial necromass-derived C and N among soil pools
To investigate the fate of the added necromass, we traced its 13C and 15N signals in microbial biomass, inorganic N (IN), dissolved organic matter, remaining necromass in the model soils, and leached fractions in the underlying subsoil (5.5-14 cm depth). By day 10, 2.68% (±0.46%) of added necromass 13C and 0.77% (±0.15%) of 15N were recovered in microbial biomass, with both significantly influenced by clay content and microbial group (P < 0.001; Fig. 4; Fig. S3, S4; Tables S5, S6). Across all treatments and sampling times, less than 0.65% of the added necromass 15N was mineralized to IN, showing only a slight increase with clay content (P = 0.064; Fig. 4; Fig. S3, S4; Table S5). Similarly, recovery in dissolved organic matter was minimal, declining from 0.38% (±0.06%) of 13C and 0.10% (±0.01%) of 15N at day 10 to 0.09% (±0.02%) of 13C and 0.02% (±0.004%) of 15N at day 386, with 13C slightly increasing and 15N decreasing with clay content (P = 0.091 and 0.030; Fig. 4; Fig. S3, S4; Table S5, S6). By day 10, 41.6% (±5.6%) of added necromass 13C and 51% (±6.3%) of added necromass 15N remained as necromass, declining rapidly thereafter. By the end of the incubation, 15.7% (±2%) of 13C and 31.2% (±2.7%) of 15N were retained in necromass, with 13C recovery significantly lower than 15N recovery (P < 0.001; Fig. 4). Both remaining necromass 13C and 15N were higher in high-clay soil than in low- and medium-clay soil (P < 0.001; Table S5, S6). The added necromass leached rapidly from the model soils into the underlying subsoil (soil depth ~8.5 cm), with 2.97-51.39% of 13C and 2.54-60.37% of 15N recovered at day 10. Leaching losses were higher in the low- and medium-clay treatments (all P < 0.001; Fig. 4). Over the 386-day incubation, 13C and 15N leaching declined in low- and medium-clay soil but increased in high-clay soil (Fig. 4; Table S6). Overall, more 13C than 15N was leached into the subsoil (P = 0.033), indicating preferential mobilization of C relative to N.

Drivers of microbial necromass retention
Despite clear chemical differences between bacterial and fungal necromass, their persistence did not differ (Fig. 3; Tables S2-S4), so the two types were combined for subsequent analyses. To identify the factors regulating necromass retention, we examined relationships between the retention of the added necromass 13C and 15N after 386 days of decomposition and a suite of potential influencing factors, including microbial community characteristics and soil properties (See Methods; Fig. 5). Necromass 13C and 15N retention was closely related to the living microbial community characteristics and soil properties (Fig. 5a, b). Furthermore, bacterial community richness (Richness-B), soil moisture, total porosity, specific surface area (SSA), fungal community richness (Richness-F) and microbial metabolic quotient (qCO2) were the most important predictors (Fig. 5b). Necromass 13C and 15N retention was positively correlated with soil moisture and SSA, but negatively correlated with richness-B, richness-F and qCO2 (Fig. 5c; Fig. S3). Moreover, qCO2 increased with bacterial and fungal richness and with air-filled porosity (AFP), but declined with soil moisture (Fig. 5a). Both bacterial and fungal richness showed the same pattern, increasing with AFP and decreasing with soil moisture (Fig. 5a). Across the clay gradient, soil moisture, SSA, and total porosity increased with clay content, whereas AFP, qCO2, bulk density, and microbial richness all decreased (Fig. 5a).

To resolve the microscale mechanisms underlying necromass stabilization, we examined the spatial arrangement and interactions between added labelled necromass, native OM, and mineral particles using scanning electron microscopy (SEM) and nanoscale secondary ion mass spectrometry (NanoSIMS) (Fig. 6). These complementary techniques allowed direct visualization of the co-localization between labelled necromass and soil constituents. Consistent with the bulk IRMS results, NanoSIMS showed higher 13C−:(12C− + 13C−) and 12C15N−:(12C14N− + 12C15N−) ratios in high-clay soil (Fig. 6d, g). Native OM, identified by the 12C⁻ and 12C14N⁻ secondary ion, was predominantly associated with minerals (79.5 ± 1.6%), as indicated by the 16O⁻ secondary ion (Fig. 6a1-c2; Fig. S5). Mineral surfaces coated with OM were generally rougher, as reflected by a higher 16O−:area ratio (Fig. 6i). Newly added necromass 13C and 15N accumulated mainly via organo-organic interactions: labelled necromass preferentially attached to native OM already associated with mineral surfaces (79.8 ± 3.2% of 13C, 87.3 ± 1.9% of 15N), rather than bare mineral surfaces (Fig. 6a1-c2, e, h). High-clay soil exhibited a larger specific surface area covered by OM-mineral associations than low- and medium-clay soil (Fig. 6f). Density fractionation further confirmed that most retained microbial necromass was located within the mineral-associated organic matter fraction (96.2 ± 0.4% of 13C, 93.3 ± 0.5% of 15N; Fig. S6).

Methods
Study site and experimental design
The study was conducted in the Changbai Mountain National Nature Reserve, located in Jilin Province in northern China (42.70° N,127.63° E). The region has a typical temperate climate, with a mean annual temperature of 3.6 ℃ and mean annual precipitation of 745 mm. The predominant coniferous species in the forest is Pinus koraiensis and the broad-leaved species are Quercus mongolica, Tilia amurensis, and Fraxinus mandshurica. The soils are Albic Luvisols developed from volcanic ash61. In October 2017, approximately 10 kg of fresh leaf litter from the forest floor, primarily consisting of Pinus koraiensis, Quercus mongolica, Fraxinus mandshurica, Tilia amurensis Rupr., and Asophora japonica Linn., was collected, dried, ground, and sterilized to use as the organic matter for building the model soils.

In May 2018, nine experimental plots (3 m × 3 m) were randomly established with > 5 m buffer zones between adjacent plots. Six soil cores (5 cm in diameter, 20 cm in depth) were randomly collected from each plot after removing the litter layer. The cores were combined into one composite sample, immediately transported to the laboratory on ice, and homogenized for microbial inoculum extraction.

We created low-clay, medium-clay, and high-clay model soils with 20%, 40%, and 60% clay content, respectively, using quartz sand, montmorillonite, and plant litter (Fig. 1). The model soils were placed in PVC tubes (5 cm in diameter and 14 cm in height) with closed bottoms using PVC pipe caps, inoculated with the microbial inoculum, and pre-incubated for 120 days in the laboratory62. After pre-incubation, the PVC tubes were installed in the experimental plots for a stabilization period of 30 days. Subsequently, 13C15N-labelled bacterial and fungal necromass were injected into the model soils as tracers and incubated for 386 days under in situ conditions (Fig. 1). The experiment consisted of nine treatments: either (a) with 13C15N-labelled bacterial necromass, (b) 13C15N-labelled fungal necromass, or (c) without necromass (controls were injected with water-only); each of which was applied to soils of three different clay contents (low, medium, and high). In each of the 6 necromass plots, 15 PVC tubes with bacterial or fungal necromass were installed, while in each of the 3 control plots, 7 PVC tubes without added necromass were installed, resulting in a total of 111 PVC tubes.

Preparation of model soils
The quartz sand (composed of 30% silt- and 70% sand-sized particles; sourced from Aladdin) was cleaned in diluted acid and combusted at 500 ℃ to remove organic carbon and then was ground and sieved to fine (50-250 μm) and coarse (250-2000 μm) particle sizes. Detailed properties of the model soil materials are listed in Table S9. A typical expandable 2:1 phyllosilicate clay (montmorillonite; sourced from Sigma-Aldrich) was mixed with quartz sand in varying proportions to form low-clay soil (sandy loam soil; 20% clay, 24% fine quartz, and 56% coarse quartz), medium-clay soil (clay loam soil; 40% clay, 18% fine quartz and 42% coarse quartz) and high-clay soil (clay soil; 60% clay, 12% fine quartz and 28% coarse quartz). The organic matter obtained from the forest dried leaf litter was mixed at 45 mg g-1 model soil. The specific surface area of the low, medium, and high-clay soil was 27.1, 59.1, and 94.9 m2 g−1 soil, respectively. The water holding capacity (WHC) of low-, medium- and high-clay soil was 0.60, 0.89, and 1.21, respectively, and increased significantly with clay content (Table S10).

Approximately 80 g of the model soil was placed in a PVC tube (5 cm in diameter, 14 cm in height) for each sample. The height and bulk density of the model soils were ~5.5 cm and 0.75 g cm−3, respectively. A total of 37 samples were prepared for each clay treatment. We created the microbial inoculum by suspending 10 g of the field-collected soil in 90 mL of deionized water46. After 3 days, each sample was inoculated with the microbial inoculum. All samples were pre-incubated at 20 ℃ and 60% WHC for 120 days to establish stable soil structure and microbial community. Litter water extract was added biweekly (1.4 mg C, 0.027 mg N g−1 soil). The litter water extract was created by mixing 150 g dried forest leaf litter with 2 L of deionized water and shaking for 24 h before filtering63.

Preparation of labelled microbial necromass
The 13C15N-labelled bacterial and fungal necromass was prepared by cultivating the microbial inoculum in a minimal M9 salts medium with 99 atom% 13C-glucose and 99.5 atom% 15NH4Cl in a rotary shaker incubator. The bacterial culture was grown in 500 mL batches for 3 days at 37 °C with the addition of a fungal inhibitor (cycloheximide). The fungal culture was grown in 500 mL batches for 4 weeks at 28 °C with the addition of bacterial inhibitors (ampicillin and streptomycin). The bacterial and fungal cultures were then collected by centrifugation, sterilized by ultraviolet irradiation, mixed thoroughly, and ground to powder. This approach yielded bacterial and fungal necromass with a much broader species composition than necromass generated from pure cultures. The atom % 13C and atom % 15N of microbial necromass exceeded 71.95% and 92.08%, respectively (Table S1). The phospholipid fatty acid (PLFA) analysis confirmed that bacterial necromass predominantly consisted of bacterial species, while fungal necromass predominantly consisted of fungal species (Table S11).

Field experiment with dual-labelled microbial necromass
After laboratory pre-incubation of the model soils, on 19 August 2018, the PVC tubes were transported to the established field plots. Plots were randomly assigned to each treatment. In each necromass plot, we first filled the top ~8.5 cm of the PVC tubes with natural subsoils (5.5-14 cm), then turned the PVC tubes over and removed the PVC caps. We then inserted 15 PVC tubes of the same clay treatment into the soil to a depth of 14 cm after removing the natural soil. During the experiment, both ends of the PVC tubes were open, and the model soils were separated from the surrounding natural soil by a 1 mm nylon mesh, which allowed physical contact while preventing direct mixing. In each control plot, 7 PVC tubes were installed using the same process. The PVC tubes were placed in the plots for about one month prior to the addition of necromass to minimize potential disturbance effects associated with long-distance transport of the model soils.

On 18 September 2018, the necromass (10 mg dry weight) was suspended in deionized water and injected with a side-port needle in a 10-point grid pattern at a depth of 3 cm in the model soils within the PVC tubes. The added microbial necromass corresponded to an average of 9% and 22% of model soil C and N, respectively. Within each necromass plot, 15 PVC tubes were injected either with 13C15N-labelled bacterial or fungal necromass, while in each control plot, 7 PVC tubes were injected with deionized water.

Three PVC tubes with bacterial necromass or three PVC tubes with fungal necromass from each necromass plot, and one control PVC tube from each control plot were collected on 28 September 2018 (10 days), 18 October 2018 (30 days), 24 April 2019 (218 days) and 9 October 2019 (386 days). The soils from the collected PVC tubes were divided into model soils and natural subsoils. A portion of model soils collected at 10 and 386 days was stored at 4 °C before measuring soil microbial biomass C and N, dissolved organic C and N, inorganic N, and water content. The natural subsoils and a portion of model soils collected at the four sample times were air-dried for soil organic C and total N analyses. The remaining model soils collected at 386 days were stored at −80 °C for microbial community DNA extraction. To estimate necromass-derived CO2-C production, three PVC tubes of each necromass and control treatment were attached to a 1 L PVC chamber on 21 September 2018 (3 days), 28 September 2018 (10 days), 6 October 2018 (18 days), 12 October 2018 (24 days), 21 October 2018 (33 days), 28 October 2018 (40 days), 22 April 2019 (216 days) and 9 October 2019 (386 days). Then, a 150 mL gas sample was taken from each chamber at 0 h and 2 h using a syringe and stored in air bags for CO2 concentration and isotope measurement. The CO2 concentration and isotope ratio were analyzed using a CO2 isotope analyzer (CCIA-36d-EP, LGR, USA).

