Alix Vidal

Claire Chenu

Manuel Blouin

Ainsi Qu'

Carsten Müller

Francisco Javier Gonzalez Vila

The fate of root and shoot litter in soil and earthworm casts

exercice des remerciements serait si difficile : n'oublier personne, mettre tout le monde dans le bon ordre, ne pas trop en faire, trouver des synonymes au mot 'merci', etc. Je me lance.

Je tiens tout d'abord à remercier les membres de mon jury pour m'avoir accordé leur temps et

'The plough is one of the most ancient and most valuable of mans inventions; but long before he existed the land was in fact regularly ploughed, and still continues to be thus ploughed by earth-worms. It may be doubted whether there are many other animals which have played so important a part in the history of the world, as have these lowly organised creatures.' Charles Robert Darwin; The formation of vegetal mould through the action of worms, with observations of their habits (1881) 'Est-ce que nous ne sommes vraiment plus capables de respecter la nature, la liberté vivante, sans aucun rendement, sans utilité, sans autre objet que de se laisser entrevoir de temps en temps ? La liberté elle-même est anachronique'.

Roman Kacew, dit Romain Gary ; Les Racines du ciel (1956) Françoise Watteau Merci à tous les thésards, post-doc, ATER, stagiaires, qui sont passés par le laboratoire et avec qui j'ai partagé de très bons moments : Kirikou (pour les fous rires, les longues discussions, mon petit François), Aurélie (qui est d'une gentillesse rare et fait des gâteaux magnifiques, en plus!), Sarah (la Miss GDGTs qui m'a souvent aidée et conseillée pendant ma thèse), Cyrielle (pour son soutien sur la dernière ligne droite et les analyses de dernière minute), Rime (pour sa douceur, et sa cuisine également!), Yannick (pour sa gentillesse et ses coups de main mémorables les dernières semaines), Antoine, Franck, Alexandre, Mathieu, Nadou.

Merci et désolée à toutes les personnes que j'ai croisées de près ou de loin pendant cette thèse et que j'ai peut-être oubliées.

Je remercie particulièrement mes parents qui ont développé mon sens de la persévérance, de la curiosité et de la fantaisie. Merci à ma soeur, Tiphaine, pour m'avoir ouvert les portes des sciences du sol et avoir toujours cru en moi. Et merci à ses deux petits monstres, Liza et Raphaël, pour leur energie et leur joie de vivre. Un tendre merci à Sam, pour sa présence réconfortante, son courage impressionnant, les longues soirées à m'attendre patiemment, puis à m'écouter longuement. Merci pour tout. Un clin d'oeil également à Christine (le 'i' se prononce 'aïe'), conseillière littéraire, oratrice à ses heures et aime les vaches, surtout.

Merci à France Inter pour avoir accompagné de manière pationnante de longues journées au laboratoire.

Enfin, je ne peux terminer sans remercier les vers de terre pour avoir donné leur vie à la science! Tant pis pour les synonymes du mot merci…

Introduction

The society of the 21 st century will have to face many challenges. Carbon dioxide (CO 2 ) concentration in the atmosphere is increasing and climatic change is now an urgent and critical international issue, as the global warming should at least reach 1.5 °C by the end of the 21 st century (IPCC, 2013). Moreover, by 2050, the world population should exceed nine billions, resulting in a need for an increase of 70% of the world food supply. In parallel, agriculture represents one of the main cause of greenhouse gas emissions since the emergence of the industrial era (IPCC, 2013).

Soils are vital for a multitude of life-supporting ecosystem services, e.g. nutrient cycling, food supply, as well as climate regulation, including carbon sequestration. Globally, soils contain three times the amount of carbon present in the atmosphere and four times the amount of above-ground biomass carbon [START_REF] Lal | Soil carbon sequestration impacts on global climate change and food security[END_REF]. They are, therefore, major players in the terrestrial carbon cycle. Limiting CO 2 discharge from the soil and/or sequestering atmospheric CO 2 into soil organic matter (SOM) through changes in agricultural practices is an appealing idea that requires less investment than other proposed methods of carbon sequestration [START_REF] Hoffert | Advanced technology paths to global climate stability: energy for a greenhouse planet[END_REF][START_REF] Kell | Large-scale sequestration of atmospheric carbon via plant roots in natural and agricultural ecosystems: why and how[END_REF]. The notion of changing agricultural practices to increase below-ground carbon stabilization is gaining scientific, economic and political credibility. For example, in 2015, the French government has announced their intent to develop agricultural practices that increase SOM stocks by 4‰ per annum. Thus, a great effort is needed to further investigate the processes implied in SOM formation and stabilization into the soil. SOM formation is mainly driven by plant inputs, either from above-ground (leaves, shoots) or below-ground (roots and their products) parts. SOM stabilization relies on numerous abiotic (e.g. climate and physico-chemical characteristics) and biotic (e.g. litter quality, activity of soil fauna and microorganisms) factors [START_REF] Oades | The retention of organic matter in soils[END_REF][START_REF] Cortez | Field decomposition of leaf litters: relationships between decomposition rates and soil moisture, soil temperature and earthworm activity[END_REF]. In the present study, we focused on two key biotic factors regulating carbon cycling -litter quality and soil fauna. The term litter quality will be used to describe the nature/composition of the litter.

Litter represents the main source for SOM. Litter quality is one of the factors determining how much carbon will be incorporated into the soil, how long litter decomposition will take and what will be the litter contribution to soil stable carbon pool. While above-ground biomass has long been considered as the main source for SOM, roots are currently recognized as essential contributors to stable organic carbon (OC). The relative contribution from root and shoot litter to SOM has been the subject of many studies during the last decades and it is now recognized that roots decompose at a slower rate compared to shoots. However, the reasons explaining the slower decomposition of roots and its high contribution to soil carbon pool remain debated. Is it driven by the chemical composition, physical protection and/or physico-chemical protection of roots in soils [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF]?

The transfer of litter into the soil and its decomposition mainly relies on the action of soil fauna and microorganisms. The present study will focus on earthworms, which are considered as the main ecosystem engineers in temperate soils [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF]. These invertebrates have a key role on soil structure and carbon cycling [START_REF] Blouin | A review of earthworm impact on soil function and ecosystem services[END_REF]. They ingest both organic (plant residues and microorganisms) and mineral (soil particles) materials, in different proportions depending on their ecological category. During ingestion, soil and plant residues are mixed, intimately associated with mucus and excreted along burrows or at the soil surface in the form of casts [START_REF] Guggenberger | Soil organic matter within earthworm casts of an anecic-endogeic tropical pasture community, Colombia[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF]. Casts are complex organomineral structures where mineralization of OC is generally enhanced at short terms, followed by its stabilization in drying and ageing casts at long terms [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF]. It has been demonstrated that earthworm feeding behavior is influenced by litter quality and palatability, but little is known about the impact of earthworms on root incorporation and decomposition in soils [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF][START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF].

The aim of this thesis is to investigate the fate of roots and shoots in soil, in the presence or absence of earthworms. A mesocosm experiment was set up to follow the incorporation of 13 C-labelled Ryegrass root and shoot litter in the soil, in the presence of epi-anecic earthworms (Lumbricus terrestris). Artificial isotopic labelling of litter allows precise monitoring of the litter incorporation and distinguishing the added organic matter (OM) from OM initially present in the soil [START_REF] Thompson | Pulse-labelling a cover crop with 13 C to follow its decomposition in soil under field conditions[END_REF][START_REF] Klumpp | Long-term steady state 13 C labelling to investigate soil carbon turnover in grasslands[END_REF][START_REF] Brüggemann | Carbon allocation and carbon isotope fluxes in the plantsoil-atmosphere continuum: a review[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF][START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF]. It is important to note that root and shoot litter were both deposited at the soil surface. Thus, this work will specifically focus on the impact of litter chemical characteristics on soil carbon cycling [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF]. The physical protection of carbon potentially induced by the localization of root vs. shoot in the soil will not be investigated in the present study. Soil samples were collected at 0-20 and 40-60 cm, as well as surface casts, at the beginning and after 1, 2, 4, 8, 24 and 54 weeks of experiment.

Soils are known to be heterogeneous across a wide range of spatio-temporal scales. Thus, soils are one of the most complex systems on Earth in terms of physical structure, chemical constitution and biodiversity. Until recently, soil science has been mainly operated on the basis of rather separated disciplines of physics, chemistry and biology. However, there is now an increasing awareness that these disciplines need to be integrated into a soil system view, i.e. using multidisciplinary approaches. In the present study, exploring how roots and shoots are incorporated and decomposed in soil and casts is achieved by combining methods across various spatial scales ranging from the molecular level up to 'bulk' isotopic level. This manuscript is divided in 5 chapters. The first chapter presents the current state of knowledge on SOM (origin, decomposition and stabilization) and earthworm implications in soil carbon cycling. In this part, the fate of roots and shoots in soil are used as guiding thread.

The second chapter describes the mesocosm experiment and the analytical methods used in order to localize, quantify and characterize the OC in the present study. The third chapter is dedicated to the general appreciation on the localization and quantification root-and shootderived carbon in cast and soil at two depths (0-20 and 40-60 cm) in mesocosms, using 'bulk' isotopic quantification. The fourth chapter focuses on the characterization of the OM in earthworm casts and is divided in three main sections. The first section investigates the incorporation and decomposition of roots and shoots in casts at the molecular level. The second section evaluates the potential of the innovative combination of transmission electron microscopy (TEM) and nano-scale secondary ion mass spectrometry (NanoSIMS) to characterize OM in casts, at the microscale. In the third section, these two methods are used to study root and shoot degradation state and the microorganisms implied in this decomposition in casts, at two key time steps. The last chapter is divided in two sections and gives a first appreciation on OM (first section) and microbial community (second section) characterization after one year of experiment in soil and casts.

Chapter 1.

State of the art

Soil organic matter and earthworm implications in soil carbon cycling [START_REF] Quéré | Global carbon budget 2014[END_REF].

The global carbon budget

In May 2013, the daily average CO 2 concentration in the atmosphere went above the symbolic value of 400 part per million (ppm) at Mauna Loa station, in Hawaiian Islands. This reflected the atmospheric CO 2 concentration increase, which has exceeded 40 % since 1750 (Figure 1), with a cumulative anthropic emissions accounting for 555 gigatons of carbon (Gt C). These emissions are the main consequences of fossil fuel combustion (375 Gt C) and, to a lesser extent, deforestation and land use modifications (180 Gt C). This discharge is directly linked to the global warming of 0.85 °C (land and ocean surface) between 1880 and 2012. Before the end of the 21 st century, global warming should reach at least 1.5 °C (IPCC, 2013).

A central climate target of the 21 st conference of parties (COP21) declaration is the restriction of temperature increase to less than 2°C and to work towards a limit of 1.5°C by the end of the 21 st century. Ocean and land surface remove a large fraction of the greenhouse gases anthropic emissions (Le [START_REF] Quéré | Global carbon budget 2014[END_REF] (Figure 2). The soil is a continental ecosystem able to store carbon. In the top three meters, the soil can store approximatively 2344 Gt of OC [START_REF] Jobbágy | The vertical distribution of soil organic carbon and its relation to climate and vegetation[END_REF], which is four times the capacity of the biotic sink and three times the atmospheric sink [START_REF] Lal | Soil carbon sequestration impacts on global climate change and food security[END_REF]. Thus, the 8.9 Gt of carbon emitted every year from fossil fuel activities represent approximatively 4‰ of the soil OC stock (Le [START_REF] Quéré | Global carbon budget 2014[END_REF] (Figure 2). Limiting CO 2 discharge from the soil to the atmosphere and/or increasing carbon storage in the soil could limit the increase in CO 2 concentration in the atmosphere.

Although the ocean represents an important sink of carbon, it was shown that increasing soil carbon pool was more cost-effective and environmentally friendly [START_REF] Lal | Soil carbon sequestration impacts on global climate change and food security[END_REF]. Based on this concept, the French government has announced in 2015 their intent to develop agricultural practices that increase soil carbon stocks by 4‰ per annum, which would stop the annual increase in CO 2 in the atmosphere.

The soil 1.2.1. Soil definition and composition

The soil is a fine superficial layer at the scale of the earth globe, generally comprised between 0.3 and 3 meters, framed by the lithosphere and the atmosphere. As a comparison, the earth crust is 20 to 70 km thick [START_REF] Gobat | Le sol vivant: bases de pédologie, biologie des sols, Gérer l'environnement[END_REF]. Soil is a dynamic, continuous and threedimensional ecological system with a liquid, gaseous and solid phase.

The arrangement of the voids and soil solid components forms the porosphere. Pores contain the liquid and gaseous phase of the soil, in addition to various microorganisms. The soil solid phase is a dynamic system composed of organic and mineral elements, giving the soil a central position in biogeochemical cycles [START_REF] Gobat | Le sol vivant: bases de pédologie, biologie des sols, Gérer l'environnement[END_REF][START_REF] Calvet | Les matières organiques des sols: Rôles agronomiques et environnementaux[END_REF][START_REF] Baize | Les sols et leurs structures: Observations à différentes échelles[END_REF]. Mineral particles originate from the physical degradation and the biochemical alteration of rocks. Organic compounds encompass living forms, their products (e.g. exudates)

and the products of their decomposition [START_REF] Gobat | Le sol vivant: bases de pédologie, biologie des sols, Gérer l'environnement[END_REF]. In the soil, the sum of the organic components forms the SOM. SOM is a complex mixture of compounds originating from plants, animals and microorganisms, under various physical and chemical forms [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF][START_REF] Bouché | Des vers de terre et des hommes, découvrir nos écosystèmes fonctionnant à l'énergie solaire[END_REF][START_REF] Quenea | Variation in lipid relative abundance and composition among different particle size fractions of a forest soil[END_REF][START_REF] Calvet | Les matières organiques des sols: Rôles agronomiques et environnementaux[END_REF] heterogeneously distributed [START_REF] Lehmann | Spatial complexity of soil organic matter forms at nanometre scales[END_REF]. It is generally agreed that SOM contains around 58 % of OC, depending on sources [START_REF] Calvet | Les matières organiques des sols: Rôles agronomiques et environnementaux[END_REF]. OM is often adsorbed onto mineral particles. The rate of adsorption relies on mineral properties (mineralogy, specific surface area, physical arrangements) [START_REF] Baldock | Role of the soil matrix and minerals in protecting natural organic materials against biological attack[END_REF] and on the presence of organic binding agents (plant exudates, microbial products, microbial residues) [START_REF] Chenu | Interactions between microorganisms and soil particles: an overview[END_REF].

Biological and/or physico-chemical processes lead to the rearrangement of particles, flocculation and cementation, forming complex organo-mineral structures called aggregates (Figure 3) [START_REF] Bronick | Soil structure and management: a review[END_REF]. Biological processes include soil microorganisms involved in the formation of the cement binding OM and minerals, while the meso and macrofauna favor organo-mineral interactions during the passage into their intestinal tractus.

Aggregate formation also results from physico-chemical processes that depend on pedoclimatic conditions and generally occurs during the dry-wetting process of clays [START_REF] Duchaufour | Introduction à la science du sol: sol, végétation, environnement[END_REF]. Aggregates can be classified according to their size: microaggregates (< 250 µm) and macroaggregates (> 250 µm) [START_REF] Oades | Aggregate hierarchy in soils[END_REF].

Bacteria

Either free organic and mineral structures or complex microstructures containing clay particles, pores, organic residues and microorganismes

Soil as a carbon sink

The quantity of carbon stabilized in soil relies on a complex equilibrium between inputs and outputs. Soil carbon inputs are mainly represented by plant-and microorganism-derived compounds, while outputs principally results from the release of carbon under gas form [START_REF] Lal | Soil carbon sequestration impacts on global climate change and food security[END_REF]. Relevant terms regarding carbon cycling were defined as they will be used in this manuscript (adapted from [START_REF] Baldock | Role of the soil matrix and minerals in protecting natural organic materials against biological attack[END_REF]:

1-Mineralization 'the conversion of OC to CO 2 via respiration' 2-Alteration/degradation 'conversion of the OC from one chemical/anatomical structure into a different one, resulting from either the mechanical, physical or chemical action of organisms'

3-Assimilation 'the incorporation of OC into tissues of decomposer organisms'

4-Decomposition 'loss of OC components resulting from the alteration/degradation, the mineralization and the assimilation'

5-Protection 'physical or physico-chemical isolation of the OC preventing its decomposition and reflecting its, at least temporal stabilization'

6-Stabilization 'the ability of OC to overcome decomposition, thanks to chemical recalcitrance, physical or physico-chemical protection'. As a compound is considered as stable when its mean residence time in soil reaches centuries [START_REF] Marschner | How relevant is recalcitrance for the stabilization of organic matter in soils?[END_REF], in the present manuscript stabilization will be termed as 'potential stabilization'.

Carbon stabilization in soils starts with the fixation of atmospheric CO 2 by plants via photosynthesis. Litter deposited on soil surface can either be mineralized, releasing CO 2 to the atmosphere, or incorporated into soil in the form of organic compounds. Litter transferred from the soil surface or introduced in soil via root growth can be mineralized, transferred to deeper layers and/or stored in soil. Litter decomposition and incorporation into soil depend on numerous biotic and abiotic factors. Abiotic factors mainly correspond to soil climate (temperature and moisture) and physico-chemical characteristics (texture and clay mineralogy). These factors have a critical influence on organo-mineral interactions, impacting OM decomposition and retention in soil. Biotic factors encompass litter quality, activity of soil fauna and microorganisms [START_REF] Oades | The retention of organic matter in soils[END_REF][START_REF] Cortez | Field decomposition of leaf litters: relationships between decomposition rates and soil moisture, soil temperature and earthworm activity[END_REF].

