# The role of earthworms in the transformation of labile plant litter into mineral-associated organic matter

Chao Song, Yacouba Zi, Marie-France Dignac, Nicolas Bottinelli, Axel Felbacq, Shanshan Song, Cornelia Rumpel

► To cite this version:

Chao Song, Yacouba Zi, Marie-France Dignac, Nicolas Bottinelli, Axel Felbacq, et al.. The role of earthworms in the transformation of labile plant litter into mineral-associated organic matter. Soil Biology and Biochemistry, 2025, 208, pp.109859. 10.1016/j.soilbio.2025.109859. hal-05328222

HAL Id: hal-05328222

https://hal.inrae.fr/hal-05328222v1

Submitted on 23 Oct 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
The role of earthworms in the transformation of labile plant litter into
mineral-associated organic matter

Check for
updates

Chao Song<sup>a,b,c,1</sup>, Yacouba Zi<sup>c,1</sup> <sup>●</sup>, Marie-France Dignac<sup>c</sup>, Nicolas Bottinelli<sup>c,d</sup> <sup>●</sup>, Axel Felbacq<sup>c</sup> <sup>●</sup>, Shanshan Song<sup>e</sup>, Cornelia Rumpel<sup>c,*</sup> <sup>●</sup>

ARTICLE INFO

Keywords:

Earthworms

Neutral sugars

Soil types

Organo-mineral complex

Cast

A B S T R A C T

Although it has been recognized that the formation of organo-mineral complexes may be fostered by soil fauna, their formation pathways are far from understood. The objective of this study was to investigate the processes involved in earthworm-mediated mineral-associated organic matter (MAOM) formation. To this end, we used microcosms to produce casts with three different temperate earthworm species (epigeic *Lumbricus castaneus* [LC], epi-anecic *Lumbricus terrestris* [LT], endogeic *Aporrectodea icterica* [AI]) fed with ¹³C-enriched *Miscanthus* litter in two contrasting soil types (Luvisol, Cambisol). To investigate MAOM formation processes, we used ¹³C isotope tracing to track litter-derived carbon origin and neutral sugar biomarkers to resolve microbial transformation pathways.

Our results indicated that cast MAOM-C concentrations increased compared to the control treatment without earthworms, with LC casts in Luvisol exhibiting the highest MAOM-C concentration (24.1 mg g⁻<fnref n="1" />). MAOM-C contribution to casts decreased in the order LC > LT > AI. Sugar biomarkers revealed species- and soil- dependent stabilization pathways: MAOM of LC casts in Luvisol contained high proportions of plant-derived arabinose, while MAOM of AI casts in Cambisol were enriched in microbial-derived hexoses (mannose) and deoxy-hexoses (rhamnose, fucose). We conclude that earthworms depending on species and soil type accelerated MAOM formation via synergistic pathways: (1) direct stabilization of plant sugars and (2) microbial trans- formation of labile litter into necromass during cast formation. We suggest that earthworm-driven MAOM for- mation depends on species-specific traits (e.g., LC's capacity for litter fragmentation) and soil properties (e.g., clay and CaCO₃ content).

1. Introduction

Soil organic matter (SOM) formation is crucial for maintaining soil health and enhancing agricultural productivity (Bossio et al., 2020; Kopittke et al., 2024), with significant implications for climate change mitigation (Van Groenigen et al., 2011; Rumpel et al., 2022). During this process, labile plant litter entering the soil as particulate organic matter (POM) may be protected from microbial degradation by occlusion into soil aggregates (Six et al., 2000) or transformed into stable SOM through interactions with the mineral phase (von Lützow et al., 2007; Cotrufo

et al., 2019). Mineral-associated organic matter (MAOM), which con-
stitutes a substantial portion of total SOM in mineral soils (Sokol et al.,
2022), has traditionally been regarded as a persistent fraction due to its
association with mineral surfaces (von Lützow et al., 2007; Cotrufo
et al., 2019; Yu et al., 2022). However, some studies suggest that MAOM
dynamics are more complex, with portions cycling on shorter timescales
depending on factors such as microbial activity, mineral composition,
and soil moisture fluctuations (Kleber et al., 2021; Sokol et al., 2022).
This highlights the dual role of MAOM in both long-term carbon stabi-
lization and dynamic cycling.

<footnote><sup>a</sup> Institute of Hydrogeology and Environmental Geology, Chinese Academy of Geological Sciences, Shijiazhuang, 050061, China</footnote>

<footnote><sup>b</sup> Key Laboratory of Quaternary Chronology and Hydro-Environmental Evolution, China Geological Survey, Shijiazhuang, 050061, China</footnote>

<footnote><sup>c</sup> IRD, CNRS, Sorbonne Université, Univ Paris Est Creteil, INRAE, Institute of Ecology and Environmental Sciences (iEES-Paris), F-75005, Paris, France</footnote>

<footnote><sup>d</sup> Department of Soil Sciences, Soils and Fertilizers Research Institute (SFRI), Hanoi, Viet Nam</footnote>

<footnote><sup>e</sup> Department of Earth System Science, Ministry of Education Key Laboratory for Earth System Modeling, Institute for Global Change Studies, Tsinghua University, Beijing, 100084, China</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: cornelia.rumpel@inrae.fr (C. Rumpel).</footnote>

<footnote n="1">¹ Both authors contributed equally to the manuscript.</footnote>
The MAOM formation involves two primary pathways: (1) direct stabilization of dissolved organic matter (DOM) or depolymerized plant compounds onto mineral surfaces and (2) stabilization of microbial necromass and microbial products (Liang et al., 2017; Sokol et al., 2019; Lavallee et al., 2020; Cotrufo and Lavallee, 2022; Whalen et al., 2022; Xiao et al., 2023; Zhu et al., 2024). Resolving the formation pathways of MAOM-C necessitates advanced tools, such as ¹³C isotope tracing to distinguish newly added litter-C from native soil-C (Vidal et al., 2017), and biomarker analyses to indicate the SOM source (plant or microbial derived) as for example hemicellulosic sugars (Rumpel et al., 2010; Gunina and Kuzyakov, 2015). Microbial processes dominate MAOM formation in most systems (Liang et al., 2017; Angst et al., 2024a); however, their efficiency might be slow. Only ~13 % of litter-derived carbon transitions into MAOM within weeks, with the majority persisting as undecomposed residues (Liang et al., 2017; Angst et al., 2024a). The efficiency of MAOM formation may be modulated by litter chemistry and soil mineralogy (Córdova et al., 2018; Cotrufo and Lavallee, 2022; Sokol et al., 2022; Xu et al., 2022; Canisares et al., 2023).

Earthworms act as ecosystem engineers and are known to be involved in MAOM formation (Njoroge et al., 2022; Vidal et al., 2023; Angst et al., 2024b), with MAOM-C accounting for >90 % of earthworm cast carbon, persisting for over 400 days under tropical conditions in contrasting landuse systems (Song et al., 2024). They were reported to accelerate MAOM formation through: (1) gut-mediated grinding of ingested plant litter into fine particles, which increases the surface area for microbial decomposition (Gan et al., 2023), (2) mucus secretion, which strengthens organo-mineral binding (Guhra et al., 2020), (3) selective ingestion, which concentrates labile organic matter thereby accelerating DOM generation (Njoroge et al., 2022; Vidal et al., 2023), and (4) stimulating microbial activity, leading to increased necromass production (Angst et al., 2022). These physiological and behavioral earthworm traits are earthworm species-specific and enable MAOM formation within hours (Zi et al., 2025), contrasting sharply with microbial pathways requiring weeks (Córdova et al., 2018). Despite the presumed major role of earthworms in MAOM formation, current SOM models emphasize microbial pathways (Cotrufo and Lavallee, 2022; Sokol et al., 2022), neglecting earthworms and their activity in different soil types (Filser et al., 2016), because the interactions between species traits and soil mineralogy remain unquantified, and the underlying mechanisms poorly understood (Angst et al., 2024b).