Physical fractionation
The model soils at day 386 were separated into four distinct OM fractions using a combined size-density fractionation protocol64. Air-dried model soil (18-20 g) was gently capillary-saturated with sodium iodide solution (NaI; 1.8 g cm−3) after 12 h, the free-floating particulate organic matter (fPOM) was collected using a vacuum pump. Occluded POM (oPOM) was released from soil aggregates by ultrasonic dispersion (Bandelin Sonopuls HD 2200; 440 J mL−1)65, enabling its separation from the heavier mineral fractions. Both fPOM and oPOM fractions were washed several times with deionized water to remove excess salt. While coarse and clay fractions were separated by wet sieving, mineral fractions < 53 µm were separated via centrifugation, and later combined as one MAOM fraction.

Necromass and soil chemical analyses
The specific surface area (SSA) of model soil materials and bulk model soils were determined by N2 adsorption at 77 K and subsequent desorption of nitrogen with a Tristar 5-point BET-instrument on freeze-dried samples. The total soil porosity was estimated from the measured bulk density and the particle density. The air-filled porosity was calculated as the difference between the total porosity and volumetric water content. The N and OC contents of the necromass and soil samples were determined by combustion using an elemental analyzer (Vario EL III; Elementar, Hanau, Germany). Inorganic N including ammonium (NH4+-N) and nitrate (NO3−-N) was extracted from fresh model soils using 2 M potassium chloride at a soil:water ratio of 1:5 and analyzed using a continuous flow injection analyzer (Autoanalyzer 3 SEAL, Bran and Luebbe, Norderstedt, Germany). The N isotope ratios of NH4+-N and NO3−-N were measured using the ammonium diffusion method 61. Microbial biomass C (MBC) and N (MBN) were determined by a fumigation-extraction method 66. Two subsamples (fumigated and non-fumigated) of fresh model soils were extracted with 0.5 M K2SO4. The non-fumigated soil extracts were used to measure dissolved organic C (DOC) and N (DON). The C and N concentrations of the extracts were determined by a TOC-500 analyzer (Shimadzu Corporation). The C or N isotope ratios of necromass C and N, soil organic C and total N, MAOM fractions, dissolved organic C and N, microbial biomass C and N, and inorganic N were analyzed using an Elementar Vario EL Cube (Elementar Analysis System GmbH) interfaced with an isotope ratio mass spectrometer (IsoPrime100, IsoPrime Limited, Stockport, UK).

To characterize the chemical composition of bacterial and fungal necromass, Fourier-transform infrared spectroscopy (FTIR) analysis was performed. Briefly, freeze-dried bacterial and fungal necromass were finely ground, mixed with KBr at a ratio of 1:100 (wt:wt), and analyzed using a Thermo Nicolet 6700 infrared spectrometer (Thermo Electron Scientific Instruments Corp., Madison, WI, USA). Absorbance spectra (4000-400 cm−1) were recorded with 64 scans and a resolution of 4 cm−1. Absorbance intensity at 700, 860, and 975 cm−1 corresponded to aromatic groups; 2855, 2930, and 3300 cm−1 were assigned to aliphatic groups; and 1075 cm−1 corresponded to polysaccharide group 67. The relative absorbance intensity (rA, %) of each of the seven bands was normalized to the percent absorbance intensity of the sum of absorbance intensities of the seven bands.

To characterize the microbial community composition of bacterial and fungal necromass, PLFA analysis was performed. Briefly, 80 mg freeze-dried necromass was extracted with a chloroform:methanol:phosphorus-buffer (volume ratio: 1:2:0.8). After the organic phase was separated, dried, purified, and subjected to methyl esterification, lipids were analyzed using an Agilent 7890B GC (Agilent Technologies, Santa Clara, CA, USA) equipped with MIDI software. Microbial composition was reflected by the biomarker PLFAs of different microbial groups68.

SEM and NanoSIMS micro-spectroscopy
To directly visualize the distribution of added microbial necromass on mineral surfaces, we used SEM and NanoSIMS to analyze the model soils at 386 days. Dried model soil samples (<0.15 mm) were dispersed in deionized water, and 10 µL of the suspension was deposited onto a germanium wafer and dried overnight in a desiccator. To avoid the charging phenomena, samples were gold-coated prior to SEM analysis by physical vapor deposition under an argon atmosphere (Emitech Sputtercoater SC7620, Gala Instrumente, Bad Schwalbach, Germany). The microscale structures of mineral–necromass associations were first examined by SEM (Jeol JSM 5900LV, Freising, Germany). Representative regions were then analyzed using a Cameca NanoSIMS 50 L (Cameca, Gennevilliers, France). For NanoSIMS analysis, a high Cs+ primary ion beam current was used to remove surface contamination and gold coating, and to implant Cs+ ions into the sample to enhance the yield of secondary ions. Secondary ions of 12C−, 13C−, 12C14N−, 12C15N−, 16O−, 28Si−, and 27Al16O− were collected simultaneously using electron multipliers with a fixed dead time of 44 ns. Ion images were acquired with a dwell time of 2 ms pixel⁻¹, at a resolution of 512 × 512 pixels over a 35 × 35 µm area in a single scan cycle. For each sample, five regions of interest were analyzed to obtain a representative dataset for quantifying ¹³C and ¹⁵N distribution.

NanoSIMS image data were processed using the OpenMIMS plugin in Fiji (Schindelin et al., 2012). All images were corrected for electron multiplier dead time (44 ns). The ion images of 12C− and 12C14N− were merged for an indicator of native OM49. Regions of interest (ROIs) on these images were selected using the threshold option with the default method. All ROIs with an area greater than 10 pixels2 were used for further calculations. The 13C−:(12C− + 13C−), 12C15N−:(12C14N− + 12C15N−) ratios, and the area of the ROIs were extracted from all images and used for further calculations. The new, necromass-derived OM defined as ROIs in the 13C−:(12C− + 13C−) and 12C15N−:(12C14N− + 12C15N−) ratio image with pixel values greater than natural abundance levels.

Amplicon sequencing and data processing
Soil genomic DNA was extracted from 0.25 g freeze-dried soil collected on the last sampling time using PowerSoil DNA Isolation Kits (MoBio Laboratories, Carlsbad, CA, U.S.A.). Bacterial and fungal communities were assigned by amplifying and sequencing the V4–V5 region of the 16S rRNA gene and the ITS1 region, respectively. After PCR amplification and purification, 16S rRNA and ITS gene sequencing were performed using the Illumina platform. Raw sequences shorter than 200 bp or with a quality score below 20 (Q < 20) were removed prior to analysis using the QIIME 2 pipeline. Quality-filtered sequences of 16S rRNA and ITS gene amplicons were clustered into operational taxonomic units (OTUs) at 97% sequence similarity using UPARSE. Taxonomic classification of representative bacterial and fungal ASVs was conducted using the SILVA database and the UNITE database, respectively. Observed OTUs and phylogenetic diversity were calculated to assess microbial alpha diversity.

Data analysis
To determine the fraction of necromass-derived C or N (Cfrac or Nfrac) in the model soils, subsoil, microbial biomass, dissolved organic matter, inorganic nitrogen, and headspace gas, we calculated the fraction of each pool that was necromass-derived by the following isotope mixing model:
Cfrac or Nfrac = (atom%Sample − atom%Control) / (atom%Necromass − atom%Control) (1)
where atom%Sample is the atom% of 13C or 15N value of the C or N pools (i. e., MBC, MBN, DOC, DON, CO2-C, IN, model soil and subsoil C or N) of a given sample; atom%Control is the atom% of 13C or 15N in the equivalent C or N pool from the control; atom%Necromass is the atom% of 13C or 15N in the labelled microbial necromass added to soils.

The 13C or 15N recovery was calculated as the percentage of labelled C or N remaining in each C or N pool (MBC, MBN, DOC, DON, IN, bulk soil, and subsoil C or N) relative to initial necromass inputs. The 13C recovery in CO2-C was calculated using linear interpolation between sample points. The 13C recovery in necromass C was calculated by subtracting the 13C recovery in MBC and DOC from the 13C recovery in model soil C. The 15N recovery in necromass N was calculated by subtracting the 15N recovery in MBN, DON, and IN from the 15N recovery in model soil N.

Due to the variability and limited sampling times for 13C and 15N recovery of labelled necromass, the commonly used one- or two-pool exponential decay models could not accurately simulate our data. Therefore, we determined the mineralization rate constant of necromass 13C or 15N (kC or kN) using a linear regression on the natural log transformation of 13C or 15N recovery69.
kC or kN = ln (St / S0) / (t − t0) (2)
where St is the recovery of necromass 13C or 15N in bulk soil at the end of the experiment (t); S0 is the recovery of necromass 13C or 15N in bulk soil at the beginning of the experiment (t0).