Soil OC stocks are highly variable around the globe due to the diversity of soil, climate and vegetation. For example, soils from temperate regions present higher OC concentrations compared to arid regions. In addition to this natural variability, anthropic practices also have a significant impact on soil OC. For example, the mean soil OC stock is higher in pastures compared to cultivated lands. Cultural practices also have an important impact on SOM, and therefore on carbon fluxes. Indeed, the decomposition of SOM by mineralization can increase CO 2 discharge in the atmosphere. On the contrary, adding or increasing the stability of SOM favors carbon storage in the soil. In order to limit CO 2 emissions and/or increase SOM stabilization, processes implied in SOM formation and storage need to be investigated [START_REF] Lal | Soil carbon sequestration impacts on global climate change and food security[END_REF][START_REF] Totsche | Biogeochemical interfaces in soil: The interdisciplinary challenge for soil science[END_REF][START_REF] Calvet | Les matières organiques des sols: Rôles agronomiques et environnementaux[END_REF][START_REF] Schmidt | Persistence of soil organic matter as an ecosystem property[END_REF][START_REF] Jandl | Current status, uncertainty and future needs in soil organic carbon monitoring[END_REF].

SOM: origin, decomposition, stabilization

1.3.1. SOM formation: traditional vs. emergent vision SOM is (1) a source of nutrients for soil organisms, (2) a key determinant of soil fertility, structuration and evolution and (3) a potential sink of carbon [START_REF] Jobbágy | The vertical distribution of soil organic carbon and its relation to climate and vegetation[END_REF].

All existing theories on SOM formation agree that it starts with the fragmentation of plant and soil fauna. However, the fate of these complex compounds in soil remains unclear [START_REF] Stockmann | The knowns, known unknowns and unknowns of sequestration of soil organic carbon[END_REF] and highly debated in the scientific community [START_REF] Schmidt | Persistence of soil organic matter as an ecosystem property[END_REF][START_REF] Lehmann | The contentious nature of soil organic matter[END_REF]. The traditional vision of SOM relies on the operational fractionation based on acid and based solubility. The fractions obtained, termed fulvic acids, humic acids and humin, have been considered for long as reflecting an increasing humification, with humin representing the most stable pool of SOM [START_REF] Stevenson | Humus chemistry: genesis, composition, reactions[END_REF]. This

view is currently debated and the existence of humic substances is even questioned [START_REF] Schmidt | Persistence of soil organic matter as an ecosystem property[END_REF][START_REF] Lehmann | The contentious nature of soil organic matter[END_REF]. Indeed, it has long been observed that the soil microbial community is able to decompose almost every organic compounds, even those considered for long as the most recalcitrant, such as lignin [START_REF] Baldrian | Fungal laccases-occurrence and properties[END_REF][START_REF] Filley | Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi[END_REF][START_REF] Tuor | Enzymes of white-rot fungi involved in lignin degradation and ecological determinants for wood decay[END_REF] or polycyclic aromatic hydrocarbons [START_REF] Gramss | Degradation of polycyclic aromatic hydrocarbons with three to seven aromatic rings by higher fungi in sterile and unsterile soils[END_REF]. The emergent view assumes that SOM is a continuum of organic fragments and microbial products [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF] that can be preserved or submitted to further degradation in soils [START_REF] Lehmann | The contentious nature of soil organic matter[END_REF]. In this view, preservation of organic compounds in the soil is not only related to its chemical composition, but also depends on multiple soil processes, such as physical disconnection, sorption/desorption and freezing/thawing [START_REF] Schmidt | Persistence of soil organic matter as an ecosystem property[END_REF] (Figure 4). 2) emerging views of organic matter formation and preservation in the soil [START_REF] Schmidt | Persistence of soil organic matter as an ecosystem property[END_REF].

The emergence of this view was made possible thanks to the development of new analytical tools, which have greatly improved the knowledge on SOM molecular composition and dynamics. A large array of techniques are used for SOM characterization as reviewed by [START_REF] Derenne | Characterizing the molecular structure of organic matter from natural environments: An analytical challenge[END_REF], and only a few will be mentioned in the present manuscript.

The precise identification of organo-soluble compounds (free lipids) is commonly achieved after solvent extraction followed by gas chromatography and mass spectrometry (GC-MS) [START_REF] Bull | Organic geochemical studies of soils from the Rothamsted classical experiments-V. The fate of lipids in different long-term experiments[END_REF][START_REF] Quenea | Variation in lipid relative abundance and composition among different particle size fractions of a forest soil[END_REF]Quenea et al., , 2006)). Lipids are used as source markers (i.e. plant or microorganisms) and OM degradation stage indicators [START_REF] Bull | Organic geochemical studies of soils from the Rothamsted classical experiments-V. The fate of lipids in different long-term experiments[END_REF]. This issue will be developed in section 1.3.2. Bulk chemical feature of macromolecular fraction of SOM can be determined by Nuclear magnetic resonance analyses (NMR) and Fourier transform infrared (FTIR) [START_REF] Derenne | Characterizing the molecular structure of organic matter from natural environments: An analytical challenge[END_REF]. However, these techniques do not give the precise molecular composition of SOM. Pyrolysis combined with GC-MS (Py-GC-MS) has been commonly used for more than 30 years to characterize SOM at the molecular level [START_REF] De | Influence de vers de terre (Lombricus terrestris) sur l'incorporation de biomasses végétales marquées ( 13 C) dans un sol en conditions contrôlées[END_REF]. Analytical pyrolysis is a thermal degradation technique based on the cracking of the covalent bonds of OM molecules. In addition to details on SOM origin, pyrolysis provides information on its state of decomposition. For example, using in situ derivatization with tetramethylammonium hydroxide (TMAH), the identification of lignin moieties, cutin/suberin and tannin markers is improved in soils. Thus, the monitoring of the relative abundance of these compounds gives information on OM decomposition state [START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF][START_REF] De | Influence de vers de terre (Lombricus terrestris) sur l'incorporation de biomasses végétales marquées ( 13 C) dans un sol en conditions contrôlées[END_REF][START_REF] Del Río | Analysis of aliphatic biopolymers using thermochemolysis with tetramethylammonium hydroxide (TMAH) and gas chromatography-mass spectrometry[END_REF][START_REF] Clifford | A new rapid technique for the characterization of lignin in vascular plants: thermochemolysis with tetramethylammonium hydroxide (TMAH)[END_REF].

The fate of plant-derived molecules in soil

Plant residues represent the main contributor to SOM, followed by microorganisms. Among plant residues, the distinction between above and below ground inputs is crucial. It was estimated that in grassland 33 % and 67 % of the annual litter inputs comes from above and below-ground, respectively [START_REF] Freschet | Linking litter decomposition of above-and below-ground organs to plant-soil feedbacks worldwide[END_REF]. In grassland and arable systems, the quality and quantity of litter inputs from shoots are highly variable given species concerned and its management. [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF] reviewed that the annual carbon above-ground input varies from 0.03 to 0.71 kg.m -2 . In similar systems, around 30% of the above-ground carbon is transferred to the below-ground, depending upon the growing stage of the plant and the concerned species. Thus, the carbon transferred below-ground represents 0.15-0.22 kg.m -2 per growing period [START_REF] Kuzyakov | Carbon input by plants into the soil[END_REF]. The relative contribution from root and shoot litter to SOM has been the subject of many studies during the last decades, revealing important discrepancies regarding their respective fate in soil [START_REF] Balesdent | Major contribution of roots to soil carbon storage inferred from maize cultivated soils[END_REF]Gale and Cambardella, 2000;[START_REF] Puget | Short-term dynamics of root-and shoot-derived carbon from a leguminous green manure[END_REF][START_REF] Lu | Carbon dynamics of rhizodeposits, root-and shootresidues in a rice soil[END_REF][START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF][START_REF] Mendez-Millan | Can cutin and suberin biomarkers be used to trace shoot and root-derived organic matter? A molecular and isotopic approach[END_REF][START_REF] Mambelli | Relative contribution of foliar and fine root pine litter to the molecular composition of soil organic matter after in situ degradation[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF]. The fate of roots and shoots in the soil will be used as guiding thread of the following part.

Plants are constituted of two main tissues submitted to decomposition while reaching the soil: parenchyma and woody tissues. Woody tissues generally contain higher concentrations of complex polymeric molecules such as lignin, compared to parenchyma. Cells from the parenchyma are encountered in leaves, young twigs and fine roots while woody tissues participate to form stems, leaf ribs and barks. During decomposition processes, plants tend to loose their anatomical characteristics. However, the molecular characteristics of plant species and tissues still provide information on SOM origins [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. Plant tissues are built of various compounds according to their localization in plant cells. Moreover, these compounds exhibit different decomposition rates. 'Metabolic' compounds, often intracellular, have a low molecular mass and are highly labile. They represent an important source of carbon and energy, easily accessible for soil microorganisms [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF][START_REF] Calvet | Les matières organiques des sols: Rôles agronomiques et environnementaux[END_REF][START_REF] Dungait | Soil organic matter turnover is governed by accessibility not recalcitrance[END_REF]. This group encompasses simple sugars and organic acids that can be directly derived from plants and/or come from exudation, excretion, death or degradation of cells from soil microorganisms. 'Structural' compounds mainly originate from cell walls and generally decompose at a slower rate compared to metabolic compounds. This group encompasses lignin, tannins, cutins and suberins [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF][START_REF] Calvet | Les matières organiques des sols: Rôles agronomiques et environnementaux[END_REF][START_REF] Dungait | Soil organic matter turnover is governed by accessibility not recalcitrance[END_REF]. Polysaccharides and lignin are the major constituents of plant residues.

 Polysaccharides are mainly represented by cellulose (a linear glucan polymer which is the major structural compound of cell walls of plants), as well as hemicelluloses and polyoses [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. Carbohydrates derived from plant generally serve as a source of energy and carbon for soil microorganisms and may thus, be rapidly recycled in microorganism-derived carbon [START_REF] Gleixner | Molecular dynamics of organic matter in a cultivated soil[END_REF]. Despite their apparent labile form, it has been demonstrated that some carbohydrate pools, initially derived from plants and recycled by microorganisms, can be preserved from biodegradation, presenting long residence time [START_REF] Gleixner | Molecular dynamics of organic matter in a cultivated soil[END_REF][START_REF] Derrien | Microbial biosyntheses of individual neutral sugars among sets of substrates and soils[END_REF].

 Tannins are polyphenols occurring either under condensate, non-hydrolysable or hydrolysable forms in higher plants. They are identified in many plant structures and are used for defense from fungi and insect by plants [START_REF] Haslam | Plant polyphenols (syn. vegetable tannins) and chemical defense-A reappraisal[END_REF]. Tannins can also precipitate proteins in aqueous substances [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF], which often make their extraction from soil samples complicated [START_REF] Yu | Evaluation of methods for measuring polyphenols in conifer foliage[END_REF]. They present an important role in nutrient cycling by affecting decomposition rates [START_REF] Kraus | Tannins in nutrient dynamics of forest ecosystems-a review[END_REF].

 Lignin is a high molecular weight tridimensional macromolecule built of phenylpropane units which provides structural rigidity to plant cell walls [START_REF] Adler | Lignin chemistry-past, present and future[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF] (Figure 5). Roots contain higher concentration of lignin than shoots. For example, roots and shoots of Ray Grass contain 14.5 and 6 % of lignin, respectively (Rasse et al., 2005). Plants synthetize lignin by oxidative copolymerization of coumaryl/hydroxyphenyl, coniferyl/guaiacyl and sinapyl/syringyl alcohols [START_REF] Hedges | The characterization of plant tissues by their lignin oxidation products[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF] (Figure 6). These precursors are present in different proportion depending on plant species. Grass lignin contains approximatively equal proportions of these three precursors [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. The sum of the molecules derived from these three alcohols is used to evaluate the quantity of lignin in soils. During lignin decomposition in soils, this sum tends to decrease and the proportion of acids over aldehydes in lignin-derived molecules tends to increase (reviewed by [START_REF] Thevenot | Fate of lignins in soils: A review[END_REF]. Due to the important concentration of lignin in plants and its relative resistance to decomposition, lignin was considered for long as a dominant element of SOM, regulating its pool-size and dynamics. Thus, it was believed that lignin accumulated in litter while water soluble compounds and non-protected cellulose controlled the initial phase of litter decomposition [START_REF] Berg | Decomposition rate and chemical changes of scots pine needle litter. II. influence of chemical composition[END_REF]. More recently, it was reported that, in the presence of bioavailable carbon, lignin could decompose in the early stages of litter degradation [START_REF] Klotzbücher | A new conceptual model for the fate of lignin in decomposing plant litter[END_REF]. [START_REF] Rasse | Lignin turnover in an agricultural field: from plant residues to soil-protected fractions[END_REF] showed that up to 90% of the lignin was metabolized in one year modeling and [START_REF] Dignac | Carbon-13 natural abundance as a tool to study the dynamics of lignin monomers in soil: an appraisal at the Closeaux experimental field (France)[END_REF] highlighted that the lignin turnover tended to be more rapid than bulk SOM.

Figure 5. Model of spruce lignin [START_REF] Adler | Lignin chemistry-past, present and future[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF].  Cutin and suberin are polyesters considered among the most resistant molecules to degradation (Figure 7). Cutin, associated with waxes, forms the cuticle which protects leaves and shoots from dehydration. Suberin is encountered in plant roots and barks [START_REF] Kolattukudy | Biopolyester membranes of plants: cutin and suberin[END_REF]. Both cutin and suberin are composed of C 16 and C 18 monomers of di and tri hydroxylalkanoic acids, as well as epoxy alkanoic acids and series of n-alkanoic acids, nalkanedioic acids and ω-hydroxyalkanoic acids. However, contrary to cutin, suberin monomers are known to comprise longer carbon chains (C ≥ 20), mainly of n-alkanedioic acids and ω-hydroxyalkanoic acids [START_REF] Kolattukudy | Biopolyester membranes of plants: cutin and suberin[END_REF][START_REF] Bernards | Demystifying suberin[END_REF]Mueller et al., 2012Mueller et al., , 2013)). Suberin also contains a significant amount of phenolic acids such as ferulic and syringyl acids [START_REF] Kolattukudy | Biopolyester membranes of plants: cutin and suberin[END_REF][START_REF] González-Vila | Pyrolytic alkylation-gas chromatography-mass spectrometry of model polymers Further insights into the mechanism and scope of the technique[END_REF][START_REF] Chefetz | Characterization of Organic Matter in Soils by Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH)[END_REF][START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF]Otto et al., 2005). Due to their distinct molecular composition, cutin and suberin are often used to characterize and monitor shoot-and root-derived OM in soils, respectively [START_REF] Del Río | Analysis of aliphatic biopolymers using thermochemolysis with tetramethylammonium hydroxide (TMAH) and gas chromatography-mass spectrometry[END_REF][START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF][START_REF] Mendez-Millan | Can cutin and suberin biomarkers be used to trace shoot and root-derived organic matter? A molecular and isotopic approach[END_REF][START_REF] Otto | Sources and composition of hydrolysable aliphatic lipids and phenols in soils from western Canada[END_REF]. [START_REF] Nierop | Rapid molecular assessment of the bioturbation extent in sandy soil horizons under pine using ester-bound lipids by on-line thermally assisted hydrolysis and methylation-gas chromatography/mass spectrometry[END_REF] also used the ratio between cutin and suberin biomarkers to evaluate the intensity of bioturbation in soils, considering that bioturbation is a mixture of above-ground tissues with soil. Cutin-and suberin-derived compounds were often identified in soils but OM composition is generally dominated by suberin-derived compounds, especially in deeper soil horizons [START_REF] Rumpel | Vertical distribution, age, and chemical composition of organic carbon in two forest soils of different pedogenesis[END_REF][START_REF] Feng | The distribution and degradation of biomarkers in Alberta grassland soil profiles[END_REF]Mueller et al., 2012). This highlights the important contribution of root-derived carbon in the soil [START_REF] Bull | Organic geochemical studies of soils from the Rothamsted classical experiments-V. The fate of lipids in different long-term experiments[END_REF]. Although cutin and suberin possess a complex molecular structures which confers them a reputation of resistant compounds, their monomers decrease rapidly in the early phase of decomposition in soils, with no distinction for cutin and suberin [START_REF] Riederer | Occurrence, distribution and fate of the lipid plant biopolymers cutin and suberin in temperate forest soils[END_REF][START_REF] Angst | The fate of cutin and suberin of decaying leaves, needles and roots -Inferences from the initial decomposition of bound fatty acids[END_REF].

Figure 7. Proposed models and monomer structures of (1) cutin and ( 2) suberin [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF][START_REF] Kolattukudy | Biopolyester membranes of plants: cutin and suberin[END_REF].

 Lipids are organic molecules present in both plants and microorganisms. They are often defined as insoluble in water and extractable with organic solvents. However, as most lipids exhibit an aliphatic character, the lipid term is also abusively used for high molecular weight aliphatic molecules, even though they are insoluble in organic solvents, such as esterbound lipids derived from cutin and suberin. Thus, in order to study vegetation history, soil processes and lipid transformations, it is recommended to distinguish free from bound lipids [START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF]. Lipids account for 4 to 8 % of SOM and represent a family of extremely diverse organic compounds [START_REF] Bull | Organic geochemical studies of soils from the Rothamsted classical experiments-V. The fate of lipids in different long-term experiments[END_REF]. The structure of lipids varies from complex organic compounds (phospholipids, sphingolipids and lipoproteins) to simple functional classes (hydrocarbon, aldehydes, ketones, alcohols and carboxylic acids) (Figure 8). These forms can be complexed to other compounds, such as wax esters, sterol esters, mono-, di-and triglycerides [START_REF] Bianchi | Surface lipid composition of C3 and C4 plants[END_REF]. Lipid hydrophobic structure and their strong affinity with soil minerals confer them a high resistance to decomposition. Thus, they play an important role in processes of incorporation, decomposition and stabilization of SOM [START_REF] Quenea | Variation in lipid relative abundance and composition among different particle size fractions of a forest soil[END_REF][START_REF] Wiesenberg | Source determination of lipids in bulk soil and soil density fractions after four years of wheat cropping[END_REF].

