# Inferring the impact of earthworms on the stability of organo-mineral associations, by Rock-Eval thermal analysis and 13C NMR spectroscopy

Guillaume Le Mer, Justine Barthod, Marie-France Dignac, Pierre Barré, François Baudin, Cornélia Rumpel

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## To cite this version:

Guillaume Le Mer, Justine Barthod, Marie-France Dignac, Pierre Barré, François Baudin, et al. Inferring the impact of earthworms on the stability of organo-mineral associations, by Rock-Eval thermal analysis and 13C NMR spectroscopy. Organic Geochemistry, 2020, 144, pp.104016. 10.1016/j.orggeochem.2020.104016. hal-02905673

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Distributed under a Creative Commons Attribution 4.0 International License
1 Inferring earthworms' impact on the stability of organo-mineral

2 associations by Rock-Eval pyrolysis and ¹³C NMR spectroscopy

3 Le Mer, Guillaume¹, Barthod, Justine¹, Dignac, Marie-France¹, Barré, Pierre²,

4 Baudin, François³, and Rumpel, Cornelia¹

5

6 ¹ Sorbonne Université, CNRS, INRA, Institute of Ecology and Environmental

7 Sciences Paris (IEES), Paris, France

8 ² Laboratoire de Géologie, UMR 8538, Ecole Normale Supérieure, PSL Research

9 University, CNRS, Paris 75005, France

10 ³ Institut des Sciences de la Terre de Paris (ISTeP), Sorbonne Université, Paris

11 75005, France
12 **Abstract**

13 Stabilization of organic matter (OM) against decomposition and its de-
14 stabilization leading to mineralization are important processes controlling
15 greenhouse gas emissions and carbon sequestration in soils. Soil organisms,
16 particularly earthworms, may greatly influence these processes through their
17 potential impact on the interaction of OM with clay minerals, a mechanism
18 which may protect OM at medium (years) to long (decades, centuries) time scales.
19 The aim of this study was to use earthworms and pure minerals in order to
20 enhance carbon sequestration through the formation of aggregates containing
21 particulate OM and organo-mineral associations during a composting
22 experiment. To this end we compared OM transformations in composting
23 treatments with and without (i) clay minerals (montmorillonite) and (ii) epigeic
24 earthworms (*Eisenia andrei* and *Eisenia foetida*). We used density fractionation
25 of the end-products to quantify the amount of aggregates including organo-
26 mineral associations formed during 196 days. Their chemical composition and
27 stability were analyzed by elemental analyses, Rock-Eval 6 pyrolysis and solid-
28 state ¹³C nuclear magnetic resonance spectrometry.
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30 Our results indicated that the addition of minerals led to the formation of heavy
31 fractions most probably containing aggregates and organo-mineral associations
32 in treatments with and without earthworms. While OM showed higher oxidative
33 transformation in all treatments, addition of earthworms changed organic carbon
34 (OC) and nitrogen (N) concentrations in specific density fractions and increased
35 the thermal stability of OM in heavy density fractions. These fractions contained
36 higher proportions of aromatic and proteinaceous material. We conclude that
37 earthworms under composting conditions may be able to effectively stabilize OM,
38 though the formation of aggregates and/or organo-mineral associations
39 containing greater proportions of microbial-derived material.
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40

41 **Keywords:** earthworms, organo-mineral associations, compost, carbon
42 sequestration, carbon forms, thermal stability
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# 1. Introduction

In the actual context of global changes, carbon sequestration on land may be one option to reduce atmospheric CO₂ concentrations (IPCC, 2018). However, up to now organic carbon (OC) is massively lost from the largest terrestrial carbon reservoir (soil), mainly due to agricultural activities (Sanderman et al., 2017). The residence time of organic compounds in soil is driven by their chemical composition (recalcitrance), their accessibility to microorganisms and the size, diversity and activity of microbial decomposer communities (Schmidt et al., 2011). Physical and physico-chemical protection of organic matter (OM), a key mechanism for OC stabilization (von Lützow et al., 2007; Kögel-Knabner et al., 2008), may be highly influenced by soil fauna, which is rarely taken into account in soil OM studies.

In most terrestrial ecosystems, earthworms are one of the largest contributors to the soil invertebrate biomass (Asshoff et al., 2010). These organisms are considered as ecological engineers because they directly or indirectly modify access to biotic or abiotic resources for other species (Jones et al., 1994). On the one hand, stable aggregates and organo-mineral associations may be formed in the earthworms' gut by mechanically mixing OM and mineral constituents and by transforming OM into microbial products. As a result, earthworms could enhance OM physico-chemical protection from mineralization (Six et al., 2004; Bossuyt et al., 2005; Angst et al., 2017, 2019). On the other hand, by ingesting OM, micro-organisms and mineral particles, earthworms may increase the rate of OM turnover by mineralization, fragmentation and stimulation of microbial activity (Lavelle et al., 1989; Don et al., 2007). Thus, earthworms may affect OM in two contrasting ways: they may (i) increase OM mineralization, by stimulating microbiota by provision of easily assimilable organic compounds and at the same time (ii) increase OM stabilization, by promoting the sorption of organic compounds onto mineral surfaces and through the formation of stable biogenic aggregates (Domínguez et al., 2010; Wu et al., 2017; Angst et al., 2019). To ultimately conclude on the net effect of earthworms on OM stabilization, both processes need to be understood (Barthod et al., 2020). While the earthworms'
effect on greenhouse gas emissions has been addressed extensively (see review by Lubbers et al., 2013), few studies are investigating the organo-mineral complexes formed in the earthworms' gut.

In this study we used composting of organic waste in the presence of minerals and earthworms as a model to investigate the effects of earthworms on OM stabilization by mineral associations. Vermicomposting in the presence of epigeic earthworms such as *Eisenia sp.* is often used for transforming household wastes (Garg et al., 2006). This practice always induces higher CO₂ emissions compared to regular composting, due to earthworms' respiration and stimulation of microbial activity (Barthod et al., 2018). Mixing organic waste with minerals during early steps of composting, the so-called co-composting, can significantly reduce CO₂ emissions and *in fine* OM mineralization (Vinceslas-Akpa and Loquet, 1997; Bolan et al., 2012; Barthod et al., 2016), most probably due to the formation of organo-mineral complexes. However, up to now, to the best of our knowledge no studies have been carried out to quantify formation of aggregates containing organo-mineral associations during compositing or vermicomposting. This information is necessary, to elucidate the processes responsible for reduction of mineralization rates after addition of minerals.

We used density fractionation to characterize the end-products of a 6-months composting experiment with montmorillonite with or without addition of earthworms. Density fractionation has often been used as a method to evaluate the protection of OM in aggregates by separating mineral-associated OM from free OM (Six et al., 2002; Wagai et al., 2018). Indeed, the differences in OM stability, composition and distribution between fractions (free OM or associated with mineral) may be related to OM residence time and mineralization dynamics at different timescales (Poeplau et al., 2018). Free OM is easily separated from mineral particles by wetting and low energy agitation, while dispersion of macroaggregates (with intermediate density), microaggregates, and separation of OM strongly associated with minerals requires energy input (e.g. sonification) (Golchin et al., 1994).
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The objective of our study was to quantify and characterize the aggregates
containing organo-mineral associations formed in the presence of earthworms
and to compare them to those formed without earthworms. We examined the
distribution of OM (carbon (C) and nitrogen (N)) in the four different density
fractions and used Rock Eval 6 (RE6) pyrolysis to analyze their thermal stability.
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Moreover, we aimed at investigating the organic compounds affected by the
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physicochemical stabilization processes. To this end, we characterised the
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chemical composition of OM in density fractions by solid-state ¹³C cross-
polarization magic-angle spinning nuclear magnetic resonance (CPMAS NMR)
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spectroscopy.
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We hypothesized that OM transformation in the presence of earthworms and
minerals will enhance carbon sequestration through the formation of organo-
mineral associations and/or aggregates. Moreover, we expected that composting
in the presence of earthworms will lead to end products with distinct thermal
stability indices. We expected significant earthworm effects on (i) the
contribution of easily decomposable compounds and (ii) the stabilization of
organic compounds in dense fractions.
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## 2. Material & Methods

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### 2.1. (*Vermi-*)composting experiment

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A mixture of organic materials was prepared consisting of (dry weight): 1.2%
lettuce, 19.6% apples, 39.1% spent coffee ground, 10.1% maize and 27.9%
cardboard. The mixture has an initial C:N ratio of 40, according to earthworms'
and microbial degradation requirements (Barthod et al., 2016). Lettuces and
apples, products of organic agriculture, were obtained from a general store. The
apple grains were removed before use. The spent coffee ground was collected
from a public cafeteria, and the aboveground maize biomass from a field in
Grignon (France).
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Epigeic earthworms generally used for vermicomposting were selected for this
experiment. *Eisenia andrei* and *E. fetida* are species able to develop in fresh OM,
which can adapt to a wide range of environmental factors and present a high rate
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of consumption, digestion and assimilation of OM (Suthar, 2006). Moreover,
these species are characterized by high reproduction rates. The earthworms were
purchased from La Ferme du Moutta (a worm farm in France) and reproduced in
the lab.