A two-way analysis of variance (ANOVA) was conducted to assess the effects of clay content (low-, medium- and high-clay) and microbial groups (bacteria and fungi) on the mineralization rate constant and cumulative gaseous emissions of necromass. A three-way ANOVA was conducted to assess the effects of clay contents, microbial groups, and time on necromass fates in different soil C or N pools. A repeated measures ANOVA model (MANOVA) was used to compare the effects of clay contents, microbial groups, and time on the retention and gaseous emissions of necromass. Statistical findings were considered significant if the confidence level was set at 95% (P < 0.05). The above statistical analyses were conducted using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Additionally, linear regressions were used to analyze the relationship between the necromass retention and bacterial community diversity (richness-B), soil moisture content, specific surface area (SSA), microbial metabolic quotient (qCO2), and air-filled porosity (AFP) at the end of incubation. Principal component analysis (PCA) was conducted to explore relationships between necromass retention and environmental variables. Principal coordinates analysis (PCoA) with Bray-Curtis distance was conducted to explore differences in microbial community composition using amplicon data. Statistical differences in the microbial community composition were tested using permutational multivariate analysis of variance (PERMANOVA). Both PCA and PCoA analyses were conducted in R (v. 4.4.3) using the vegan package70. The hierarchical partitioning method was employed to quantify the contribution of microbial community and soil properties to necromass retention in R (v. 4.4.3) using the “rdacca.hp” package71.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This is an important study to investigate the effect of clay content on microbial necromass persistence, in experiments conducted in situ in real soils. I think that the work is interesting to a broad readership, but I think the authors miss an opportunity to provide new information on why high clay content increases microbial necromass persistence. Based on previous work I feel that the take home message that high clay content increases microbial necromass persistence is rather expected, and the work as it stands serves as a confirmation of this expectation, rather than providing a substantial advance in understanding of this expectation. For this reason, I think the work as it stands is perhaps not ready for publication in Nature Communications but could be with some extra analyses that allow the authors to more rigorously interrogate some of the speculative explanations that they provide for their results. I have some more minor comments on the introduction and results below, and then I exemplify my main point at several sections of the discussion below.\n\nIntroduction:\nLine 22: clay minerals are not usually described as the most reactive soil particles (metal (oxyhydr)oxides are much more reactive and play a disproportionately important role in elemental cycling in soils), but they are among the most reactive soil particles – so I suggest rephrasing as: “as among the most reactive soil particles”\nLine 48: adsorption not absorption\nLine 50: how does soil clay content affect the living microbial community and why does this suggest that soils with high clay content could alternatively exhibit lower necromass persistence? – this is later explained in lines 68-82 but I feel there needs to be better connection between this statement and the eventual explanation later on.\nLine 53: do the authors mean clay minerals are characterized by their high charge and specific surface area? (also their SSA is nowhere near as high as iron (oxyhydr)oxides, for example).\nLine 97: I feel that a short description of the field experiments using model soils is warranted here – its quite difficult in the methods to follow what was actually done – whilst the experiments were conducted in situ in a field plot, the authors use model artificial soils, so I think that this needs to be clearly explained – a flow diagram or some sort of schematic in the main text or supplementary information would be really helpful to show the reader what was done (Fig 1 and Fig S8 are helpful but two separate figures makes it difficult to follow – I feel that these could be combined).\nLine 88 and 101: whilst the higher N content of bacterial necromass may also result in tighter binding to minerals, the chitin content of fungal necromass is known to degrade into carboxyl-containing compounds which adsorb very strongly to soil minerals, so perhaps this needs to be considered wrt this hypothesis – at least upon its introduction around line 88.\n\nResults:\nLine 115: how much of the added C was partially mineralised (to DOC)? – I think this would be instructive to add at this point of the results.\nLine 166: this sentence doesn’t make sense – “Biochemical characterization using FTIR analyses indicated that had a higher relative abundance of polysaccharides and a lower relative abundance of polysaccharides compared to fungal necromass (Table S1).” – I think that Table S1 shows that bacteria necromass has a lower abundance of polysaccharides compared to fungal necromass?\n\nDiscussion:\nLine 177-198: concerning the main result that as clay content increases, a greater proportion of the added necromass 13C and nitrogen 15N is retained in the model soil, despite the concurrent increase in microbial biomass: whilst this may be the first study to demonstrate this outcome in situ, which is important (at least to the extent that this is truly an in situ experiment), I think that the outcome is to be expected based on previous work, so I feel that this work misses an opportunity to provide new information on why this expectation holds true in situ in soils. I think that the authors do a decent job in providing potential explanations for their result (their language is necessarily speculative), but they are unable to pinpoint the actual explanation without more detailed examination of the clay-necromass association. In this regard I feel that the overarching take home message is an important one, but is rather expected, and as such the work at the moment probably does not provide as substantial an advance in understanding as it could do.\nLine 216-229: concerning the result that there was substantial leaching of necromass: did the authors measure / approximate molecular weight of the necromass and/or measure the pore volume of the clays? – this would provide validation of their speculative explanations.\nLine 240-241: concerning the result that bacterial and fungal necromass exhibited similar mineralization rates: this section and exemplified at the cited lines is very speculative. I feel that this work would make a much stronger contribution to the literature if the authors provided a more in-depth analysis of the mineral-necromass associations – the speculation that bacterial necromass is more strongly adsorbed by minerals than fungal necromass need not be speculation – there are many wet chemical techniques that are routinely used to operationally define strength of adsorption that could be used here, and/or direct observational techniques, including FTIR that the authors already employ elsewhere in the work.\nLine 257-261: I feel that this needs more explanation and discussion – it was indeed one of the issues I wanted to raise wrt the experiments. Are the experiments still relevant for evaluating in situ mineralisation rates? Does this aspect of the experimental design affect the relative differences between the mineralisation rates of bacterial and fungal necromass or just the absolute rates?", "reviewer_2": "Wang et al. carried out an experiment of intermediate nature in which synthetic soils with three different clay contents were\ninoculated with microbial necromass with labelled 13C and 15N and with living microorganisms, and the synthetic soils\nwere placed in an experimental plot for up to one year. Intermediate times were sampled to follow the progress of several\nvariables. The final goal was to investigate whether soil clay content or the type of necromass (bacterial or fungal) affected\nthe preservation of this necromass. The authors conclude that clay caused a significant retention of necromass in the soil,\nand there is no difference between bacterial and fungal necromass.\nThe goals are of great interest, the experiments are carefully designed and there are numerous analyses to constrain the\nresults. However, as the result analyses demonstrate, the experimental design would have required still further care to\naddress all the possible pitfalls that may invalidate results. I am left with the question whether the authors simply or mainly\nmeasured transfer of OM down the column, so that, as expected, more OM remained where the clay content was greater; or\nwhether their different amounts of clay genrated a different microbial population, which modified the results of the\nexperiments. The discussion is poor and shows in greater contrast the limitations and possible pitfalls of the study. In my\nopinion, the ms should be rejected.\nMain issues\nLines 38-39. The definition of microbial necromass is unclear and causes trouble with the experiments themselves. Should\nwe understand that “plant inputs” are “plant products”? Is microbial necromass defined as plant products modified by\nmicroorganisms? If so, this is different from the microbial necromass used in the experiments, which consists of organic\ndebris of bacteria and fungi. This issue affects the discussion in several places. First, it is not clear whether the authors are\nconsidering the transformation sequence as: living cells necromass mineralized OM or: living cells mineralized\nOM necromass. Second, the authors argue in relation to their microbial necromass from results related to necromass from\nplant litter (lines 219-222), but do these two types of necromass have similar molecular weights and charged moieties that\nwill affect their mobility within the soil?\nThe authors call “mineralization rate” what really is the “mineralization rate constant”. In a chemical process the rate is\nproportional to the rate constant, but both are not identical. The rate constant will multiply the reagent concentrations and/or\nsome other variable(s) affecting the chance of the reagents to come in contact and react. This mistake or misconception is\nfound in the entire paper.\nThe tables in the supplementary information about statistical analysis provide the p values of the ANOVA tests, but do not\nprovide any information on whether the correlations between variables were positive or negative. We learn about some of\nthe variable pairs in some of the figures, but not about the others. This leaves important information untold. For example, in\nTable S5, the correlation between preserved N and C in microbial biomass with clay contents has a very low p and, as we\nknow from other parts of the article, the correlation is positive. If this is the case, we would expect clay contents to correlate\nnegatively with dissolved N and C, and with inorganic N. However, the high p values for the latter suggest that there is no\ncorrelation. In any case, do dissolved C and N increase with clay content? Apparently, Fig. 5 contains relevant information,\nwhich is not mentioned or discussed.\nThe presentation of the PCA results (lines 172-174) is too brief and does not discern between the meaning of the alignment\nof the several variables. The authors are trying to identify the drivers of necromass persistence. These can be only SSA,\nWHC and SM. The other variables aligning with High clay content are an effect of the preservation, and not its cause.\nThe discussion section has important problems that make it weak and inconclusive:\n1) The entire discussion appears to be a speculation, as stated by the authors: “may be”, “we speculate”, etc. The study\ngathered a lot of quantitative data that are not used to carry out a quantitative discussion.\n2) Lines 187-189 are a repetition of ideas mentioned in the introduction and they are not further discussed. Are there new\nideas for the discussion?\n3) 191-192: These two lines are the statement of the discrepancy, not a possible explanation of why it takes place.\n4) Line 195: where is it shown that there is an increase in soil C?\n5) Line 207: Why is survival only linked to mineralization? Reproduction also needs energy and respiration, with consequent\nCO2 loss.\n6) The idea that water content may affect gas diffusion is reasonable. However, what mathematical expression would\nrepresent such effect: linear, exponential, …? In other words, how can we relate the data in figure S5 with the data in figures\n2, 3, S4, etc?\n7) Lines 211-212. In my opinion, the authors hit here on the key issue that may explain all the data. The increase of clay\nmodifies the microbial community, which is what controls many of the other modifications observed. This issue, however, is\nnot followed or discussed further.\n8) Lines 216-219. The observed rapid leaching of necromass from the synthetic soil to the underlying subsoil would depend\non precipitation (rain) during the experiment. Did the authors measure precipitation during the experiment? This may be\na crucial point.\n9) Line 224: “the low leaching amounts…” Based on Fig. 4b,d (360 day experiments), this statement requires qualification:\ncan one say that the values of leached C and N (that I take to be those in subsoil) in the high-clay experiments are lower\nthan those in the low- and medium-clay experiments?\n10) Line 232: “accessibility and availability and accessibility” does not correspond to what is explained in the following lines.\n11) Lines 233-235: Why does higher N content in the necromass make it more readily used by microorganisms? Also, this\nassertion contradicts that in lines 249-250, that N-rich necromass was more resistant to mineralization.\n12) Lines 238-240. This statement contradicts the previous one in lines 230-231, that there is no significant retention\ndifference between bacterial and fungal necromass.\n13) Lines 252-254: can the authors explain why these facts indicate N limitation in the synthetic soils?\n14) The limitation mentioned in lines 257-261 appears as a potentially serious one that may invalidate the data. As the\nauthors have the values of leached necromass, can they try to discuss this issue quantitatively, rather than pointing it out\nas an afterthought? How does clay arrange itself in sandy soils? How about the silt and clay already present in the sand before\nthe addition of the smectite? (see below).\nThe methods are difficult to follow due to the complexity of the experiments and lack of clarity in the text. I would advise to\nuse a temporal sequence, rather than the present distribution based on the several elements of the experiments.