To address these issues, we employed sugar biomarker analyses of casts formed by three earthworm species (*Lumbricus castaneus*, *Lumbricus terrestris*, *Aporrectodea icterica*) with two soil types (Luvisol, Cambisol) and plant litter naturally enriched in ¹³C. Our objectives were to: (1) compare mechanisms and efficiency of microbial- vs. earthworm-driven MAOM formation and (2) quantify species- and soil-specific differences in litter-C stabilization. We hypothesized that (1) earthworm species influence MAOM formation differently, as their feeding behaviors determine the extent of mineral particle ingestion and organic matter processing. For example, litter-ingesting species (e.g., *L. castaneus*) may produce more plant-derived MAOM, while soil-ingesting species (e.g., *A. icterica*) may promote microbial-derived MAOM formation. Moreover, we hypothesized that (2) soil type controls MAOM formation capacity, with Luvisol promoting greater MAOM accumulation compared to Cambisol because of its higher clay and CaCO₃ contents.

## 2. Methods

### 2.1. Sampling of earthworm species, soil and plant litter

Three earthworm species were sampled in two experimental orchards of INRAE (43°54′55.8"N, 4°52′42.3"E) in Avignon, Vaucluse, France, where the average annual rainfall and temperature are 614 mm and 11 °C, respectively. The three species were: the epigeic *Lumbricus castaneus* (LC) (Bouché, 1972), the epi-anecic *Lumbricus terrestris* (LT) (Bouché, 1972; Bottinelli et al., 2020a), and the endogeic *Aporrectodea*

*icterica* (AI) (Bouché, 1972). Before cast production, all earthworms were starved for three days in a Petri dish filled with a wet paper towel to empty their guts.

Two types of soil (one Luvisol and one Cambisol) were used for cast production under laboratory conditions. The Luvisol was obtained from the same location as the earthworms, whereas the Cambisol was collected from a different site under agricultural use at the INRAE experimental field in Lusignan, Nouvelle-Aquitaine, France (46°25′12.9"N, 0°7′29.4"E), where mean annual temperature was 12.4 °C and mean annual precipitation 781 mm. The geochemical parameters of these soils are presented in Table 1. Prior to the incubation experiment, both soil samples were air-dried and sieved to a particle size of less than 2 mm.

As a food source, *Miscanthus giganteus* plant litter (stems, roots and leaves) was collected from the experimental station of INRAE in Grignon, France, and then crushed to particles smaller than 2 mm. The components were subsequently mixed in equal proportions: one-third stems, one-third roots, and one-third leaves, and used in microcosms to produce earthworm casts. Litter C concentration was 453.7 mg g⁻¹.

### 2.2. Preparation of microcosms, cast production and incubation

The methodology of cast production was described in detail by Le Mer et al. (2022). Briefly, we used microcosms (10 cm × 10 cm × 3 cm) made from plastic boxes with perforated lids to contain the earthworms. The shallow depth of the microcosms may represent a limitation for deep digging earthworm species like LT, which need to adapt their live strategies. However, our experiment was designed to maximize cast production and to favor the observation of MAOM formation mechanisms. To this end, plant litter was added to the soil at 30 mg g⁻¹ soil, equivalent to 3 g of dry plant mixture per 97 g of soil. To ensure a continuous and abundant supply of fresh organic matter for the earthworms, half of the litter material was mixed directly with the soil, while the other half was placed on the soil surface. The soils in the microcosms were adjusted to a bulk density of 1 g cm⁻³, and distilled water was added to reach 80 % of the water-holding capacity, creating optimal conditions for earthworm activity and cast production.

To explore the differences and similarities between earthworm-driven MAOM formation and the traditional conception of microbial-mediated MAOM formation, we designed four treatments: three with earthworm species (soil and litter with LC, LT, and AI, representing earthworm-driven MAOM formation) and one without earthworms as the control treatment (soil and litter only, representing microbial-mediated MAOM formation). Although the soil of the control treatment may have been affected by earthworm activity, we consider that this bias may be neglectable.

Earthworms were introduced into the microcosms at varying densities resulting in similar biomass—2 individuals for anecic, 4 for endogeic, and 8 for epigeic species. This strategy based on the species' individual weights was chosen to optimize cast output. Each treatment was replicated five times. All treatments were maintained in the dark at 14 °C.

Considering that the digestion times for these three earthworm species ranged from 5 to 10 h (Zi et al., 2025), fresh casts were collected from the soil surface daily for the first two weeks and every two days thereafter, over a total period of four weeks. The casts were manually sorted to remove any adhering soil and organic debris. In the control treatments, soil was sampled at the same intervals. Both the casts and the control soils were air-dried and stored for further analysis.

### 2.3. The separation of MAOM fractions

To isolate MAOM from air-dried control soil or cast samples, 10 g of each sample were submerged in 80 mL of sodium polytungstate solution (density 1.60 g cm⁻³) and sonicated to disrupt aggregates with an Analog 450 probe-type sonicator (Branson Sonifier, Branson Sonic
<table><caption>**Table 1**

Characteristics of soil types in the study.</caption><thead><tr><th rowspan="2">Soil types</th><th rowspan="2">pH</th><th rowspan="2">SOC<br>/g kg<sup>-1</sup></th><th rowspan="2">MAOM-C<br>/g kg<sup>-1</sup></th><th rowspan="2">CaCO<sub>3</sub><br>/g kg<sup>-1</sup></th><th rowspan="2">N<br>/g kg<sup>-1</sup></th><th rowspan="2">P<br>/g kg<sup>-1</sup></th><th colspan="5">Soil texture/g kg<sup>-1</sup></th></tr><tr><th>Clay</th><th>Fine silt</th><th>Coarse silt</th><th>Fine sand</th><th>Coarse sand</th></tr></thead><tbody><tr><td>Luvisol</td><td>8.3</td><td>15.4</td><td>12.05</td><td>378</td><td>1.8</td><td>0.02</td><td>292</td><td>372</td><td>152</td><td>158</td><td>26</td></tr><tr><td>Cambisol</td><td>5.9</td><td>11.9</td><td>9.43</td><td>-</td><td>1.2</td><td>0.01</td><td>167</td><td>381</td><td>285</td><td>63</td><td>104</td></tr></tbody></table>

Power Co., Danbury, CT, USA) and a calibrated energy input of 400 J mL⁻¹. After centrifugation, the floating particulate organic matter (POM) was discarded and the MAOM residue was rinsed with de-ionized water until the conductivity was below 50 µS cm⁻¹ to remove sodium polytungstate. To this end, we utilized multiple centrifugation and decantation steps. The MAOM fractions were freeze-dried, weighed, ground, and stored before analysis.

## 2.4. Analyses of total organic carbon (OC) and stable carbon isotope composition

After removal of carbonates with HCl by acid fumigation (Harris et al., 2001), the OC concentration of MAOM fractions was measured with an elemental analyzer (Vario El III Elementary, Elementar). The stable carbon isotope signature of MAOM was determined with a Flash HT elemental analyser (Thermofisher, Bremen, Germany) coupled to a Delta V mass spectrometer (Thermofisher). The results were reported as δ¹³C (‰), the isotopic ratio (¹³C/¹²C) relative to the international VPDB standard (Equation (1)).

$$ \delta^{13}C = \left( \frac{\left( \frac{^{13}C}{^{12}C} \right)_{\text{sample}}}{\left( \frac{^{13}C}{^{12}C} \right)_{\text{VPDB}}} - 1 \right) \times 1000 \quad \text{Eq. (1)} $$

Analytical precision was better than ± 0.05 ‰. The proportion of litter-derived MAOM-C in casts (C<sub>litter</sub>), was calculated with the isotopic mass balance and expressed in percentage by applying Eq. (2) (Cerri et al., 1985; Vidal et al., 2017):

$$ C_{\text{litter}} (\%) = \frac{\delta^{13}C_{\text{casts}} - \delta^{13}C_{\text{control soil}}}{\delta^{13}C_{\text{litter}} - \delta^{13}C_{\text{control soil}}} \times 100 \quad \text{Eq. (2)} $$

where δ¹³C<sub>casts</sub>, δ¹³C<sub>control soil</sub>, and δ¹³C<sub>litter</sub> stand for the δ¹³C values of cast MAOM, control soil MAOM, and litter, respectively.