Lipids in soil are commonly used as source (plants and/or microorganisms) markers (Table 1). Lipids derived from plants present long chain even carbon-numbered n-alkanoic acids and n-alkanols (C > 20) [START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments-IV. Preliminary results from a study of the effect of soil pH on organic matter decay[END_REF][START_REF] Quenea | Variation in lipid relative abundance and composition among different particle size fractions of a forest soil[END_REF]Quenea et al., , 2006)), as well as long chain n-alkanes (odd carbon-numbered, mainly C 29 , C 31 and C 33 ) [START_REF] Marseille | n-Alkanes and free fatty acids in humus and A1 horizons of soils under beech, spruce and grass in the Massif-Central (Mont-Lozère), France[END_REF][START_REF] Bull | Organic geochemical studies of soils from the Rothamsted classical experiments-V. The fate of lipids in different long-term experiments[END_REF][START_REF] Wiesenberg | Source and turnover of organic matter in agricultural soils derived from n-alkane/n-carboxylic acid compositions and C-isotope signatures[END_REF]. Soil n-alkanes can also be derived from the reduction or decarboxylation of n-alkenes, n-alkanols and n-alkanoic acids [START_REF] Lichtfouse | Isotope and biosynthetic evidence for the origin of long-chain aliphatic lipids in soils[END_REF][START_REF] Amelung | Combining biomarker with stable isotope analyses for assessing the transformation and turnover of soil organic matter[END_REF]. Lipid extracts generally also contain ω-hydroxy acids resulting from the hydrolysis of cutins and suberins (Quenea et al., 2012) that can be used to reveal shoot and root contribution, respectively [START_REF] Bull | Organic geochemical studies of soils from the Rothamsted classical experiments-V. The fate of lipids in different long-term experiments[END_REF], as described above. However, lipids are highly variable among plant species and organs (Mueller et al., 2012), thus it is important to determine both root and shoot lipid compositions to understand their contribution to SOM.

Lipids originating from microorganisms generally present a high proportion of short chain nalkanoic acids (C < 20) [START_REF] Jandl | Contribution of primary organic matter to the fatty acid pool in agricultural soils[END_REF][START_REF] Quenea | Variation in lipid relative abundance and composition among different particle size fractions of a forest soil[END_REF]Quenea et al., , 2012)). While n-alkanoic acids C 16 and C 18 are ubiquitous in most living organisms, the presence of iso and anteiso C 15 and C 17 acids clearly indicates a bacterial origins [START_REF] Wannigama | A comparison of lipid components of the fresh and dead leaves and pneumatophores of the mangrove Avicennia marina[END_REF]Quenea et al., 2012).

Lipids can also be used to indicate the state of decomposition of the OM. For example, upon decomposition of vegetal biomass, unsaturated acids are degraded faster than saturated nalkanoic acids [START_REF] Wiesenberg | Source determination of lipids in bulk soil and soil density fractions after four years of wheat cropping[END_REF]. The presence of mono-unsaturated C 16:1 and C 18:1 and di-unsaturated C 18:2 n-alkanoic acids is a sign of OM preservation in the soil [START_REF] Wiesenberg | Source determination of lipids in bulk soil and soil density fractions after four years of wheat cropping[END_REF]Quenea et al., 2012).

Figure 8.

Major components of lipids in plant and microorganisms identified in soils [START_REF] Dinel | Soil lipids: origin, nature, content, decomposition, and effect on soil physical properties[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF].

Table 1. Aliphatic lipids identified in soil samples and their principal sources (Otto and Simpson, 2005).

The fate of roots and shoots in soil

Litter has long been restricted to leaf inputs, principally for the sake of experimental convenience. It is currently admitted that roots highly contribute to SOM, with different decomposition rates compared to shoots. It is now widely recognized that roots decompose at a slower rate compared to shoots and root-derived carbon represents a larger pool of carbon in soils [START_REF] Balesdent | Major contribution of roots to soil carbon storage inferred from maize cultivated soils[END_REF]Gale and Cambardella, 2000;[START_REF] Puget | Short-term dynamics of root-and shoot-derived carbon from a leguminous green manure[END_REF][START_REF] Lu | Carbon dynamics of rhizodeposits, root-and shootresidues in a rice soil[END_REF]. In a meta-analysis, [START_REF] Freschet | Linking litter decomposition of above-and below-ground organs to plant-soil feedbacks worldwide[END_REF] reported that in grassland systems, roots decomposed 1.8 times slower compared to shoots. [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF] showed that final root-over shoot-derived OC in soils is almost always superior to 1, whatever the experimental protocol used to study root and shoot decomposition. However, reasons explaining the slower decomposition of roots and its high contribution to soil carbon pool remains debated.

Root and shoot decomposition process have partly been explained thanks to methods based on radioactive ( 14 C) and stable ( 13 C and 15 N) isotopes. In this manuscript, the focus will be made on stable carbon isotopes. Several isotope ratio mass spectrometry (IRMS) methods exist for bulk (e.g. Elemental analyzer (EA)-IRMS, Stable Isotope Proving, Secondary Ion Mass Spectrometry (SIMS)) and compound specific (e.g. Gas Chromatography-Combustion-Mass Spectrometry (GC-C-IRMS), Pyrolysis-GC-C-IRMS) stable isotope analyses [START_REF] Amelung | Combining biomarker with stable isotope analyses for assessing the transformation and turnover of soil organic matter[END_REF]. In this paragraph, we will focus on bulk analysis results. Carbon can be encountered under two stable forms in the environment: 12 C and 13 C, which represent 98.89 % and 1.11 % of carbon isotopes, respectively. The isotopic composition is commonly expressed in δ 13 C (‰) which expresses the ratio between 13 C and 12 C, relative to a standard (Pee Dee Bee Belemnite is the most common). δ 13 C values vary within the carbon cycle. Atmospheric CO 2 δ 13 C is equal to -8 ‰. The fractioning occurring during photosynthesis leads to a decrease of 13 C proportion. The intensity of this decrease varies depending on the type of photosynthetic cycle. C3 and C4 plant present a δ 13 C of -27 ‰ and -13 ‰, respectively [START_REF] Morgun | Prospects and problems of using the methods of geochemistry of stable carbon isotopes in soil studies[END_REF]. During decomposition of complex substrates, such as plants, the mean δ 13 C value of the OM depends on the contribution to δ 13 C of the substances they contain (Werth and Kuzyakov, 2010). The natural concentration in stable carbon isotopes (δ 13 C) in the soil was often used to investigate the origin and decomposition state of the vegetal cover over the time, along with the turnover of SOM [START_REF] Balesdent | Natural 13 C abundance as a tracer for studies of soil organic matter dynamics[END_REF]Mariotti and Balesdent, 1990;[START_REF] Martin | Estimate of organic matter turnover rate in a savanna soil by 13 C natural abundance measurements[END_REF][START_REF] Bernoux | The use of stable carbon isotopes for estimating soil organic matter turnover rates[END_REF][START_REF] Lynch | Use of 13C and 15N natural abundance techniques to characterize carbon and nitrogen dynamics in composting and in compost-amended soils[END_REF][START_REF] Millard | Quantifying the contribution of soil organic matter turnover to forest soil respiration, using natural abundance δ 13 C[END_REF]. However, the use of natural stable isotope labelling to follow SOM has revealed complicated by the multiplicity of OM sources and the low isotopic variability between OM and soil [START_REF] Staddon | Carbon isotopes in functional soil ecology[END_REF]Werth and Kuzyakov, 2010). During the last decades, artificial labelling has been developed and widely applied to follow SOM fluxes [START_REF] Thompson | Pulse-labelling a cover crop with 13 C to follow its decomposition in soil under field conditions[END_REF][START_REF] Klumpp | Long-term steady state 13 C labelling to investigate soil carbon turnover in grasslands[END_REF][START_REF] Brüggemann | Carbon allocation and carbon isotope fluxes in the plantsoil-atmosphere continuum: a review[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF][START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF]. Based on this method, [START_REF] Puget | Short-term dynamics of root-and shoot-derived carbon from a leguminous green manure[END_REF] followed the incorporation of 13 C labelled Fabaceae roots and shoots, during corn production. They observed that 50 % and 13 % of the carbon was derived from roots and shoots, respectively, at the end of corn growth. [START_REF] Bird | 13 C and 15 N stabilization dynamics in soil organic matter fractions during needle and fine root decomposition[END_REF] followed 13 C labelled pine needle and fine root decomposition in forest of conifers in the United States.

They showed that, after 1.5 years, 28 % more 13 C from roots had been retained in the soil compared to needles.

These results have been interpreted through different angles. Some authors underline the high chemical recalcitrance, leading to an increased stability of molecules originating from roots compared to shoots [START_REF] Balesdent | Major contribution of roots to soil carbon storage inferred from maize cultivated soils[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF]. Indeed, plant resistance to decomposition is generally related to the presence of lignin and as mentioned before, roots contain higher concentrations of lignin compared to shoots [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF].

Some authors consider that the chemical recalcitrance is not the only key to explain the slower carbon turnover originating from roots compared to shoots. Two additional important mechanisms can explain the increased stabilization of root-derived carbon: (1) the physicochemical protection of OM adsorbed onto the soil mineral phase and (2) the physical protection against microbial decomposition due to the inaccessibility and the anoxic conditions in the micropores containing root hairs (Figure 9) [START_REF] Puget | Short-term dynamics of root-and shoot-derived carbon from a leguminous green manure[END_REF][START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF][START_REF] Bird | 13 C and 15 N stabilization dynamics in soil organic matter fractions during needle and fine root decomposition[END_REF][START_REF] Mambelli | Relative contribution of foliar and fine root pine litter to the molecular composition of soil organic matter after in situ degradation[END_REF][START_REF] Stockmann | The knowns, known unknowns and unknowns of sequestration of soil organic carbon[END_REF].

Figure 9.

Processes explaining the protection of root derived carbon in the soil [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF].

Figure 10. The size spectrum of soil constituents [START_REF] Totsche | Biogeochemical interfaces in soil: The interdisciplinary challenge for soil science[END_REF].

The heterogeneous distribution of soil organic matter

Soil is a heterogeneous and complex system, and the spatial organization of SOM at the microscale can significantly influence its decomposition and stabilization in soil. Soil complexity can partly be explained by the diversity of organic and mineral compounds, which present a wide size spectrum, from molecules (10 -10 m) to rock fragments (up to 10 m) (Figure 10) [START_REF] Totsche | Biogeochemical interfaces in soil: The interdisciplinary challenge for soil science[END_REF]. The arrangement of organic and mineral compounds increase this complexity by forming aggregates of different size and stability [START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF]. The occlusion of OM inside aggregates affects its decomposition and stabilization in soils (Von [START_REF] Lützow | Stabilization of organic matter in temperate soils: mechanisms and their relevance under different soil conditions-a review[END_REF]. SOM tends to be increasingly protected in microaggregates, even very small ones (< 2 µm) [START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF][START_REF] Chenu | Clay-sized organo-mineral complexes in a cultivation chronosequence: revisiting the concept of the "primary organo-mineral complex[END_REF][START_REF] Lützow | Stabilization of organic matter in temperate soils: mechanisms and their relevance under different soil conditions-a review[END_REF], compared to macroaggregates [START_REF] Lützow | SOM fractionation methods: Relevance to functional pools and to stabilization mechanisms[END_REF]. This can be explained by the inaccessibility of microorganisms to OM located in micropores and the adsorption of OM on minerals, which prevent OM from microbial degradation [START_REF] Chenu | Interactions between microorganisms and soil particles: an overview[END_REF]. Indeed, organo-mineral interactions, mainly defined by mineral properties and organic compounds acting as binding agents [START_REF] Baldock | Role of the soil matrix and minerals in protecting natural organic materials against biological attack[END_REF][START_REF] Chenu | Interactions between microorganisms and soil particles: an overview[END_REF], probably account for the key factors explaining long term protection of OM (reviewed by [START_REF] Kögel-Knabner | Organo-mineral associations in temperate soils: Integrating biology, mineralogy, and organic matter chemistry[END_REF]. The heterogeneous distribution of SOM into complex organo-mineral arrangements enhance the development of hot spots and hot moments of elemental cycling, including carbon. Hot spots are defined as 'small soil volumes with much higher process rates and intensive interactions compared to the average soil conditions'. Hot moments are 'are short-term events or sequences of events inducing accelerated process rates as compared to the average rates' [START_REF] Kuzyakov | Microbial hotspots and hot moments in soil: Concept & review[END_REF].

Thus, the use of high spatial resolution technics is required to study the fate of OM in soil samples, including spectroscopic, microscopic and tomographic methods, which are described in [START_REF] Totsche | Biogeochemical interfaces in soil: The interdisciplinary challenge for soil science[END_REF] Regarding the methods used in the present manuscript, the focus will be made on nano-scale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy (TEM). NanoSIMS provides elemental and isotopic maps of organic and/or mineral materials, with a submicron spatial resolution. NanoSIMS brings the capacity to spatially track an isotopic label, hence identifying specific locations of components [START_REF] Clode | In situ mapping of nutrient uptake in the rhizosphere using nanoscale secondary ion mass spectrometry[END_REF][START_REF] Vogel | Submicron structures provide preferential spots for carbon and nitrogen sequestration in soils[END_REF]. It has mainly been used in cosmochemistry, material science, biology and geology [START_REF] Mcmahon | Quantitative imaging of cells with multiisotope imaging mass spectrometry (MIMS)-Nanoautography with stable isotope tracers[END_REF]Herrmann et al., 2007a;[START_REF] Hoppe | NanoSIMS: technical aspects and applications in cosmochemistry and biological geochemistry[END_REF]. It has recently been applied to soil science, first focusing on soil microorganisms (Herrmann et al., 2007b) and then to characterize organo-mineral associations in soil [START_REF] Hatton | A multi-scale approach to determine accurate elemental and isotopic ratios by nano-scale secondary ion mass spectrometry imaging[END_REF][START_REF] Heister | NanoSIMS as a tool for characterizing soil model compounds and organomineral associations in artificial soils[END_REF]Mueller et al., 2012Mueller et al., , 2013;;[START_REF] Remusat | NanoSIMS study of organic matter associated with soil aggregates: advantages, limitations, and combination with STXM[END_REF][START_REF] Vogel | Submicron structures provide preferential spots for carbon and nitrogen sequestration in soils[END_REF][START_REF] Rumpel | Nanoscale evidence of contrasted processes for rootderived organic matter stabilization by mineral interactions depending on soil depth[END_REF]. Most of the efforts have focused on developing methodologies for calibration [START_REF] Hatton | A multi-scale approach to determine accurate elemental and isotopic ratios by nano-scale secondary ion mass spectrometry imaging[END_REF], sample preparation (Mueller et al., 2012) or quantification of compounds in an artificial soil [START_REF] Heister | NanoSIMS as a tool for characterizing soil model compounds and organomineral associations in artificial soils[END_REF]. [START_REF] Vogel | Submicron structures provide preferential spots for carbon and nitrogen sequestration in soils[END_REF] went a step further by reporting encouraging results on the proportion of OM associated to mineral particles in soil in the presence of labelled litter. A recent study compared the incorporation, after three years, of 13 C and 15 N labelled roots at two soil depths and demonstrated, thanks to the NanoSIMS, contrasting processes of stabilization depending on soil depths [START_REF] Rumpel | Nanoscale evidence of contrasted processes for rootderived organic matter stabilization by mineral interactions depending on soil depth[END_REF]. Despite the submicron spatial resolution of the NanoSIMS, the identification of SOM as plant residues or microorganisms (e.g. bacteria and fungi) remains challenging. Combinations of NanoSIMS with other microscopic techniques is required [START_REF] Moore | Elemental imaging at the nanoscale: NanoSIMS and complementary techniques for element localisation in plants[END_REF][START_REF] Remusat | NanoSIMS study of organic matter associated with soil aggregates: advantages, limitations, and combination with STXM[END_REF][START_REF] Poch | Micromorphology Techniques for Soil Organic Carbon Studies[END_REF]. The coupling of NanoSIMS with scanning electron microscopy (SEM) [START_REF] Heister | NanoSIMS as a tool for characterizing soil model compounds and organomineral associations in artificial soils[END_REF] or scanning transmission X-ray microscopy (STXM) and near edge Xray absorption fine structure spectroscopy (NEXAFS) [START_REF] Remusat | NanoSIMS study of organic matter associated with soil aggregates: advantages, limitations, and combination with STXM[END_REF] is helpful to identify the nature of the OM sampled by NanoSIMS. The combination with the nm-scale resolution of the transmission electron microscopy (TEM) can be highly powerful in identifying microstructures [START_REF] Moore | Elemental imaging at the nanoscale: NanoSIMS and complementary techniques for element localisation in plants[END_REF]. TEM has been used with NanoSIMS to study the structural and chemical properties in plant physiology [START_REF] Clode | In situ mapping of nutrient uptake in the rhizosphere using nanoscale secondary ion mass spectrometry[END_REF][START_REF] Misson | Use of phosphate to avoid uranium toxicity in Arabidopsis thaliana leads to alterations of morphological and physiological responses regulated by phosphate availability[END_REF][START_REF] Smart | High-resolution elemental localization in vacuolate plant cells by nanoscale secondary ion mass spectrometry[END_REF][START_REF] Moore | High-resolution secondary ion mass spectrometry reveals the contrasting subcellular distribution of arsenic and silicon in rice roots[END_REF].

Microorganism implications in the soil carbon cycle

Microorganisms and litter decomposition

Soil complex organization is a source of uncountable habitats for a diversified and abundant microbial community. A few grams of fertile soil can contain 10 12 bacteria, 10 4 protozoa, 10 4 nematodes, 25 km of fungi mycelium and a considerable amount of other species that remain unidentified and unquantified [START_REF] Young | Interactions and self-organization in the soil-microbe complex[END_REF]. Soil microorganisms mediate processes of organic compound mineralization and transformation in soil. Thus, they have a direct implication on litter decomposition [START_REF] Kuzyakov | Microbial hotspots and hot moments in soil: Concept & review[END_REF]. It has to be noted that fresh litter also represents a source of easily available substrate for microorganisms which accelerate the decomposition of OM originally present in soils. This process is called the priming effect (reviewed by [START_REF] Kuzyakov | Priming effects: Interactions between living and dead organic matter[END_REF]Kuzyakov et al., 2000). It has been reported that microorganisms mediate about 85-90% of OM decomposition in soil, while soil fauna utilize approximatively 10-15% of OC derived energy [START_REF] Wolters | Invertebrate control of soil organic matter stability[END_REF][START_REF] Ekschmitt | Soil-carbon preservation through habitat constraints and biological limitations on decomposer activity[END_REF]. However, OM must be fragmented and degraded by enzyme (exo-enzyme) to reach a size that enables their transport across microorganism cell walls (reviewed by [START_REF] Lehmann | The contentious nature of soil organic matter[END_REF]. Thus, in order to gain energy and maintain their biomass, soil heterotrophic organisms oxidize OC source into CO 2 . The availability of OC is not always related to an increased carbon mineralization [START_REF] Ekschmitt | Soil-carbon preservation through habitat constraints and biological limitations on decomposer activity[END_REF]. Indeed, in order to face competition for food resource in soil, different organisms have developed specialization of different resources [START_REF] Craig Maclean | Adaptive radiation in microbial microcosms[END_REF]. Thus, microorganisms are able to exploit almost every organic sources (Table 2) [START_REF] Ekschmitt | Strategies used by soil biota to overcome soil organic matter stability-why is dead organic matter left over in the soil?[END_REF].