\nThe quartz “sand” had a 30% of silt. This is a high value. Did the authors investigate the mineralogical composition of the\nsilt? It may have had some clay. How is it possible that both the sand-sized quartz and the silt-sized quartz had the same\nSSA (Table S9)? By definition, for the same substance, smaller particle size means higher SSA. Was the SSA controlled by\nsome mineral other than quartz? The SSA of 0.17 m2 / g is low for quartz, although the 74 m2 / g for montmorillonite seems\ncorrect. There is no indication of how these SSA values were measured.\nThere is no indication whether the cylinders in the experiments were open or closed at the bottom; i.e., were the synthetic\nsoils and sampled subsoils in contact or somehow connected with the soils outside the cylinders?\nSpecific issues\nLines 77-78 are unclear and confusing. High clay content would cause higher water retention, and the latter cause a\ndecrease of oxygen diffusion; I am not aware that dry clay can decrease air diffusion to the point of generating reducing\nconditions. Further, how does higher water content cause necromass desorption from the clay?\nLines 105-106 need to be rewritten for clarity.\nLine 113: “rapidly”. Not really because the % values are small. The authors perhaps mean that the necromass was initially\nconsumed rapidly but the rate of consumption soon decreased substantially.\nLines 124-125. Wrong expression: ratio of retained C to retained N means C/N, which is always <1 in Fig. S2 (i.e., N>C).\nThe section “Fate of microbial necromass” is poorly presented, with very convoluted, repetitive phrases difficult to follow. I\nwould advise to simplify the phrases, to focus first on the main points for clarity, and finally move to the interesting details.\nLines 166-168 do not make sense.\nLines 289-290. It would be better to provide the soil taxonomic information following one of the standard methods or to give a\nmore complete description.\nLines 326-327: density is mass/volume, not volume/mass.\nLine 353: at what depth was the subsoil collected?\nSome of the figures have incomplete information in the captions. There are some errors such as the use of “loss” for “lost”.\nAxes titles are sometimes wrong or inexact.\nFigure 2 is confusing. The coloured surfaces suggest that the data are represented by the surfaces, while in reality the\nauthors intend that the data are those represented by the lines at the top of each surface. The plots needs to be changed to\nsingle lines like those in Fig. 3a,b. Further, how is the “respired necromass” calculated? Is it calculated from 13C in CO2? If\nsuch is the case, Figs. 2 and 3 are incompatible.\nAs stated before, the y-variable in Fig 3c is not mineralization rate, but rate constant of the mineralization process.\nFig. 4: “Fate” is not a physical or chemical variable that can be represented in a plot. The values Necromass C and N, MBC,\nMBN, DOC, TDN and IN are supposedly to refer to the synthesized soil, but this is not indicated anywhere. The results are\nnot presented clearly."}, "author_response": "Response to Reviewer #1:\nComment: This is an important study to investigate the effect of clay content on\nmicrobial necromass persistence, in experiments conducted in situ in real soils. I think\nthat the work is interesting to a broad readership, but I think the authors miss an\nopportunity to provide new information on why high clay content increases microbial\nnecromass persistence. Based on previous work I feel that the take home message that\nhigh clay content increases microbial necromass persistence is rather expected, and the\nwork as it stands serves as a confirmation of this expectation, rather than providing a\nsubstantial advance in understanding of this expectation. For this reason, I think the\nwork as it stands is perhaps not ready for publication in Nature Communications but\ncould be with some extra analyses that allow the authors to more rigorously interrogate\nsome of the speculative explanations that they provide for their results. I have some\nmore minor comments on the introduction and results below, and then I exemplify my\nmain point at several sections of the discussion below.\nResponse: We thank the reviewer for recognizing the importance of our study and for\nthe insightful comments encouraging a deeper mechanistic interpretation. In response,\nwe conducted additional Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS)\nanalyses to directly visualize clay-necromass associations, which show that newly\nformed microbial necromass is preferentially associated with organic matter coatings\non rough mineral surfaces, rather than bare minerals. Complementary density\nfractionation analyses further revealed that microbial necromass is predominantly\npresent within the mineral-associated organic matter (MAOM) fraction. Together, these\nresults provide the first direct experimental evidence that mineral-organic matter-\nnecromass interactions are a key mechanism underlying the stabilization of microbial\nnecromass in soils. We also emphasize that while it is expected clay content will\nincrease necromass retention, this concept is based primarily on the binding capacity of\nclay and overlooks other, potentially competing factors, that can occur in a clay-rich\nenvironment, such as elevated decomposition associated with higher biomass, and\nfurther does not account for other protection pathways such as reductions in leaching.\nCombined, our study, shows that when examined in situ, multiple factors associated\nwith clay content emerge as important controls on loss and retention pathways.\nComment: Line 22: clay minerals are not usually described as the most reactive soil\nparticles (metal (oxyhydr)oxides are much more reactive and play a disproportionately\nimportant role in elemental cycling in soils), but they are among the most reactive soil\nparticles – so I suggest rephrasing as: “as among the most reactive soil particles”\nResponse: Thanks! We have revised the sentence to “Microbial necromass is\nincreasingly recognized as a major source of stable soil organic matter (SOM), and its\npersistence is often attributed to interactions with clay-sized minerals.” in the revision\n(Lines 21-22).\nComment: Line 48: adsorption not absorption\nResponse: Thank you for pointing this out. We have corrected “absorption” to\n“adsorption” throughout the manuscript.\nComment: Line 50: how does soil clay content affect the living microbial community\nand why does this suggest that soils with high clay content could alternatively exhibit\nlower necromass persistence? –this is later explained in lines 68-82 but I feel there\nneeds to be better connection between this statement and the eventual explanation later\non.\nResponse: We thank the reviewer for this helpful suggestion. In the revision, we\nimproved the link between the statement and the subsequent explanation by combining\nthe direct mineral effects and the microbially mediated effects of clay on necromass\npersistence into a single paragraph. This reorganization clearly shows how clay\ninfluences multiple (sometimes opposing) mechanisms to either promote or hinder\nnecromass persistence. The revised text is provided follows:\n“Soil clay content is thought to influence microbial necromass persistence through\nmultiple, and sometimes competing, mechanisms16,17. On one hand, clay particles\npossess high surface charge and specific surface area, promoting strong adsorption of\nlow-molecular-weight necromass and forming abundant necromass-mineral\nassociations that limit microbial access10,18. The fine pores and microaggregates\ncharacteristic of clay-rich soils can further occlude necromass and physically restrict its\ncontact with microbes and extracellular enzymes19-22, while high water retention\nreduces leaching losses23,24. Additionally, reduced oxygen diffusion in moisture-rich,\nhigh-clay soils can enhance reducing conditions and promote partial Fe(III) reduction,\nweakening mineral sorption and altering necromass mobility25. On the other hand, these\nsame clay-rich environments can suppress microbial activity that slows necromass\nmineralization26. Abundant fine pores create microhabitats that support microbial\ngrowth and protect microbes from predation22,27,28, while elevated nutrient and moisture\navailability alleviate resource and water limitations23,29-31, thereby enhancing microbial\nactivity and diversity. Clay content also influences microbial community\ncomposition32,33, which in turn shapes both necromass production and decomposition.\nOverall, clay can simultaneously promote necromass protection and stimulate\nnecromass loss, underscoring the need to quantify necromass fate under in situ\nconditions where these protective and degradative pathways interact.” (Lines 51-68)\nComment: Line 53: do the authors mean clay minerals are characterized by their high\ncharge and specific surface area? (also their SSA is nowhere near as high as iron\n(oxyhydr)oxides, for example).\nResponse: Thanks for bringing up this point. We agree that the specific surface area\n(SSA) of clay minerals is generally lower than that of Fe and Al (oxyhydr)oxides. Our\nintention was to highlight that, relative to silt- and sand-sized soil particles, clay-sized\nminerals possess substantially higher charge density and SSA, which increases the\navailability of reactive sites for necromass adsorption. To avoid confusion, we have\nrevised the sentence accordingly:\n“On one hand, clay particles possess high surface charge and specific surface area,\npromoting strong adsorption of low-molecular-weight necromass and forming\nabundant mineral-necromass associations that limit microbial access10,18.” (Lines 52-\n55)\nComment: Line 97: I feel that a short description of the field experiments using model\nsoils is warranted here – its quite difficult in the methods to follow what was actually\ndone – whilst the experiments were conducted in situ in a field plot, the authors use\nmodel artificial soils, so I think that this needs to be clearly explained – a flow diagram\nor some sort of schematic in the main text or supplementary information would be really\nhelpful to show the reader what was done (Fig 1 and Fig S8 are helpful but two separate\nfigures makes it difficult to follow – I feel that these could be combined).\nResponse: We appreciate this very insightful comment. In the revised manuscript, we\nhave combined the previous Fig. 1 and Fig. S8 into a flow diagram (now Fig. 1, See\nbelow Fig. R1) that clearly illustrates the overall experimental design and workflow. In\naddition, we added a brief description of the experimental procedure in this section to\nclarify how the how the artificial soils were constructed, and transferred to the field\nplots for incubation with labelled necromass and list it as follows:\n“Here, we established an experimental clay gradient to directly assess how\nvariation in clay content regulates in situ microbial necromass de composition and\nretention (Fig. 1). We constructed artificial soils with contrasting clay contents by\nmixing montmorillonite clay, quartz sand, and plant litter while maintaining identical\norganic matter input and clay mineral. After a pre-incubation period, model soils were\nplaced into a natural forest site and amended with dual isotope-labelled (13C and 15N)\nbacterial and fungal necromass.” (Lines 88-93)\nFig. R1 Experimental design of in situ incubation with 13C15N-labelled microbial\nnecromass across a clay gradient.\nComment: Line 88 and 101: whilst the higher N content of bacterial necromass may\nalso result in tighter binding to minerals, the chitin content of fungal necromass is\nknown to degrade into carboxyl containing compounds which adsorb very strongly to\nsoil minerals, so perhaps this needs to be considered wrt this hypothesis – at least upon\nits introduction around line 88.\nResponse: We thank the reviewer for this helpful comment. To address the point, we\nhave added a comment about the strong binding of chitin degradation products to\nminerals and list it as follows:\n“In contrast, fungal necromass contains chitin and other complex polysaccharides\nthat can degrade into carboxyl-rich compounds capable of forming strong inner-sphere\ncomplexes with Fe- and Al-oxide minerals41,42” (Lines 76-78)\nComment: Line 115: how much of the added C was partially mineralised (to DOC)? –\nI think this would be instructive to add at this point of the results.\nResponse: Good point! In the revised manuscript, we added analyses for the added\nnecromass 13C and 15N recovered in dissolved organic matter pool and list it as follows\n(see below Fig. R2).\n“Similarly, recovery in dissolved organic matter was minimal, declining from\n0.38% (±0.06%) of 13C and 0.10% (±0.01%) of 15N at day 10 to 0.09% (±0.02%) of\n13C and 0.02% (±0.004%) of 15N at day 386, with 13C slightly increasing and 15N\ndecreasing with clay content (P = 0.091 and 0.030; Fig. 4; Fig. S3, S4; Table S5, S6).”\n(Lines 140-143)\nFig. R2 The added microbial necromass recovered in dissolved organic matter.\nThe amount of added (a, c) bacterial and (b, d) fungal necromass 13C and 15N recovered\nin dissolved organic matter. Values represent means and standard error (n = 3).\nComment: Line 166: this sentence doesn’t make sense – “Biochemical characterization\nusing FTIR analyses indicated that had a higher relative abundance of polysaccharides\nand a lower relative abundance of polysaccharides compared to fungal necromass\n(Table S1).” – I think that Table S1 shows that bacteria necromass has a lower\nabundance of polysaccharides compared to fungal necromass?\nResponse: We have changed “Biochemical characterization using FTIR analyses\nindicated that had a higher relative abundance of polysaccharides and a lower relative\nabundance of polysaccharides compared to fungal necromass (Table S1).” to “Fourier-\ntransform infrared spectroscopy (FTIR) analysis further showed that bacterial\nnecromass was enriched in aliphatic compounds but depleted in aromatics and\npolysaccharides relative to fungal necromass (Table S1).” (Lines 106-108)\nComment: Line 177-198: concerning the main result that as clay content increases, a\ngreater proportion of the added necromass 13C and nitrogen 15N is retained in the\nmodel soil, despite the concurrent increase in microbial biomass: whilst this may be the\nfirst study to demonstrate this outcome in situ, which is important (at least to the extent\nthat this is truly an in situ experiment), I think that the outcome is to be expected based\non previous work, so I feel that this work misses an opportunity to provide new\ninformation on why this expectation holds true in situ in soils. I think that the authors\ndo a decent job in providing potential explanations for their result (their language is\nnecessarily speculative), but they are unable to pinpoint the actual explanation without\nmore detailed examination of the clay-necromass association. In this regard I feel that\nthe overarching take home message is an important one, but is rather expected, and as\nsuch the work at the moment probably does not provide as substantial an advance in\nunderstanding as it could do.