## 2.5. Analyses of neutral sugars and calculation of earthworm-induced mineral-bonded sugar compounds

Neutral sugars in cast MAOM fractions, and in litter samples were analyzed as alditol acetates using a method described by Rumpel and Dignac (2006) and modified by Eder et al. (2010). The procedure involved hydrolysis of non-cellulosic polysaccharides in 250 mg of soil with 5 ml 4 M trifluoroacetic acid (TFA) at 105 °C for 4 h, and addition of methyl-glucose as a recovery standard. After cooling down the samples were filtered to remove soil particles and the TFA was evaporated using a rotary evaporator. The samples were frozen overnight, redissolved in H₂O containing myo-inositol used as internal standard to calculate the response factor after GC/FID analysis, and acidified. To inhibit co-precipitation of organics and metals, samples were treated with EDTA before acidification. Reduction of polysaccharides to alditolts was achieved using sodium borohydride in dimethyl sulfoxide (NaBH₄-DMSO), followed by acetylation. The acetylation process involved the addition of acetic anhydride and glacial acetic acid, with methyl-imidazole serving as the catalyst. The reaction was halted after 10 min by adding ice-cold deionized water. The derivatized sugar monomers were extracted by liquid-liquid extraction using dichloromethane for subsequent GC analysis (HP 6890 GC equipped with a flame ionization detector). Separation of the monosaccharide units was achieved with a 60 m fused silica capillary column (BPX 70, 0.32 mm

internal diameter, 0.25 µm film thickness) and the following temperature program: from 200 °C to 250 °C at 3 °C/min, 15 min isothermal at 250 °C. Carrier gas was Helium with a flow rate of 1.0 ml min⁻¹. The injector was kept at 250 °C and the detector at 260 °C. The method achieved recovery rates exceeding 75 % for myo-inositol and over 95 % for neutral sugars relative to the total soil samples. This analysis yielded a suite of monosaccharides being composed of pentose (C5) and hexoses (C6). Pentoses (arabinose, xylose) are primarily plant-derived, while hexoses (mannose, galactose) and deoxy-hexoses (rhamnose, fucose) generally indicate microbial processing (Rumpel et al., 2010; Gunina and Kuzyakov, 2015).

The sugar concentration of MAOM fractions was expressed as (1) milligrams of sugar per gram of MAOM, and (2) milligrams of sugar-C per gram of MAOM-C. To eliminate the initial variability in MAOM-C and sugar-C measurements across different microcosms and evaluate the influence of earthworm activity on sugar distribution within the soil, we calculated two parameters: (i) the Earthworm-Induced Enrichment Coefficient (EC<sub>EI</sub>) for sugar content in MAOM or C content in MAOM (MAOM-C) and (ii) the Newly-formed (NF<sub>EI</sub>) sugar content in MAOM or newly formed C content in MAOM (MAOM-C) added through earthworm transformation, as described by Eqs. (3) and (4).

$$ EC_{EI-sugar \text{ or } -MAOM C} = \frac{\text{Sugar or MAOM-C}_{\text{cast}}}{\text{Sugar or MAOM-C}_{\text{ctrl}}} \quad \text{Eq. (3)} $$

$$ NF_{EI-sugar \text{ or } -MAOM C} = \text{Sugar or MAOM-C}_{\text{cast}} - \text{Sugar or MAOM-C}_{\text{ctrl}} \quad \text{Eq. (4)} $$

where EC is the enrichment coefficient and NF is the amount of newly formed compounds, sugar is the sugar concentration in MAOM, and MAOM-C is the C concentration in MAOM, Sugar or MAOM-C<sub>cast</sub> is the sugar or MAOM-C concentration of casts, and Sugar or MAOM-C<sub>ctrl</sub> is the sugar or MAOM-C concentration of the control soil.

The replacement of native MAOM-C by newly formed litter-derived MAOM-C induced by earthworms was quantified using Eq. (5).

$$ \text{Substituted MAOM-C} = C_{\text{litter}} \times \text{MAOM-C}_{\text{cast}} - \text{NF}_{EI-MAOM C} \quad \text{Eq. (5)} $$

where Substituted MAOM-C is the amount of MAOM-C replaced by newly formed MAOM-C, C<sub>litter</sub> the proportion of litter-derived MAOM-C in casts (Eq. (2)) and NF<sub>EI-MAOM C</sub> Newly-formed C content in MAOM (Eq. (4)).

To evaluate the potential changes in the total amount and composition of sugars due to earthworm activities, we compared the expected to the actual total sugar (or monosaccharide) concentrations in casts. The expected total sugar (or monosaccharide)-C concentration in cast MAOM (ET<sub>sugar</sub>) was calculated using Equation (6).

$$ ET_{\text{sugar}} = \text{Sugar}_{\text{ctrl}} \text{MAOM} \times (1 - C_{\text{litter}}) + \text{Sugar}_{\text{litter}} \times C_{\text{litter}} \quad \text{Eq. (6)} $$

where Sugar<sub>ctrl MAOM</sub> represents the total sugar (or monosaccharide)-C concentration in control soil MAOM fractions, Sugar<sub>litter</sub> represents the total sugar (or monosaccharide)-C concentration of litter, C<sub>litter</sub> is the litter-derived carbon in the cast MAOM (Eq. (2)).

The parameter ET<sub>sugar</sub> corresponds to an expected total sugar (or monosaccharide) concentration in cast MAOM assuming direct incorporation of all sugars present in litter into this fraction. Using ET<sub>sugar</sub> and the actual total sugar (or monosaccharide)-C concentration of MAOM, we calculated the Sugar Transformation Ratio (STR) for total sugars or specific monosaccharides (equation (7)).
$$STR = \frac{\text{Measured sugar}}{\text{ET}_{\text{sugar}}} \qquad \text{Eq. (7)}$$

where measured sugar is the sugar content determined in MOAM and ET<sub>sugar</sub> is the expected total sugar- or monosaccharide-C concentration in cast MAOM (Eq. (6)).

An STR less than 1 indicated lower total sugar or monosaccharide concentrations compared to the expected ones, suggesting preferential assimilation by earthworms and/or microbial turnover. Conversely, an STR value of 1 signified a match between actual and expected total sugar- or monosaccharide-C concentrations, indicating a neutral impact of earthworm activity on sugar composition. An STR greater than 1 suggested an enrichment of sugars or monosaccharides in cast MAOM, indicative of the preferential stabilization of sugars derived from plant-litter during cast formation. The STR was calculated for total sugars and individual monosaccharides to allow for the assessment of transformation of plant sugars (arabinose and galactose in the case of *Miscantus* litter) into microbial ones (mannose, rhamnose and fucose). This data interpretation allowed for insights into the role of earthworms in the dynamics of labile plant litter compounds.