Montmorillonite was purchased from Sigma Aldrich in powder form with a
purity of 99% and an average particle size of < 80 nm.

Laboratory composting and vermicomposting experiments were carried out in the dark at 20°C for 196 days. Both treatments were set up with or without minerals in four containers (10 L) closed with a plastic lid with holes to ensure aeration. For the treatments with minerals (co-compost and co-vermicompost), the organic material was thoroughly mixed with montmorillonite in 7:3 (OM:M) weight proportions. Water was added to the clay treatment in order to reach a similar water content (75% of dry material) as in the regular compost and vermicompost mixtures. 20 earthworms were added to the vermicomposting and co-vermicomposting containers. The number of earthworms needed for complete digestion of the OM had been determined in a preliminary experiment. Samples were turned every 15 days. After six months, samples were harvested, At the end of the composting period no precursor compounds could be recognized in any treatments. The materials were well decomposed, minerals had been incorporated into OM, and earthworms had reproduced. Adult earthworms and cocoons were manually removed from the corresponding treatments. Samples were air-dried before density fractionation. Elemental parameters of bulk samples are presented in Table 1.

2.2. *Density fractionation*

A sequential density fractionation was conducted for all treatments using
deionized water and sodium polytungstate solutions (SPT-0 grade, Sometsu,
Germany). This fractionation was carried out on 3 subsamples of each treatment
(with or without earthworm). Briefly, 2 g of the compost products were
sequentially isolated to obtain four fractions. F0 corresponded to dissolved OM or
particles with a density < 1 g.cm⁻³. F1 corresponded to OM < 1.6 g.cm⁻³ and two
fractions with mineral associated OM (F2 with a density between 1.6 and 1.8 g.cm⁻³ and F3 with a density > 1.8 g.cm⁻³) were also isolated. Density cut-offs have been chosen based on the literature for the isolation of particulate OM fractions (exp. Golchin et al., 1994). The fractionation was carried out without ultrasonic dispersion to avoid disruption of the aggregates (Golchin et al., 1994; Wagai et al., 2018). Between each fractionation step, samples were mechanically shaken in SPT solution for one hour. As sodium polytungstate is a salt solution, this procedure dispersed unstable macroaggregates.

After mechanical shaking, SPT solutions were centrifugated (10 min; 10000 RPM) to collect supernatants, which were filtered through a glass fiber filter (Whatman, GF/F) using a vacuum pump. The material remaining on the filter paper was rinsed with deionized water to remove SPT. Pellets remaining in the centrifugation tubes were resuspended in SPT solutions with a higher density. The steps were repeated sequentially using the same sample to separate the 3 fractions. All fractions were freeze-dried, weighted and stored. An aliquot was mechanically grounded (< 250 µm) to prepare the samples before analyses. In earthworm treatments with minerals, the heaviest fraction (F3) always contained macroscopic black granules (1 mm diameter). These were removed from the F3 fractions and separately analyzed (elemental analysis and Raman spectroscopy).

## 2.3. *Elemental analysis and Raman spectroscopy*

Total organic-C (OC) and total nitrogen (N) concentrations of all fractions and also the black granules were analyzed by dry combustion using a CHN analyzer (CHN NA 1500, Carlo Erba).

Raman spectra (Renishaw InVia) were obtained from black granules of the F3 fraction of co-vermicompost treatments. The analysis was carried out on intact particles (surface analysis) and on crushed ones (internal composition).

## 2.4. *Rock-Eval 6 pyrolysis*
The thermal stability of OM in the density fractions was investigated with a Rock-Eval 6 turbo device (Vinci Technologies). For each fraction, around 60 mg of finely ground material (60 mg of organo-mineral fractions (F2 and F3) or sand mixed with 10 mg of OM fractions for the F0 and F1 fractions) were exposed to thermal treatments. Rock Eval 6 pyrolysis consists of a stepwise pyrolysis under N₂ atmosphere (S2 phase, 200-650 °C; 30 °C·min⁻¹ thermal ramping rate) followed by a combustion-oxidation under laboratory air atmosphere (300-850 °C; 20 °C·min⁻¹ thermal ramping rate). For both steps, CO and CO₂ were monitored by infrared (IR) detection. Free hydrocarbon (HC) effluents were also measured during the pyrolysis step by a flame ionization detector (FID). Several standard parameters referring to OM chemistry were calculated (Disnar et al., 2003; Schomburg et al., 2018): Total organic carbon (TOC), pyrolysable carbon (PC), residual carbon (RC), hydrogen index (HI) and oxygen index (OI). Several additional RE6 parameters indicative of OM stability were also calculated. The I- and the R-index were calculated from the S2 signal (Sebag et al., 2016). The R-index corresponds to the relative contribution of thermally stable OM with refractory compounds released at higher temperatures and the I-index gives information on the pool of thermally labile immature OM with compounds released at lower temperatures (Sebag et al., 2016). They were both related to compost maturity (Albrecht et al., 2015) and the proportion of OC in particulate OM fractions isolated from soil (Soucémarianadin et al., 2018). An additional RE6 parameter was calculated using the S3 signal: the T₅₀ CO₂ PYR corresponds to the temperature at which 50% of the CO₂ had evolved. The T₅₀ CO₂ PYR has been related to the proportion of centennial persistent OC in soils (Cécillon et al., 2018). We also determined the temperature at which 50% of the residual OM was oxidized to CO₂ during the oxidation phase (T₅₀ CO₂ OX). This variable was used as an index of the overall biogeochemical stability of OM (Barré et al., 2016).

## 2.5. Solid-state ¹³C NMR spectrometry

The chemical composition of bulk OM and density fractions was analyzed by solid-state ¹³C CPMAS NMR spectroscopy using a Bruker DSX-200 NMR spectrometer. The F0 fraction could not be analyzed due to limited amount of
material. For bulk samples and all other fractions (F1, F2 & F3), NMR spectra of two replicates were recorded. Cross polarization with magic angle spinning (CPMAS) (Schaefer and Stejskal, 1976) was applied at 6.8 kHz. The ¹³C chemical shifts were referenced to tetramethylsilane. A contact time of 1 ms was used and pulse delay was 800 ms. Solid-state ¹³C CPMAS NMR signals were recorded as free induction decay (FID) and Fourier transformed to yield the CPMAS NMR spectra. A line broadening of 0 was used for every spectrum. For fractions and bulk samples of all treatments, the area (% of total area) under the curve corresponding to five chemical shift regions (0-45 ppm, 45-110 ppm, 110-140 ppm, 140-160 ppm and 160-220 ppm) was integrated using the integration routine of the spectrometer. These regions were respectively assigned to alkyl C, O/N-alkyl C, C substituted aryl C, O substituted aryl C and carboxylic C (Wilson, 1987).

An estimation of natural molecules contributing to OM composition was performed with ¹³C CPMAS NMR spectroscopic data through a molecular mixing model based on coefficients reflecting their terrestrial distribution (Nelson and Baldock, 2005). For terrestrial samples mostly composed of plant-derived materials and microbial compounds, this predictive model represents more than 95% of the total intensity of the ¹³C CPMAS NMR signal (Nelson and Baldock, 2005). For each density fraction of co-(vermi)compost treatments, the relative contribution of four biomolecule components (carbohydrates, proteins, lignins, aliphatic compounds) and two additional components (carbonyls and char) was estimated. To investigate the impact of earthworms on their distribution in the density fractions, the response ratio between treatments ('co-vermicompost response' to 'co-compost response' ratio) was calculated for each component using logarithmic transformation.

## 2.6. Statistical analysis

Prior to analysis, normality and homogeneity of variances were inspected using respectively Shapiro-Wilk's test and Levene's tests. Even after data log-transformation, conditions to perform mean comparison parametric tests were not fulfilled.
In consequence the non-parametric Kruskal-Wallis test was performed with Bonferroni correction to assess differences in (i) mass distribution, (ii) OC and N concentrations, (iii) C:N ratios and (iv) thermal stability indices, among the different fractions and treatments (agricolae package). Simple mean comparisons between two treatments were performed using the U-Mann & Whitney test to assess significant differences. Significance was declared when p-values were lower than 0.05. Correlations between variables were statistically tested with the Pearson correlation test. Only relevant and significant correlations were presented. All statistical calculations were carried out using the R software (R core team, 2005). Graphics were computed using the *ggplot2* package.

The estimation of the molecular composition from NMR data was performed on Microsoft® Excel Solver for all fractions of co-composts and co-vermicomposts, using coefficients provided by Nelson and Baldock (2005). Response ratios of each variable in the fractions were displayed using the *forestplot* package in R software (R core team, 2005).

## 3. Results and discussion

### 3.1. *Effect of epigeic earthworms on OC and N distributions in density fractions*

Density fractionation of treatments without minerals (compost and vermicompost) yielded only light fractions < 1.6 g.cm⁻³, containing soluble (F0) and free (F1) OM. The fractions F0 and F1 represented 5.4% and 94.6% of total mass for compost and 4.8% and 95.2% for vermicompost. In both treatments, mass recovery was 94 ± 3 % (mean ± sd) on average (n = 6). Samples with minerals (co-compost and co-vermicompost) were density fractionated in 4 fractions with a recovery of 91 ± 5 % (n = 6). We recovered 85 ± 2 % of OC and 99 ± 1 % of N present in the bulk samples (n = 6).