\nResponse: We appreciate this very insightful comment! In the revised manuscript, we\nadded NanoSIMS and density fractionation analyses to further examine the clay-\nnecromass association. The NanoSIMS results showed that most soil organic matter\nwas distributed on mineral surfaces, particularly on rough surface regions (Fig. S3).\nThe added microbial necromass was almost always associated with organic matter\nalready bound to these rough mineral surfaces (79.8% of necromass 13C, 87.3% of\nnecromass 15N), while little necromass was observed on bare mineral surfaces (0.43%\nof necromass 13C, 1.86% of necromass 15N). This finding provides the first direct\nevidence that microbial necromass in soils is primarily preserved through mineral-\norganic matter-necromass associations, rather than through directly adsorbed onto the\nmineral surface. Consistent with this, density fractionation results indicated that\nmicrobial necromass was mainly stabilized through mineral protection rather than\nthrough physical occlusion within aggregates (Fig. S9). Soils with higher clay contents\nretained more microbial necromass due to a greater abundance of rough mineral\nsurfaces (Fig. S3). We have now added these new results into the main text and\ndiscussed these results in the context of other information in the literature.\n“To resolve the microscale mechanisms underlying necromass stabilization, we\nexamined the spatial arrangement and interactions between added labelled necromass,\nnative OM, and mineral particles using scanning electron microscopy (SEM) and\nnanoscale secondary ion mass spectrometry (NanoSIMS) (Fig. 6). These\ncomplementary techniques allowed direct visualization of the co-localization between\nlabelled necromass and soil constituents. Consistent with the bulk IRMS results,\nNanoSIMS showed higher 13C−:(12C− + 13C−) and 12C15N−:(12C14N− + 12C15N−) ratios in\nhigh-clay soil (Fig. 6d, g). Native OM, identified by the 12C⁻ and 12C14N⁻ secondary ion,\nwas predominantly associated with minerals (79.5 ± 1.6%), as indicated by the 16O⁻\nsecondary ion (Fig. 6a1-c2; Fig. S5). Mineral surfaces coated with OM were generally\nrougher, as determined by the higher ratio of 16O−:area (Fig. 6i). Newly added\nnecromass 13C and 15N accumulated via organo-organic interactions: labelled\nnecromass preferentially attached to native OM already associated with mineral\nsurfaces (79.8 ± 3.2% of 13C, 87.3 ± 1.9% of 15N), rather to bare mineral surfaces (Fig.\n6a1-c2, e, h). High-clay soils exhibited a larger specific surface area covered by OM-\nmineral associations than low- and medium-clay soils (Fig. 6f). Density fractionation\nfurther confirmed that most retained microbial necromass was predominantly within\nthe mineral-associated organic matter fraction (96.2 ± 0.4% of 13C, 93.3 ± 0.5% of 15N;\nFig. S6).” (Lines 174-190)\nComment: Line 216-229: concerning the result that there was substantial leaching of\nnecromass: did the authors measure approximate molecular weight of the necromass\nand/or measure the pore volume of the clays? – this would provide validation of their\nspeculative explanations.\nResponse: We thank the reviewer for this helpful suggestion. In this study, we\nmeasured the total porosity of the three model soils (see below Fig. R3). The results\nshowed low-clay soil had lowest total porosity, where its large leaching after 10 days\nof in situ incubation may be a function of larger pore size typical of sandier soils.\nFurthermore, we compared the FTIR spectra of the microbial necromass and plant litter.\nThe microbial necromass contained higher proportions of low-molecular-weight\naliphatic compounds and lower proportions of high-molecular-weight aromatic and\npolysaccharide compounds. This finding supports our hypothesis that the microbial\nnecromass has a greater leachable fraction due to their lower molecular weight (see\nbelow Fig. R4), particularly in low-clay soils with relatively low total porosity (which\nusually coincides with a lower proportion of micropores) (see below Fig. R3). We have\nnow added these new results into the main text and discussed these results in the context\nof other information in the literature.\n“This substantial early leaching likely reflects the low molecular weight of\nmicrobial necromass, which confers high mobility54. Supporting this, FTIR analysis\nshowed that the microbial necromass contained higher proportions of low-molecular-\nweight lipid compounds and lower proportions of high-molecular-weight aromatic and\npolysaccharide compounds (Fig. S15) compared with plant litter55. These findings\nsuggest that the previously reported high proportion of necromass in subsoil organic\nmatter14 partly originates from the leaching of microbial processing products of surface\nplant litter. The pronounced reduction in necromass in the subsoil of low-clay soils\nfrom day 10 to day 386 indicates continued leaching into deeper soil layers, further\nsupporting our conclusion.” (Lines 228-236)\nReference:\n14. Ni X, et al. The vertical distribution and control of microbial necromass carbon in\nforest soils. Global Ecol Biogeogr 29, 1829-1839 (2020).\n56. Spence A, et al. The degradation characteristics of microbial biomass in soil.\nGeochim Cosmochim Acta 75, 2571-2581 (2011).\n57. Yang N, et al. Leaf litter species identity influences biochemical composition of\nectomycorrhizal fungi. Mycorrhiza 29, 85-96 (2019).\nFig. R3 Soil porosity. (a) Total porosity and (b) air-filled porosity of low-, medium-\nand high-clay soils after 386 days of incubation. Values represent means and standard\nerror (n = 3). Different lowercase letters indicate significant differences among clay\ntreatments.\nFig. R4 Biochemical characteristics of microbial necromass and plant leaf. Relative\nabundance of functional groups in bacterial and fungal necromass (this study) and\nBeech (Fagus sylvatica), lime (Tilia cordata), maple (Acer pseudoplatanus), and ash\n(Fraxinus excelsior) leaf litter (data from Yang and Butenschoen¹) determined by\nFourier transform infrared spectroscopy (FTIR).\nComment: Line 240-241: concerning the result that bacterial and fungal necromass\nexhibited similar mineralization rates: this section and exemplified at the cited lines is\nvery speculative. I feel that this work would make a much stronger contribution to the\nliterature if the authors provided a more in-depth analysis of the mineral-necromass\nassociations – the speculation that bacterial necromass is more strongly adsorbed by\nminerals than fungal necromass need not be speculation – there are many wet chemical\ntechniques that are routinely used to operationally define strength of adsorption that\ncould be used here, and/or direct observational techniques, including FTIR that the\nauthors already employ elsewhere in the work.\nResponse: We thank the reviewer for this valuable comment. In this study, we\nconducted additional Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS)\nanalyses to directly visualize the spatial associations between microbial necromass and\nminerals. Contrary to our initial hypothesis, most of the added necromass did not\ndirectly adhere to the mineral surface but was instead predominantly associated with\norganic matter already bound to rough mineral surfaces (Fig. 6e and h, same as the\nbelow Fig. R5). The proportions of bacteria and fungi necromass-derived carbon and\nnitrogen distributed on the surface of organic matter already bound to rough mineral\nsurfaces were comparable (C: P = 0.39, N: P = 0.24). This similarity likely reflects the\ndominant role of organic–organic interactions in necromass stabilization. Specifically,\nN-rich bacterial necromass and chitin-rich fungal necromass can interact with pre-\nexisting organic matter on mineral surfaces through ionic, hydrogen bonds and\nhydrophobic interactions, resulting in a comparable degree of organic–organic\nassociation. We have now added these new results into the main text and discussed\nthese results in the context of other information in the literature.\n“Inconsistent with the second hypothesis, our results suggest that the source of\nmicrobial necromass is not a strong determinant of its retention in soil45,56, as bacterial\nand fungal necromass showed comparable recovery and mineralization rate constants.\nThis suggests that broad compositional differences were not decisive in governing\nnecromass fate in this study. Stabilization likely depends on finer-scale molecular\nproperties that are shared across necromass sources, such as their low molecular weight\nand abundance of polar functional groups10,56,57, which facilitate association with OM-\ncoated mineral surfaces36,44. This result implies that shifts in microbial community\ncomposition may influence necromass persistence primarily through changes in activity\nor efficiency rather than changes in the inherent sorption potential of the necromass\nproduced.” (Lines 242-251)\nFig. R5 Spatial distribution of retained necromass and native OM on mineral surfaces.\nComment: Line 257-261: I feel that this needs more explanation and discussion – it\nwas indeed one of the issues I wanted to raise wrt the experiments. Are the experiments\nstill relevant for evaluating in situ mineralisation rates? Does this aspect of the\nexperimental design affect the relative differences between the mineralisation rates of\nbacterial and fungal necromass or just the absolute rates.\nResponse: We thank the reviewer for raising this important point. The adsorption of\nmicrobial necromass by minerals and organic matter is a rapid process, typically\ncompleted within seconds to hours. Previous study has also shown that in short-term\nincubations (e.g., within 3 days), the majority of microbial necromass become\nassociated with mineral surfaces or other necromass. Accordingly, the necromass we\nadded would have been quickly adsorbed by minerals or organic matter. Furthermore,\nour fractionation results indicate that at the end of the incubation, most of the necromass\nwas retained within mineral-associated organic matter (~96.4% necromass 13C and\n93.4% necromass 15N), whereas only a small fraction stabilized within soil aggregate\n(~1.2% necromass 13C and 2.3% necromass 15N) (Fig. S6, same as below Fig. R6). The\ninitially added necromass were not protected by aggregates and therefore had only a\nminor influence on the experimental results. The high leaching observed in low-clay\nsoil is attributable to their relatively lower amount of micropores and soil surface charge\nresulting from the low clay content, as evidenced by the much lower leaching rates in\nhigh-clay soil (~7% necromass 13C and 7.5% necromass 15N) (Fig. 4). In summary,\nwhile absolute mineralization or leaching rates may differ from field conditions, our\nexperimental design reliably captures the influence of clay content and necromass\nsource on necromass preservation. These clarifications have now been incorporated\ninto the revision and list as follows:\n“Although our study provides clear mechanistic insights, several limitations\nshould be acknowledged. First, the injected necromass was not initially associated with\nminerals as naturally occurring necromass would be, making a portion more susceptible\nto early leaching. However, mineral adsorption appears to occur rapidly36, and\naggregate protection played only a minor role under our conditions, suggesting that this\neffect did not substantially bias the overall patterns of necromass retention and\ndecomposition. Consistent with this, leaching in the high-clay treatment quickly\ndeclined to low levels, with only about 7% of necromass-derived ¹³C and 7.5% of ¹⁵N\nmoving into the subsoil after 10 days, corresponding to a possible overestimation of the\ndecomposition rate constant by 0.068 and 0.073 yr⁻¹, respectively.” (Lines 261-269)\nReference:\n36. Buckeridge KM, et al. Sticky dead microbes: Rapid abiotic retention of microbial\nnecromass in soil. Soil Biol Biochem 149, 107929 (2020).\nFig. R6 Allocation of necromass-derived C and N to OM fractions.\nResponses to Reviewer #2:\nComment: Wang et al. carried out an experiment of intermediate nature in which\nsynthetic soils with three different clay contents were inoculated with microbial\nnecromass with labelled 13C and 15N and with living microorganisms, and the synthetic\nsoils were placed in an experimental plot for up to one year. Intermediate times were\nsampled to follow the progress of several variables. The final goal was to investigate\nwhether soil clay content or the type of necromass (bacterial or fungal) affected the\npreservation of this necromass. The authors conclude that clay caused a significant\nretention of necromass in the soil, and there is no difference between bacterial and\nfungal necromass.\nThe goals are of great interest, the experiments are carefully designed and there are\nnumerous analyses to constrain the results. However, as the result analyses demonstrate,\nthe experimental design would have required still further care to address all the possible\npitfalls that may invalidate results. I am left with the question whether the authors\nsimply or mainly measured transfer of OM down the column, so that, as expected, more\nOM remained where the clay content was greater; or whether their different amounts\nof clay genrated a different microbial population, which modified the results of the\nexperiments. The discussion is poor and shows in greater contrast the limitations and\npossible pitfalls of the study. In my opinion, the ms should be rejected.\nResponse: We sincerely thank the reviewer for your thorough assessment and\nconstructive comments. We greatly appreciate the recognition that our work is well-\ndesigned and timely, and we also acknowledge the reviewer’s concerns regarding\npresentation and potential limitations in our conclusions. Below, we provide\nclarifications and additional evidence addressing the reviewer’s concerns.\nOur experimental system was carefully designed not only to capture the variation\nin leaching, but also to disentangle the effects of adsorption processes and microbially\nmediated mineralization both driven by differences in clay content on necromass\nretention. Our results clearly demonstrate that necromass retention was strongly\nassociated with both microbial community characteristics and soil physicochemical\nproperties. Among these, bacterial community richness (Richness-B), soil moisture,\ntotal porosity, specific surface area (SSA), fungal community richness (Richness-F) and\nmicrobial metabolic quotient (qCO ) were identified as key predictors.