## 2.6. Statistical analysis

Two-way ANOVA was performed to assess differences among earthworm casts and the control soils, after testing data for normality and homogeneity of variances using the Shapiro-Wilk and Levene's tests, respectively. For data not meeting normality, log transformation was applied; however, in cases of normality or homogeneity of variances violations, a Kruskal-Wallis test was used instead to ensure robust analysis. Differences among treatments were considered significant at the 0.05 probability level, with LSD post hoc tests for ANOVA. T-tests were used for assessing differences between measured and δ<sup>13</sup>C-predicted sugar components. After normalization (Z-score), we also performed between-class analyses (BCA), a multivariate statistical technique that uses class labels to guide the analyses with the aim to maximize the separation between groups and Monte Carlo permutation tests (999 permutations). This analysis was used to investigate the dependence of MAOM composition on soil type and earthworm species. All statistical analyses and visualizations were executed in R, utilizing packages like "car", "ade4", and "ggplot2".

## 3. Results

### 3.1. MAOM-C concentration, earthworm-induced MAOM-C enrichment, stable carbon isotope signature and litter-derived OC in the MAOM fraction

The MAOM fraction of casts and control soil represented between 75.1 and 99.6% of total mass and its carbon concentrations ranged from 10 to 24 mg g<sup>-1</sup> (Table 2). Cast formation by all earthworm species led to increased MAOM-C concentrations compared to control treatments for both soil types (Table 2). Specifically, the LC casts from Luvisol exhibited the highest MAOM-C concentration and the lowest MAOM contribution to cast (75.1%) (Table 2). The MAOM-C concentrations for the earthworm casts decreased in the following order: LC Luvisol > LC Cambisol ≈ LT Cambisol > LT Luvisol > AI Luvisol > AI Cambisol, displaying a general trend of LC > LT > AI, regardless the soil type (Table 2 and Fig. 1). The proportion of C derived from *Miscanthus* litter incorporated in MAOM (C<sub>litter</sub>) ranged between 10% and 57% (Table 2). It decreased in the order: LC Luvisol > LC Cambisol > LT Cambisol > LT Luvisol > AI Luvisol > AI Cambisol and was impacted by earthworm species, soil type and their interaction (P < 0.05, two-way ANOVA, Table 2).

The carbon enrichment of the MAOM fraction induced by earthworm activity (EC<sub>El-MAOM C</sub>) represented up to 2.1 times the amount of carbon found in the MAOM fraction of control soils, reaching a concentration of newly-formed MAOM C (NF<sub>El-MAOM C</sub>) of 12.6 mg g<sup>-1</sup>. Both parameters decreased in the order LC > LT > AI in the Luvisol soil and LC ≈ LT > AI in the Cambisol soil (Fig. 1). Notably, no earthworm-induced MAOM-C enrichment was observed for AI in the Cambisol soil (Fig. 1). The NF<sub>El-MAOM C</sub> values for casts produced by LC species in both soils were consistently lower than the values calculated using the <sup>13</sup>C enrichment (see Substituted MAOM-C, P < 0.05, Fig. 1). The analysis from the two-way ANOVA indicated that the MAOM-C concentration was significantly affected by both, earthworm species and soil type, with no significant interactions between these two factors (P > 0.05, Table 2).

### 3.2. Sugar concentration and contribution to MAOM-C

The total sugar concentration of the MAOM fractions ranged between 4.1 and 35.2 mg g<sup>-1</sup> and was higher in casts than in control soils (Table 2). The LC casts from Luvisol showed the highest sugar concentration. Sugar concentrations decreased in the order LC casts Luvisol > AI casts Luvisol > LT casts Cambisol > AI casts Cambisol > LC casts Cambisol > LT casts Luvisol (Table 2). Sugar-C represented less than 20

<caption>**Table 2**</caption>

MAOM contribution (% of cast/soil), MAOM carbon concentration (mg g<sup>-1</sup> MAOM), δ<sup>13</sup>C value, the proportion of litter-derived C in casts (C<sub>litter</sub>), sugar concentration (mg g<sup>-1</sup> and mg C g<sup>-1</sup> MAOM-C) of mineral-associated organic matter (MAOM) fractions across different earthworm (EW) casts in Luvisol (Luvi.) and Cambisol (Camb.) control soil. Data are presented as means ± standard deviation (n = 5). The different letters indicate significant differences in different treatment (two-way ANOVA, ns > 0.05 > * > 0.01 > ** > 0.001 > ***, P < 0.05).

<table><thead><tr><th rowspan="2">Treatment</th><th rowspan="2">MAOM %</th><th rowspan="2">MAOM-C mg g<sup>-1</sup></th><th rowspan="2">δ<sup>13</sup>C<sub>MAOM</sub><br>%</th><th rowspan="2">C<sub>litter</sub><br>%</th><th colspan="2">Sugar concentration</th></tr><tr><th>mg g<sup>-1</sup></th><th>mg C g<sup>-1</sup> MAOM-C</th></tr></thead><tbody><tr><td>Litter</td><td>-</td><td>-</td><td>-13.30</td><td>-</td><td>£402.43 ± 68.57</td><td>-</td></tr><tr><td>Ctrl-Luvi.</td><td>99.6 ± 0.2<sup>a</sup></td><td>11.56 ± 0.46<sup>bcd</sup></td><td>-25.40 ± 0.08<sup>d</sup></td><td>-</td><td>4.59 ± 0.45<sup>c</sup></td><td>158.27 ± 23.53<sup>c</sup></td></tr><tr><td>LC-Luvi.</td><td>75.1 ± 1.3<sup>e</sup></td><td>24.14 ± 1.45<sup>a</sup></td><td>-18.55 ± 0.46<sup>a</sup></td><td>57 ± 4</td><td>35.12 ± 6.76<sup>a</sup></td><td>580.14 ± 103.41<sup>a</sup></td></tr><tr><td>LT-Luvi.</td><td>94.6 ± 0.4<sup>de</sup></td><td>16.43 ± 1.32<sup>ab</sup></td><td>-22.13 ± 0.48<sup>bc</sup></td><td>27 ± 4</td><td>9.83 ± 2.08<sup>b</sup></td><td>231.52 ± 27.70<sup>bc</sup></td></tr><tr><td>AI-Luvi.</td><td>97.4 ± 0.8<sup>bc</sup></td><td>14.37 ± 0.73<sup>bc</sup></td><td>-23.22 ± 0.59<sup>bc</sup></td><td>18 ± 5</td><td>13.3 ± 1.37<sup>b</sup></td><td>380.70 ± 55.98<sup>abc</sup></td></tr><tr><td>Ctrl-Camb.</td><td>98.3 ± 0.1<sup>ab</sup></td><td>10.26 ± 1.15<sup>d</sup></td><td>-25.78 ± 0.15<sup>d</sup></td><td>-</td><td>4.16 ± 0.61<sup>c</sup></td><td>177.20 ± 23.95<sup>c</sup></td></tr><tr><td>LC-Camb.</td><td>96.1 ± 0.1<sup>cd</sup></td><td>15.90 ± 2.75<sup>bc</sup></td><td>-20.96 ± 0.99<sup>b</sup></td><td>39 ± 8</td><td>10.83 ± 1.18<sup>b</sup></td><td>298.94 ± 46.68<sup>bc</sup></td></tr><tr><td>LT-Camb.</td><td>95.1 ± 1.0<sup>cd</sup></td><td>15.77 ± 1.86<sup>b</sup></td><td>-21.20 ± 0.30<sup>b</sup></td><td>37 ± 2</td><td>12.63 ± 1.28<sup>b</sup></td><td>325.89 ± 20.27<sup>bc</sup></td></tr><tr><td>AI-Camb.</td><td>96.4 ± 0.3<sup>cd</sup></td><td>10.27 ± 0.56<sup>cd</sup></td><td>-24.56 ± 0.20<sup>cd</sup></td><td>10 ± 2</td><td>10.86 ± 1.57<sup>b</sup></td><td>430.30 ± 66.41<sup>ab</sup></td></tr><tr><td colspan="7">Two-way ANOVA F and p values</td></tr><tr><td>EW species</td><td>** 48.4 ***</td><td>16.2 ***</td><td>75.5 ***</td><td>-</td><td>16.3 ***</td><td>10.9 ***</td></tr><tr><td>Soil type (ST)</td><td>** 2.6 ns</td><td>14.4 ***</td><td>4.6 *</td><td>-</td><td>10.2 **</td><td>0.6 ns</td></tr><tr><td>Species*ST</td><td>** 9.6 ***</td><td>2.4 ns</td><td>4.1 *</td><td>-</td><td>10.5 ***</td><td>5.1 **</td></tr></tbody></table>