Table 2. Capacity of soil microorganisms to decompose complex molecular structures [START_REF] Ekschmitt | Strategies used by soil biota to overcome soil organic matter stability-why is dead organic matter left over in the soil?[END_REF].

Fungi and bacteria represent more than 90% of the soil microbial biomass [START_REF] Six | Bacterial and fungal contributions to carbon sequestration in agroecosystems[END_REF] and present different resource exploitation strategies. For example, fungi can directly assimilate carbon in the litter soil layer and also utilize phenolic compounds such as lignin.

However, fungi activity is mainly restricted to the soil surface (< 20cm) [START_REF] Ekschmitt | Soil-carbon preservation through habitat constraints and biological limitations on decomposer activity[END_REF] as the decomposition of these complex substrates requires oxygen [START_REF] Baldrian | Fungal laccases-occurrence and properties[END_REF]. On the contrary, most bacteria are highly dependent on soluble carbon availability and transport through the soil [START_REF] Poll | Mechanisms of solute transport affect small-scale abundance and function of soil microorganisms in the detritusphere[END_REF]. When the soil structure enables labile carbon translocation to soil deeper parts, some bacteria are able to grow and develop in deeper soil horizons [START_REF] Fierer | Variations in microbial community composition through two soil depth profiles[END_REF].

Microorganism contribution to soil stable carbon pool

Microorganisms can participate indirectly to carbon stabilization by emphasizing mineral adsorption, aggregate formation and stabilization in soils. Indeed, during OM decomposition process, living microorganisms produce polysaccharides that enhance adsorption of minerals, increase inter-particle cohesion, thus serving as binding agents in soil aggregates [START_REF] Chenu | Interactions between microorganisms and soil particles: an overview[END_REF].

Microorganisms are increasingly recognized as participating also directly to the soil stable carbon pool. Even though plant residues are the primary source of SOM, a large proportion of compounds derived from residues pass through the microbial biomass and it is increasingly recognized that microorganisms highly contribute to stable soil carbon [START_REF] Lichtfouse | Isotope evidence for soil organic carbon pools with distinct turnover rates-II. Humic substances[END_REF][START_REF] Gleixner | Molecular dynamics of organic matter in a cultivated soil[END_REF][START_REF] Derrien | Microbial biosyntheses of individual neutral sugars among sets of substrates and soils[END_REF][START_REF] Liang | Microbial production of recalcitrant organic matter in global soils: implications for productivity and climate policy[END_REF].

The carbon introduced in the soil via microbial biomass can be (1) mineralized, (2) used by other microorganisms and thus incorporated into their biomass and/or (3) stabilized under non-living SOM [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF][START_REF] Schurig | Microbial cell-envelope fragments and the formation of soil organic matter: a case study from a glacier forefield[END_REF]. It was recently reviewed by [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF] that microbial contribution to stable SOM is not mainly achieved by the living biomass but preferentially by the microbial necromass. Thus, up to 40% of the bacterial biomass carbon might be transformed to non-living SOM and the bacterial necromass was found to be 40 times higher than the living microbial biomass [START_REF] Liang | Microbial production of recalcitrant organic matter in global soils: implications for productivity and climate policy[END_REF]. [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF] nicely concluded that 'soils are not a garden of living microbes but rather a mass cemetery of microbes with some active ones left'. The same authors proposed a pathway for the stabilization of necromass-derived carbon, named the 'patchy fragment formation cycle' (Figure 11). First, microorganisms develop on plant residues which constitute an important source of energy and carbon. Over time, resources decrease and microorganisms start starving and dying. Their internal cell content is rapidly released in the soil, while cell walls are fragmented and potentially stabilized in soils.

It was demonstrated that SOM chemical composition, organo-mineral interactions and microorganisms play a central role on SOM formation, decomposition and stabilization.

However, all these factors are under the strong influence of soil invertebrates [START_REF] Wolters | Invertebrate control of soil organic matter stability[END_REF] which tremendously impact their environment [START_REF] Lavelle | Soil invertebrates and ecosystem services[END_REF].

Figure 11. Formation of non-living SOM from microbial necromass [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF].

Earthworms

Soil fauna encompasses all the main taxonomic groups described in terrestrial ecosystems, comprising organisms from a few micrometers (i.e. some bacteria) to several meters (i.e. some earthworms) [START_REF] Nielsen | Soil Biodiversity and the Environment[END_REF]. Earthworms, ants and termites are considered as the main ecosystem engineers, having significant impact on their environment [START_REF] Lavelle | Soil invertebrates and ecosystem services[END_REF]. In temperate regions, earthworms account for the main invertebrate biomass in soils [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF][START_REF] Edwards | The Importance of Earthworms as Key Representatives of the Soil Fauna[END_REF]. Earthworms play a key role in soils thanks to their (1) physical action on the soil structure and (2) biochemical action on nutrient cycling [START_REF] Lavelle | Large-scale effects of earthworms on soil organic matter and nutrient dynamics[END_REF][START_REF] Bossuyt | Protection of soil carbon by microaggregates within earthworm casts[END_REF][START_REF] Blouin | A review of earthworm impact on soil function and ecosystem services[END_REF].

Earthworms belong to the Annelida phylum, the Oligochaeta class, the Haplotaxida subclass and the suborder of Lumbricina which contains 20 families including Lumbricidae. 7000 species of earthworms have been described in the world, with 400 in Europe. Earthworms are classified according to their feeding habits and characteristics of their burrows. Three main ecological categories exist: (1) Epigenic earthworms feed on the litter and mainly impact the soil surface. These earthworms feed on the litter deposited at the soil surface, which is transferred along vertical burrows which can reach 1 to 2 meters depth. They are generally considered as detritivores [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF][START_REF] Bouché | Des vers de terre et des hommes, découvrir nos écosystèmes fonctionnant à l'énergie solaire[END_REF].

Earthworms and soil aggregation

Earthworms are saprophagous invertebrates ingesting both organic (plant residues and microorganisms) and mineral (soil particles) materials, in different proportions depending on their ecological category. During ingestion, residues are fragmented and the preexisting soil microstructures destroyed. Mineral and organic elements are mixed, complexed with mucus, partly mineralized and released in the form of organo-mineral aggregates called casts [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF]) (Figure 12). This process creates new aggregates and nuclei for the further formation of organo-mineral aggregates in soil. In the presence of earthworms, the soil contains a larger proportion of macroaggregates (> 250 µm) compared to soil with no earthworms [START_REF] Bossuyt | Protection of soil carbon by microaggregates within earthworm casts[END_REF]. Moreover, aggregates formed upon earthworm activity are generally more stable than non-biogenic aggregates [START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF][START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF]. For example, [START_REF] Bossuyt | Protection of soil carbon by microaggregates within earthworm casts[END_REF] showed that the proportion of large macroaggregates (> 2000 µm) was 3.6 fold higher in the presence of earthworms in a cultivated soil incubated in laboratory. This increase generally occurs in short periods of time.

For example, [START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF] showed in a laboratory study, that the proportion of macroaggregates (> 1 cm) increased from 17.8 % to 42.1 % after two months of experiment with earthworms compared to the bulk soil. Earthworms also have a significant impact on microaggregates formation and composition [START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF]. [START_REF] Bossuyt | Rapid incorporation of carbon from fresh residues into newly formed stable microaggregates within earthworm casts[END_REF] incubated earthworms in a soil in the presence of 13 C labelled residues for 12 days and showed that the production of microaggregates within macroaggregates was four times higher in the presence vs. absence of earthworms [START_REF] Bossuyt | Rapid incorporation of carbon from fresh residues into newly formed stable microaggregates within earthworm casts[END_REF].

Earthworms and soil carbon

The physical action applied by earthworms during ingestion of organic and mineral compounds is also coupled to a chemical action, decisive for the fate of the OC in the soil.

Molecules easily decomposed by the intestinal enzymes produced in the alimentary canal of earthworms pass through the intestinal membrane and are directly assimilated by the earthworm (around 27 % of the OC ingested). Non-assimilable elements (around 50 % of the OC ingested) are excreted at the soil surface or along burrows in the form of casts. The last part of the C org (around 23 % of the OC ingested) is lost as CO 2 . Few months after cast production, non-assimilated elements contained in casts are partly degraded by microorganisms, enabling earthworms to re-ingest their own casts [START_REF] Cortez | Field decomposition of leaf litters: earthwormmicroorganism interactions -the ploughing-in effect[END_REF] (Figure 13). The annual production of casts by earthworms at the soil surface reach 30-50 t/ha [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF], representing at least 5.4 t/ha of OC [START_REF] Bouché | Des vers de terre et des hommes, découvrir nos écosystèmes fonctionnant à l'énergie solaire[END_REF].

Figure 13. Approximate organic carbon (t.ha -1 .an -1 ) balance during its ingestion, assimilation and dejection by earthworms (in blue: the fate of organic carbon after cast formation) (Adapted from [START_REF] Bouché | Des vers de terre et des hommes, découvrir nos écosystèmes fonctionnant à l'énergie solaire[END_REF].

As earthworms ingest residues containing higher concentration of OC than soil, they impact the incorporation and the stock of carbon in aggregates [START_REF] Fonte | Influence of earthworm activity on aggregate-associated carbon and nitrogen dynamics differs with agroecosystem management[END_REF][START_REF] Fonte | Interactive effects of plants and earthworms on the physical stabilization of soil organic matter in aggregates[END_REF][START_REF] Hong | How do earthworms influence organic matter quantity and quality in tropical soils?[END_REF][START_REF] Arai | Changes in soil aggregate carbon dynamics under no-tillage with respect to earthworm biomass revealed by radiocarbon analysis[END_REF] and along the soil profile [START_REF] Jégou | Burrowing activity of the earthworms Lumbricus terrestris and Aporrectodea giardi and consequences on C transfers in soil[END_REF]. The net impact of earthworms on soil carbon strongly varies depending on the studied spatial and time scale.

1-At short time scale -As most earthworms feed on the litter, the addition of earthworms in a soil with plant residues at its surface leads, in a few hours, to a decrease in litter quantities, causing a decrease in the global carbon stock [START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF]. During the gut transit, mineralization of OC is accelerated. Indeed, in earthworm gut, the physical protections of the OM originally present in the soil are broken and plant residues are fragmented, increasing the surface area available for microbial decomposition [START_REF] Lavelle | Small-scale and large-scale effects of endogeic earthworms on soil organic matter dynamics in soils of the humid tropics[END_REF]. Moreover, the presence of water and intestinal mucus creates conditions favorable to mineralization by the microorganisms present in the earthworm gut and fresh casts [START_REF] Martin | Les mucus intestinaux de ver de terre moteur de leurs interactions avec la microflore[END_REF][START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF][START_REF] Drake | As the worm turns: The earthworm gut as a transient habitat for soil microbial biomes[END_REF] (Figure 14). After a few days or weeks, earthworms will produce fresh casts. As plant residues contain more OC compared to the soil, it is generally admitted that casts, burrows and their periphery contain more OC compared to the surrounding soil [START_REF] Jégou | Burrowing activity of the earthworms Lumbricus terrestris and Aporrectodea giardi and consequences on C transfers in soil[END_REF][START_REF] Fonte | Interactive effects of plants and earthworms on the physical stabilization of soil organic matter in aggregates[END_REF] or to aggregates formed by physicochemical or microbial processes [START_REF] Fonte | Interactive effects of plants and earthworms on the physical stabilization of soil organic matter in aggregates[END_REF]. In fresh casts, the microbial activity continues to be enhanced by the presence of easily accessible compounds contained in mucus and fresh residues [START_REF] Lavelle | Small-scale and large-scale effects of endogeic earthworms on soil organic matter dynamics in soils of the humid tropics[END_REF].

Figure 14. Simplified representation of the effect of earthworms on the soil organic carbon (OC) at different time and spatial scale (Adapted from [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF].

2-At the scale of months or years, casts are drying and ageing, leading to the stabilization of carbon (Figure 14) [START_REF] Martin | Short-and long-term effects of the endogeic earthworm Millsonia anomala (Omodeo) (Megascolecidae, Oligochaeta) of tropical savannas, on soil organic matter[END_REF][START_REF] Lavelle | Small-scale and large-scale effects of endogeic earthworms on soil organic matter dynamics in soils of the humid tropics[END_REF][START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF]. Plant derived carbon which has been incorporated and embedded within microaggregates by earthworms, tends to be protected from degradation [START_REF] Bossuyt | Protection of soil carbon by microaggregates within earthworm casts[END_REF][START_REF] Pulleman | Earthworms and management affect organic matter incorporation and microaggregate formation in agricultural soils[END_REF]. [START_REF] Martin | Short-and long-term effects of the endogeic earthworm Millsonia anomala (Omodeo) (Megascolecidae, Oligochaeta) of tropical savannas, on soil organic matter[END_REF] who incubated casts from an endogenic tropical earthworm, showed that carbon mineralization was about half time lower than that of control soil, after one year. According to [START_REF] Shipitalo | Chemistry and micromorphology of aggregation in earthworm casts[END_REF], this is mainly explained by the appearance of clay-polyvalent cation-OM linkages in the ageing and drying casts, strengthening organo-mineral bonds and reducing microorganism accessibility. Thus, at a larger time scale, the translocation of OC from the surface to the subsurface by earthworms

Structures

tends to decrease its vulnerability to mineralization and favor its stabilization in deeper soil layers [START_REF] Don | Organic carbon sequestration in earthworm burrows[END_REF][START_REF] Stromberger | Distinct microbial and faunal communities and translocated carbon in Lumbricus terrestris drilospheres[END_REF]. This phenomena is added to favorable conditions for carbon stabilization in subsoils (e.g. reduced microbial activity compared to the soil surface) [START_REF] Dungait | Soil organic matter turnover is governed by accessibility not recalcitrance[END_REF].

It has to be noted, that the short and long time scale were separated in order to understand the process of production and transformation of one series of casts. However, at the field scale, the regular enrichment in fresh litter makes this process much more complex and erratic.

Earthworm feeding habits

Residue decomposition rate by earthworms depends on residue palatability [START_REF] Cortez | C and N transfer in soil with or without earthworms fed with 14 C-and 15 N-labelled wheat straw[END_REF]. It is generally admitted that earthworms feed mainly on organic-enriched compounds [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF], mainly in the form of dead plant tissues. Earthworms prefer litter with higher soluble carbohydrate and protein content and lower lignin and tannin concentrations. For example, it has been observed that earthworms preferentially fed on shoots compared to roots, which generally contain higher lignin concentrations [START_REF] Bouché | Fonction des lombriciens; II: Recherches méthodologiques pour l'analyse du sol ingéré (étude du peuplement de la station RCP-165/PBI)[END_REF][START_REF] Curry | The feeding ecology of earthworms -A review[END_REF]. However, little is known about the incorporation and decomposition of root residues by earthworms in soil [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF][START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF].

According to [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF], earthworm species feeding in deep soil horizons tend to consume important quantities of dead roots although in smaller proportion than shoots [START_REF] Watkin | Some factors affecting earthworm populations under pasture[END_REF]. [START_REF] Cortez | Do earthworms eat living roots?[END_REF] showed that young living roots were palatable for L.terrestris, but according to [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF], this ingestion is incidentally induced while feeding on rhizosphere soil.

Litter ingestion by earthworms also depends on its microbial colonization. [START_REF] Moody | Selective consumption of decomposing wheat straw by earthworms[END_REF] showed that earthworms preferred wheat straw inoculated with specific fungi compared to non-inoculated straw. [START_REF] Zirbes | Earthworms use odor cues to locate and feed on microorganisms in soil[END_REF] highlighted the importance of odor cues produced by fungi to attract earthworms on specific organic resources, suggesting a form of 'chemical communication' between earthworms and microorganisms. Moreover, the production of complex and diversified enzymes (i.e. chitinase, protease, phosphatase cellulose, glucosidase, peroxidase, etc.) in the gut enables the digestion and assimilation of microorganisms by earthworms [START_REF] Tracey | Cellulase and Chitinase of Earthworms[END_REF][START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF][START_REF] Zhang | Activity and origin of digestive enzymes in gut of the tropical earthworm Pontoscolex corethrurus[END_REF][START_REF] Lattaud | Enzymatic digestive capabilities in geophagous earthworms-origin and activities of cellulolytic enzymes[END_REF]. Thus, earthworms have the capacity to feed on microorganisms, especially fungal communities [START_REF] Wolters | Invertebrate control of soil organic matter stability[END_REF][START_REF] Curry | The feeding ecology of earthworms -A review[END_REF][START_REF] Shan | Digestion and residue stabilization of bacterial and fungal cells, protein, peptidoglycan, and chitin by the geophagous earthworm Metaphire guillelmi[END_REF].

Earthworms and soil microorganisms

The volume of soil occupied by microbial community is particularly low (<< 1%) and heterogeneously distributed [START_REF] Schmidt | Persistence of soil organic matter as an ecosystem property[END_REF]. Microorganisms are able to decompose most of the organic compounds but due to their limited capacities to move into the soil, they are often physically disconnected from their resource [START_REF] Ekschmitt | Soil-carbon preservation through habitat constraints and biological limitations on decomposer activity[END_REF]. Earthworms tend to maintain mutualistic relationships with soil microorganisms, based on the 'sleeping beauty paradox' proposed by [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF] for endogenic earthworms. At the scale of few centimeters, the spatial distribution of often dormant microorganisms ('Sleeping Beauties') is modified by earthworms inducing heterogeneous resource distribution [START_REF] Decaëns | Macroecological patterns in soil communities[END_REF]. Earthworms (the 'Prince Charming') are highly mobile into the soil and create favorable conditions for microorganism activation in their gut, fresh casts and along burrows.