\ncomposition of microbial communities under different clay content and assessed how\ntheir variations affected necromass retention. Additionally, we added new Nano-scale\nSecondary Ion Mass Spectrometry (NanoSIMS) and density fractionation analyses to\nbetter elucidate the role of clay content in adsorption processes. These results provide\nthe first direct evidence that necromass in soils is primarily preserved through\nassociated with organic matter already bound to these rough mineral surfaces, rather\nthan through directly adsorption onto the mineral surface. Soils with higher clay\ncontents retained more microbial necromass due to their greater abundance of rough\nmineral surfaces.\nFinally, we have thoroughly revised the Discussion section to provide a more\ncomprehensive interpretation of our findings and to explicitly address the study’s\nlimitations. We emphasize that these limitations have only a minor influence on our\nmain conclusions. We believe these revisions have substantially improved the clarity\nand robustness of the manuscript.\nComment: Lines 38-39. The definition of microbial necromass is unclear and causes\ntrouble with the experiments themselves. Should we understand that “plant inputs” are\n“plant products”? Is microbial necromass defined as plant products modified by\nmicroorganisms? If so, this is different from the microbial necromass used in the\nexperiments, which consists of organic debris of bacteria and fungi. This issue affects\nthe discussion in several places. First, it is not clear whether the authors are\nconsidering the transformation sequence as: living cells-necromass-mineralized OM or: living cells-\nmineralized OM-necromass. Second, the authors argue in relation to their microbial\nnecromass from results related to necromass from plant litter (lines 219-222), but do\nthese two types of necromass have similar molecular weights and charged moieties that\nwill affect their mobility within the soil?\nResponse: Thank you for pointing this out. We have redefined microbial necromass in\nthe revision as dead microbial biomass, including intact cells, cell fragments, and\nintracellular components released upon lysis. While plant-derived and microbial-\nderived necromass differ in molecular weight and chemical composition, flourier\ntransform infrared spectroscopy (FTIR) indicated that microbial necromass contains\nhigher proportions of aliphatic compounds and lower aromatic and polysaccharide\ncompounds compared to plant leaf litter (Fig. S15, same as below Fig. R7). Because\naliphatic compounds generally have lower molecular weight and polarity, microbial\nnecromass tends to exhibit higher mobility in soil than plant-derived necromass. We\nalso have revised the relevant accordingly:\n“Microbial necromass consists of the dead microbial biomass, including intact cells,\ncell fragments, and intracellular components released upon lysis” (Lines 41-42)\n“This substantial early leaching likely reflects the low molecular weight of\nmicrobial necromass, which confers high mobility54. Supporting this, FTIR analysis\nshowed that the microbial necromass contained higher proportions of low-molecular-\nweight lipid compounds and lower proportions of high-molecular-weight aromatic and\npolysaccharide compounds (Fig. S15) compared with plant litter55” (Lines 228-232)\nFig. R7 Biochemical characteristics of microbial necromass and plant leaf. Relative\nabundance of functional groups in bacterial and fungal necromass (this study) and\nBeech (Fagus sylvatica), lime (Tilia cordata), maple (Acer pseudoplatanus), and ash\n(Fraxinus excelsior) leaf litter (data from Yang and Butenschoen¹) determined by\nFourier transform infrared spectroscopy (FTIR).\nComment: The authors call “mineralization rate” what really is the “mineralization\nrate constant”. In a chemical process the rate is proportional to the rate constant, but\nboth are not identical. The rate constant will multiply the reagent concentrations and/or\nsome other variable(s) affecting the chance of the reagents to come in contact and react.\nThis mistake or misconception is found in the entire paper.\nResponse: We appreciate the reviewer’s insightful clarification. We agree that\n“mineralization rate” and “mineralization rate constant” are not interchangeable, and\nthat our study quantifies the first-order rate constant of necromass mineralization rather\nthan the mineralization rate itself. To ensure conceptual accuracy, we have replaced all\ninstances of “mineralization rate” with “mineralization rate constant” throughout the\nmanuscript and figure legends.\nComment: The tables in the supplementary information about statistical analysis\nprovide the p values of the ANOVA tests, but do not provide any information on\nwhether the correlations between variables were positive or negative. We learn about\nsome of the variable pairs in some of the figures, but not about the others. This leaves\nimportant information untold. For example, in Table S5, the correlation between\npreserved N and C in microbial biomass with clay contents has a very low p and, as we\nknow from other parts of the article, the correlation is positive. If this is the case, we\nwould expect clay contents to correlate negatively with dissolved N and C, and with\ninorganic N. However, the high p values for the latter suggest that there is no\ncorrelation. In any case, do dissolved C and N increase with clay content? Apparently,\nFig. 5 contains relevant information, which is not mentioned or discussed.\nResponse: We thank the reviewer for this insightful comment. We have included a\ncorrelation analysis between necromass retention, its fate, and the influencing factors\nto provide a clearer understanding of these relationships (See below Fig. R8).\nOur results showed that the recovery in dissolved organic matter was minimal,\ndeclining from 0.38% (±0.06%) of 13C and 0.10% (±0.01%) of 15N at day 10 to 0.09%\n(±0.02%) of 13C and 0.02% (±0.004%) of 15N at day 386, with 13C slightly increasing\nand 15N decreasing with clay content (P = 0.091 and 0.030; Fig. 4; Fig. S3, S4; Table\nS5, S6). We have now added these new results into the main text and discussed these\nresults in the context of other information in the literature. (Lines 140-143)\nFig. R8 Investigate the correlation between necromass retention, its fate, and\ninfluencing factors after 386 days of in situ incubation. Heatmap of correlation\nbetween added necromass 13C and 15N retention, its recovery in necromass C and N\n(13C-necromass C, 15N-necromass N), microbial biomass C and N (13C-MBC, 15N-\nMBN), dissolved organic C and N (13C-DOC, 15N-DON), subsoil C and N (13C-subsoil\nC, 15N-subsoil N), CO (13C-CO ) and inorganic N (15N-inorganic N) and Richness-B\n2 2\nand Richness-F (bacterial and fungal community richness), PCoA1-B and PCoA1-F\n(bacterial and fungal community compositions), qCO (microbial metabolic quotient),\n2\nsoil moisture, Total porosity, SSA (specific surface area), AFP (air-filled porosity), and\nbulk density. Significance levels are indicated as follows: •, P < 0.1; *, P < 0.05; **, P\n< 0.01; ***, P < 0.001.\nComment: The presentation of the PCA results (lines 172-174) is too brief and does\nnot discern between the meaning of the alignment of the several variables. The authors\nare trying to identify the drivers of necromass persistence. These can be only SSA,\nWHC and SM. The other variables aligning with High clay content are an effect of the\npreservation, and not its cause.\nResponse: We thank reviewer for this constructive comment. In the revised manuscript,\nwe have provided a more detailed discussion of the relationships among variables,\nemphasizing how clay content influences these variables and, in turn, affects necromass\nretention. We also performed a hierarchical partitioning analysis to quantify the relative\ncontributions of these factors to necromass retention. The text has been revised\naccordingly.\n“The retention of the added necromass 13C and 15N was closely related to the living\nmicrobial community characteristics and soil properties (Fig. 5a, b). Furthermore,\nbacterial community richness (Richness-B), soil moisture, total porosity, specific\nsurface area (SSA), fungal community richness (Richness-F) and microbial metabolic\nquotient (qCO ) were the most important predictors (Fig. 5b). The retention of the\n2\nadded necromass 13C and 15N was positively correlated with soil moisture and SSA, but\nnegatively correlated with richness-B, richness-F and qCO (Fig. 5c; Fig. S3).\n2\nporosity (AFP), but declined with soil moisture (Fig. 5a). Both bacterial and fungal\nrichness showed the same pattern, increasing with AFP and decreasing with soil\nmoisture (Fig. 5a). Across the clay gradient, soil moisture, SSA, and total porosity\nincreased with clay content, whereas AFP, qCO , bulk density, and microbial richness\n2\nall decreased (Fig. 5a).” (Lines 162–173)\nThe discussion section has important problems that make it weak and inconclusive:\nComment: 1) The entire discussion appears to be a speculation, as stated by the authors:\n“may be”, “we speculate”, etc. The study gathered a lot of quantitative data that are not\nused to carry out a quantitative discussion.\nResponse: Agreed. In the revised manuscript, we strengthened the discussion by\nincorporating additional quantitative evidence derived from our correlation and\nvariance partitioning analyses, as well as newly added NanoSIMS and density\nfractionation data. These results provide clearer numerical support for the proposed\nmechanisms, reducing speculative language and enhancing the robustness of our\ninterpretations.\nComment: 2) Lines 187-189 are a repetition of ideas mentioned in the introduction and\nthey are not further discussed. Are there new ideas for the discussion?\nResponse: Good comment! In the revised manuscript, we have revised this passage and\npresented new insights based on the NanoSIME results as follows:\n“Our quantitative NanoSIMS imaging revealed that almost the newly accumulated\nnecromass C and N was associated with OM already bound to these rough mineral\nsurfaces, whereas only a minor fraction occurred on bare mineral surfaces. Compared\nwith smooth mineral surfaces, rough mineral surfaces exhibited stronger 16O− ion\nintensities, indicative of enrichment in hydroxyl groups, oxides, and hydrated layers\nthat offer abundant reactive sites for organic matter association18,48,49. High-clay soils\ncontain more rough mineral surfaces, which promote the formation of mineral-OM\nassociations and provide more reactive sites for new necromass preservation. These\nresults imply that, although only a limited proportion of clay mineral surfaces involved\nin necromass sequestration, they can promote the formation of multilayered, onion-like\nstructures via the mineral-OM-necromass association, thereby enabling the\nstabilization of substantial microbial necromass. This mechanistic insight complements\ntraditional sorption-based concepts by showing that organo-organic interactions on\nOM-coated mineral surfaces are key pathways for new necromass accumulation44.”\n(Lines 199–211)\nComment: 3) 191-192: These two lines are the statement of the discrepancy, not a\npossible explanation of why it takes place.\nResponse: Thanks! We have now removed the statements in Lines 191-192 and revised\nthe text to provide a mechanistic explanation centered on clay-necromass associations.\nComment: 4) Line 195: where is it shown that there is an increase in soil C?\nResponse: We thank the reviewer for pointing this out. We have removed the\ncorresponding statement on soil C from the text.\nComment: 5) Line 207: Why is survival only linked to mineralization? Reproduction\nalso needs energy and respiration, with consequent CO loss.\nmicrobial survival and reproduction require energy and involve respiratory CO loss.\nexhibit higher nutrient use efficiency, allowing them to allocate a greater proportion of\nassimilated necromass to biomass synthesis rather than to mineralization losses. In the\nrevised manuscript, we have changed this statement “This means that the necromass C\nand N assimilated by microbes are allocated more to reproduction than survival (i.e.,\nless mineralization but more biomass).”\nto\n“Instead, the higher soil moisture under high clay we observed likely increased\nmicrobial nutrient use efficiency by reducing substrate diffusion constraints (Fig. S7;\nTable S7, S8)50,51.” (Lines 213-215)\nComment: 6) The idea that water content may affect gas diffusion is reasonable.\nHowever, what mathematical expression would represent such effect: linear,\nexponential, …? In other words, how can we relate the data in figure S5 with the data\nin figures 2, 3, S4, etc?\nResponse: Excellent suggestions! We have added an analysis of soil porosity\ncharacteristics (below Fig. R9), which shows that increasing clay content leads to a\nrapid rise in water content and a concomitant decline in air-filled porosity (AFP).\nWe also analyzed the relationship between AFP and necromass retention and respiratory\nloss (below Fig. R10). These analyses indicate that the decrease in air-filled porosity\nmay limit oxygen diffusion within the pore network, thereby suppressing microbial\nactivity and respiration and necromass decomposition. The text and figure have been\nupdated accordingly as follows:\n“Elevated moisture also sharply reduced air-filled porosity (Fig. S8), restricting\noxygen diffusion and consequently suppressing microbial activity)52, respiration53, and\nnecromass decomposition (Fig. S9, S10).” (Lines 215-218)\nFig. R9 Soil porosity. (a) Total porosity and (b) air-filled porosity of low-, medium-\nand high-clay soils after 386 days of in situ incubation. Values represent means and\nstandard error (n = 3). Different lowercase letters indicate significant differences among\nclay treatments.\nFig. R10 Linkage between air-filled porosity (AFP) and necromass retention and\nrespiratory loss. Linear regressions (n = 18) between air-filled porosity (AFP) and (a)\nnecromass 13C, (b) 15N retention, (c) necromass respiratory loss, (d) necromass derived-\nCO -13C efflux rate, (e) CO -C efflux rate, (f) microbial biomass C (MBC), and (g)\n2 2\nmicrobial metabolic quotient (qCO ) after 10 days or 386 days of in situ incubation.\nmay explain all the data. The increase of clay modifies the microbial community, which\nis what controls many of the other modifications observed. This issue, however, is not\nfollowed or discussed further.\nResponse: We appreciate the reviewer’s insightful comment. In the revised manuscript,\nwe provided the changes in microbial composition (below Fig. R11) and analyzed the\nrelationships among microbial composition with microbial necromass retention (below\nFig. R12). we have expanded the discussion to emphasize that clay-induced changes in\nmicrobial communities are a key driver of necromass stabilization and list as follows:\n“These oxygen-limited conditions further reduced bacterial diversity and\ndecreased the abundance of copiotrophic taxa such as Firmicutes and Actinobacteria\nthat are involved in the turnover of necromass-derived polysaccharides and proteins29,\ncollectively reinforcing necromass preservation (Fig. S11-13)” (Lines 218-221)\nFig. R11 Relative abundance of microbial community across soils. Relative\nabundance of the ten most abundant phyla for (a) bacterial and (b) fungal community\nin low-, medium- and high-clay soils after 386 days of in situ incubation (n = 3).