£: mg sugar g<sup>-1</sup> litter (n = 3), and the contribution of sugar-C to litter-C was 355.17 ± 60.56 mg sugar-C g<sup>-1</sup> litter-C; ##: Due to violations of normality of variances assumptions, a Kruskal-Wallis test was employed for the contribution parameter instead of two-way ANOVA.
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Earthworm-induced newly-formed MAOM-C (NF<sub>EI-MAOM C</sub>), Enrichment Coefficient (EC<sub>EI-MAOM C</sub>) and Substituted MAOM-C (C<sub>litter</sub> × MAOM-C<sub>cast</sub> – NF<sub>EI-MAOM C</sub>, representing the disruption of native MAOM due to priming effects and substitution by ingested litter) across various earthworm species and soil types. The different letters indicate significant statistical differences in MAOM-C among species (epigeic LC, epi-anecic LT and endogeic AI) under different soil conditions (P < 0.05). Stars represent the extent of deviation from a baseline value of soil control for Newly-formed MAOM-C and Substituted MAOM-C under each specific species-soil type combination (t-test, ns > 0.05>*>0.01>**>0.001>***, P < 0.05).</figcaption></figure>

% of total C in control soils, while their contribution increased in casts, where they represented up to 58% of total C. The sugar concentration in the MAOM fractions was influenced by earthworm species, soil type, and their interaction (P < 0.001, two-way ANOVA, Table 2).

Fig. 2 displays the variations in sugar-C enrichment (EC<sub>EI-sugar C</sub>) across different cast types. Specifically, LC casts produced with Luvisol had the highest EC<sub>EI-sugar C</sub> value of 3.7 and the highest concentration of newly formed sugars (NF<sub>EI-sugar C</sub>) amounting to 421.9 mg sugar-C g<sup>-1</sup> MAOM-C (P < 0.05, Fig. 2). The NF<sub>EI-sugar C</sub> decreased following the order LC > AI > LT. On the other hand, in the Cambisol soil, both EC<sub>EI-sugar C</sub> and NF<sub>EI-sugar C</sub> decreased in the order AI > LT > LC (Fig. 2), which

<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Earthworm-induced newly-formed sugar-C (NF<sub>EI-sugar C</sub>) and Enrichment Coefficient (EC<sub>EI-sugar C</sub>) across various earthworm species and soil types. The different letters indicate significant statistical differences in total sugar-C among species (epigeic LC, epi-anecic LT and endogeic AI) under different soil conditions (P < 0.05). Stars represent the extent of deviation from a baseline value of control treatment for newly-formed sugar-C under each specific species-soil type combination (t-test, 0.05; * < 0.05; ** < 0.01; *< 0.001*<).</figcaption></figure>

is inconsistent with the casts' MAOM-C concentrations (see Fig. 1).

### 3.3. Composition of sugars

The sugar composition of plant litter and the MAOM fractions of control soils and casts is presented in Fig. 3 and the newly formed sugars in casts are presented in Fig. 4. Interestingly, *Miscanthus* litter showed an important contribution of the pentose galactose in addition to arabinose and xylose (Fig. 3). While the sugar composition was similar for MAOM of the two control soils, the contribution of different monosaccharides to MAOM-C of casts differed among soil types and among earthworm species (Fig. 3). For example, in the control soils of Luvisol, which exhibited the lowest proportion of sugar-C in the MAOM fraction, xylose was the most abundant, followed by galactose (Fig. 3). For LC and LT casts from this soil, newly-formed sugar-C resulted from a significant increase in both, pentoses and hexoses (Fig. 4), arranged in the order of arabinose > xylose ≈ galactose > ribose ≈ glucose ≈ mannose. Notably, in LT casts, the only newly formed monosaccharide was arabinose (Fig. 4).

In Cambisol, cast MAOM showed significant increases of all monosaccharides (P < 0.05), with the exception of rhamnose and fucose in LT casts, which did not show significant changes (P > 0.05, Fig. 4). The newly-formed monosaccharides included arabinose, ribose and galactose. Specifically, in cast MAOM of the AI species, we observed a more balanced sugar profile, with contributions of xylose, arabinose, galactose and glucose across both soil types (Fig. 3). Furthermore, the activities of LC and AI species resulted in the presence of newly formed deoxy-hexoses in cast MAOM from Cambisol (Fig. 4).

### 3.4. Sugar transformation ratio (STR) of monosaccharides

The STR of total sugar and monosaccharides is presented in Fig. 5. It illustrates the substantial influence of earthworm species and soil type on the transformation and/or incorporation pattern of sugars into cast MAOM. Specifically, in Luvisol, STR values of LC casts were significantly above the expected value 1 (P < 0.05) as a result of higher than expected incorporation of ribose, arabinose, mannose, and glucose into MAOM (P < 0.05). A similar STR pattern was noted for AI, with significantly enhanced incorporation of ribose, mannose, and glucose, but with additional incorporation of xylose and without the accumulation of arabinose, in contrast to what was noted for LC casts. Contrastingly, LT cast MAOM showed STR of 1 or even lower than 1 for rhamnose, fucose and galactose (P < 0.05), indicating low levels of sugar transformation and/or incorporation.

For Cambisol, the STR values of mannose in cast MAOM from all three species were significantly greater than 1 (P < 0.05), indicating that contrasting earthworms lead to a preferential enrichment of mannose within this fraction. Moreover, for the Cambisol casts of LT and AI, the STR values of monosaccharides, with the exception of arabinose and galactose, were significantly greater than 1 (P < 0.05). This indicated an enhanced transformation and/or incorporation rate for other than *Miscanthus* litter-derived sugars.

### 3.5. Between-class analysis (BCA)

To assess the influence of earthworm species and soil type on MAOM formation and sugar dynamics in this fraction, we conducted a between-class analysis (BCA) integrating newly-formed MAOM-C, monosaccharides, and the proportion of litter-derived MAOM-C (C<sub>litter</sub>) (Fig. 6). The analysis revealed distinct clustering of earthworm-soil combinations along Axis 1 (72.05% variance explained) and Axis 2 (20.5% variance explained), with a significant group separation (Monte Carlo test: p = 0.004). The results indicated different characteristics for the same earthworm species in the two soil types, with their effects on MAOM formation and sugar dynamics being soil type dependent. Earthworm species LC, LT, and AI showed distinct patterns in Luvisol
<figure><img src="image_4.png" /><figcaption>Fig. 3. Monosaccharide C concentrations of MAOM formed from different control soils with different earthworm species (mg C g⁻¹ MAOM C) and of litter (mg C g⁻¹ litter C).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Fig. 4. Earthworm-induced newly-formed monosaccharides (NF<sub>EI</sub>-monosaccharides = Monosaccharides<sub>cast</sub> – Monosaccharides<sub>Ctrl</sub>, as shown in Eq (4)) of cast MAOM fractions in different soil types, stars indicate the extent of deviation from a baseline value of 0 for each specific species-soil type combination (t-test, P < 0.05; *< 0.05; **< 0.01; ***< 0.001).</figcaption></figure>

(Luvi) and Cambisol (Camb), with LT and LC having some overlap, but AI being separate (Fig. 6a). In Luvisol, LC showed a strong positive association with C<sub>litter</sub> and newly formed MAOM-C, as well as monosaccharides like arabinose, galactose, ribose, and glucose (Fig. 6b). Conversely, in Cambisol, AI was linked negatively to C<sub>litter</sub> and positively to the presence of rhamnose and fucose. LT exhibited intermediate effects in both soil types.