The lightest fractions (F0 & F1) showed similar mass contributions for co-compost and co-vermicompost ranging from 1.7 to 17.8 % (Table 2). The fraction with a density between 1.6 and 1.8 g.cm⁻³ (F2) was the largest fraction in both treatments, comprising from 51 to 68% of mass. This fraction most probably
contains occluded OM (Wagai et al., 2008) and organo-mineral associations (Baisden et al., 2002). It tended to be higher in co-vermicompost as compared to co-compost (Table 1), suggesting that epigeic earthworms promote the formation of aggregates (Lavelle and Martin, 1992; Six et al., 2000).

The heaviest fraction > 1.8 g.cm⁻³ (F3) had a significantly lower contribution in the co-vermicompost than in the co-compost treatment. This could be due to the continuous incorporation of pre-existing microaggregates into new macroaggregates during the composting process. These structures present high stability with microbial products acting as glue (Bossuyt et al., 2004; Angst et al., 2019).

Moreover, the >1.8 g.cm⁻³ fraction of the co-vermicompost treatment was characterized by the presence of black granules, which were separately collected. Raman spectroscopic analysis of these granules showed that they are probably made of amorphous silica (Fig. 1a), with no organic signal, as confirmed by elemental analysis (Table 1). Scanning Electron Microscopy (SEM) images of the granules showed a flat silica surface with holes (Fig. 1b). Previous studies have also indicated biotransformation processes involving silica during vermicomposting (Gago-Duport et al., 2008; Méndez et al., 2008). Extracellular silica nanoparticles produced by micro-organisms may be remobilized in the worm gut to form macrostructures (Espíndola-Gonzalez et al., 2014). This biotransformation through microbial metabolism takes place in the earthworm gut and may impact the silica cycle (Bityutskii et al., 2017). We did not observe calcium carbonate granules generally found with earthworm activity because of the calcium glands present in these invertebrates (Canti, 1998).

The OC and N distributions in the density fractions of co-compost and co-vermicompost (Fig. 2) did not show any significant differences between treatments (p-value > 0.05). OC concentrations in fractions ranged from 72.7 to 374.0 mg.g⁻¹ (Table 2). They were significantly higher in F0 fractions of the vermicompost treatment compared to the F0 fraction of the compost treatment (+6%). In contrast, lower OC concentrations (-35%) were recorded in the F3
fractions of co-vermicomposts as compared to co-composts. N concentrations in the fractions ranged from 6.7 to 30.8 mg.g⁻¹ (Table 1). Light fractions (F0 and F1) of the vermicompost treatment showed lower N concentrations than those of the compost treatment. Consequently, they also showed higher C:N ratios in lightest fractions, ranging between 19 and 29 as compared to 10 and 13, for the compost treatment (Table 1). Assuming that materials with low C:N ratios are characterized by a higher decomposition degree than materials with high C:N ratios (Manzoni et al., 2008), our data indicate that the addition of earthworms and minerals during composting had an influence on the decomposition rate of OM and the biogeochemical properties of the endproducts. The stoichiometric ratios indicated that the non-occluded F1 fraction might have a significantly lower decomposability in the co-vermicompost treatment compared to the co-compost treatment. The C:N ratios decreased with regards to the initial OM. This could be related to the decrease of labile OM and plant biopolymers such as cellulose and hemicellulose (Albrecht et al., 2015). However, the higher C:N ratios of F0 and F1 fractions of vermicompost as compared to compost were not in agreement with a higher decomposition degree usually observed in the presence of epigeic earthworms. These earthworms generally accelerate the decomposition and transformation of OM during vermicomposting (Huang and Xia, 2018). In order to characterize the impact of these transformations on the OM thermal stability, we performed Rock-Eval 6 pyrolysis, which may provide some indications about dynamics of OM (Soucémarianadin et al., 2018).

## 3.2. *OM thermal signature of bulk samples*

Rock-Eval 6 (RE6) thermal analysis of bulk samples showed a significant effect of composting practices on thermal stability indices (Table 3). Average Hydrogen Index (HI) and Oxygen Index (OI) corresponding to the elemental composition of the samples were (mg [HC or CO₂] g⁻¹ TOC⁻¹): 391 and 165 for compost, 403 and 161 for vermicompost, 359 and 208 for co-compost and 341 and 178 for co-vermicompost (Fig. 3). According to Fig. 3, these indices were affected by the addition of montmorillonite during the (vermi)composting processes (p-value < 0.05). The HI decreased and, conversely, the OI increased with the
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addition of montmorillonite. Furthermore, the T<sub>50_CO2_PYR</sub> index, which may serve
as an OM stability indicator, was significantly lower in the co-compost treatment.
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The T<sub>50_CO2_OX</sub> index also tended to be higher in earthworm treatments (Table 3).
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Mean values of other thermal parameters for all composting practices were 0.44
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for the recalcitrance index (R), 0.40 for the index of the contribution of immature
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OM (I), 0.39 for pyrolysable C (PC:TOC ratio), 0.62 for residual C (RC:TOC ratio)
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and 0.63 for the ratio of PC:RC (Table 3). These last RE6 parameters did not
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show any significant differences among treatments.
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The RE6 indices suggest that loss of H bonds led to a significant
dehydrogenation of organic compounds, which was enhanced in treatments with
earthworms and minerals (Table 3). Contrasting HI may reflect differences of
OM decomposition processes in the presence of earthworms and minerals
(Barthod et al., 2020). The HI is related to the H:C atomic ratio, which decreases
following the decomposition of OM (Barré et al., 2016). Moreover, OI values
increased in co-compost and co-vermicompost treatments as compared to compost
and vermicompost treatments (Table 3). This could be an indicator of the
enhanced oxidative transformation of OM in the presence of minerals (von
Lützow and Kögel-Knabner, 2010). The OI was previously found to be positively
correlated with the proportion of persistent OC pools in temperate soils (Cécillon
et al., 2018) and thus rather stable compounds. During the degradation of OM in
presence of minerals and earthworms (co-vermicompost), Rock eval parameters
indicated that dehydrogenation (HI decrease) and oxidation (OI increase) were
occurring in both treatments.
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Regarding T<sub>50_CO2_PYR</sub>, it was interesting to note that the proportion of
persistent OC pools (Cécillon et al., 2018) seemed to be lower in co-composts than
in other treatments. According to Barthod et al. (2020), the addition of minerals
during composting may significantly reduce microbial biomass during the
degradation of OM, indicating that minerals may have a negative effect on
microbial exoenzyme activities (Lähdesmäki and Pnspanen, 1992). Our data
show that earthworm activity seemed to compensate this negative effect.
Moreover, earthworm presence during (co-)vermicomposting seemed to increase
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the biogeochemical stability of OM, as the final product was characterized by higher $T_{50\_CO_2\_OX}$ values. This effect was even stronger without addition of mineral and might thus not reflect increased stabilization of OM by interaction with mineral particles. In the following we quantified OM thermal stability in density fractions.

### 3.3. *OM thermal signature of density fractions*

For density fractions of co-compost and co-vermicompost standard RE6 parameters ranged from 248 to 484 mg HC g⁻¹ TOC⁻¹ for HI, 135 to 189 HC g⁻¹ TOC⁻¹ for OI, 0.29 to 0.47 for PC:TOC, 0.54 to 0.71 for RC:TOC, and 0.41 to 0.87 for PC:RC (Table 3). HI values of light fractions (F0 & F1) isolated from co-composts were significantly higher (461 mg HC g⁻¹ TOC⁻¹) compared to heavy fractions (F2 & F3) and also to co-vermicompost fractions (Fig. 4). OI values were higher for F0 and F3 (177 ± 12 mg CO₂ g⁻¹ TOC⁻¹) than for F1 and F2 (138 ± 7 mg CO₂ g⁻¹ TOC⁻¹) fractions of both treatments (Fig. 4). Co-compost products showed higher PC content and lower RC content in light fractions (F0 & F1) than in heavy fractions (F2 & F3) (Table 3). We recorded highest HI values in the light fractions. This may indicate that decomposition occurs preferentially in the heavier fractions. Earthworms homogenized pyrolysable (PC) and residual (RC) carbon throughout the fractions (0.30 ± 0.02 for PC:TOC and of 0.70 ± 0.02 for RC:TOC). Easily decomposable OM compounds (characterized by high PC:RC ratio and HI, Schomburg et al., 2018) are generally present in the light fractions F0 and F1. Earthworms may preferentially feed on this material (Brown et al., 2000) and may create an interaction with minerals during the gut transit (Angst et al., 2017), explaining the lower HI values in lightest fractions and the homogeneity of these parameters in all other fractions.