\nFig. R12 Relationships among necromass retention, soil properties, microbial\nactivity, and microbial community composition after 386 days of in situ incubation.\nPrincipal component analyses (PCA) of necromass retention, soil properties, microbial\nactivity, and (a) bacterial or (c) fungal community composition (n = 6). Correlations\nbetween necromass retention, soil properties, microbial activity, and (b) bacterial or (d)\nfungal community composition (n = 6). AFP, air-filled porosity; qCO₂, microbial\nmetabolic quotient; MBC, microbial biomass C; DOC, dissolved organic C; DON,\ndissolved organic N.\nComment: 8) Lines 216-219. The observed rapid leaching of necromass from the\nsynthetic soil to the underlying subsoil would depend on precipitation (rain) during the\nexperiment. Did the authors measure precipitation during the experiment? This may be\na crucial point.\nResponse: We appreciate the reviewer’s constructive comment. Precipitation and\ntemperature were recorded during the field incubation experiment. On the fifth and\nsixth days after necromass addition, 12 mm and 2 mm of rainfall occurred, respectively\n(Fig. S14, see below Fig. R13), which may have contributed to the leaching of\nnecromass in the low-clay soils with lower water-holding capacity. These data have\nbeen included in the revised manuscript, and we have noted in the text that precipitation\nevents likely contributed to the observed downward movement of necromass to the\nsubsoil. (Lines 226-227)\nFig. R13 Precipitation and temperature conditions during the first month after\nmicrobial necromass addition.\nComment: 9) Line 224: “the low leaching amounts…” Based on Fig. 4b,d (360 day\nexperiments), this statement requires qualification: can one say that the values of\nleached C and N (that I take to be those in subsoil) in the high-clay experiments are\nlower than those in the low- and medium-clay experiments?\nResponse: Thank you for pointing this out. In the revised manuscript, we have changed\nthis statement “The significant reduction in necromass in the subsoil at day 386\ncompared to day 10, likely due to leaching into deeper soil, further supports our\nstatement.” to\n“The pronounced reduction in necromass in the subsoil of low-clay soils from\nday 10 to day 386 indicates continued leaching into deeper soil layers, further\nsupporting our conclusion.” (Lines 234-236)\nComment: 10) Line 232: “accessibility and availability” does not correspond to what\nis explained in the following lines.\nResponse: In the revised manuscript, we have removed this statement and discussed\nthe absence of differences in the retention of bacterial and fungal necromass from the\ndegree of association between necromass and minerals, as revealed by the NanoSIMS\nresults. Corresponding modifications have been made in the revised manuscript and list\nas follows:\n“Inconsistent with the second hypothesis, our results suggest that the source of\nmicrobial necromass is not a strong determinant of its retention in soil45,56, as bacterial\nand fungal necromass showed comparable recovery and mineralization rate constants.\nThis suggests that broad compositional differences were not decisive in governing\nnecromass fate in this study. Stabilization likely depends on finer-scale molecular\nproperties that are shared across necromass sources, such as their low molecular weight\nand abundance of polar functional groups10,56,57, which facilitate association with OM-\ncoated mineral surfaces36,44. This result implies that shifts in microbial community\ncomposition may influence necromass persistence primarily through changes in activity\nor efficiency rather than changes in the inherent sorption potential of the necromass\nproduced.” (Lines 242-251)\nComment: 11) Lines 233-235: Why does higher N content in the necromass make it\nmore readily used by microorganisms? Also, this assertion contradicts that in lines 249-\n250, that N-rich necromass was more resistant to mineralization.\nResponse: Sorry for this incorrect statement. Our intention was to indicate that there is\na positive relationship between the N content and the utilization preferences of\nmicroorganisms under nitrogen limitation conditions60. To avoid potential\nmisunderstanding, we have removed the corresponding statement in the revised text.\nReference:\n60. Mooshammer M, et al. Adjustment of microbial nitrogen use efficiency to\ncarbon:Nitrogen imbalances regulates soil nitrogen cycling. Nat Commun 5, 3694\n(2014).\nComment: 12) Lines 238-240. This statement contradicts the previous one in lines 230-\n231, that there is no significant retention difference between bacterial and fungal\nnecromass.\nResponse: We thank the reviewer for pointing this out. Our intention was to emphasize\nthe change in necromass retention resulting from the increase in clay content from 20%\nto 60%. However, since there was no overall difference in the persistence of bacterial\nand fungal necromass, we have removed the original statement in the revised\nmanuscript to prevent any potential misunderstanding.\nComment: 13) Lines 252-254: can the authors explain why these facts indicate N\nlimitation in the synthetic soils?\nResponse: In our model soils, the occurrence of N limitation is supported by multiple\nobservations. First, the soil C:N ratio exceeds the commonly recognized threshold of\n~20, above which microbial growth typically shifts from C limitation to N limitation62.\nUnder such conditions, microorganisms retain more nitrogen from decomposed\nmicrobial necromass for their own biomass synthesis, leading to increased necromass-\nderived N accumulation. Consistently, we observed that only a small fraction of\nnecromass N was recovered in the inorganic N pool, further indicating the microbial N\nlimitation in the model soils. Corresponding modifications have been made in the\nrevised manuscript and list as follows:\n“The C:N ratio of our model soils exceeded the commonly recognized threshold\nof ~20 (Table S9), above which microbial growth typically shifts from C limitation to\nN limitation59. Under such N-limited conditions, microorganisms preferentially retain\nN released from necromass decomposition to support biomass synthesis, resulting in\ngreater necromass-derived N accumulation60. Consistently, only a small fraction of\nnecromass N (0.23-0.65%) was recovered in the inorganic N pool (Fig. 4), further\nindicating microbial N limitation in our model soils.” (Lines 253-259)\nReference:\n59. Mooshammer M, Wanek W, Zechmeister-Boltenstern S, Richter A. Stoichiometric\nimbalances between terrestrial decomposer communities and their resources:\nmechanisms and implications of microbial adaptations to their resources. Front\nMicrobiol 5, 22 (2014).\n60. Mooshammer M, et al. Adjustment of microbial nitrogen use efficiency to\ncarbon:Nitrogen imbalances regulates soil nitrogen cycling. Nat Commun 5, 3694\n(2014).\nComment: 14) The limitation mentioned in lines 257-261 appears as a potentially\nserious one that may invalidate the data. As the authors have the values of leached\nnecromass, can they try to discuss this issue quantitatively, rather than pointing it out\nas an afterthought? How does clay arrange itself in sandy soils? How about the silt and\nclay already present in the sand before the addition of the smectite? (see below).\nResponse: We thank the reviewer for raising this important point. To address the\npotential concern regarding the method of necromass addition, we examined the\nleached necromass data and found that at day 10 necromass leaching significant\ndecreased with clay content; in high-clay treatment, only about 7% of 13C and 7.5% of\n15N were leached to the subsoil. This indicates that the majority of necromass leaching\nin the low-clay treatment is primarily determined by the intrinsic properties of the soil\nrather than the method of necromass addition. Consequently, our estimates of\nnecromass C and N stability may be slightly underestimated by ~7 and ~7.5%,\nrespectively, and the decomposition rate constant of necromass may be overestimated\nby 0.068 and 0.073 yr⁻¹, respectively. These quantitative considerations have now been\nincorporated into the revised manuscript and list as follows:\n“However, mineral adsorption appears to occur rapidly36, and aggregate protection\nplayed only a minor role under our conditions, suggesting that this effect did not\nsubstantially bias the overall patterns of necromass retention and decomposition.\nConsistent with this, leaching in the high-clay treatment quickly declined to low levels,\nwith only about 7% of necromass-derived ¹³C and 7.5% of ¹⁵N moving into the subsoil\nafter 10 days, corresponding to a possible overestimation of the decomposition rate\nconstant by 0.068 and 0.073 yr⁻¹, respectively.” (Lines 264-269)\nWe also appreciate the reviewer’s question regarding the arrangement of clay in\nthe sandy matrix. Upon reviewing the experimental records, we found that the actual\n“silt-sized” and “sand-sized” quartz fractions used in this study were 50-250 μm and\n250-2000 μm, respectively. The quartz sand was analytical-grade pure quartz, which\nwas ground and sieved to obtain these two size fractions. These fractions were\nsubsequently mixed with clay-sized montmorillonite, allowing the added clay to\ndisperse among the sand particles and partially mimic its spatial arrangement in natural\nsandy soils.\nMethod\nComment: The methods are difficult to follow due to the complexity of the experiments\nand lack of clarity in the text. I would advise to use a temporal sequence, rather than\nthe present distribution based on the several elements of the experiments.\nResponse: We thank the reviewer for the helpful suggestion. The experimental flow\ndiagram has been reorganized following a temporal sequence (now Fig. 1), with\nadditional explanations added in the text to clarify the experimental procedures.\nComment: The quartz “sand” had a 30% of silt. This is a high value. Did the authors\ninvestigate the mineralogical composition of the silt? It may have had some clay. How\nis it possible that both the sand-sized quartz and the silt-sized quartz had the same\nSSA (Table S9)? By definition, for the same substance, smaller particle size means higher\nSSA. Was the SSA controlled by some mineral other than quartz? The SSA of 0.17 m2\n/ g is low for quartz, although the 74 m2 / g for montmorillonite seems correct. There\nis no indication of how these SSA values were measured.\nResponse: We thank the reviewer for this pointing out and apologize for the previous\ninaccurate description. As noted in our previous response, the actual “silt-sized” and\n“sand-sized” quartz fractions used in this study were 50-250 μm and 250-2000 μm,\nrespectively. The quartz sand was analytical-grade pure quartz, which was ground and\nsieved to obtain these two size fractions. The quartz sand was separated into these two\nsize fractions, excluding very silt, to simulate the natural soil’s particle-size\ncomposition while minimizing interference with the clay fraction’s experimental\neffects. The SSA of the model soil materials and bulk model soils were determined\nusing a standard method for measuring surface area in soils based on the physisorption\nof N gas on a sample at 77 K using the Brunauer-Emmett-Teller equation. Because\n2\nboth two quartz sand fractions are relatively coarse, they exhibit similarly low specific\nsurface areas (~0.170 and 0.167 m2 g-1), consistent with previous studies reporting ~0.1\nm² g-¹ for quartz sand7 (0.1 m2 g-1). These details have now been clarified in the revised\nMethods section and list as follows:\n“The quartz sand (composed of 30% silt- and 70% sand-sized particles; sourced\nfrom Aladdin) was cleaned in diluted acid and combusted at 500 ℃ to remove organic\ncarbon and then ground and sieved to fine (50-250 μm) and coarse (250-2000 μm)\nparticle sizes. Detailed properties of the model soil materials are listed in Table S9. A\ntypical expandible 2:1 layer silicate clay (montmorillonite; sourced from Sigma-\nAldrich) was mixed with quartz sand in varying proportions to form low-clay soil\n(sandy loam soil; 20% clay, 24% fine quartz, and 56% coarse quartz), medium-clay soil\n(clay loam soil; 40% clay, 18% fine quartz and 42% coarse quartz) and high-clay soil\n(clay soil; 60% clay, 12% fine quartz and 28% coarse quartz).” (Lines 320-327)\nComment: There is no indication whether the cylinders in the experiments were open\nor closed at the bottom; i.e., were the synthetic soils and sampled subsoils in contact or\nsomehow connected with the soils outside the cylinders?\nResponse: During the experiment, both the top and bottom of the PVC cylinders were\nopen, and the model soils were separated from the surrounding natural soil by a 1 mm\nnylon mesh, which allowed physical contact while preventing direct mixing. These\nclarifications have also been incorporated into the revised manuscript. (Lines 364-366)\nSpecific issues\nComment: Lines 77-78 are unclear and confusing. High clay content would cause\nhigher water retention, and the latter cause a decrease of oxygen diffusion; I am not\naware that dry clay can decrease air diffusion to the point of generating reducing\nconditions. Further, how does higher water content cause necromass desorption from\nthe clay?\nResponse: Thanks for bringing up this point. In the revised manuscript, we have\nchanged this statement “These smaller pore sizes further decrease O diffusion and\n2\navailability, inhibiting microbial activity and potentially leading to slower\ndecomposition of necromass.” To\n“Additionally, reduced oxygen diffusion in moisture-rich, high-clay soils can\nenhance reducing conditions and promote partial Fe(III) reduction, weakening mineral\nsorption and altering necromass mobility25” (Lines 57-60)\nComment: Lines 105-106 need to be rewritten for clarity.\nResponse: In the revised manuscript, we have changed this statement “The model soils\nwere artificially constructed mixtures of clay minerals, quartz sand, and plant litter,\nwith initially the same organic matter and clay mineral composition but varying clay\ncontent.” to\n“We constructed artificial soils with contrasting clay contents by mixing\nmontmorillonite clay, quartz sand, and plant litter while maintaining identical organic\nmatter input and clay mineral.” (Lines 89-91)\nComment: Line 113: “rapidly”. Not really because the % values are small. The authors\nperhaps mean that the necromass was initially consumed rapidly but the rate of\nconsumption soon decreased substantially.