During the gut transit, the contact between OM and microorganisms is increased. Moreover, the intestinal mucus ('the Kiss') they secrete is a mixture of easily assimilated compounds: amino-acids, sugars and glycoproteins [START_REF] Martin | Les mucus intestinaux de ver de terre moteur de leurs interactions avec la microflore[END_REF]. As a result of these favorable conditions, structures produced by earthworms generally present higher microbial activity compared to bulk soil [START_REF] Amador | Microbiological characterization of the structures built by earthworms and ants in an agricultural field[END_REF][START_REF] Jayasinghe | Earthworms as the vectors of actinomycetes antagonistic to litter decomposer fungi[END_REF][START_REF] Frouz | Microbial properties of soil aggregates created by earthworms and other factors: spherical and prismatic soil aggregates from unreclaimed post-mining sites[END_REF]. For example, [START_REF] Jayasinghe | Earthworms as the vectors of actinomycetes antagonistic to litter decomposer fungi[END_REF] detected a significantly higher number of actinomycetes in earthworm casts compared to bulk soil. Thus, the presence of earthworm can potentially induce hotspots of microbial activity [START_REF] Decaëns | Macroecological patterns in soil communities[END_REF][START_REF] Kuzyakov | Microbial hotspots and hot moments in soil: Concept & review[END_REF]. However, in the case of casts, the microbial community characteristics are particularly influenced by the age and drying state [START_REF] Tiunov | Microbial biomass, biovolume and respiration in Lumbricus terrestris L. cast material of different age[END_REF]. Moreover, the microbial biomass and respiration generally gradually decrease a few days after cast production [START_REF] Parle | A Microbiological Study of Earthworm Casts[END_REF][START_REF] Wolters | Microbial carbon turnover in beech forest soils worked by Aporrectodea caliginosa (Savigny) (Oligochaeta:Lumbricidae)[END_REF][START_REF] Tiunov | Microbial biomass, biovolume and respiration in Lumbricus terrestris L. cast material of different age[END_REF].

Data on the composition and diversity of the microbial community found in earthworm gut and burrows are contradictory. Some authors showed that microorganisms identified in earthworm gut were similar to those observed in soil and litter [START_REF] Drake | As the worm turns: The earthworm gut as a transient habitat for soil microbial biomes[END_REF][START_REF] Jayasinghe | Earthworms as the vectors of actinomycetes antagonistic to litter decomposer fungi[END_REF]. Others highlighted some difference between the gut microbial communities compared to the soil [START_REF] Sampedro | Changes in the fatty acid profiles through the digestive tract of the earthworm Lumbricus terrestris L[END_REF][START_REF] Byzov | Culturable microorganisms from the earthworm digestive tract[END_REF]. Indeed, the earthworm gut can be assimilated to a mobile anoxic microarea to which all microorganisms might not survive and/or that might induce sporulation [START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF][START_REF] Drake | As the worm turns: The earthworm gut as a transient habitat for soil microbial biomes[END_REF], depending on the transit time [START_REF] Hartenstein | Production and carrying capacity for the earthworm Lumbricus terrestris in culture[END_REF].

Objectives

In this state of knowledge, we reported that SOM mainly originates from plant above-and below-ground tissues which are composed of a high diversity of molecules, from simple carbohydrates to complex lignin-derived compounds. Litter quality (root vs. shoot in the present study) can influence SOM composition and the contribution to stable carbon pools.

Numerous studies have compared the fate of root vs. shoot litter-derived carbon in soil [START_REF] Blair | Decomposition of 13 C and 15 N labelled plant residue materials in two different soil types and its impact on soil carbon, nitrogen, aggregate stability, and aggregate formation[END_REF][START_REF] Williams | Distribution and fate of 13 C-labeled root and straw residues from ryegrass and crimson clover in soil under western Oregon field conditions[END_REF][START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13C labelled-leaf litter experiment[END_REF][START_REF] Mambelli | Relative contribution of foliar and fine root pine litter to the molecular composition of soil organic matter after in situ degradation[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF] and shown that roots decomposed at a slower rate compared to shoots. However, it is still strongly debated whether this slower decomposition depends on chemical composition, physical or physico-chemical protection. This debate is even more complex if we consider the role of soil fauna in litter decomposition. One of the key soil engineers in temperate soil are earthworms, which have a significant impact on soil structure, carbon cycling and microbial community regulation. Many studies have investigated the role of earthworms on the fate of above-ground litter in soils [START_REF] Fonte | Influence of earthworm activity on aggregate-associated carbon and nitrogen dynamics differs with agroecosystem management[END_REF][START_REF] Stromberger | Distinct microbial and faunal communities and translocated carbon in Lumbricus terrestris drilospheres[END_REF][START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF].

However, there are still some knowledge gaps on the interaction between earthworms and root litter. Earthworms form casts, which are the result of the ingestion/incorporation of both litter and soil. These complex organo-mineral structures generally contain high OC concentrations [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF], which is partly mineralized at short terms and tent to be stabilized at long terms [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF]. Despite their prominent role in carbon cycling, earthworm casts implication in SOM formation and transformation, in the presence of root vs. shoot litter have rarely been studied, particularly at the fine scale (molecular and microscopic level).

The aim of this PhD thesis was to investigate the fate of roots vs. shoots in soil and earthworm casts. One of the keys to unravel carbon cycling processes in the soil is to combine analytical methods using multidisciplinary approaches. Thus, a one year mesocosm experiment was carried out and samples were analyzed with various analytical tools to locate, quantify and characterize SOM at different time and spatial levels, in soil and earthworm casts (Figure 15).

We used to the following questions as guiding thread in the present manuscript:

Where is incorporated root-and shoot-derived carbon along the year of experiment?

What impact do earthworms have on the incorporation and/or decomposition of roots and shoots?

Are the structures formed by earthworms (casts) playing a specific role in root-and shootderived carbon cycling?

What forms do root-and shoot-derived carbon take after one year of experiment in soil and casts? Chapter 2.

Materials and Methods

Experimental design, sampling and analytical methods

Experimental set up and sampling

Soil characteristics and experimental design

The soil was collected on a permanent grassland in the North of France. It is a loamy-sand soil with characteristics presented in Table 3. Six PVC containers (length -80 cm, diameter -40 cm) were filled with approximatively 75 L of soil . The soil was previously sieved to 4 mm for homogenization and large plant residues and mesofauna were removed. Soil containers were pre-incubated in a greenhouse during 6 months so as to pass the bacterial flush. Each container was weekly vaporized with water and seedlings or mosses developing in mesocosms were eliminated. The temperature and humidity was measured weekly in soils during the year of experiment and an average of 13°C (± 1.3) and 23 % (± 1.9) was observed, respectively.

Table 3. Initial composition of the soil used in the experiment.

In the entire manuscript, the six mesocosms will be identified as (1) Root-E for the mesocosm with root residues and earthworms (2) Root-NE for the mesocosm with root residues and no earthworm (3) Shoot-E, for the mesocosm with shoot and earthworms (4) Shoot-NE for the mesocosm with shoot and no earthworm (5) Control-E, for the mesocosm without plant residues but earthworms and (6) Control-NE for the mesocosm with no plant residues and no earthworms (Figure 16.A and B). At the beginning of the experiment, six earthworms per container were added in three of the mesocosms. Mean living mass was 26.8 ± 0.5 g per container. A low density of earthworms was chosen to favor their survival in conditions closest to reality [START_REF] Fründ | Using earthworms as model organisms in the laboratory: Recommendations for experimental implementations[END_REF].

A B

This work focuses on the epi-anecic earthworm Lombricus terrestis. Earthworms were grown by the SARL Lombri'carraz (France) and were received at adult stage, with an average weight of 4.5 g (Figure 17).

Figure 17. Illustration of the earthworms deposited at the soil surface at the beginning of the experiment.

Sampling

Samples were collected at seven time steps: before and 1, 2, 4, 8, 24 and 54 weeks after adding roots or shoots and earthworms. For the sake of simplification, the 54 weeks will often be mentioned as 'a one year experiment' in this manuscript. At each time step and for each mesocosm, three replicates of soil core were collected with an aluminum auger (diameter: 2.5 cm), after putting aside plant residues. Two depth intervals (0-20 and 40-60 cm) were sampled from these cores. One composite sample of earthworm casts was also collected on the soil surface, in the mesocosms containing earthworms (Figure 18). Casts samples collected in the mesocosms Root-E, Shoot-E and Control-E will be termed Cast-Root, Cast-Shoot and Cast-Control, respectively.

Analytical methods

Various analytical tools were applied to initial litter, soil and/or cast samples, at each or selected time steps. The corresponding results are presented in one or several chapters as summarized in Table 5. The OC concentrations and the δ 13 C were measured on initial litter, soil and cast samples for all time steps (Chapter 3) (Table 5). Soil and cast samples were freeze-dried and ground prior to analysis using a Vario pyro cube -Micromass Isoprime Elemental Analysis -Isotope Ratio Mass Spectrometry (EA-IRMS). The standard gas was calibrated with respect to the international standard (CO 2 in PeeDee Belemnite-PDB). For casts, the analysis was repeated three times. All samples were decarbonated before analysis, using the method developed by [START_REF] Harris | Acid fumigation of soils to remove carbonates prior to total organic carbon or CARBON-13 isotopic analysis[END_REF]. Briefly, subsamples of soil (~ 40 mg) and casts (~ 15 mg) were placed in open silver capsules (8*5 mm). Samples were placed in a microtiter plate and 15 µL of ultrapure water were added. They were placed in a vacuum desiccator containing a 100 mL beaker of HCl (12 M). Samples were exposed to HCl vapors during 6 hours. Excesses of HCl vapors were removed under 1 hour vacuum, after removing the HCl beaker. Samples were then transferred to an oven at 40°C for 15 hours.

Sample isotopic composition is expressed in δ 13 C relative to the PDB standard (Equation 1):

δ 13 C (‰) = [(R sample -R standard ) / R standard ] * 1000 (1)
Where R is the 13 C/ 12 C ratio of the sample or the standard.

The proportion of carbon derived from the labelled litter in the soil or casts (C lab ), expressed in percentage, was obtained by applying equation 2:

C lab (%) = [(δ s -δ c ) / (δ l -δ c )] * 100 (2)
Where δ s is the δ 13 C value of the soil or cast sample with litter, δ c is the δ 13 C value for the control soil sample or the control cast, δ l is the δ 13 C value for the litter.

In order to overcome the initial variability of the soil OC measured in the different mesocosms before the beginning of the experiment, all the OC content were transformed applying equation 3:

(RC org ) x = C x / C 0 (3)
Where (RC org ) x represents the evolution ratio of the OC content after x weeks of experiment, C x is the OC content at the same time step and C 0 is the OC content before adding earthworms and residues, for the corresponding mesocosm. Thus, values lower than one express a decrease in OC with respect to the beginning of the experiment, and values higher than one express an increase in OC in comparison with the beginning of the experiment.

A similar formula was applied for the casts, where C x is the OC content in cast after x weeks of experiment and C 0 is the OC content in the soil before adding earthworms and residues, for the corresponding mesocosm. Thus, values higher than one indicate that the OC content is higher in the casts compared to soil.

Statistical analyses

Statistical analyses were performed δ 13 C values and OC content using the R statistical software. Samples from different mesocosms were compared using the non-parametric Kruskal-Wallis test followed by a pairwise comparison of groups from the package named 'RcmdrPlugin.coin'. For all analyses, statistical significance was set as α = 0.05.

Molecular analyses

Molecular analyses were performed on both the lipid fraction and the macromolecular one.

Lipids

Lipid extracts from initial litter, soil and casts were analyzed after one year of experiment (Chapter 5) (Table 5).

Samples were extracted with a dichloromethane (DCM)/methanol mixture (2:1, v:v) (GC) fitted with a 30 m, 0.25 mm, film thickness 0.5 µm RXi5SIlMS capillary column (Restek). After 30 s at 80 °C, the GC oven temperature was programmed from 80 °C to 100 °C at 10 °C/min, from 100 °C to 320 °C at 4 °C/min and fixed at 320 °C for 30 min. The GC was coupled to a mass spectrometer (Agilent MS 5973) operating at 70 eV.

Macromolecular fraction

Cast samples from all time steps (except after 1 week of experiment -only Cast-Shoot could be observed and collected at this time step) were analyzed using TMAH-Py-GC-MS (Chapter 4). Lipid-free soil and cast samples were also analyzed after one year of experiment (Chapter 5).

Samples were submitted to Curie point pyrolysis in the presence of tetramethylammonium hydroxide (TMAH). Samples (5-9 mg, depending on OC content) were impregnated with a few drops of TMAH in MeOH at 25% (1/1, w/w) prior to loading into ferromagnetic tubes.

Samples were pyrolysed with a Curie temperature of 650 °C for 10 s using a pyrolysis unit (Pilodist), coupled to a gas chromatograph (GC Trace Thermo) and a mass spectrometer (MS DSQ Thermo). After 10 min at 50 °C, the GC oven temperature was programmed from 50 °C to 310 °C at 2 °C/min.

For semi-quantitative purpose, peaks of interest were selected. The peak area of each compound was obtained by integration of the total ion current (TIC). As TMAH-Py-GC-MS is semi-quantitative, the relative abundance of each compound was obtained by dividing its peak area by the sum of those of the selected pyrolysis products. Note that the same set of pyrolysis products was used for all quantification.

Statistical analysis

Statistical analysis was performed using the R statistical software. For the cast samples (Chapter 4), a principal component analysis (PCA) was performed on 22 selected variables of the 15 cast samples using the multivariate analysis package 'FactoMineR'. Root and shoot litter samples were used as illustrative samples. For soil and cast samples collected after 54 weeks of experiment (Chapter 5), two PCA were performed: (1) on free lipids (23 variables) and ( 2) on lipid-free (15 variables) for the 9 samples. The Shapiro-Wilk test was applied to all variables. As most of the variables were not normally distributed, values were transformed in ranks before analysis.

Microscale observations

Transmission electron microscopy (TEM) and nano-scale secondary ion mass spectrometry (NanoSIMS) analyses were mainly performed on initial litter and cast samples (Chapter 4).

Selected soil images were also used to introduce Chapter 5 (Table 5).

Transmission electron microscopy

In order to preserve to initial organo-mineral associations, samples were processed directly after sampling without drying, grinding or chemically extracting SOM [START_REF] Watteau | 0-20 μm aggregate typology based on the nature of aggregative organic materials in a cultivated silty topsoil[END_REF].

Soil samples were fractionated with distilled water in order to obtain 0-20 and 20-50 µm fractions. Approximatively 10 g of fresh soil were dispersed in 65 mL of distilled water and stirred gently for 1 h. Fractions were obtained using sequential wet-sieving (20, 50 and 200 µm meshes). The dispersed soil was poured on the 200 µm sieve and gently sprayed with distilled water. The 20-50 µm fraction was collected on the sieve. The 0-20 µm fraction was collected after centrifugation (8000 RPM, 20 min). Cast samples were not fractionated.

Initial litter, soil and fresh casts (2 grams) were chemically fixed with osmium tetroxide and soil and cast structures were physically preserved with agar [START_REF] Watteau | Root impact on the stability and types of micro-aggregates in silty soil under maize[END_REF]. About ten cubes of few mm 3 were cut in the core of the sample, dehydrated in graded acetone series, and embedded in epoxy resin (Epon 812) until complete polymerization. Five repetitions were thus obtained for each sample. Ultrathin sections of 80-100 nm were obtained using a diamond knife on a Leica Ultracut S ultramicrotome. The sections were stained with uranyl acetate and lead citrate and analyzed with a JEOL EMXII transmission electron microscope operating at 80kV. Each section was investigated in order to obtain a general view of the considered section and the occurrence of representative structures.

Nano-scale secondary ion mass spectrometry

For NanoSIMS analyses, ultrathin sections of 100-200 nm from the same block of dehydrated osmium-fixed samples as for TEM were used in order to obtain twin sections, making possible the identification of the structures with nanoSIMS. To ensure charge dissipation, samples were coated with 10 nm of gold. Images were acquired using the NanoSIMS 50 (Cameca, France) located at Museum National d'Histoire Naturelle in Paris, France. Sample surface was sputtered by a 1.5 pA Cs + beam to obtain a 20×20 µm 2 images divided into 256×256 pixels, at 1 ms/pixel raster speed, with an approximate spatial resolution of 100 nm.

Secondary images of 12 C -, 12 C 14 N -, 13 C 14 N -, 16 O -and 28 Si -were simultaneously collected;

between 50 to 70 frames were accumulated. It must be noted that the beam never got through the 200 nm thick samples, implying a very low sputtering rate (less than 0.04 nm/s). Prior to each acquisition, the sample surface was presputtered over 30×30 µm 2 using a 100pA Cs + beam for 10 minutes to remove gold coating and surface contamination and to reach sputtering steady-state. The images were processed using the L'IMAGE® software (L.

Nittler, Carnegie Institution, USA). The 13 C 14 N -/ 12 C 14 N -ratio was used to generate 13 C isotopic maps, relative to the PDB standard. The 13 C -/ 12 C -was not used in order to avoid potential biases created by the resin signal contain carbon. For the sake of clarity, the values will be termed δ 13 C in the following. It must be noted that 12 C 14 N -emission from the epoxy resin is negligible compared to the OM signal due to the virtual lack of nitrogen in the resin.

A kerogen standard was used to check for instrumental stability over the course of the analytical session. As the instrumental fractionation was lower than 5‰ during the session, it was neglected for the δ 13 C correction. The resin was masked on the δ 13 C maps using a threshold defined thanks to the 12 C 14 N pixel distribution. In brief, we determined that the resin count rate was about 10,000 counts/s. Thus, we could mask the resin-rich areas and kept, for the isotopic images, only areas with the largest count rates. Regions of interest (ROI) were manually drawn to select microorganisms or plant residues and determine their isotopic composition. The initial labelled litter was analyzed and images were used for the interpretation of cast images.

Microbial analyses

Microbial analyses were performed on soil and cast samples after one year of experiment (Chapter 5) (Table 5). Quantification of microbial communities and catabolic profiles were achieved.