Moreover, in density fractions, R-indices varied between 0.39 and 0.67 while I-indices ranged between 0.09 and 0.49 (Table 4). The Fig. 5 depicts R-indices plotted against I-indices for bulk samples and density fraction of co-composts and co-vermicomposts. A negative correlation ($R^2$ = -0.99, p-value < 0.0001) shows that R-indices increased while I-indices decreased from the lightest fractions to
the heaviest fractions (F0 to F3, Fig. 5). No difference of thermal stability indices was observed for bulk samples (Table 2). The presence of earthworms during the composting process with minerals significantly impacted R- and I-indices of the F2 and F3 fractions. Higher R-indices were observed for the F2 fraction and lower I-indices for the F2 and F3 fractions in vermicompost treatments compared to the treatment without earthworms (Table 3). The S2 signal, released during the pyrolysis phase, showed peaks corresponding to the successive cracking of organic compounds of different thermal stability (Disnar et al., 2003; Copard et al., 2006; Saenger et al., 2013). The R-index is related to the contribution of OM compounds with higher thermal stability. In contrast, the I-index refers to contribution of OM pools presenting lower thermal stability (Sebag et al., 2016). The I-indices recorded for bulk samples and fractions of compost and vermicompost material with and without minerals are in the range of values recorded by Schomburg et al. (2018) for decomposed OM in sediments. Bulk samples and fractions collected in the present experiment, had R-index and I-index values fitting with SOM pools defined by Sebag et al. (2016). Bulk samples and F0 and F1 fractions were in the range of values recorded for litters and organic horizons (Albrecht et al., 2008, 2009), while F2 and F3 fractions were similar to "inherited OM" corresponding to organo-mineral and mineral soil horizons (Sebag et al., 2016). The presence of more thermally immature OM in lighter fractions (F0 & F1) and more thermally refractory OM in heavier fractions (F2 & F3) corresponds to the presence of (i) undecomposed particulate OM in F0 and F1 and (ii) more decomposed OM in aggregates and organo-mineral associations.

Earthworms seem to change the allocation of OM to density fractions with less OM in the F3 fraction of co-vermicompost than co-compost treatments (Fig.2). They also change the OM composition, as we observed more thermally recalcitrant OM pools in the F2 fraction and less thermally labile OM in F2 & F3 fractions of co-vermicompost than co-compost treatments (Table 4). T<sub>50_CO2_PYR</sub> was significantly higher in the heaviest fraction (F3) of co-vermicompost as compared to co-compost (Table 3), and could thus indicate a higher contribution of persistent OC (Cécillon et al., 2018). Moreover, T<sub>50_CO2_OX</sub> was significantly
higher in the lightest fractions (F0 & F1) of co-vermicompost compared to co-
compost (Table 3). This result suggests that OM will be decomposed more slowly
because it needs more energy input for a low energy benefit (Rovira et al., 2008;
Barré et al., 2016). We thus suggest that the presence of earthworms during
composting with minerals may lead to the formation of thermally recalcitrant
material. We analyzed the chemical composition of the different fractions with
¹³C CPMAS NMR spectroscopy in order to understand if these changes in
thermal stability along the density gradient may be explained by accumulation of
chemically recalcitrant OM compounds.

3.4. *Chemical composition of OM in density fractions*

The ¹³C CPMAS NMR spectrum of the initial OM was dominated by signals in
the O/N-alkyl C region, which represented 76.9 ± 0.2% of the signal intensity
(Fig. 6). Major differences between the spectra of initial OM and density fractions
were observed, with a decrease of the O/N-alkyl C region and an increase of alkyl
C, C substituted aryl C, O substituted aryl C and carboxylic C regions (Fig. 6). A
larger decrease of the contribution of signal intensity in the 45-110 ppm region
was observed for the co-compost treatment (-45.9%) compared to the co-
vermicompost treatment (-37.6%). The sharpest peak in this region, observed
around 75 ppm, may be assigned to oxygenated carbon of carbohydrates (Golchin
et al., 1994). It was the most important signal in all fractions regardless the
treatment (Fig. 6).

The alkyl C region (0-45 ppm), representing most likely aliphatic compounds
such as lipids, cutin, suberin and other biomacromolecules, increased in all
fractions of co-compost and co-vermicompost compared to initial OM (Fig. 6). The
emergence of a prominent peak at around 30-35 ppm corresponds to the presence
of aliphatic carbon in long chain polymethylene structures (Golchin et al., 1994)
originating most likely from the accumulation of plant-cuticle derived aliphatic
compounds during the co-(vermi)composting process. The enrichment of long-
chain aliphatic material in the F1 fraction containing free particulate OM may be
due to selective preservation of chemically recalcitrant plant litter compounds
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(Pisani et al., 2014). In the F2 and F3 fraction, it could also be explained by direct stabilization of plant-derived OM compounds (Rumpel et al., 2015).

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The proportion of aromatic C tended to be slightly higher in co-vermicompost compared to co-compost (Fig. 6). The higher contribution of the alkyl C and C and O substituted aryl C, along with lower O/N-alkyl C contribution to end-products of the compost treatment (Fig. 6) might be due to the degradation of easily decomposable carbohydrates and the selective preservation of more recalcitrant material (Kögel-Knabner, 2002; Shiau et al., 2017) by effects of micro-organisms and earthworms. Enrichment of aromatic material during OM decomposition in the presence of earthworms and minerals has been observed before (Barthod et al., 2020) and may be explained by organo-mineral interactions via mucus secretions by earthworms and/or by incomplete degradation of OM due to the preferential feeding of earthworms on specific microbial groups (Bonkowski et al., 2000). Moreover, anaerobic conditions in the earthworm gut could have led to associations between recalcitrant aromatic compounds and minerals, as shown by Huang et al. (2019) for lignin decomposition products and redox-sensitive iron minerals.

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In order to investigate the chemical recalcitrance of OM in the density fractions, we calculated ratios of signal intensities, indicative of the degree of OM decomposition: (i) 'alkyl C / O-alkyl C', (ii) 'aromatic C / O-alkyl C' and (iii) '(alkyl C + aromatic C) / O-alkyl C' (Baldock et al., 1992; Ono et al., 2013; Wang et al., 2013). Strong linear relationships were obtained for co-compost and co-vermicompost treatments between the 'aromatic C / O-alkyl C' ratio and the thermal stability R-index ($r^2$ = -0.96 for co-compost and $r^2$ = -0.98 for co-vermicompost, Fig. 7). A similar relationship was obtained between thermal stability indices and the '(alkyl C + aromatic C) / O-alkyl C' ratio with a lower correlation coefficient ($r^2$ = -0.68, p-value < 0.05) (data not shown). These correlations between the OM decomposition degree and the OM thermal stability indicate that regardless the treatment chemically stable OC forms are generally enriched in the light fractions with a low thermal stability while more labile and decomposable OC forms are present in the heavy fractions with a high thermal
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stability. These results are similar to those obtained by Deng et al. (2019) for
density fractions isolated from soil. We therefore suggest that the thermal
stability of OM in co-(vermi)compost treatments measured with R- and I-indices
is explained by protection of labile OM in aggregates and/or organo-mineral
associations rather than by the accumulation of chemically recalcitrant OM
compounds.

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The negative correlation of the R-index with the 'aromatic C / O-alkyl C' ratio presented contrasting slopes in treatments with and without earthworms. For a similar R-index, heavy fractions of co-compost presented lower 'aromatic C to O-alkyl C' ratio than co-vermicompost (Fig. 7). Moreover, for a similar 'aromatic C to O-alkyl C' ratio, earthworm presence significantly increased the thermal stability of OM in F2 and F3 fractions. These results support the hypothesis that earthworms create more tight organo-mineral associations and/or more stable aggregates requiring higher amounts of energy to be broken and thus resulting here in greater R-index values. Moreover, the enrichment of aromatic compounds in heavy fractions of co-vermicompost as illustrated by a higher 'aromatic C to O-alkyl C' in the heaviest fraction (F3) compared to co-compost (Fig. 7) could partly explain these results.

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We used the ¹³C CPMAS NMR spectroscopic data to estimate the molecular
composition of natural organic compounds with the molecular mixing model
proposed by Nelson and Baldock (2005). Compared to the control treatment,
earthworms positively affected the relative abundance of lignin-derived
compounds in all fractions (Fig. 8). Previous work has shown that lignin
compounds present in earthworm casts may be originating from decomposed and
undecomposed compounds (Vidal et al., 2016b, 2016a). The decrease of aliphatic
compounds in all fractions is consistent with the consumption of OM derived
from fresh OM compounds during composting (Fig. 3). The presence of
earthworms induced a high decrease of protein relative abundance in free OM
fraction (F1) and its concurrent increase in heavy fractions (F2 & F3),
highlighting the importance of earthworm activity for fresh OM degradation and
microbial-derived compounds stabilization in aggregates and/or organo-mineral
associations. Indeed, earthworms may accelerate the degradation of plant litter and its transformation into microbial necromass within stabilized soil OM pools (Angst et al., 2019). These processes, which involve the passage of labile OM through microbial biomass may lead to higher OM stability through accumulation of persistent microbial material, susceptible to be stabilized within mineral associations (Miltner et al., 2012). Our results indicate a significant effect of earthworms on the stabilization of organic compounds (derived from microbial activity) in the heavy fractions, which could explain their higher thermal stability as compared to treatments without earthworms.