## 4. Discussion

### 4.1. Earthworm-driven MAOM formation: quantity and efficiency

In this study with temperate earthworm species, MAOM-C was enriched in casts produced by all species compared to control treatments across two contrasting soil types, except for the AI species in Cambisol (Fig. 1). This aligns with observations for anecic earthworm species
<figure><img src="image_6.png" /><figcaption>**Fig. 5.** Sugar Transformation Ratio (STR = Measured Sugar/Expected Transformative Sugar) for total sugar and monosaccharides in cast MAOM fractions across different soil types. Significance levels are indicated as follows: \*\*\*< 0.05 \*\*<0.01< \*\*<0.00. These data indicate the extent of deviation from a baseline value of 1 for each specific species-soil type combination (t-test, P < 0.05).</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 6.** Between components analysis (BCA) with newly-formed MAOM C and monosaccharides, and litter-derived C proportion in casts (C<sub>litter</sub>) showed differences across earthworm species (LC, LT, and AI) and soil types (Luvi = Luvisol; Camb = Cambisol). Group significance was assessed by Monte Carlo tests.</figcaption></figure>

(Vidal et al., 2019; Bottinelli et al., 2020b) and epigeic earthworm species (Le Mer et al., 2020), where newly-formed MAOM-C in casts reached up to 12.6 mg g<sup>-1</sup> (Fig. 1), which is within the same range as values reported recorded for tropical earthworm casts (Song et al., 2024).

Our findings indicated that the C concentration in MAOM of the control treatment did not significantly differ from that of the initial soil materials (Tables 1 and 2). This suggests that the C transformation from dry *Miscanthus giganteus* litter into MAOM in the absence of earthworms was negligible within the experimental period. This lack of MAOM

formation on a daily-weekly timescale might be ascribed to the low quality of *Miscanthus giganteus* litter, low microbial activities, and/or the inefficient entry point of OM input into the soil (Córdova et al., 2018; Sokol et al., 2019, 2022; Canisares et al., 2023).

In contrast, earthworms accelerated MAOM formation. They caused up to a 2.1-fold enrichment of C in MAOM within 24 h of cast production, which included 5–10 h of digestion (Zi et al., 2025) and the post-egestion periods. The high efficiency of earthworm-driven MAOM formation highlights earthworms’ function as bioreactors and catalysts for stabilization of labile carbon (Angst et al., 2019, 2022).
## 4.2. Earthworm-induced MAOM formation processes revealed by the combined application of ¹³C and sugar biomarkers

While the sugar concentration of control soil MAOM is consistent with the sugar concentration recorded in the MAOM fraction of crop soils (Llorente et al., 2017), after cast formation, the sugar concentrations fell within the range observed in topsoils of broad-leaved forests (7.0–82.8 mg g⁻¹), coniferous forests (1.1–90.1 mg g⁻¹), and shrubs (2.5–80.6 mg g⁻¹) (Liu et al., 2019), and even in the O horizons (Cui et al., 2016). This observation indicates a preferential stabilization of sugars by mineral interactions during cast formation.

The isotopic tracing of litter-derived carbon (C_litter) confirmed that newly-formed MAOM-C predominantly originated from ingested plant litter. However, relying solely on the ¹³C data, it is impossible to trace the transformation processes. Hence, we characterized the transformation of labile litter compounds through the analysis of hemi-cellulosic sugars as biomarkers for microbial- and plant-derived compounds to disentangle direct stabilization of plant litter compounds from stabilization of plant-derived C after microbial transformation. For LT and AI casts produced with Cambisol, the STR values exceeded 1 across most monosaccharides (Fig. 5), indicating preferential incorporation plant and microbial sugars into MAOM during cast formation. Although earthworms ingested arabinose- and galactose-rich *Miscanthus giganteus* (Fig. 3), the STR values of these sugars remained <1 in all casts (except for arabinose in LC-Luvisol; Fig. 5). This suggests that these abundant easily decomposable plant sugars are either preferentially assimilated by earthworms or experience rapid microbial turnover. The enrichment of arabinose most probably originating from the *Miscanthus* food source in LC casts provides evidence for the direct stabilization of plant-derived sugars (Kellerová et al., 2024) through the activity of this species. In contrast, elevated STR values (>1.5) for hexoses like mannose (in LC and AI casts) and deoxy-sugars (e.g., rhamnose, fucose in Cambisol) reflect the preferential accumulation of these monosaccharides, characteristic of microbial necromass and microbial products (Gunina and Kuzyakov, 2015; Liang et al., 2017) in MAOM. This highlights the microbial transformation of litter-derived precursors during MAOM formation by earthworm activity. We therefore suggest that earthworm gut passage accelerates both formation pathways possibly by fragmenting litter into fine particles (Fusaro et al., 2018; Sinha et al., 2010), enhancing microbial colonization (Angst et al., 2024b), labile carbon supply by mucus secretion (Wü;st et al., 2009) and presence of electrolytes (Pan et al., 2010) (e.g., Ca²⁺) within the earthworms' gut. All these conditions most probably stimulate microbial activity and organo-mineral bonding (Guhra et al., 2020, 2024).

## 4.3. Earthworm species and soil type dependence of MAOM formation

The between-class analysis (BCA, Fig. 6) of newly formed MAOM and its components revealed distinct clustering separating earthworm species associated with specific soil types. Indeed, LC, LT, and AI casts formed separate groups in Luvisol and Cambisol, indicating that the amount and composition of newly formed MAOM is species specific. This might be explained by species traits, as it was shown that cast characteristics were influenced by mucus secretion, selective ingestion of labile OM, and mechanical grinding of litter (Zi et al., 2025), which synergistically enhanced MAOM formation. Notably, in LC cast, the high carbon concentration of MAOM contrasts with its low contribution in casts compared to the other species. These results might be explained by specific behavioral traits of LC. Despite being primarily litter-feeding, LC likely ingested mineral particles during foraging for essential nutrients, facilitating organo-mineral interactions that enhanced stabilization in the Luvisol. In contrast, LC's efficiency diminished in Cambisol (15.9 mg g⁻¹ MAOM-C). This reduced stabilization aligns with the lower clay content (Table 1) and lower Ca²⁺ availability in Cambisol compared to Luvisol, which contained substantial amounts of CaCO₃ (Table 1). Previous studies have shown that clay surfaces and Ca²⁺ ions enhance

organo-mineral bonding (Guhra et al., 2020; Zhang et al., 2022; Ruiz et al., 2024), which supports our observation of species- and soil-dependent MAOM formation. The AI casts in Cambisol showed a negligible enrichment of MAOM-C (10.3 mg g⁻¹), reflecting their inefficiency in formation of organo-mineral complexes as epigeic litter dweller. These results are in contrast with a recent study showing that epigeic *Eisenia* species were able to form organo-mineral associations when provided with organic matter and clay mineral (Le Mer et al., 2020). Thus, species-specific traits should be taken into consideration when investigating the effect of earthworms on biogeochemical cycling. Moreover, our findings emphasize that soil mineralogy acts as a regulatory factor, modulating the species-specific efficiency of earthworms in MAOM formation. We therefore suggest that MAOM formation through the activities of earthworms emerges from the interaction between species-specific traits and the physicochemical and mineralogical soil characteristics—a critical consideration for soil carbon models aiming to predict stabilization across ecosystems.