Quantification of microbial communities by real-time PCR

The microbial communities (bacteria and fungi) were quantified by real time Polymerase

Chain Reaction (PCR) in the soil and cast samples, after one year of experiment. Total genomic DNA was extracted from 250 mg of fresh soil maintained at -80 °C, using the Bacterial and fungal abundances were thus expressed in number of copies of 16S and ITS genes per gram of soil, respectively.

Catabolic profiles of microbial communities by Biolog® EcoPlate™

The metabolic diversity of the microbial community was characterized using the Biolog® EcoPlate™. Briefly, 2 g of fresh soil were added to 200 ml saline solution (0.9% NaCl) and shaken at 200 rpm for 20 min using a rotary shaker. The preparation stood for 3 to 10 hours until soil particles had settled. The soil supernatant was then pipetted and 10 fold diluted with saline solution and inoculated in wells of Biolog® microplates (150 µl). Biolog TM microplates contained triplicate wells of 31 single carbon substrates and tetrazolium dye (Figure 19).

Further details on carbon sources in each well are reported in Appendix 1. The functional richness (FR) was expressed by the number of well presenting a corrected absorbance value > 0.25.

The metabolic potential of microbial communities was represented using polygons.

Absorbance values were summed for each groups of molecules and divided by the number of substrates for each molecular groups.

The meaning of these indicators will be detailed in section 5.2.

Statistical analyses

Statistical analysis was performed using the R statistical software. For AWCD, FR, bacterial and fungal abundances, samples from different mesocosms and depths were compared using the non-parametric Kruskal-Wallis test followed by a pairwise comparison of groups from the package named 'RcmdrPlugin.coin'. For all analyses, statistical significance was set as α = 0.05. A PCA was carried out using 12 selected individual carbon sources as variables, which best represented metabolic differences between samples. The corrected absorbance obtained for the 12 substrates was divided by the AWCD obtained for each replicates.

The knowledge on litter incorporation in soil has greatly increased using artificial isotopic labelling of the source of OM. It allows precise monitoring of the introduced litter (at a given date and place) and distinguishing introduced OM from that initially present in the soil [START_REF] Thompson | Pulse-labelling a cover crop with 13 C to follow its decomposition in soil under field conditions[END_REF][START_REF] Klumpp | Long-term steady state 13 C labelling to investigate soil carbon turnover in grasslands[END_REF][START_REF] Brüggemann | Carbon allocation and carbon isotope fluxes in the plantsoil-atmosphere continuum: a review[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF][START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF]. Previous studies using artificial labelling have focused on (1) the impact of residue quality (roots vs. shoots or plant species) on OC incorporation and decomposition into soil [START_REF] Blair | Decomposition of 13 C and 15 N labelled plant residue materials in two different soil types and its impact on soil carbon, nitrogen, aggregate stability, and aggregate formation[END_REF][START_REF] Williams | Distribution and fate of 13 C-labeled root and straw residues from ryegrass and crimson clover in soil under western Oregon field conditions[END_REF][START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13C labelled-leaf litter experiment[END_REF][START_REF] Mambelli | Relative contribution of foliar and fine root pine litter to the molecular composition of soil organic matter after in situ degradation[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF] or ( 2) the influence of earthworms on shoot decomposition and incorporation into soil and the consequences on soil carbon storage [START_REF] Fonte | Influence of earthworm activity on aggregate-associated carbon and nitrogen dynamics differs with agroecosystem management[END_REF][START_REF] Stromberger | Distinct microbial and faunal communities and translocated carbon in Lumbricus terrestris drilospheres[END_REF][START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF]. Taken separately, these two biotic factors (earthworm and residue quality) were proven to have a crucial impact on soil carbon cycling (Chapter 1). However, is the diverging fate of root and shoot residues in soil modified in the presence of earthworms? In this chapter, the combined effect of earthworms and root vs. shoot on the fate of OC in the soil profile (0-20 cm and 40-60 cm) and casts was investigated. The aim is to acquire a general appreciation of the localization and quantity of carbon derived from labelled roots and shoots. To meet this objective, all the samples collected from the mesocosm experiment (Section 2.1) were analyzed for OC and 13 C as described in section 2.2.1.

Results

Macroscopic observation of mesocosms

The macroscopic observation of mesocosms surfaces during the year of experiment gave indications on the incorporation of the labelled residues. After 24 weeks of experiment, no more shoot residues were observed in the Shoot-E mesocosm, whereas in the Root-E mesocosm, root residues were still observed at the soil surface, occupying less than 10 % of the surface. After one year of experiment, no more residues were observed at the soil surface in the two mesocosms containing earthworms (Figure 20). On the contrary, after one year, some litters were still observed at the soil surface, in mesocoms without earthworms, for either roots or shoots. These residues were collected and weighted: they represented around 30 % (in mass) of the initial litter mass. No casts could be identified after one week in the mesocosm with root residues, explaining the absence of data for Cast-Root at this time step.

In spite of regular removal of seedling and mosses during the experiment, some mosses developed in the Control-NE mesocosm, from 24 weeks until the end of the experiment.

Weeks

Carbon derived from root and shoot residues

Soil samples

The carbon derived from the labelled litter (C lab ) increased continuously in the 0-20 cm soil layer along the year of experiment, reaching between 0.96 and 11.4 % after 54 weeks, depending on the mesocosm (Figure 21.A). From the beginning of the experiment to the 8 th week, there were no significant differences between mesocosms, with a mean C lab of 0.40 %.

From 24 weeks, mesocosms presented significantly different C lab . After 24 weeks, C lab was significantly lower for Shoot-E (0.85 %) than Root-E (3.00 %). After 54 weeks, C lab was significantly higher in Root-NE (11.4 %) than in the other mesocosms. Two general trends could be observed after 24 and 54 weeks: (1) C lab in Root-NE and Shoot-NE tended to be higher than Root-E and Shoot-E, respectively and ( 2

Earthworm casts

For all the time steps, C lab measured in casts was higher than C lab in soil, with a mean C lab of 37 % with respect to 1.3 % for soils, all time steps and mesocosms included (Figure 22). C lab increased rapidly in Cast-Shoot, reaching 34.9 % in average during the first month of experiment. C lab reached a maximum after 8 weeks for shoots (58.1 %) and after 8 and 24 weeks for roots (51.8 %). C lab then decreased during the last six months of experiment, reaching around 12 % for both Cast-Root and Cast-Shoot after 54 weeks.

Organic carbon analyses

Soil samples

In shoot mesocosms, with or without earthworms, OC content tended to decrease compared with the OC content before the experiment (RC org < 1) (see section 2.2.1, for formula details) except after 54 weeks (Figure 23). On the contrary, in root mesocosms, with or withoutt earthworms, OC content tended to increase compared with the beginning of the experiment (RC org > 1). This increase tended to be delayed without earthworms and the RC org reached 1.2 after 54 weeks in Root-NE. For all the time steps, except after 54 weeks, the RC org in Root-E was higher than 1. The RC org was significantly different for Root-E and Shoot-E after 24 weeks (1.1 vs 0.85).This observation was also made between Root-NE and Shoot-NE but to a

Earthworm casts

The OC measured in casts was always substantially higher than the soil OC measured before the experiment (RC org >>1) (Figure 24). For Cast-Control, the mean RC org for all the time steps was 1.3. In both Cast-Root and Cast-Shoot, RC org was maintained at around 1.8, during the first month of experiment, before increasing to reach a maximum after 8 weeks with a RC org of 3.6 and 2.7, respectively. The RC org then decreased during the last six months of experiment, to 1.5 and 1.6 for Cast-Root and Cast-Shoot, respectively. The RC org measured for Cast-Root was higher than Cast-Shoot after 8 (3.6 vs 2.7) and 24 (2.8 vs 2.

Discussion

Litters placed on the soil surface can either be (1) totally mineralized, ( 2) left intact at the soil surface and/or (3) transferred to the soil either in form of soluble compounds or as undecomposed fragmented pieces [START_REF] Cortez | Field decomposition of leaf litters: earthwormmicroorganism interactions -the ploughing-in effect[END_REF][START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13C labelled-leaf litter experiment[END_REF]. The amount of litter incorporated in the 0-20 cm soil layer (Figure 21.A) are in agreement with other studies [START_REF] Kammer | Mineralisation, leaching and stabilisation of 13 C-labelled leaf and twig litter in a beech forest soil[END_REF][START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13C labelled-leaf litter experiment[END_REF]. For example, in a similar study in a field experiment with 13 C labelled litter from Populus nigra, [START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13C labelled-leaf litter experiment[END_REF] showed that 5% of the total C in the 0-2 cm layer of the soil was derived from leaf litter after 11 months. The absence of significant earthworm impact during the first 8 weeks can be explained by the experimental conditions. Indeed, the low density of earthworms and the localized non-destructive sampling to preserve soil structure in mesocosms, explain the great variability of data and the late significant impact of earthworms. The influence of root or shoot litter was only visible after 6 months. We can hypothesize that the soluble compounds derived from labelled residues introduced into the soil during the first 8 weeks are rapidly used and mineralized by the microorganisms, as soluble compounds are the main source of microbial products [START_REF] Cotrufo | The Microbial Efficiency-Matrix Stabilization (MEMS) framework integrates plant litter decomposition with soil organic matter stabilization: do labile plant inputs form stable soil organic matter?[END_REF].

Root vs. shoot effect

For the last two time steps, the C lab of mesocosms containing roots was higher than those with shoots, although the difference was not always statistically significant. Without earthworms, after 54 weeks, C lab in Root-NE and Shoot-NE represents 11.4 % and 3.1 %, respectively (Figure 21.A). Moreover, the RC org in Root-NE and Shoot-NE was 1.20 and 1.05, respectively, after 54 weeks, underlining the higher contribution of root residues compared to shoot ones to SOM. These results are consistent with those of William et al. (2006) who found, after incubating roots and shoots of ryegrass in soil for 10 months, a carbon contribution of 5.5 % and 1.5 %, respectively. Two explanations can be put forward to explain the higher root-derived carbon in soil. One explanation is that roots were more transferred into soil compared to shoots. More likely, root-derived carbon was decomposed at a slower rate than shoot-derived one, as evidenced by [START_REF] Scheu | Decomposition of roots and twigs: Effects of wood type (beech and ash), diameter, site of exposure and macrofauna exclusion[END_REF]Beuch et al., 2000, Gale andCambardella, 2000;[START_REF] Shi | Decomposition of roots and shoots of perennial grasses and annual barley-separately or in two residue mixes[END_REF][START_REF] Steffens | Translocation of 13 C-labeled leaf or root litter carbon of beech (Fagus sylvatica L.) and ash (Fraxinus excelsior L.) during decomposition -A laboratory incubation experiment[END_REF]. Indeed, the higher shoot-derived carbon mineralization with respect to root residues can be explained by (1) the higher water soluble carbon concentration in shoots [START_REF] Beuch | Effect of the organic residues of Miscanthus giganteus on the soil organic matter level of arable soils[END_REF][START_REF] Bird | Fine Roots vs. Needles: A Comparison of 13 C and 15 N Dynamics in a Ponderosa Pine Forest Soil[END_REF][START_REF] Shi | Decomposition of roots and shoots of perennial grasses and annual barley-separately or in two residue mixes[END_REF], which is rapidly transferred to the soil and decomposed by microorganisms [START_REF] Shi | Decomposition of roots and shoots of perennial grasses and annual barley-separately or in two residue mixes[END_REF]; ( 2) the higher chemical recalcitrance of roots due to higher concentrations of resistant molecules such as lignin, tannins and suberins [START_REF] Gleixner | 1.15 -Plant compounds and their turnover and stabilization as soil organic matter[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF][START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF]) and ( 3) the higher N concentration of shoots compared to roots (2.8 vs 1.3 in our study), which can increase the initial rate of decomposition [START_REF] Hobbie | Contrasting Effects of Substrate and Fertilizer Nitrogen on the Early Stages of Litter Decomposition[END_REF]. OM derived from roots might also benefit from higher physical protection in soil aggregates than shoot-derived OM (Gale et al., 2000;[START_REF] Puget | Short-term dynamics of root-and shoot-derived carbon from a leguminous green manure[END_REF].

However, this probably played only a minor role in this study since both root and shoot residues were deposited on the soil surface. Thus, these results highlight the importance of the chemical composition on residue decomposition.

Effect of earthworms on residue incorporation and decomposition

Earthworms accelerate residue incorporation and decomposition

Earthworms accelerated incorporation of residues in soil, as no more residues were observed at the soil surface after the year of experiment in the mesocosm containing earthworms compared to the one without earthworms. Thus, residues had been incorporated in soil or casts and/or mineralized. Earthworms also accelerated decomposition of residues in soil. After 6 months of experimentation, the C lab in soil tended to be lower in the presence of earthworms, for both root and shoot residues (Figure 21.A). This can be explained by the direct impact of earthworms which assimilate and mineralize part of the labelled OC during the ingestion of residues. Daniel (1991) showed that under favorable conditions, L.terrestris earthworms could assimilate between 43 and 55 % of a dandelion litter they had ingested.

Moreover, decomposition and mineralization are governed by microorganisms acting directly on particulate residues or on soluble compounds in the soil matrix [START_REF] Gaillard | Carbon, nitrogen and microbial gradients induced by plant residues decomposing in soil[END_REF]. In the presence of earthworms, this phenomenon can be amplified as the ingestion of residues by earthworms favors the increase of the exchange surface between microorganisms and residues. Moreover, the mucus produced by earthworms, favors proliferation of bacteria and further decomposition of residues [START_REF] Parle | A Microbiological Study of Earthworm Casts[END_REF][START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF][START_REF] Amador | Microbiological characterization of the structures built by earthworms and ants in an agricultural field[END_REF][START_REF] Frouz | Microbial properties of soil aggregates created by earthworms and other factors: spherical and prismatic soil aggregates from unreclaimed post-mining sites[END_REF][START_REF] Vidal | Incorporation of 13 C labelled shoot residues in Lumbricus terrestris casts: A combination of transmission electron microscopy and nanoscale secondary ion mass spectrometry[END_REF].

Casts -a key role for residue decomposition

The incorporation and decomposition of OC in earthworm casts present a completely different path compared to soil samples. As expected, the C lab and OC content are higher in casts compared to soil. This can be explained by the feeding behavior of earthworms, especially anecic ones, which incorporate fresh residues and/or organic rich compounds in casts [START_REF] Decaëns | Carbon and nitrogen dynamics in ageing earthworm casts in grasslands of the eastern plains of Colombia[END_REF][START_REF] Zhang | Comparison of organic compounds in the particle-size fractions of earthworm casts and surrounding soil in humid Laos[END_REF][START_REF] Frouz | Microbial properties of soil aggregates created by earthworms and other factors: spherical and prismatic soil aggregates from unreclaimed post-mining sites[END_REF][START_REF] Hong | How do earthworms influence organic matter quantity and quality in tropical soils?[END_REF][START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF]. C lab reached a maximum at 58.1% in Cast-Shoot, after 8 weeks of incubation. This result is in agreement with [START_REF] Jégou | Effects of four ecological categories of earthworms on carbon transfer in soil[END_REF], who showed that half of the carbon in L.terrestris casts was derived from labelled litter after 246 days of experiment, adding labelled litter every day. A chronology can be proposed for residue incorporation by earthworms in casts, based on C lab and RC org results (Figure 22 and Figure 24). During the first four weeks of experiment, both C lab and RC org increased in casts, likely due to the combined direct and indirect incorporation of residues by eathworms. Upon direct incorporation, residues are ingested and pass through the earthworm gut [START_REF] Shipitalo | Chemistry and micromorphology of aggregation in earthworm casts[END_REF]. Indirect incorporation, also called 'pre-oral litter decomposition', consists in a 'ploughing-in' of residues: casts are deposited on residues, without ingestion, initiating microbial decomposition [START_REF] Cortez | Field decomposition of leaf litters: earthwormmicroorganism interactions -the ploughing-in effect[END_REF]. After 4 weeks and until 24 weeks, C lab and RC org continued to increase. Earthworms are able, after few months to re-ingest their casts in which residues have been partially decomposed by microorganisms [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF].

Thus, between 4 and 8 weeks of experiment, the 'ploughing-in' and ingestion of undecomposed labelled residues still present at the soil surface were probably associated with the re-ingestion of previously labelled casts (from 1 to 4 weeks), increasing the C lab . After 24 weeks, C lab and RC org highly decreased. At 54 weeks, no more litter is present at the surface of the mesocosm containing earthworms and litter, indicating that all the residues have been either incorporated in the soil and casts, or mineralized. Thus, the decrease of C lab and RC org indicated that labelled residues incorporated in casts sampled at 54 weeks have been partially

decomposed. This decomposition was enhanced by bacteria and fungi as observed on Cast-Shoot NanoSIMS images in a previous study, after 24 weeks of experiment (Vidal et al., 2016).

A slight effect in the 40-60 cm soil layer

No significant increase in C lab was observed in the 40-60 cm soil layer during the 54 weeks.

This phenomenon is observed in other studies which reported that below 2 or 5 cm, labelled litter incorporation was not significant, at least for short time scales [START_REF] Jégou | Effects of four ecological categories of earthworms on carbon transfer in soil[END_REF][START_REF] Rubino | Carbon input belowground is the major C flux contributing to leaf litter mass loss: Evidences from a 13C labelled-leaf litter experiment[END_REF][START_REF] Kammer | Mineralisation, leaching and stabilisation of 13 C-labelled leaf and twig litter in a beech forest soil[END_REF], mainly due to the little migration of soluble carbon in deeper soil layers [START_REF] Thompson | Pulse-labelling a cover crop with 13 C to follow its decomposition in soil under field conditions[END_REF]. In the present study, a slight increase in C lab , although not statistically significant, was observed for both root and shoot residues in the presence of earthworms (Figure 21.B). The burrows of L. terrestris are considered as permanent structures that can reach deep soil layers (> 1 m) [START_REF] Jégou | Burrowing activity of the earthworms Lumbricus terrestris and Aporrectodea giardi and consequences on C transfers in soil[END_REF][START_REF] Don | Organic carbon sequestration in earthworm burrows[END_REF]. Thus, earthworms may have contributed to the incorporation of labelled organic compounds into the 40-60 cm soil layer directly, by covering their burrow walls with mucus and below ground casts enriched in 13 C [START_REF] Jégou | Burrowing activity of the earthworms Lumbricus terrestris and Aporrectodea giardi and consequences on C transfers in soil[END_REF] or to a lesser extent, indirectly, by creating preferential paths for labelled water soluble carbon circulation [START_REF] Don | Organic carbon sequestration in earthworm burrows[END_REF]. The C lab increase was probably not significant because earthworm influence is localized on burrow walls, periphery and cast [START_REF] Don | Organic carbon sequestration in earthworm burrows[END_REF]. Jegou et al. (2000) showed in an experiment using 13 C labelled litter that below the 2 cm soil layer, the carbon litter enrichment in the soil surrounding burrows was barely detectable. Thus, the 13 C signal was probably diluted in the soil as earthworm burrows were not sampled separately.