## 4. Conclusion

This study investigated the role of earthworms for the formation of stable OM compounds in aggregates and/or organo-mineral associations of composts produced in the presence of clay minerals. We used density fractionation to separate free and mineral-associated OM pools and investigated their thermal stability and chemical composition.

Our results indicated that isolation of OM pools was necessary to elucidate the impact of earthworms on OM transformation.

Mineral addition during composting led to the formation of aggregates containing particulate OM and/or organo-mineral associations with higher thermal stability. The presence of earthworms during co-vermicomposting changed the contribution of the free/dissolved OM pools and mineral associated OM pools. Earthworms were found to affect elemental concentration and stoichiometry of all fractions. We could validate our hypotheses, as their presence led to the formation of more thermally stable OM and changes in OM chemical composition as compared to the treatment without earthworms. We conclude that earthworms' presence in the co-vermicompost treatments favored the accumulation of proteinaceous compounds most probably derived from microbial activity in aggregates and/or organo-mineral associations with high thermal stability.
558 Acknowledgments

559 This project was financially supported by the Vermisol research project under the
560 framework of the DOSTE program of ADEME and by Sorbonne Université
561 through the thesis of G. Le Mer. We acknowledge the technical assistance of
562 Florence Savignac for the Rock-Eval analyses. We are grateful to Damien
563 Deldicque, " Yohan Marcangeli and Valérie Pouteau for technical support of this
564 study.

565 References

566 Albrecht, R., Joffre, R., Gros, R., Le Petit, J., Terrom, G., Périssol, C., 2008. Efficiency of near-infrared reflectance spectroscopy to assess and predict the stage of transformation of organic matter in the composting process. *Bioresource Technology* 99, 448–455.

570 Albrecht, R., Joffre, R., Petit, J. Le, Terrom, G., Périssol, C., 2009. Calibration of chemical and biological changes in cocomposting of biowastes using near-infrared spectroscopy. *Environmental Science and Technology* 43, 804–811.

573 Albrecht, R., Sebag, D., Verrecchia, E., 2015. Organic matter decomposition: bridging the gap between Rock-Eval pyrolysis and chemical characterization (CPMAS13C NMR). *Biogeochemistry* 122, 101–111.

576 Angst, G., Mueller, C.W., Prater, I., Angst, Š., Peterse, F., Nierop, K.G.J., 2019. Earthworms act as biochemical reactors to convert labile plant compounds into stabilized soil microbial necromass. *Nature Communications* Biology 1–7.

580 Angst, Š., Mueller, C.W., Cajthaml, T., Angst, G., Lhotáková, Z., Bartuška, M., Špaldoňová, A., Frouz, J., 2017. Stabilization of soil organic matter by earthworms is connected with physical protection rather than with chemical changes of organic matter. *Geoderma* 289, 29–35.

584 Asshoff, R., Scheu, S., Eisenhauer, N., 2010. Different earthworm ecological
585 groups interactively impact seedling establishment. European Journal of Soil
586 Biology 46, 330–334.

587 Baisden, W.T., Amundson, R., Brenner, D.L., Cook, A.C., Kendall, C., Harden, J.W., 2002. A multiisotope C and N modeling analysis of soil organic matter turnover and transport as a function of soil depth in a California annual grassland soil chronosequence. Global Biogeochemical Cycles 16, 82-1-82–26.

591 Baldock, J.A., Oades, J.M., Waters, A.G., Peng, X., Vassallo, A.M., Wilson, M.A., 1992. Aspects of the chemical structure of soil organic materials as revealed by solid-state13C NMR spectroscopy. Biogeochemistry 16, 1–42.

594 Barré, P., Plante, A.F., Cécillon, L., Lutfalla, S., Baudin, F., Bernard, S., Christensen, B.T., Eglin, T., Fernandez, J.M., Houot, S., Kätterer, T., Le Guillou, C., Macdonald, A., van Oort, F., Chenu, C., 2016. The energetic and chemical signatures of persistent soil organic matter. Biogeochemistry 130. doi:10.1007/s10533-016-0246-0

599 Barthod, J., Dignac, M.-F., Le Mer, G., Bottinelli, N., Watteau, F., Kögel-Knabner, I., Rumpel, C., 2020. How do earthworms affect organic matter decomposition in the presence of clay-sized minerals? Soil Biology and Biochemistry 107730.

603 Barthod, J., Rumpel, C., Dignac, M.F., 2018. Composting with additives to improve organic amendments. A review. Agronomy for Sustainable Development 38. doi:10.1007/s13593-018-0491-9

606 Barthod, J., Rumpel, C., Paradelo, R., Dignac, M.F., 2016. The effects of worms, clay and biochar on CO2 emissions during production and soil application of co-composts. Soil 2, 673–683.

609 Bityutskii, N., Yakkonen, K., Loskutov, I., 2017. Content of iron, zinc and manganese in grains of Triticum aestivum, Secale cereale, Hordeum vulgare and Avena sativa cultivars registered in Russia. Genetic Resources and Crop Evolution 64, 1955–1961.

613 Bolan, N.S., Kunhikrishnan, A., Choppala, G.K., Thangarajan, R., Chung, J.W., 2012. Stabilization of carbon in composts and biochars in relation to carbon
615 sequestration and soil fertility. Science of The Total Environment 424, 264-
616 270.

617 Bonkowski, M., Griffith, B.S., Ritz, K., 2000. Food preferences of earthworms for
618 soil fungi. Pedobiologia, 44, 666-676.

619 Bossuyt, H., Six, J., Hendrix, P.F., 2004. Rapid incorporation of carbon from fresh
620 residues into newly formed stable microaggregates within earthworm casts.
621 European Journal of Soil Science 55, 393-399.

622 Bossuyt, H., Six, J., Hendrix, P.F., 2005. Protection of soil carbon by
623 microaggregates within earthworm casts. Soil Biology and Biochemistry 37,
624 251-258.

625 Brown, G.G., Barois, I., Lavelle, P., 2000. Regulation of soil organic matter
626 dynamics and microbial activity in the drilosphere and the role of
627 interactions with other edaphic functional domains. European Journal of Soil
628 Biology 36, 177-198.

629 Canti, M., 1998. Origin of Calcium carbonate granules found in buried soils and
630 Quaternary deposits. Boreas 27, 275-288.

631 Cécillon, L., Baudin, F., Chenu, C., Houot, S., Jolivet, R., Kätterer, T., Lutfalla,
632 S., Macdonald, A., Van Oort, F., Plante, A.F., Savignac, F., Soucémarianadin,
633 L.N., Barré, P., 2018. A model based on Rock-Eval thermal analysis to
634 quantify the size of the centennialy persistent organic carbon pool in
635 temperate soils. Biogeosciences 15, 2835-2849.

636 Copard, Y., Giovanni, C. Di, Martaud, T., Albéric, P., Olivier, J., Copard, Y.,
637 Giovanni, C. Di, Martaud, T., Albéric, P., Olivier, J., 2006. Using Rock-Eval 6
638 pyrolysis for tracking fossil organic carbon in modern environments :
639 implications for the roles of erosion and weathering. Earth Surface Processes
640 and Landforms 2, 135-153.

641 Deng, J., Zhu, W., Zhou, Y., Yin, Y., 2019. Soil organic carbon chemical functional
642 groups under different revegetation types are coupled with changes in the
643 microbial community composition and the functional genes. Forests 10.
644 doi:10.3390/f10030240
645 Disnar, J.R., Guillet, B., Keravis, D., Di-Giovanni, C., Sebag, D., 2003. Soil organic matter (SOM) characterization by Rock-Eval pyrolysis: Scope and limitations. Organic Geochemistry 34, 327-343.

648 Domínguez, A., Bedano, J.C., Becker, A.R., 2010. Negative effects of no-till on soil macrofauna and litter decomposition in Argentina as compared with natural grasslands. Soil and Tillage Research 110, 51-59.

651 Don, A., Schumacher, J., Scherer-Lorenzen, M., Scholten, T., Schulze, E.D., 2007. Spatial and vertical variation of soil carbon at two grassland sites - Implications for measuring soil carbon stocks. Geoderma 141, 272-282.

654 Espíndola-Gonzalez, A., Fuentes-Ramirez, R., Martínez-Hernández, A.L., Castaño, V.M., Velasco-Santos, C., 2014. Structural characterization of silica particles extracted from grass stenotaphrum secundatum: Biotransformation via annelids. Advances in Materials Science and Engineering 2014. doi:10.1155/2014/956945

659 Gago-Duport, L., Briones, M.J.I., Rodríguez, J.B., Covelo, B., 2008. Amorphous calcium carbonate biomineralization in the earthworm's calciferous gland: Pathways to the formation of crystalline phases. Journal of Structural Biology 162, 422-435.

663 Garg, P., Gupta, A., Satya, S., 2006. Vermicomposting of different types of waste using Eisenia foetida: A comparative study. Bioresource Technology 97, 391-395.