## 5. Conclusions and perspectives

This study investigated the processes involved in MOAM formation through earthworm activity in a controlled laboratory experiment. It revealed that earthworms rapidly transformed plant litter-derived carbon into MAOM, which represented >75 % of the casts' mass. Our data further showed that two processes were involved in earthworm-mediated MAOM formation. Earthworm activity led to (1) direct stabilization of plant litter compounds and (2) stabilization of litter-derived carbon after microbial transformation. The amount and composition of MAOM was found to be dependent on earthworm species and soil type. Current soil carbon models should therefore integrate earthworm activity, particularly in systems dominated by litter-feeding species or reactive soils. Future work should investigate the long-term persistence of earthworm-mediated MAOM and its interactions with recalcitrant plant compounds, such as lignin, to refine soil carbon models predicting stabilization dynamics under climate change.

### CRediT authorship contribution statement

**Chao Song:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. **Yacouba Zi:** Writing – review & editing, Methodology, Investigation, Data curation. **Marie-France Dignac:** Writing – review & editing, Validation, Supervision, Investigation, Conceptualization. **Nicolas Bottinelli:** Supervision, Investigation, Funding acquisition, Conceptualization. **Axel Felbacq:** Supervision, Methodology, Data curation. **Shanshan Song:** Methodology, Investigation, Formal analysis. **Cornelia Rumpel:** Writing – review & editing, Validation, Supervision, Investigation, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

We would like to thank two anonymous referees, and the handling editor for constructive comments and suggestions to improve the manuscript. This project was financially supported by the ANR project U2 worm (ANR-20-CE01-0015-01). C. S. was supported by China Scholarship Council (CSC) Grant #202004180033 and Natural Science Foundation of China (NSFC) grant #41877398.
## References

Angst, G., Angst, S., Frouz, J., Jabinski, S., Jílková, V., Kukla, J., Li, M., Meador, T.B., Angel, R., 2024a. Stabilized microbial necromass in soil is more strongly coupled with microbial diversity than the bioavailability of plant inputs. Soil Biology and Biochemistry 190, 109323.

Angst, G., Frouz, J., van Groenigen, J.W., Scheu, S., Kögel Knabner, I., Eisenhauer, N., 2022. Earthworms as catalysts in the formation and stabilization of soil microbial necromass. Global Change Biology 28, 4775-4782.

Angst, G., Mueller, C.W., Prater, I., Angst, S., Frouz, J., Jílková, V., Peterse, F., Nierop, K. G., 2019. Earthworms act as biochemical reactors to convert labile plant compounds into stabilized soil microbial necromass. Communications Biology 2, 1-7.

Angst, G., Potapov, A., Joly, F.X., Angst, S., Frouz, J., Ganault, P., Eisenhauer, N., 2024b. Conceptualizing soil fauna effects on labile and stabilized soil organic matter. Nature Communications 15, 5005.

Bossio, D.A., Cook-Patton, S.C., Ellis, P.W., Fargione, J., Sanderman, J., Smith, P., Wood, S., Zomer, R.J., von Unger, M., Emmer, I.M., Griscom, B.W., 2020. The role of soil carbon in natural climate solutions. Nature Sustainability 3, 391-398.

Bottinelli, N., Hedde, M., Jouquet, P., Capowiez, Y., 2020a. An explicit definition of earthworm ecological categories - Marcel Bouchés triangle revisited. Geoderma 372, 114361.

Bottinelli, N., Kaupenjohann, M., Märten, M., Jouquet, P., Soucémarianadin, L., Baudin, F., Tran, T.M., Rumpel, C., 2020b. Age matters: fate of soil organic matter during ageing of earthworm casts produced by the anecic earthworm Amyntha khami. Soil Biology and Biochemistry 148, 107906.

Bouché, M.B., 1972. Lombriciens de France. Ecologie et systématique. Inra Editions.

Canisares, L.P., Banet, T., Rinehart, B., McNear, D., Poffenbarger, H., 2023. Litter quality and living roots affected the formation of new mineral-associated organic carbon but did not affect total mineral-associated organic carbon in a short-term incubation. Geoderma 430, 116302.

Cerri, C., Christian, F., Jerome, B., Reynaldo, V., A, P., 1985. Application du traçage isotopique naturel en ¹³C à l'étude de la dynamique de la matière organique dans les sols. Comptes Rendus De l'académie Des Sciences De Paris 300, 423-428.

Córdova, S.C., Olk, D.C., Dietzel, R.N., Mueller, K.E., Archontouilis, S.V., Castellano, M. J., 2018. Litter plant quality affects the accumulation rate, composition, and stability of mineral-associated soil organic matter. Soil Biology and Biochemistry 125, 115-124.

Cotrufo, M.F., Lalavee, J.M., 2022. Soil organic matter formation, persistence, and functioning: a synthesis of current understanding to inform its conservation and regeneration. Advances in Agronomy 172, 1-66.

Cotrufo, M.F., Ranalli, M.G., Haddix, M.L., Six, J., Lugato, E., 2019. Soil carbon storage informed by particulate and mineral-associated organic matter. Nature Geoscience 12, 989-994.

Cui, L., Liang, C., Duncan, D.S., Bao, X., Xie, H., He, H., Wickings, K., Zhang, X., Chen, F., 2016. Impacts of vegetation type and climatic zone on neutral sugar distribution in natural forest soils. Geoderma 282, 139-146.

Eder, E., Spielvogel, S., Köbl, A., Albert, G., Kögel-Knabner, I., 2010. Analysis of hydrolysable neutral sugars in mineral soils: improvement of alditol acetylation for gas chromatographic separation and measurement. Organic Geochemistry 41, 580-585.

Filser, J., Faber, J.H., Tiunov, A.V., Brussaard, L., Frouz, J., De Deyn, G., Uvarov, A.V., Berg, M.P., Lavelle, P., Loreau, M., Wall, D.H., Querner, P., Eijsackers, H., Jiménez, J.J., 2016. Soil fauna: key to new carbon models. Soil 2, 565-582.

Fusaro, S., Gavinelli, F., Lazzarini, F., Paoletti, M.G., 2018. Soil biological quality index based on earthworms (QBS-e). A new way to use earthworms as bioindicators in agroecosystems. Ecological Indicators 93, 1276-1292.

Gan, S.H., McTavish, M.J., Bourchier, R.S., Smith, S.M., 2023. Soil grit influences the effects of earthworms (Lumbricus terrestris) on seed damage. Applied Soil Ecology 185, 104807.

Guhra, T., Stolze, K., Schweizer, S., Totsche, K., 2020. Earthworm mucus contributes to the formation of organo-mineral associations in soil. Soil Biology and Biochemistry, 107785.

Guhra, T., Wonneberger, A., Stolze, K., Ritschel, T., Totsche, K.U., 2024. The functional role of earthworm mucus during aggregation. Journal of Plant Nutrition and Soil Science 187, 63-76.

Gunina, A., Kuzyakov, Y., 2015. Sugars in soil and sweets for microorganisms: review of origin, content, composition and fate. Soil Biology and Biochemistry 90, 87-100.

Harris, D., Horwáth, W.R., Van Kessel, C., 2001. Acid fumigation of soils to remove carbonates prior to total organic carbon or carbon-13 isotopic analysis. Soil Science Society of America Journal 65, 1853-1856.

Kellerová, A., Angst, G., Jílková, V., 2024. Earthworms facilitate stabilization of both more-available maize biomass and more-recalcitrant maize biochar on mineral particles in an agricultural soil. Soil Biology and Biochemistry 189, 109278.

Kleber, M., Bourg, I.C., Coward, E.K., Hansel, C.M., Myneni, S.C.B., Nunan, N., Sveriges, L., 2021. Dynamic interactions at the mineral-organic matter interface. Nature Reviews Earth & Environment 2, 402-421.

Kopittke, P.M., Minasny, B., Pendall, E., Rumpel, C., McKenna, B.A., 2024. Healthy soil for healthy humans and a healthy planet. Critical Reviews in Environmental Science and Technology 54, 210-221.