Combined effect of earthworms and root vs. shoot

Root and shoot differences are minimized in the presence of earthworms

In soil samples, the difference in C lab and RC org between root and shoot mesocosms without earthworms, after 54 weeks, was minimized in the presence of earthworms (Figure 21.A and Figure 23), underlying their ability to enhance root decomposition, as mentioned by [START_REF] Cortez | Do earthworms eat living roots?[END_REF]. [START_REF] Hopkins | Lumbricus terrestris counteract the effects of modified lignin biosynthesis on the decomposition of tobacco plant residues[END_REF] have compared decomposition of tobacco roots and modified tobacco roots with altered lignin structure and composition. They found that in the presence of earthworms, the difference in carbon mineralization between both types of residues was reduced. Lavelle et al. (1989) showed that after 5-10 weeks of experiment in the presence of earthworms, the quality of OM tended to homogenize whatever the origin of this OM. Earthworms can enhance the decomposition of recalcitrant residues and smooth the difference between residues of various qualities, probably by facilitating the access of plant residues to microbial decomposition.

The difference between root and shoot residues was also minimized in casts. Cast-Root and Cast-Shoot both followed the same general path of formation and decomposition for C lab and RC org . However, principally based on the observation of the mesocosms, differences were detected. After one week of experiment, no casts were observed on the soil surface in the mesocosm containing roots and the litter showed no evolution pattern. After 24 weeks, while shoots had totally disappeared from the soil surface in Shoot-E, roots were still visible in Root-E. Thus, in the present experimental conditions, dead roots in early stage of decomposition seemed less palatable for earthworms than shoots, confirming observations of [START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF]. [START_REF] Coq | Earthworm activity affects soil aggregation and organic matter dynamics according to the quality and localization of crop residues-An experimental study (Madagascar)[END_REF] also observed that less casts were produced in woody twigs of soybean compared with the small straws of rice, underlining the importance of residues palatability for earthworm ingestion. This can be explained by the high C/N value of roots in comparison to shoots (31 vs 15 in this study -Table 4), as it is known that earthworms preferentially consume organic compounds with low C/N [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF]. However, after 2 weeks, the C lab increases rapidly in Cast-Root, underlying the capacity of earthworms to process also roots (Cortez and Bouche, 1992). After 54 weeks, no more litter was observable in both mesocosms and the C lab and RC org were not significantly different for casts containing roots or shoots. Thus, even if earthworms prefer feeding on shoots, they have the capacity to incorporate roots in casts, where the decomposition is activated at a comparable rate than shoots.

Fate of organic matter initially present in soil

In the presence of earthworms, during the first 6 months, the RC org is higher in Root-E than in Shoot-E, with a significant difference after 6 months of experiment. Moreover, the C org content in Shoot-E is lower than the OC content measured before the experiment in the same mesocosm (RC org < 1) (Figure 23). A priming effect has probably been induced in this mesocosm, leading to a high mineralization of the OM originally present in the soil. This is also observed to a lesser extent with shoots but without earthworms. On the contrary, in the presence of earthworms and roots, the OC content is higher than the one measured before the experiment (RC org > 1). Thus, in this case, the OM originally present in the soil tended to be preserved, or at least, if this OM was mineralized, it was compensated by OC inputs from labelled residues. The enhanced SOM mineralization in the presence of fresh residues has been reported in many studies [START_REF] Kuzyakov | Priming effects: Interactions between living and dead organic matter[END_REF]. Indeed, microorganisms depend on the energy present in fresh residues to decompose recalcitrant OM present in the soil [START_REF] Fontaine | Fungi mediate long term sequestration of carbon and nitrogen in soil through their priming effect[END_REF][START_REF] Fontaine | Stability of organic carbon in deep soil layers controlled by fresh carbon supply[END_REF]. For example, [START_REF] Lu | Carbon dynamics of rhizodeposits, root-and shootresidues in a rice soil[END_REF] observed that the simple presence of fresh residues, either root or shoot, favored the degradation of native soil OM after 8 weeks of experiment, probably due to the stimulation of the microbial activity. [START_REF] Coq | Earthworm activity affects soil aggregation and organic matter dynamics according to the quality and localization of crop residues-An experimental study (Madagascar)[END_REF] showed that total soil carbon had decreased after 5 months of incubation of rice or soybean shoot residues, in the presence of endogeic earthworms. [START_REF] Fontaine | Fungi mediate long term sequestration of carbon and nitrogen in soil through their priming effect[END_REF] measured the effect of a 13 C labelled cellulose input in a soil during 161 days. They showed that, after one month, respiration of unlabeled CO 2 reached a maximum, revealing a priming effect. A continuous and slow decrease of unlabeled CO 2 respiration was then observed until the end of the experiment. This is in agreement with the present results showing that, after one year, RC org was around 1 for Control-E, Root-E, Shoot-E and Shoot-NE. Even though anecic earthworms preferentially feed on surface fresh litter, L.terrestris also assimilate carbon from older OM [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF][START_REF] Ferlian | Carbon food resources of earthworms of different ecological groups as indicated by 13 C compound-specific stable isotope analysis[END_REF], as underlined by the RC Org lower than 1 in the Control-E and higher than 1 in Cast-Control. However, in the presence of roots, this phenomenon may be compensated by the higher preservation of root derived carbon coupled to reduced activation of microbial activity compared to shoot residues [START_REF] Shi | Decomposition of roots and shoots of perennial grasses and annual barley-separately or in two residue mixes[END_REF]. Thus, in the present study, the priming effect induced by root residues seems to be lower compared to the one of shoot residues during the first six months of experiment.

However, this process is smoothed after one year; supporting [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF] comment that the preferential shoot priming effect on SOM mineralization cannot explain the higher root-derived carbon in the soil, as observed after one year in the present study (Figure 21.A).

Conclusion

This mesocosm experiment allowed monitoring the fate of root and shoot residues in soil and earthworm casts. Residues were continuously incorporated in the 0-20 cm soil layer during the year of experiment. In agreement with previous studies, root residues incorporated in the 0-20 cm soil layer tended to decompose at a slower rate than shoot residues, highlighting the essential role of litter chemical composition in the OC fate. Earthworms tended to accelerate both root and shoot residue decomposition in soil. The activity of earthworms also induced a slight transfer of carbon derived from residues in the deeper soil layer (40-60 cm), without any distinction between roots and shoots. In earthworm casts, root and shoot residues presented similar trends in incorporation and decomposition of OC. A chronology for cast formation and evolution was proposed, based on OC content and isotopic composition. Three phases were identified: (1) the first month: mainly incorporation of fresh residues in casts, (2) until 6 months: re-ingestion of labelled casts and incorporation of fresh residues and finally

(3) after 6 months: mainly microbial decomposition of residues in casts. This study provides new elements on the role of earthworms on the incorporation and decomposition of OC from roots and shoots into soil. Earthworms minimized the diverging fate of root and shoot residues, after the year of experiment, in both soil and cast samples. Thus, the presence of earthworms tended to enhance the decomposition of structures recognized as more resistant to decomposition (i.e. roots). Moreover, during the first six months of experiment, the presence of earthworms and shoot residues enhanced the decomposition of soil OC initially present in the soil. After one year, earthworms had accelerated the incorporation and decomposition of plant residues in the soil. However, after this period, the δ 13 C signal had not decreased in the soil and had only started to decrease in casts. Thus, longer term experiments are needed to investigate the stabilization of carbon-derived from root and shoot residues in the presence of earthworms.

These results gave an overview of the fate of OC, derived or not from the labelled litter, in the soil and surface casts during the year of experimentation. However, these analyses raised one main question: what forms takes the OC when incorporated in soil and casts? To answer this question, both microscale and molecular approaches were used in the following chapters.

As this chapter highlighted that after one year, the percentage of labelled litter derived carbon was very low at the 40-60 cm depth, Chapter 4 and 5 will mainly focus on samples from the 0-20 cm layer of soil and surface casts. Moreover, casts contained important concentration of OC derived from the labelled litter, which significantly evolved during the year of experiment. Thus, Chapter 4 will focus on cast analyses at molecular and microscopic scales.

Chapter 4.

Organic matter characterization in earthworm casts

Molecular characterization of earthworm casts

It is now recognized that roots decompose at a slower rate than shoots, so root-derived carbon may play a key role in soil carbon sequestration [START_REF] Chefetz | Characterization of Organic Matter in Soils by Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH)[END_REF][START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF][START_REF] Feng | The distribution and degradation of biomarkers in Alberta grassland soil profiles[END_REF]. However, reasons accounting for the difference in decomposition rate between roots and shoots in soils remain debated. Part of the explanation should rely on their contrasted molecular composition. Indeed, leaf cuticles and plant roots comprise cutin and suberin, respectively, and these macromolecules exhibit some chemical differences [START_REF] Kolattukudy | Structure, Biosynthesis, and Biodegradation of Cutin and Suberin[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. Incorporation and decomposition of roots and shoots in soil are highly dependent on the action of the soil fauna. More specifically, earthworms play a major role in SOM dynamics in temperate areas [START_REF] Edwards | The Importance of Earthworms as Key Representatives of the Soil Fauna[END_REF]. Most earthworms feed on both soil mineral matter/OM and fresh plant residues deposited at the soil surface. It has been demonstrated that earthworm feeding behavior is influenced by litter quality and palatability, but little is known about the impact of earthworms on root incorporation and decomposition in soils [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF][START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF]. Studies using molecular analyses to investigate the general impact of earthworms on SOM composition and dynamics are scarce [START_REF] Filley | Comparison of the chemical alteration trajectory of Liriodendron tulipifera L. leaf litter among forests with different earthworm abundance[END_REF][START_REF] Crow | Earthworms, stand age, and species composition interact to influence particulate organic matter chemistry during forest succession[END_REF]. [START_REF] Filley | Comparison of the chemical alteration trajectory of Liriodendron tulipifera L. leaf litter among forests with different earthworm abundance[END_REF] and [START_REF] Crow | Earthworms, stand age, and species composition interact to influence particulate organic matter chemistry during forest succession[END_REF] used CuO oxidation to characterize the influence of earthworm invasion in a US forest on the molecular composition of litter residues and soil particulate OM, respectively. The influence of earthworms on SOM has been mainly studied using total carbon content and/or carbon isotopes [START_REF] Lavelle | Large-scale effects of earthworms on soil organic matter and nutrient dynamics[END_REF][START_REF] Jégou | Burrowing activity of the earthworms Lumbricus terrestris and Aporrectodea giardi and consequences on C transfers in soil[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF][START_REF] Fonte | Interactive effects of plants and earthworms on the physical stabilization of soil organic matter in aggregates[END_REF][START_REF] Stromberger | Distinct microbial and faunal communities and translocated carbon in Lumbricus terrestris drilospheres[END_REF][START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF]. In contrast, the number of studies investigating the chemical composition of casts is limited [START_REF] Guggenberger | Soil organic matter within earthworm casts of an anecic-endogeic tropical pasture community, Colombia[END_REF][START_REF] Hong | How do earthworms influence organic matter quantity and quality in tropical soils?[END_REF] and mainly aimed at comparing casts and bulk soil. For example, [START_REF] Guggenberger | Soil organic matter within earthworm casts of an anecic-endogeic tropical pasture community, Colombia[END_REF] characterized OM in casts and surrounding soil collected in Colombia using CuO oxidation, acid hydrolysis and 13 C NMR. They highlighted the abundance of microbial-derived and lignin-derived molecules in casts compared to soil. [START_REF] Hong | How do earthworms influence organic matter quantity and quality in tropical soils?[END_REF] used CuO oxidation and pyrolysis coupled to gas chromatography and mass spectrometry (Py-GC-MS) to compare soil and casts from Vietman, according to their aggregate size. They showed that the protection of OM is enhanced in small size aggregates of casts. However, to the best of our knowledge, the short and longer term dynamics of litter incorporation and decomposition in casts has not been studied at the molecular level.

The aim of the present section was to monitor the incorporation and decomposition of root or shoot litter in earthworm casts using molecular characterization. For this purpose, the cast samples collected at all the time steps (with the exception of 1 week, as no casts were observed in the mesocosm with roots at this time step) were analyzed with Py-GC-MS (Section 2.2.2). Indeed, Py-GC-MS is a powerful tool in soil sciences, as reported in chapter 1.

Root and shoot litter molecular characteristics

The recognition of specific indicators of roots and shoots is a prerequisite for monitoring their fate in earthworm casts. It was achieved through TMAH-Py-GC-MS. Indeed, suberin and cutin are commonly considered as typical biopolymers of root and shoot tissues, respectively [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF] and their monomers have been used to trace the root-and shootderived OM in soil [START_REF] Del Río | Analysis of aliphatic biopolymers using thermochemolysis with tetramethylammonium hydroxide (TMAH) and gas chromatography-mass spectrometry[END_REF][START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF][START_REF] Otto | Sources and composition of hydrolysable aliphatic lipids and phenols in soils from western Canada[END_REF][START_REF] Mendez-Millan | Can cutin and suberin biomarkers be used to trace shoot and root-derived organic matter? A molecular and isotopic approach[END_REF]. However, the monomeric composition of these macromolecules is different depending on the vegetation type [START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF].The thermochemolysates of the root and shoot litter samples comprised two main types of products (Figure 25.A and B) as commonly reported in TMAH-Py-GC-MS analyses of plant biomass and soils (e.g. [START_REF] Clifford | A new rapid technique for the characterization of lignin in vascular plants: thermochemolysis with tetramethylammonium hydroxide (TMAH)[END_REF][START_REF] Del Río | Analysis of aliphatic biopolymers using thermochemolysis with tetramethylammonium hydroxide (TMAH) and gas chromatography-mass spectrometry[END_REF][START_REF] Chefetz | Characterization of Organic Matter in Soils by Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH)[END_REF][START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF]: (i) aromatic compounds which encompassed mainly lignin-and/or suberin-derived compounds from pcoumaryls (H), guaiacyls (G) and syringyls (S), as well as more ubiquitous phenols (P) and

(ii) aliphatic compounds, which were represented by n-alkanoic acids, n-alkanedioic acids and ω-hydroxyalkanoic acids, as well as n-alkanols and n-alkanes. In the present study, aliphatic compounds can derive from free lipids, bound lipids, cutin and/or suberin.

The first part of the GC traces comprised aromatic products commonly assigned to lignin and tannins, although a contribution from the aromatic domain of suberin [START_REF] Bernards | Demystifying suberin[END_REF] should also be considered. Aromatic products were dominated by guaiacyls (G), in both root and shoot samples and especially the 3-(3,4-dimethoxyphenyl) 2-propenoic acid methyl ester (G5). This compound, along with the 3-(4-methoxyphenyl) 2-propenoic acid methyl ester (H1), are typical for non-woody lignin [START_REF] Clifford | A new rapid technique for the characterization of lignin in vascular plants: thermochemolysis with tetramethylammonium hydroxide (TMAH)[END_REF], in agreement with the ryegrass origin of the root and shoot litter. Syringyl derivatives [3,4,5-trimethoxybenzaldehyde (S1) and 3,4,5-trimethoxybenzoic acid methyl ester (S2)] were also identified in the root sample, but contributed less to the shoot sample. The other main difference between root and shoot samples lay in the high relative abundance of 4-methoxybenzoic acid methyl ester (P3) with respect to 1,4-dimethoxybenzene (P1) in the root sample. P3 is classified as a lignin-derived compound (e.g. [START_REF] Chefetz | Characterization of Organic Matter in Soils by Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH)[END_REF][START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF] and was previously identified in root rye grass [START_REF] Whitehead | Composition and decomposition of roots of ryegrass and red clover[END_REF]. In contrast, P1 is not a lignin pyrolysis product [START_REF] Chefetz | Characterization of Organic Matter in Soils by Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH)[END_REF].

The same aromatic products were observed in both root and shoot litter thermochemolysates but their relative abundance with respect to aliphatic compounds was higher in the root sample. The higher concentration of aromatic compounds in root tissues has been reported in various studies (e.g. [START_REF] Dignac | Carbon-13 natural abundance as a tool to study the dynamics of lignin monomers in soil: an appraisal at the Closeaux experimental field (France)[END_REF][START_REF] Hoyos-Santillan | Getting to the root of the problem: litter decomposition and peat formation in lowland Neotropical peatlands[END_REF] and considered to reflect the contribution of the aromatic domain of suberin [START_REF] Bernards | Demystifying suberin[END_REF]Otto et al., 2005).

Thus, the evolution of the relative abundance of the aromatic compounds and the fate of some specific pyrolysis products, especially abundant in roots, such as P3, were used below to monitor root incorporation and decomposition in casts.