666 Golchin, A., Oades, J.M., Skjemstad, J.O., Clarke, P., 1994. Soil structure and carbon cycling. Australian Journal of Soil Research 32, 1043-1063.

668 Huang, K., Xia, H., 2018. Role of earthworms' mucus in vermicomposting system: Biodegradation tests based on humification and microbial activity. Science of the Total Environment 610-611, 703-708.

671 Huang, W., Hammel, K.E., Hao, J., Thompson, A., Timokhin, V.I., Hall, S.J., 2019. Enrichment of Lignin-Derived Carbon in Mineral-Associated Soil Organic Matter. Environmental Science & Technology 53, 7522-7531.

674 IPCC, 2018. Global warming of 1.5°C. An IPCC Special Report on the impacts of
675 global warming of 1.5°C above pre-industrial levels and related global
676 greenhouse gas emission pathways, in the context of strengthening the
677 global response to the threat of climate change.. In Press.

678 Jégou, D., Cluzeau, D., Balesdent, J., Tréhen, P., 1998. Effects of four ecological
679 categories of earthworms on carbon transfer in soil. Applied Soil Ecology 9,
680 249-255.

681 Jones, C.G., Lawton, J.H., Shachak, M., 1994. Nordic Society Oikos Organisms as
682 Ecosystem Engineers. Source: Oikos 69, 373-386.

683 Kleber, M., Sollins, P., Sutton, R., 2007. A conceptual model of organo-mineral
684 interactions in soils: Self-assembly of organic molecular fragments into zonal
685 structures on mineral surfaces. Biogeochemistry 85, 9-24.

686 Kögel-Knabner, I., 2002. The macromolecular organic composition of plant and
687 microbial residues as inputs to soil organic matter. Soil Biology and
688 Biochemistry 105, A3-A8.

689 Kögel-Knabner, I., Guggenberger, G., Kleber, M., Kandeler, E., Kalbitz, K.,
690 Scheu, S., Eusterhues, K., Leinweber, P., 2008. Organo-mineral associations
691 in temperate soils: Integrating biology, mineralogy, and organic matter
692 chemistry. Journal of Plant Nutrition and Soil Science 171, 61-82.

693 Lähdesmäki, P., Pnspanen, R., 1992. Soil enzymology: Role of protective colloid
694 systems in the preservation of exoenzyme activities in soil. Soil Biology and
695 Biochemistry 24, 1173-1177.

696 Lavelle, P., Barois, I., Martin, A., Zaidi, Z., Schaefer, R., 1989. Management of
697 earthworm populations in agro-ecosystems: A possible way to maintain soil
698 quality?, in: Clarholm, M., Bergström, L. (Eds.), Ecology of Arable Land ---
699 Perspectives and Challenges: Proceeding of an International Symposium, 9-
700 12 June 1987 Swedish University of Agricultural Sciences, Uppsala, Sweden.
701 Springer Netherlands, Dordrecht, pp. 109-122.

702 Lavelle, P., Martin, A., 1992. Small-scale and large-scale effects of endogeic
703 earthworms on soil organic matter dynamics in soils of the humid tropics.
704 Soil Biology and Biochemistry 24, 1491-1498.
705 Le Bayon, R.-C., Milleret, R., 2009. Effects of earthworms on phosphorus
dynamics – A review. *Dynamic Soil, Dynamic Plant*. Global Science Books
21–27.

708 Lubbers, I.M., van Groenigen, K.J., Fonte, S.J., Six, J., Brussaard, L., van
709 Groenigen, J.W., 2013. Greenhouse-gas emissions from soils increased by
710 earthworms. *Nature Climate Change* 3, 187–194.

711 Manzoni, S., Jackson, R.B., Trofymow, J.A., Porporato, A., 2008. The global
712 stoichiometry of litter nitrogen mineralization. *Science* 321, 684–686.

713 Méndez, J., Rodríguez-González, J.B., Alvarez-Otero, R., Briones, M.J.I., Gago-
714 Duport, L., 2008. Crystallization stages of the CaCO3 deposits in the
715 earthworm's calciferous gland, in: Aretz, A., Hermanns-Sachweh, B., Mayer,
716 J. (Eds.), EMC 2008 14th European Microscopy Congress 1–5 September
717 2008, Aachen, Germany. Springer Berlin Heidelberg, Berlin, Heidelberg, pp.
718 127–128.

719 Miltner, A., Bombach, P., Schmidt-Brücken, B., Kästner, M., 2012. SOM genesis:
720 microbial biomass as a significant source. *Biogeochemistry*, 111, 41-55.

721 Nelson, P.N., Baldock, J.A., 2005. Estimating the molecular composition of a
722 diverse range of natural organic materials from solid-state 13 C NMR and
723 elemental analyses. *Biogeochemistry* 72, 1–34.

724 Ono, K., Hiradate, S., Morita, S., Hirai, K., 2013. Fate of organic carbon during
725 decomposition of different litter types in Japan. *Biogeochemistry* 112, 7–21.

726 Pisani, O., Hills, K.M., Courtier-Murias, D., Haddix, M.L., Paul, E.A., Conant,
727 R.T., Simpson, A.J., Arhonditsis, G.B., Simpson, M.J., 2014. Accumulation of
728 aliphatic compounds in soil with increasing mean annual temperature.
729 Organic Geochemistry 76, 118–127.

730 Poeplau, C., Don, A., Six, J., Kaiser, M., Benbi, D., Chenu, C., Cotrufo, M.F.,
731 Derrien, D., Gioacchini, P., Grand, S., Gregorich, E., Griepentrog, M.,
732 Gunina, A., Haddix, M., Kuzyakov, Y., Kühnel, A., Macdonald, L.M., Soong,
733 J., Trigalet, S., Vermeire, M.-L., Rovira, P., van Wesemael, B., Wiesmeier,
734 M., Yeasmin, S., Yevdokimov, I., Nieder, R., 2018. Isolating organic carbon
fractions with varying turnover rates in temperate agricultural soils – A comprehensive method comparison. Soil Biology and Biochemistry 125, 10–26.

R, D.C.T., 2005. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.

Rovira, P., Kurz-Besson, C., Coûteaux, M.M., Ramón Vallejo, V., 2008. Changes in litter properties during decomposition: A study by differential thermogravimetry and scanning calorimetry. Soil Biology and Biochemistry 40, 172–185.

Rumpel, C., Baumann, K., Remusat, L., Dignac, M.F., Barré, P., Deldicque, D., Glasser, G., Lieberwirth, I., Chabbi, A., 2015. Nanoscale evidence of contrasted processes for root-derived organic matter stabilization by mineral interactions depending on soil depth. Soil Biology and Biochemistry 85, 82–88.

Saenger, A., Cécillon, L., Sebag, D., Brun, J.J., 2013. Soil organic carbon quantity, chemistry and thermal stability in a mountainous landscape: A Rock-Eval pyrolysis survey. Organic Geochemistry 54, 101–114.

Sanderman, J., Creamer, C., Baisden, W.T., Farrell, M., Fallon, S., 2017. Greater soil carbon stocks and faster turnover rates with increasing agricultural productivity. Soil 3, 1–16.

Schaefer, J., Stejskal, E.O., 1976. Carbon-13 Nuclear Magnetic Resonance of Polymers Spinning at the Magic Angle. Journal of the American Chemical Society 98, 1031–1032.

Schmidt, M.W.I., Torn, M.S., Abiven, S., Dittmar, T., Guggenberger, G., Janssens, I.A., Kleber, M., Kögel-Knabner, I., Lehmann, J., Manning, D.A.C., Nannipieri, P., Rasse, D.P., Weiner, S., Trumbore, S.E., 2011. Persistence of soil organic matter as an ecosystem property. Nature 478, 49–56.

Schomburg, A., Verrecchia, E.P., Guenat, C., Brunner, P., Sebag, D., Le Bayon, R.C., 2018. Rock-Eval pyrolysis discriminates soil macro-aggregates formed by plants and earthworms. Soil Biology and Biochemistry 117, 117–124.
765 Schulten, H.-R., Leinweber, P., 1993. Pyrolysis-field ionization mass spectrometry of agricultural soils and humic substances: Effect of cropping systems and influence of the mineral matrix. Plant and Soil 151, 77-90.

768 Sebag, D., Verrecchia, E.P., Cécillon, L., Adatte, T., Albrecht, R., Aubert, M., Bureau, F., Cailleau, G., Copard, Y., Decaens, T., Disnar, J.R., Hetényi, M., Nyilas, T., Trombino, L., 2016. Dynamics of soil organic matter based on new Rock-Eval indices. Geoderma 284, 185-203.

772 Shiau, Y.J., Wang, H.C., Chen, T.H., Jien, S.H., Tian, G., Chiu, C.Y., 2017. Improvement in the biochemical and chemical properties of badland soils by thorny bamboo. Scientific Reports 7, 1-10.

775 Six, J., Bossuyt, H., Degryze, S., Denef, K., 2004. A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics. Soil and Tillage Research 79, 7-31.