Lavalee, J.M., Soong, J.L., Cotrufo, M.F., 2020. Conceptualizing soil organic matter into particulate and mineral-associated forms to address global change in the 21st century. Global Change Biology 26, 261-273.

Le Mer, G., Barthod, J., Dignac, M., Barré, P., Baudin, F., Rumpel, C., 2020. Inferring the impact of earthworms on the stability of organo-mineral associations, by Rock-Eval thermal analysis and ¹³C NMR spectroscopy. Organic Geochemistry 144, 104016.

Le Mer, G., Bottinelli, N., Dignac, M., Capowiez, Y., Jouquet, P., Mazurier, A., Baudin, F., Caner, L., Rumpel, C., 2022. Exploring the control of earthworm cast macro- and micro-scale features on soil organic carbon mineralization across species and ecological categories. Geoderma 427, 116151.

Liang, C., Schimel, J.P., Jastrow, J.D., 2017. The importance of anabolism in microbial control over soil carbon storage. Nature Microbiology 2, 1-6.

Liu, C., Jia, J., Dai, G., Ma, T., Feng, X., 2019. Origin and distribution of neutral sugars in soils. Chinese Journal of Plant Ecology 43, 284.

Llorente, M., Glaser, B., Turrión, M., 2017. Effect of land use change on contents and distribution of monosaccharides within density fractions of calcareous soil. Soil Biology and Biochemistry 107, 260-268.

Njoroge, D.M., Chen, S.C., Zuo, J., Dossa, G.G.O., Cornelissen, J.H.C., 2022. Soil fauna accelerate litter mixture decomposition globally, especially in dry environments. Journal of Ecology 110, 659-672.

Pan, X., Song, W., Zhang, D., 2010. Earthworms (Eisenia foetida, Savigny) mucus as complexing ligand for imidacloprid. Biology and Fertility of Soils 46, 845-850.

Ruiz, F., Barreto, M.S.C., Rumpel, C., Nóbrega, G.N., Oliveira, H.A., Menandro, A.S., Péres, L.O., Montes, C.R., Ferreira, T.O., 2024. Adsorption and thermal stability of dissolved organic matter on Ca- and Mg-exchanged montmorillonite: implications for persistence in soils and sediments. Chemical Geology 643, 121813.

Rumpel, C., Amiraslani, F., Bossio, D., Chenu, C., Henry, B., Espinoza, A.F., Koutika, L., Ladha, J., Madari, B., Minasyn, B., 2022. The role of soil carbon sequestration in enhancing human resilience in tackling global crises including pandemics. Soil Security 8, 100069.

Rumpel, C., Dignac, M., 2006. Gas chromatographic analysis of monosaccharides in a forest soil profile: analysis by gas chromatography after trifluoroacetic acid hydrolysis and reduction-acetylation. Soil Biology and Biochemistry 38, 1478-1481.

Rumpel, C., Eusterhues, K., Kögel-Knabner, I., 2010. Non-cellulosic neutral sugar contribution to mineral associated organic matter in top- and subsoil horizons of two acid forest soils. Soil Biology and Biochemistry 42, 379-382.

Sinha, R.K., Agarwal, S., Chauhan, K., Chandran, V., Soni, B.K., 2010. Vermiculture technology: reviving the dreams of Sir Charles Darwin for scientific rise of earthworms in sustainable development programs. Technology and Investment 01 155-172.

Six, J., Paustian, K., Elliott, E.T., Combrink, C., 2000. Soil structure and organic matter: I. Distribution of aggregate-size classes and aggregate-associated carbon. Soil Science Society of America Journal 64, 681-689.

Sokol, N.W., Sanderman, J., Bradford, M.A., 2019. Pathways of mineral-associated soil organic matter formation: integrating the role of plant carbon source, chemistry, and point of entry. Global Change Biology 25, 12-24.

Sokol, N.W., Whalen, E.D., Jilling, A., Kallenbach, C., Pett Ridge, J., Georgiou, K., 2022. Global distribution, formation and fate of mineral-associated soil organic matter under a changing climate: a trait-based perspective. Functional Ecology 36, 1411-1429.

Song, C., Bottinelli, N., Tran, T., Ruiz, F., Colombini, G., Zi, Y., Jouquet, P., Rumpel, C., 2024. Land use determines the composition and stability of organic carbon in earthworm casts under tropical conditions. Soil Biology and Biochemistry 190, 109291.

Van Groenigen, K.J., Osenberg, C.W., Hungate, B.A., 2011. Increased soil emissions of potent greenhouse gases under increased atmospheric CO2. Nature 475, 214-216.

Vidal, A., Blouin, M., Lubbers, I., Capowiez, Y., Sanchez-Hernandez, J.C., Calogiuri, T., van Groenigen, J.W., 2023. The role of earthworms in agronomy: consensus, novel insights and remaining challenges. Advances in Agronomy 181, 1-78.

Vidal, A., Quenea, K., Alexis, M., Nguyen Tu, T.T., Mathieu, J., Vaury, V., Derenne, S., 2017. Fate of ¹³C labelled root and shoot residues in soil and anecic earthworm casts: a mesocosm experiment. Geoderma 285, 9-18.

Vidal, A., Watteau, F., Remusat, L., Mueller, C.W., Nguyen Tu, T., Buegger, F., Derenne, S., Quenea, K., 2019. Earthworm cast formation and development: a shift from plant litter to mineral associated organic matter. Frontiers in Environmental Science 7, 55.

von Lützny, M., Kogel-Knabner, I., Ekschmitt, K., Flessa, H., Guggenberger, G., Matzner, E., Marschner, B., 2007. SOM fractionation methods: relevance to functional pools and to stabilization mechanisms. Soil Biology and Biochemistry 39, 2183-2207.

Whalen, E.D., Grandy, A.S., Sokol, N.W., Keiluweit, M., Ernakovich, J., Smith, R.G., Frey, S.D., 2022. Clarifying the evidence for microbial- and plant-derived soil organic matter, and the path toward a more quantitative understanding. Global Change Biology 28, 7167-7185.

Wüst, P.K., Horn, M.A., Drake, H.L., 2009. In situ hydrogen and nitrous oxide as indicators of concomitant fermentation and denitrification in the alimentary canal of the EarthwormLumbricus terrestris. Applied and Environmental Microbiology 75, 1852-1859.

Xiao, K., Zhao, Y., Liang, C., Zhao, M., Moore, O.W., Otero-Fariña, A., Zhu, Y., Johnson, K., Peacock, C.L., 2023. Introducing the soil mineral carbon pump. Nature Reviews Earth & Environment 4, 135-136.

Xu, Y., Liu, K., Yao, S., Zhang, Y., Zhang, X., He, H., Feng, W., Ndzana, G.M., Chenu, C., Olk, D.C., Mao, J., Zhang, B., 2022. Formation efficiency of soil organic matter from plant litter is governed by clay mineral type more than plant litter quality. Geoderma 412, 115727.

Yu, W., Huang, W., Weintraub-Leff, S.R., Hall, S.J., 2022. Where and why do particulate organic matter (POM) and mineral-associated organic matter (MAOM) differ among diverse soils? Soil Biology and Biochemistry 172, 108756.
Zhang, F., Chen, X., Yao, S., Ye, Y., Zhang, B., 2022. Responses of soil mineral-associated and particulate organic carbon to carbon input: a meta-analysis. Science of the Total Environment 829, 154626.

Zhu, X., Kong, W., Huang, Y., Xiao, K., Luo, Y., An, S., Liang, C., 2024. Soil microbial carbon pump conceptual framework 2.0. Chinese Journal of Applied Ecology 35, 102–110.

Zi, Y., Bottinelli, N., Razafindrakoto, M., Capowiez, Y., Florio, A., Song, C., Dignac, M., Rumpel, C., 2025. Let's get functional: relationship between earthworm traits and cast properties. Soil Biology and Biochemistry, 109809.