\nResponse: In the revised manuscript, we have changed this statement “The added\nnecromass was rapidly mineralized by microorganisms (Fig. 2a and c), with 13C-CO\n2\nflux peaking at 3-18 days and then declining (Fig. S1).” to\n“When necromass was added in field soil, it was rapidly utilized by\nmicroorganisms during the initial stage of incubation (Fig. 2a, c), as reflected by the\npeak in 13C-CO flux at 3-18 days. After this peak, the mineralization rate decreased\n2\nsubstantially (Fig. S1).” (Lines 108-111)\nComment: Lines 124-125. Wrong expression: ratio of retained C to retained N means\nC/N, which is always <1 in Fig. S2 (i.e., N>C).\nResponse: We appreciate the reviewer’s careful reading. In the revised manuscript, we\nhave changed this statement “The ratios of retained added necromass 13C to 15N for all\nclay contents were above 1 (Fig. S2).” to\n“Accordingly, 15N :13C retention ratios were > 1 for all clay contents (Fig. S2).”\n(Lines 121-122)\nComment: The section “Fate of microbial necromass” is poorly presented, with very\nconvoluted, repetitive phrases difficult to follow. I would advise to simplify the phrases,\nto focus first on the main points for clarity, and finally move to the interesting details.\nLines 166-168 do not make sense.\nResponse: We thank the reviewer for the constructive comment. We have revised the\n“Fate of microbial necromass” section to improve clarity and logical flow. The revised\ntext now first presents the main observations on necromass retention, microbial\nincorporation, and leaching, followed by detailed patterns across clay and necromass\ntreatments and over time. Redundant and convoluted phrases have been removed, and\nall numerical data are presented clearly with consistent statistical references. (Lines\n131-154)\nComment: Lines 289-290. It would be better to provide the soil taxonomic information\nfollowing one of the standard methods or to give a more complete description.\nResponse: We appreciate the reviewer’s suggestion. The studied soil is classified as a\nHaplic Luvisol according to the World Reference Base for Soil Resources (WRB, 2022).\nIt features a brown to dark brown topsoil layer and exhibits pronounced clay illuviation\nin the subsurface (Bt horizon) developed from volcanic ash. This classification has been\nadded to the revised manuscript and list as follows:\n“The soils are Albic Luvisols developed from volcanic ash61.” (Lines 294-295)\nComment: Lines 326-327: density is mass/volume, not volume/mass.\nResponse: Thank you for pointing this out. We have corrected the statement as follows:\n“The height and bulk density of the model soils were ~5.5 cm and 0.75 g−1 cm3,\nrespectively.” (Lines 334-335)\nComment: Line 353: at what depth was the subsoil collected?\nResponse: The natural subsoil used to fill the upper part of the PVC tubes was collected\nfrom a depth of 5.5–14 cm. This information has been added to the revised manuscript.\n(Lines 361-362)\nComment: Some of the figures have incomplete information in the captions. There are\nsome errors such as the use of “loss” for “lost”. Axes titles are sometimes wrong or\ninexact.\nResponse: We appreciate the reviewer’s careful reading. We have carefully revised all\nfigure captions to ensure they provide complete and accurate information. Specifically,\nwe corrected the use of “loss” to “lost” where appropriate, checked and updated axes\ntitles for accuracy, and clarified any missing or ambiguous details. These revisions have\nbeen implemented throughout the manuscript to improve clarity and consistency.\nComment: Figure 2 is confusing. The coloured surfaces suggest that the data are\nrepresented by the surfaces, while in reality the authors intend that the data are those\nrepresented by the lines at the top of each surface. The plots need to be changed to\nsingle lines like those in Fig. 3a,b. Further, how is the “respired necromass” calculated?\nIs it calculated from 13C in CO2? If such is the case, Figs. 2 and 3 are incompatible.\nResponse: We thank the reviewer for the valuable comments. We have revised Figure\n2 to display single-line plots, similar to those in Figure 3a,b, to clearly represent the\nmeasured data. Respired necromass was calculated from 13C recovered as CO -C using\n2\nlinear interpolation between sampling points. To ensure consistency, we have revised\nthe figure legends and related text. For clarity, Figure 2 shows the cumulative carbon\nlost as CO from the added necromass, while Figure 3 shows the amounts of necromass-\n2\nderived carbon and nitrogen remaining in the model soils. The sums of the two do not\nequal 100% due to downward leaching into subsoil layers or absorbed by plants (for N).\nComment: As stated before, the y-variable in Fig 3c is not mineralization rate, but rate\nconstant of the mineralization process.\nResponse: Thank you for the comment. The y-axis label in Fig. 3c and its legend have\nbeen corrected to indicate the rate constant of the mineralization process.\nComment: Fig. 4: “Fate” is not a physical or chemical variable that can be represented\nin a plot. The values Necromass C and N, MBC, MBN, DOC, TDN and IN are\nsupposedly to refer to the synthesized soil, but this is not indicated anywhere. The\nresults are not presented clearly.\nResponse: We thank the reviewer for the comment. Figure 4 and its caption have been\nrevised accordingly. “Fate of microbial necromass” has been replaced with “Allocation\nof necromass 13C and 15N among soil pools in the PVC tubes”, and it is now clearly\nindicated that necromass C and N, MBC, MBN, DOC, TDN, and IN refer to the model\nsoils.\nResponse to Reviewer #1:\nComment: I think that the authors have done a good job addressing my comments. In\nparticular i think that their improved focus on mechanistic insights to explain their,\nsometimes expected, observations presents an important advance for understanding\nSOM dynamics. I would be pleased to see this manuscript accepted for publication.\nI recommend that the whole manuscript is carefully checked for English language -\nthere are a few places where sentence structure might be improved, and there appear to\nbe some missing words or missing plurals here and there, also the tense needs\nstandardising in places (to either past or present). In particular the paragraph starting at\nL199.\nI guess this is a stylistic comment but i would avoid claiming \"firsts\" (even if these are\nto the authors' knowledge) - in this respect i think L192 reads oddly and should be\nrephrased. This sentence was possibly instructive for reviewers during the\nreview process, but if this work is accepted for publication in Nature Communications,\nreaders will already appreciate that it is presenting something new that makes an important\ncontribution to the literature, without having to explicitly claim \"firsts\".\nResponse: We sincerely thank the reviewer for the careful evaluation of our revised\nmanuscript and for the positive assessment of our efforts to strengthen the mechanistic\ninterpretation of our findings.\nWe have carefully checked the entire manuscript for English language and grammar,\nwith particular attention to sentence structure, plural forms, and tense consistency. In\nparticular, the paragraph beginning at former L199 has been carefully rewritten to\nimprove clarity and readability.\nWe have removed the explicit claim of being the “first study” and rephrased the\ncorresponding sentence as follows: “Our in situ necromass incubation experiment\nshowed that higher clay content promoted the retention of necromass 13C and 15N in\nsoil.” (Lines187-188)\nWe thank the reviewer again for the constructive comments, which have helped us\nfurther improve the clarity and presentation of the manuscript.\nROUND 1 REVIEWER 1 ATTACHMENT:\nThis is an important study to investigate the effect of clay content on microbial necromass\npersistence, in experiments conducted in situ in real soils. I think that the work is interesting to a\nbroad readership, but I think the authors miss an opportunity to provide new information on why\nhigh clay content increases microbial necromass persistence. Based on previous work I feel that\nthe take home message that high clay content increases microbial necromass persistence is rather\nexpected, and the work as it stands serves as a confirmation of this expectation, rather than\nproviding a substantial advance in understanding of this expectation. For this reason, I think the\nwork as it stands is perhaps not ready for publication in Nature Communications but could be with\nsome extra analyses that allow the authors to more rigorously interrogate some of the speculative\nexplanations that they provide for their results. I have some more minor comments on the\nintroduction and results below, and then I exemplify my main point at several sections of the\ndiscussion below.\nIntroduction:\nLine 22: clay minerals are not usually described as the most reactive soil particles (metal\n(oxyhydr)oxides are much more reactive and play a disproportionately important role in elemental\ncycling in soils), but they are among the most reactive soil particles – so I suggest rephrasing as: “as\namong the most reactive soil particles”\nLine 48: adsorption not absorption\nLine 50: how does soil clay content affect the living microbial community and why does this\nsuggest that soils with high clay content could alternatively exhibit lower necromass persistence? –\nthis is later explained in lines 68-82 but I feel there needs to be better connection between this\nstatement and the eventual explanation later on.\nLine 53: do the authors mean clay minerals are characterized by their high charge and specific\nsurface area? (also their SSA is nowhere near as high as iron (oxyhydr)oxides, for example).\nLine 97: I feel that a short description of the field experiments using model soils is warranted here\n– its quite difficult in the methods to follow what was actually done – whilst the experiments were\nconducted in situ in a field plot, the authors use model artificial soils, so I think that this needs to\nbe clearly explained – a flow diagram or some sort of schematic in the main text or supplementary\ninformation would be really helpful to show the reader what was done (Fig 1 and Fig S8 are helpful\nbut two separate figures makes it difficult to follow – I feel that these could be combined).\nLine 88 and 101: whilst the higher N content of bacterial necromass may also result in tighter\nbinding to minerals, the chitin content of fungal necromass is known to degrade into carboxyl-\ncontaining compounds which adsorb very strongly to soil minerals, so perhaps this needs to be\nconsidered wrt this hypothesis – at least upon its introduction around line 88.\nResults:\nLine 115: how much of the added C was partially mineralised (to DOC)? – I think this would be\ninstructive to add at this point of the results.\nLine 166: this sentence doesn’t make sense – “Biochemical characterization using FTIR analyses\nindicated that had a higher relative abundance of polysaccharides and a lower relative abundance\nof polysaccharides compared to fungal necromass (Table S1).” – I think that Table S1 shows that\nbacteria necromass has a lower abundance of polysaccharides compared to fungal necromass?\nDiscussion:\nLine 177-198: concerning the main result that as clay content increases, a greater proportion of the\nadded necromass 13C and nitrogen 15N is retained in the model soil, despite the concurrent\nincrease in microbial biomass: whilst this may be the first study to demonstrate this outcome in\nsitu, which is important (at least to the extent that this is truly an in situ experiment), I think that\nthe outcome is to be expected based on previous work, so I feel that this work misses an\nopportunity to provide new information on why this expectation holds true in situ in soils. I think\nthat the authors do a decent job in providing potential explanations for their result (their language\nis necessarily speculative), but they are unable to pinpoint the actual explanation without\nmore detailed examination of the clay-necromass association. In this regard I feel that the\noverarching take home message is an important one, but is rather expected, and as such the work at\nthe moment probably does not provide as substantial an advance in understanding as it could do.\nLine 216-229: concerning the result that there was substantial leaching of necromass: did the\nauthors measure / approximate molecular weight of the necromass and/or measure the pore\nvolume of the clays? – this would provide validation of their speculative explanations.\nLine 240-241: concerning the result that bacterial and fungal necromass exhibited similar\nmineralization rates: this section and exemplified at the cited lines is very speculative. I feel that\nthis work would make a much stronger contribution to the literature if the authors provided a\nmore in-depth analysis of the mineral-necromass associations – the speculation that bacterial\nnecromass is more strongly adsorbed by minerals than fungal necromass need not be speculation\n– there are many wet chemical techniques that are routinely used to operationally define strength\nof adsorption that could be used here, and/or direct observational techniques, including FTIR that\nthe authors already employ elsewhere in the work.\nLine 257-261: I feel that this needs more explanation and discussion – it was indeed one of the\nissues I wanted to raise wrt the experiments. Are the experiments still relevant for evaluating in\nsitu mineralisation rates? Does this aspect of the experimental design affect the relative\ndifferences between the mineralisation rates of bacterial and fungal necromass or just the\nabsolute rates?"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I think that the authors have done a good job addressing my comments. In particular i think that their improved focus on\nmechanistic insights to explain their, sometimes expected, observations presents an important advance for understanding\nSOM dynamics. I would be pleased to see this manuscript accepted for publication.\nI recommend that the whole manuscript is carefully checked for English language - there are a few places where sentence\nstructure might be improved, and there appear to be some missing words or missing plurals here and there, also the tense\nneeds standardising in places (to either past or present). In particular the paragraph starting at L199.\nI guess this is a stylistic comment but i would avoid claiming \"firsts\" (even if these are to the authors' knowledge) - in this\nrespect i think L192 reads oddly and should be rephrased. This sentence was possibly instructive for reviewers during the\nreview process, but if this work is accepted for publication in Nature Communications, readers will already appreciate that it\nis presenting something new that makes an important contribution to the literature, without having to explicitly claim \"firsts\"."}, "author_response": ""}]

Decision letter