778 Six, J., Callewaert, P., Lenders, S., De Gryz, S., Morris, S.J., Gregorich, E.G., Paul, E.A., Paustian, K., 2002. Measuring and understanding carbon storage in afforested soils by physical fractionation. Soil Science Society of America Journal 66, 1981-1987.

782 Six, J., Elliott, E.T., Paustian, K., 2000. Soil structure and soil organic matter: II. A normalized stability index and the effect of mineralogy. Soil Science Society of America Journal 64, 1042-1049.

785 Soucémarianadin, L., Cécillon, L., Chenu, C., Baudin, F., Nicolas, M., Girardin, C., Barré, P., 2018. Is Rock-Eval 6 thermal analysis a good indicator of soil organic carbon lability? – A method-comparison study in forest soils. Soil Biology and Biochemistry 117, 108-116.

789 Suthar, S., 2006. Potential utilization of guar gum industrial waste in vermicompost production. Bioresource Technology 97, 2474-2477.

791 Vidal, A., Quenea, K., Alexis, M., Derenne, S., 2016a. Molecular fate of root and shoot litter on incorporation and decomposition in earthworm casts. Organic Geochemistry 101, 1-10.

794 Vidal, A., Remusat, L., Watteau, F., Derenne, S., Quenea, K., 2016b.
795

Incorporation of 13C labelled shoot residues in Lumbricus terrestris casts: A combination of transmission electron microscopy and nanoscale secondary ion mass spectrometry. Soil Biology and Biochemistry 93, 8–16.

796

797

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Vinceslas-Akpa, M., Loquet, M., 1997. Organic matter transformations in lignocellulosic waste products composted or vermicomposted (Eisenia fetida andrei): Chemical analysis and 13C CPMAS NMR spectroscopy. Soil Biology and Biochemistry 29, 751–758.

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801

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von Lützow, M., Kögel-Knabner, I., 2010. Response to the Concept paper: ‘What is recalcitrant soil organic matter?’ by Markus Kleber. Environmental Chemistry 7, 333–335.

803

804

805

von Lützow, M., Kögel-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.

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807

808

809

Wagai, R., Kajiura, M., Uchida, M., Asano, M., 2018. Distinctive Roles of Two Aggregate Binding Agents in Allophanic Andisols: Young Carbon and Poorly-Crystalline Metal Phases with Old Carbon. Soil Systems 2, 29.

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Wagai, R., Mayer, L.M., Kitayama, K., Knicker, H., 2008. Climate and parent material controls on organic matter storage in surface soils: A three-pool, density-separation approach. Geoderma 147, 23–33.

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Wang, H., Liu, S., Wang, J., Shi, Z., Lu, L., Guo, W., Jia, H., Cai, D., 2013. Dynamics and speciation of organic carbon during decomposition of leaf litter and fine roots in four subtropical plantations of China. Forest Ecology and Management 300, 43–52.

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Wilson, M.A., 1987. N.M.R. Techniques and Applications in Geochemistry and Soil Chemistry, Pergamon P. ed. Oxford.

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Wu, J., Li, H., Zhang, W., Li, F., Huang, J., Mo, Q., Xia, H., 2017. Contrasting impacts of two subtropical earthworm species on leaf litter carbon sequestration into soil aggregates. Journal of Soils and Sediments 17, 1672–1681.

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823

824
825 Tables

826 Tab.1 Organic carbon (OC) and nitrogen (N) concentrations, and C:N ratio of
827 bulk samples. Data are presented as means (n=3). Different letters show
828 significant differences between treatments

<table><thead><tr><th></th><th>OC<br>mg/g</th><th>N<br>mg/g</th><th>C:N</th></tr></thead><tbody><tr><td>Compost</td><td>372.4<sup>a</sup></td><td>32.4<sup>a</sup></td><td>11.5</td></tr><tr><td>Vermicompost</td><td>344.7<sup>b</sup></td><td>36.1<sup>a</sup></td><td>9.6</td></tr><tr><td>Co-compost</td><td>164.9<sup>c</sup></td><td>15.7<sup>c</sup></td><td>10.5</td></tr><tr><td>Co-vermicompost</td><td>150.8<sup>c</sup></td><td>16.5<sup>b</sup></td><td>9.1</td></tr><tr><td>p-value</td><td>&lt;0.05</td><td>&lt;0.05</td><td>ns</td></tr></tbody></table>

829
830
831
832
833
834
835

836

837

838

839

<table><caption>**Tab.2** Mass, organic carbon (OC) and nitrogen (N) concentrations, and C:N ratios of density fractions separated from co-compost (co-C) and co-vermicompost (co-V). Data are presented as means (n=3). Different letters show significant differences among treatments and fractions</caption><thead><tr><th rowspan="3">Fractions</th><th colspan="2">Mass</th><th colspan="2">OC</th><th colspan="2">N</th><th colspan="2">C:N</th></tr><tr><th colspan="2">%</th><th colspan="2">mg/g</th><th colspan="2">mg/g</th><th colspan="2">mg/g</th></tr><tr><th>Co-C</th><th>Co-V</th><th>Co-C</th><th>Co-V</th><th>Co-C</th><th>Co-V</th><th>Co-C</th><th>Co-V</th></tr></thead><tbody><tr><td>F0</td><td>3.9<sup>de</sup></td><td>1.7<sup>e</sup></td><td>351.7<sup>b</sup></td><td>374.0<sup>a</sup></td><td>27.9<sup>a</sup></td><td>13.4<sup>c</sup></td><td>12.6<sup>a</sup></td><td>29.1<sup>a</sup></td></tr><tr><td>F1</td><td>11.8<sup>c</sup></td><td>11.3<sup>c</sup></td><td>320.9<sup>c</sup></td><td>312.0<sup>c</sup></td><td>30.8<sup>a</sup></td><td>16.2<sup>c</sup></td><td>10.4<sup>b</sup></td><td>19.4<sup>a</sup></td></tr><tr><td>F2</td><td>51.5<sup>ab</sup></td><td>67.9<sup>a</sup></td><td>195.7<sup>d</sup></td><td>201.2<sup>d</sup></td><td>18.6<sup>b</sup></td><td>18.7<sup>b</sup></td><td>10.5<sup>b</sup></td><td>10.8<sup>b</sup></td></tr><tr><td>F3</td><td>32.8<sup>b</sup></td><td>10.9<sup>c</sup></td><td>97.9<sup>e</sup></td><td>72.7<sup>f</sup></td><td>9.2<sup>d</sup></td><td>6.7<sup>d</sup></td><td>10.7<sup>b</sup></td><td>10.8<sup>b</sup></td></tr><tr><td>Silica granules</td><td>BDL</td><td>8.2<sup>d</sup></td><td>BDL</td><td>&lt; 1</td><td>BDL</td><td>&lt; 1</td><td>NA</td><td>NA</td></tr><tr><td>p-value</td><td colspan="2">&lt; 0.05</td><td colspan="2">&lt; 0.01</td><td colspan="2">&lt; 0.01</td><td colspan="2">&lt; 0.05</td></tr></tbody></table>

840

BDL = below detection limit; NA = not analysed

841
Tab. 3 Rock-Eval 6 thermal parameters of compost (C), vermicompost (V), co-compost (co-C) and co-vermicompost (co-V). Data are presented as means (n=3). P-values and letters show significant parameter differences between treatments (n.s.: not significant).

<table><thead><tr><th>Parameters</th><th>Unit</th><th>C</th><th>V</th><th>Co-C</th><th>Co-V</th><th>p-value</th></tr></thead><tbody><tr><td>HI</td><td>mg HC g<sup>-1</sup> TOC<sup>-1</sup></td><td>391<sup>a</sup></td><td>403<sup>a</sup></td><td>359<sup>ab</sup></td><td>341<sup>b</sup></td><td>&lt; 0.05</td></tr><tr><td>OI</td><td>mg CO<sub>2</sub> g<sup>-1</sup> TOC<sup>-1</sup></td><td>165<sup>a</sup></td><td>161<sup>a</sup></td><td>208<sup>b</sup></td><td>178<sup>b</sup></td><td>&lt; 0.05</td></tr><tr><td>R-index</td><td></td><td>0.43</td><td>0.42</td><td>0.47</td><td>0.45</td><td>n.s.</td></tr><tr><td>I-index</td><td></td><td>0.40</td><td>0.44</td><td>0.36</td><td>0.39</td><td>n.s.</td></tr><tr><td>PC/TOC ratio</td><td></td><td>0.39</td><td>0.41</td><td>0.38</td><td>0.36</td><td>n.s.</td></tr><tr><td>RC/TOC ratio</td><td></td><td>0.61</td><td>0.59</td><td>0.62</td><td>0.64</td><td>n.s.</td></tr><tr><td>PC/RC ratio</td><td></td><td>0.65</td><td>0.68</td><td>0.63</td><td>0.56</td><td>n.s.</td></tr><tr><td>T<sub>50_CO2_PYR</sub></td><td>°C</td><td>348<sup>a</sup></td><td>347<sup>a</sup></td><td>342<sup>b</sup></td><td>349<sup>a</sup></td><td>&lt; 0.05</td></tr><tr><td>T<sub>50_CO2_OX</sub></td><td>°C</td><td>338<sup>b</sup></td><td>481<sup>a</sup></td><td>454<sup>b</sup></td><td>463<sup>ab</sup></td><td>&lt; 0.05</td></tr></tbody></table>

OC = organic carbon concentration, N = nitrogen concentration, HI = hydrogen index, OI = oxygen index, R-index = refractory index, I-index = immature index, PC = pyrolysable carbon, RC = residual carbon, TOC = total organic carbon, "T50_CO2_PYR" = 50% of CO2 gas evolution during the pyrolysis stage and "T50_CO2_OX" = 50% of CO2 gas evolution during the oxidation stage.
850
851

<table><caption>**Tab. 4** Rock-Eval 6 thermal parameters of density fractions. Data are presented as means (n=3). P-values and letters show significant parameter differences among treatments and fractions.</caption><thead><tr><th rowspan="2">Parameters</th><th rowspan="2">Unit</th><th colspan="4">Co-compost</th><th colspan="4">Co-vermicompost</th><th rowspan="2">p-value</th></tr><tr><th>F0</th><th>F1</th><th>F2</th><th>F3</th><th>F0</th><th>F1</th><th>F2</th><th>F3</th></tr></thead><tbody><tr><td>HI</td><td>mg HC g<sup>-1</sup> TOC<sup>-1</sup></td><td>438<sup>ab</sup></td><td>484<sup>a</sup></td><td>295<sup>bc</sup></td><td>291<sup>cd</sup></td><td>248<sup>d</sup></td><td>294<sup>bc</sup></td><td>265<sup>cd</sup></td><td>263<sup>cd</sup></td><td>&lt; 0.05</td></tr><tr><td>OI</td><td>mg CO<sub>2</sub> g<sup>-1</sup> TOC<sup>-1</sup></td><td>189<sup>a</sup></td><td>135<sup>c</sup></td><td>135<sup>c</sup></td><td>182<sup>ab</sup></td><td>165<sup>b</sup></td><td>137<sup>c</sup></td><td>145<sup>c</sup></td><td>171<sup>ab</sup></td><td>&lt; 0.001</td></tr><tr><td>R-index</td><td></td><td>0.39<sup>e</sup></td><td>0.49<sup>d</sup></td><td>0.54<sup>c</sup></td><td>0.63<sup>ab</sup></td><td>0.39<sup>e</sup></td><td>0.49<sup>d</sup></td><td>0.59<sup>b</sup></td><td>0.67<sup>a</sup></td><td>&lt; 0.001</td></tr><tr><td>I-index</td><td></td><td>0.48<sup>a</sup></td><td>0.29<sup>b</sup></td><td>0.19<sup>c</sup></td><td>0.09<sup>d</sup></td><td>0.49<sup>a</sup></td><td>0.29<sup>b</sup></td><td>0.13<sup>d</sup></td><td>0.03<sup>e</sup></td><td>&lt; 0.001</td></tr><tr><td>PC:TOC ratio</td><td></td><td>0.45<sup>ab</sup></td><td>0.47<sup>a</sup></td><td>0.31<sup>cd</sup></td><td>0.34<sup>bc</sup></td><td>0.29<sup>d</sup></td><td>0.32<sup>cd</sup></td><td>0.29<sup>d</sup></td><td>0.31<sup>cd</sup></td><td>&lt; 0.05</td></tr><tr><td>RC:TOC ratio</td><td></td><td>0.55<sup>cd</sup></td><td>0.54<sup>d</sup></td><td>0.69<sup>ab</sup></td><td>0.67<sup>bc</sup></td><td>0.71<sup>a</sup></td><td>0.69<sup>ab</sup></td><td>0.71<sup>a</sup></td><td>0.69<sup>ab</sup></td><td>&lt; 0.05</td></tr><tr><td>PC:RC ratio</td><td></td><td>0.82<sup>ab</sup></td><td>0.87<sup>a</sup></td><td>0.45<sup>cd</sup></td><td>0.51<sup>bc</sup></td><td>0.41<sup>d</sup></td><td>0.46<sup>cd</sup></td><td>0.40<sup>d</sup></td><td>0.45<sup>cd</sup></td><td>&lt; 0.05</td></tr><tr><td>T<sub>50_CO2_PYR</sub></td><td>°C</td><td>343.7<sup>c</sup></td><td>355.3<sup>ab</sup></td><td>353.3<sup>bc</sup></td><td>353.3<sup>bc</sup></td><td>355.0<sup>ab</sup></td><td>356.3<sup>ab</sup></td><td>356.7<sup>ab</sup></td><td>359.7<sup>a</sup></td><td>&lt; 0.05</td></tr><tr><td>T<sub>50_CO2_OX</sub></td><td>°C</td><td>424.7<sup>cd</sup></td><td>411.0<sup>d</sup></td><td>440.3<sup>ab</sup></td><td>442.7<sup>a</sup></td><td>443.3<sup>ab</sup></td><td>427.0<sup>cd</sup></td><td>432.3<sup>bc</sup></td><td>443.0<sup>a</sup></td><td>&lt; 0.05</td></tr></tbody></table>

852
853
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855

“HI” for hydrogen index, “OI” for oxygen index, “R-index” for refractory index, “I-index” for immature index, “PC” for pyrolysable carbon, “RC” for residual carbon, “TOC” for total organic carbon, “T<sub>50_CO2_PYR</sub>” corresponding to 50% of CO<sub>2</sub> gas evolution during the pyrolysis stage and “T<sub>50_CO2_OX</sub>” corresponding to 50% of CO<sub>2</sub> gas evolution during the oxidation stage.
<figure><img src="image_3.png" /></figure>

857

858 **Fig. 1** Raman spectrum measured on silica granules present in heavy

859 fractions of co-vermicompost treatment.
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Total organic carbon (a) and nitrogen (b) distributions in density fractions (n = 3) of compost (C), vermicompost (V), co-compost (Co-C) and co-vermicompost (Co-V).</figcaption></figure>
<figure><img src="image_5.png" /></figure>

C = compost; Co-C = co-compost; V = Vermicompost; co-V = co-vermicompost

**Fig. 3** Hydrogen Index (a) and Oxygen Index (b) of compost (C), vermicompost (V), co-compost (Co-C) and co-vermicompost (Co-V). Different letters indicate significant differences (n=3)

864

865

866

867

868

869
<figure><img src="image_6.png" /></figure>

870

871

* = p < 0.05; ** = p < 0.01; *** = p < 0.001

872 **Fig. 4** Hydrogen Index (a) and Oxygen Index (b) of density fractions

873 separated from co-compost and co-vermicompost end-products (n = 3).

874 \*indicate significant differences between compost and co-compost

875 treatments

876
<figure><img src="image_7.png" /></figure>

877

Fig. 5 I-index plotted against R-index as indicators of OM thermal stability of co-compost (circle) and co-vermicompost (triangle) bulk samples and density fractions (n = 3). Thermal stability increases when the I-index decreases and the R-index increases.

882
Initial OM

<figure><img src="image_8.png" /><figcaption>**Fig. 6** $^{13}$C CPMAS NMR spectra of initial OM and density fractions of co-compost and co-vermicompost, with relative contribution of chemical shift regions (%).</figcaption></figure>

883

884

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886

887
<figure><img src="image_9.png" /><figcaption>**Fig. 7** Thermal stability R-index plotted against 'Aromatic C / O-alkyl C' ratio as an indicator of OM chemical decomposition degree in density fractions of co-compost (circle) and co-vermicompost (triangle)</figcaption></figure>

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892
<table><thead><tr><th>Variables</th><th>Fractions</th><th></th></tr></thead><tbody><tr><td rowspan="3">Carbohydrate</td><td>F1</td><td>•</td></tr><tr><td>F2</td><td>•</td></tr><tr><td>F3</td><td>•</td></tr><tr><td rowspan="3">Protein</td><td>F1</td><td>—</td></tr><tr><td>F2</td><td>—</td></tr><tr><td>F3</td><td>—</td></tr><tr><td rowspan="3">Lignin</td><td>F1</td><td>—</td></tr><tr><td>F2</td><td>—</td></tr><tr><td>F3</td><td>—</td></tr><tr><td rowspan="3">Aliphatic</td><td>F1</td><td>•</td></tr><tr><td>F2</td><td>•</td></tr><tr><td>F3</td><td>—</td></tr><tr><td rowspan="3">Carbonyl</td><td>F1</td><td>•</td></tr><tr><td>F2</td><td>•</td></tr><tr><td>F3</td><td>—</td></tr><tr><td rowspan="3">Char</td><td>F1</td><td>•</td></tr><tr><td>F2</td><td>•</td></tr><tr><td>F3</td><td>•</td></tr></tbody></table>

<figure><img src="image_10.png" /></figure>

**Fig. 8** Impact of the earthworm species *Eisenia sp.* during co-composting practices on estimated molecular composition of natural organic compounds, in the four density fractions (F0: dissolved OM, F1: free OM particles and F2 & F3: mineral associated OM), compared to results from control treatment. The response ratio was calculated as log (co-vermicompost : co-compost).