C 16 to C 32 n-alkanoic acids, along with their unsaturated counterparts C 18:1-2 , were observed in both roots and shoots, with a maximum at C 16 . C 16 and C 18 n-alkanoic acids are ubiquitous in living organisms [START_REF] Del Río | Analysis of aliphatic biopolymers using thermochemolysis with tetramethylammonium hydroxide (TMAH) and gas chromatography-mass spectrometry[END_REF][START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF]. However, the C 20 and C 24 nalkanoic acids were more abundant in roots compared to shoots. Moreover, the C 22 , C 24 and C 26 ω-hydroxyalkanoic acids could be identified in the root sample (Figure 25.A), whereas they were virtually absent from the shoot sample (Figure 25.B). In contrast, C 26 and C 28 nalkanols, as well as C 29 , C 31 and C 33 n-alkanes, commonly present in plant biomass [START_REF] Bull | Organic geochemical studies of soils from the Rothamsted classical experiments-V. The fate of lipids in different long-term experiments[END_REF][START_REF] Wiesenberg | Source and turnover of organic matter in agricultural soils derived from n-alkane/n-carboxylic acid compositions and C-isotope signatures[END_REF], were more abundant in shoots than roots. Thus, aliphatic compounds showed major differences between root and shoot residues, as reported in previous studies [START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF][START_REF] Otto | Sources and composition of hydrolysable aliphatic lipids and phenols in soils from western Canada[END_REF][START_REF] Mendez-Millan | Can cutin and suberin biomarkers be used to trace shoot and root-derived organic matter? A molecular and isotopic approach[END_REF]. This likely reflected the differences in molecular composition between cutin and suberin biopolymers [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. Suberin monomers are known to comprise longer carbon chains (C ≥ 20) than cutin [START_REF] Kolattukudy | Biopolyester membranes of plants: cutin and suberin[END_REF]. The relative abundance of n-alkanes + n-alkanols and long chain ω-hydroxyalkanoic acids could be used to monitor the fate of shoots and roots in casts, respectively.

Cast-Control molecular contribution

The pyrolysis products from Cast-Control samples could (i) originate from the OM initially present in the soil and/or (ii) be produced by earthworms during gut transit. Thus, the analysis of Cast-Control samples collected in the mesocosm without shoot or root litter allowed assessment of the contribution of the SOM and/or earthworm products to the casts, independently from the incorporation of shoot or root constituents. The GC trace of the Cast-Control sample collected after two weeks of experiment is presented in Figure 25.C. All the compounds identified in root and shoot litter samples were present in the Cast-Control sample. However, the relative contribution of aliphatic compounds to the total pyrolysis products was significantly higher than for root and shoot samples. First, this could reflect the selection of aliphatic compounds in soil by earthworms or the production of these compounds during gut transit. It has been shown that earthworms tend to avoid plant residues with high lignin content [START_REF] Tian | Breakdown of plant residues with contrasting chemical compositions under humid tropical conditions: Effects of earthworms and millipedes[END_REF] and are not able to decompose lignin without microorganism participation [START_REF] Neuhauser | Soil invertebrates and the degradation of vanillin, cinnamic acid, and lignins[END_REF][START_REF] Curry | The feeding ecology of earthworms -A review[END_REF]. [START_REF] Filley | Comparison of the chemical alteration trajectory of Liriodendron tulipifera L. leaf litter among forests with different earthworm abundance[END_REF] showed in a litter bag experiment, that the proportion of aliphatic compounds decreased in tulip poplar leaves litter, in the presence of earthworms after six months of decay. This suggests that earthworms might favor aliphatic compounds incorporation in soils. However, we are not aware of any work on the selection or production of aliphatic compounds by earthworms.

Second, it could also reflect the predominance of aliphatic compounds in the soil and their ingestion by earthworms. This is in agreement with a common view [START_REF] Riederer | Occurrence, distribution and fate of the lipid plant biopolymers cutin and suberin in temperate forest soils[END_REF][START_REF] Feng | The distribution and degradation of biomarkers in Alberta grassland soil profiles[END_REF][START_REF] Lorenz | Strengthening the soil organic carbon pool by increasing contributions from recalcitrant aliphatic bio(macro)molecules[END_REF], according to which aliphatics are less degraded than aromatic compounds in soil. Hence, they represent an important pool of stable OM.

The 8,16 -9,16 and 10,16 dihydroxyhexadecanoic acids (DiOH16) and 9,10,18 trihydroxyoctadecanoic acid (TriOH) were identified in the Cast-Control trace. Under the conditions used here, the three isomers of DiOH16 coeluted [START_REF] Del Río | Analysis of aliphatic biopolymers using thermochemolysis with tetramethylammonium hydroxide (TMAH) and gas chromatography-mass spectrometry[END_REF].

Along with the TriOH, the DiOH16 is a pyrolysis product of cuticles (Del Río and [START_REF] Del Río | Analysis of aliphatic biopolymers using thermochemolysis with tetramethylammonium hydroxide (TMAH) and gas chromatography-mass spectrometry[END_REF] and they were commonly identified in cutin and/or suberin products [START_REF] Otto | Sources and composition of hydrolysable aliphatic lipids and phenols in soils from western Canada[END_REF]. ω-Hydroxyalkanoic acids ranging from C 16 to C 26 and maximizing at C 22 were the dominant series in the chromatogram. Along with ω-hydroxyalkanoic acids, the occurrence of C 22 and C 24 n-alkanedioic acids supported a suberin origin for these compounds [START_REF] Kolattukudy | Biopolyester membranes of plants: cutin and suberin[END_REF][START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF]. This was confirmed by the dominant aromatic compound P3, as observed in the root sample, although to a lesser extent (Figure 25.A). The next dominant aromatic compounds were 1,2,4-trimethoxy benzene (P2), S2 and G5, which were observed in both root and shoot samples but in higher relative abundance in roots for S2 and

G5.

All the compounds in the Cast-Control sample reflected the cast OM originating mainly from higher plants. This is consistent with the feeding habits of earthworms, which select soil fractions rich in OM [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF]. More specifically, the Cast-Control sample reflected an important contribution from root-derived compounds. This higher contribution from root vs. shoot derived compounds has frequently been observed in soil samples [START_REF] Chefetz | Characterization of Organic Matter in Soils by Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH)[END_REF][START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF][START_REF] Feng | The distribution and degradation of biomarkers in Alberta grassland soil profiles[END_REF][START_REF] Wiesenberg | Source determination of lipids in bulk soil and soil density fractions after four years of wheat cropping[END_REF]. Thus, cast molecular composition may reflect the earlier vegetation contribution to the soil used in the experiment, as reported in many studies [START_REF] Chefetz | Characterization of Organic Matter in Soils by Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH)[END_REF][START_REF] Nierop | Composition of plant tissues and soil organic matter in the first stages of a vegetation succession[END_REF], and in agreement with the > 50 yr permanent pasture land use of the soil collected for the experiment. For confirmation, earthworm casts should be compared with bulk soil, which was beyond the goal of the present study.

Fate of root and shoot residues in earthworm casts

Cast-Root and Cast-Shoot samples released the same pyrolysis products as Cast-Control, whatever the time interval, but in different relative abundances. In order to highlight potential differences in OM composition between the different casts, a PCA was carried out. During the last decade, such multivariate analyses on pyrolysis products have revealed a powerful statistical tool for characterizing SOM origin and evolution [START_REF] Buurman | Soil organic matter chemistry in allophanic soils: a pyrolysis-GC/MS study of a Costa Rican Andosol catena[END_REF][START_REF] Carr | Biome-scale characterisation and differentiation of semi-arid and arid zone soil organic matter compositions using pyrolysis-GC/MS analysis[END_REF][START_REF] Schellekens | Soil organic matter dynamics in Mediterranean A-horizons-The use of analytical pyrolysis to ascertain land-use history[END_REF][START_REF] Hoyos-Santillan | Getting to the root of the problem: litter decomposition and peat formation in lowland Neotropical peatlands[END_REF]. Prior to the application of PCA, one chromatogram from Cast-Root and Cast-Shoot was entirely described and the most abundant compounds were assigned and integrated in all chromatograms. In the PCA, aromatic compounds and aliphatic compounds were distinguished (Table 6). The 11 selected aromatic compounds were used as 11 separated variables as most of them had specific origins and different contributions to root and shoot litter samples. For example, 4-methoxybenzoic acid methyl ester (P3) had a significantly higher relative abundance in root vs. shoot sample products. In total, 40 aliphatic compounds were used in the PCA. In order to make data interpretation easier (i.e. limiting the number of variables), aliphatic compounds were considered either individually or gathered according to their common origin, leading to 11 aliphatic variables. For example, long even chain n-alkanoic acids (≥ C 20 ) were gathered to form a higher plant indicator (FAeven; [START_REF] Chefetz | Characterization of Organic Matter in Soils by Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH)[END_REF]. n-Alkanoic acids C 16 (FA16) and C 18 were used as two variables, as C 16 is one of the dominant compounds in all chromatograms and unlike long chain n-alkanoic acids, C 16 and C 18 are present in most organisms and can be derived from both cutin and suberin, as well as plant waxes [START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF]. Details concerning the variables (compositions and main origins) are presented in Table 6.

Root and shoot molecular differences are reflected in casts

The PCA generated two factors (F1and F2) explaining 65% of the variance (Figure 26 7). This observation corroborates the feeding habit of earthworms, which consume fresh residues at the soil surface and incorporate them into casts [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF]. Results illustrated the ability of earthworms to ingest both types of litter. Table 6. Pyrolysis products used for the PCA.Most of the common names for the non-methylated form of aromatic compounds are indicated between brackets; the probable origins and/or significance of each product or group of products, with key references are also mentioned.

Incorporation of fresh residues into casts and decomposition

Besides reflecting roots and shoots being incorporated by earthworms, the PCA also provided information about the rate of incorporation of fresh residues into casts and decomposition therein. In order to facilitate the interpretation and representation of the PCA results, the latter were coupled to histograms assessing the relative abundance of compounds or groups of compounds over the year of experiment, representative of Cast-Control, Cast-Root and Cast-Shoot samples, respectively (Figure 27). n-Alkanedioic acids (Dia) and ω-hydroxyacids A positive priming effect could be hypothesized in casts, although a possible increase of the relative contribution from other compounds cannot be excluded. The priming effect consists of significant mineralization of the OM originally present in the soil, induced by an input of fresh OM [START_REF] Fontaine | Stability of organic carbon in deep soil layers controlled by fresh carbon supply[END_REF][START_REF] Fontaine | Fungi mediate long term sequestration of carbon and nitrogen in soil through their priming effect[END_REF][START_REF] Kuzyakov | Priming effects: Interactions between living and dead organic matter[END_REF]. This mechanism is based on the fact that microorganisms depend on the energy available to decompose the recalcitrant OM present in the soil. As no fresh plants were added to the Cast-Control, this priming effect was probably induced by the action of earthworms. It has been shown that earthworms increase the contact between SOM and microorganisms during ingestion [START_REF] Lavelle | Small-scale and large-scale effects of endogeic earthworms on soil organic matter dynamics in soils of the humid tropics[END_REF]. Moreover, it was proven that the mucus, produced in high amount by earthworms during ingestion, contains high concentrations of easily available compounds, such as carbohydrates [START_REF] Martin | Les mucus intestinaux de ver de terre moteur de leurs interactions avec la microflore[END_REF][START_REF] Schmidt | Carbon and nitrogen stable isotope ratios in body tissue and mucus of feeding and fasting earthworms (Lumbricus festivus)[END_REF][START_REF] Pan | Earthworms (Eisenia foetida, Savigny) mucus as complexing ligand for imidacloprid[END_REF].These factors tend to stimulate microbial activity and induce a priming effect in the presence of earthworms [START_REF] Lu | Carbon dynamics of rhizodeposits, root-and shootresidues in a rice soil[END_REF][START_REF] Coq | Earthworm activity affects soil aggregation and organic matter dynamics according to the quality and localization of crop residues-An experimental study (Madagascar)[END_REF].

In the PCA, both Cast-Root and Cast-Shoot samples could be separated into three groups for Cast-Root and Cast-Shoot, respectively. Moreover, an increase in the δ 13 C value between 4 and 8 weeks for both Cast-Root (from 351 to 673‰) and Cast-Shoot (505 to 938 ‰) was observed (Table 7). Molecular analyses, together with isotopic analyses and OC content displayed increasing concentration of the OM deriving from fresh plant residues in casts. Two mechanisms can account for these results: (i) incorporation of fresh litter and/or (ii) reingestion of older casts. Indeed, after a few months, earthworms are able to re-ingest their casts in which litter has been partially decomposed by microorganisms [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF].

The third group in the PCA corresponded to samples collected after 54 weeks (Figure 26.B). 7), the molecular characterization revealed a slower evolution of the Cast-Root samples. The lesser decomposition of Cast-Root samples probably reflected the combination of two factors:

Cast

1. Decomposition was slightly masked by the extended incorporation of roots after 24 weeks.

Indeed, after 24 weeks, the mesocosm containing roots still had a small quantity of roots at the soil surface, while shoot litter had entirely disappeared from the soil surface in the mesocosm containing shoots. This would reflect the lower "palatability" of root vs. shoot litter for earthworms [START_REF] Bouché | Fonction des lombriciens; II: Recherches méthodologiques pour l'analyse du sol ingéré (étude du peuplement de la station RCP-165/PBI)[END_REF][START_REF] Curry | The feeding ecology of earthworms -A review[END_REF] 2. Roots incorporated into casts decomposed at a slower rate than shoot residues, partly because of their chemical composition, as reviewed by [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF].

The decrease in lignin-derived compound relative abundance, such as P3 (Figure 27.B), in Cast-Root samples between 8 and 54 weeks, is consistent with previous results obtained in soils, revealing that lignin derived from fresh residues decomposed rapidly and was not preferentially preserved in soil [START_REF] Rasse | Lignin turnover in an agricultural field: from plant residues to soil-protected fractions[END_REF][START_REF] Feng | The distribution and degradation of biomarkers in Alberta grassland soil profiles[END_REF][START_REF] Lorenz | Strengthening the soil organic carbon pool by increasing contributions from recalcitrant aliphatic bio(macro)molecules[END_REF].The ability of soil microorganisms, especially fungi, to alter and/or decompose lignin has been long acknowledged [START_REF] Tuor | Enzymes of white-rot fungi involved in lignin degradation and ecological determinants for wood decay[END_REF][START_REF] Filley | Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi[END_REF][START_REF] Dignac | Carbon-13 natural abundance as a tool to study the dynamics of lignin monomers in soil: an appraisal at the Closeaux experimental field (France)[END_REF].

Moreover, the decomposition of root residues in casts between 8 and 54 weeks was reflected by the oxidation of lignin-derived compounds. Indeed, in the PCA (Figure 26), while the Cast-Root sample after 8 weeks of experiment tended to be highly represented by 3,4dimethoxy benzaldehyde (G3), after 54 weeks it was represented mainly by its acid counterpart (G4). Similarly a slight increase in the syringyl acid (S2) contribution vs. that of the aldehyde (S1) was observed between 8 and 54 weeks from the Cast-Root samples. The oxidation of guaiacyl and syringyl moieties is known to reflect the degradation of lignin [START_REF] Mulder | An analytical pyrolysis mass spectrometric study of Eucryphia cordifolia wood decayed by white-rot and brown-rot fungi[END_REF][START_REF] Hatcher | Comparison of two thermochemolytic methods for the analysis of lignin in decomposing gymnosperm wood: the CuO oxidation method and the method of thermochemolysis with tetramethylammonium hydroxide (TMAH)[END_REF][START_REF] Huang | monitoring Biomacromolecular degradation of Calluna Vulgaris in a 23 year field experiment using solid state 13 C-NMR and pyrolysis-GC/MS[END_REF][START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF].This process has been especially observed when carried out by fungi (Saiz-Jimenez and de [START_REF] Saiz-Jimenez | Pyrolysis-gas chromatography-mass spectrometry of isolated, synthetic and degraded lignins[END_REF][START_REF] Mulder | An analytical pyrolysis mass spectrometric study of Eucryphia cordifolia wood decayed by white-rot and brown-rot fungi[END_REF][START_REF] Goñi | Fungal degradation of wood lignins: Geochemical perspectives from CuO-derived phenolic dimers and monomers[END_REF]. Fungi, especially in the case of anecic earthworms, develop rapidly in casts, where they are abundant and can easily assimilate organic compounds [START_REF] Tiwari | Fungal abundance and diversity in earthworm casts and in uningested soil[END_REF][START_REF] Moody | Selective consumption of decomposing wheat straw by earthworms[END_REF][START_REF] Vidal | Incorporation of 13 C labelled shoot residues in Lumbricus terrestris casts: A combination of transmission electron microscopy and nanoscale secondary ion mass spectrometry[END_REF]. They are therefore likely to be active in the degradation of lignin compounds contained in litter incorporated into earthworm casts. In the longer term, oxidized lignin components (G4 and S2) might represent a more stabilized pool of casts and finally of SOM. Indeed, according to [START_REF] Rasse | Lignin turnover in an agricultural field: from plant residues to soil-protected fractions[END_REF], lignin derived from undecomposed plants (rapid turnover) evolved in the short term towards oxidized lignin preferentially adsorbed onto soil minerals (slower turnover) and thus, potentially preserved from decomposition [START_REF] Filley | Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi[END_REF]. For confirmation, longer term analyses should therefore be performed on earthworm casts.

The [START_REF] Feng | The distribution and degradation of biomarkers in Alberta grassland soil profiles[END_REF] showed that hydroxyalkanoic acids were present in bound lipids of deep soil horizons, emphasizing their preferential preservation in soils with respect to lignin compounds. All these compounds showed a greater degree of oxidation than other aliphatic compounds and such oxidized compounds could be preferentially stabilized in casts via adsorption onto soil minerals [START_REF] Eusterhues | Stabilisation of soil organic matter by interactions with minerals as revealed by mineral dissolution and oxidative degradation[END_REF][START_REF] Feng | Chemical and mineralogical controls on humic acid sorption to clay mineral surfaces[END_REF][START_REF] Chefetz | Relative Role of Aliphatic and Aromatic Moieties as Sorption Domains for Organic Compounds: A Review[END_REF]. For example, [START_REF] Rumpel | Location and chemical composition of stabilized organic carbon in topsoil and subsoil horizons of two acid forest soils[END_REF] observed that hydroxyalkanoic acids were particularly preserved in fine particle size soil fractions, with high adsorption surface area.

Moreover, it was reported that earthworms tended to protect compounds from decomposition by incorporating them into microaggregates [START_REF] Guggenberger | Soil organic matter within earthworm casts of an anecic-endogeic tropical pasture community, Colombia[END_REF][START_REF] Hong | How do earthworms influence organic matter quantity and quality in tropical soils?[END_REF].

Thus, oxidized compounds were probably protected from decomposition in earthworm casts and might be preferentially stabilized in soil on a longer term.

Conclusion

TMAH-Py-GC-MS analysis followed by a multivariate analysis enabled the molecular composition dynamics of earthworm casts, to be expressed at various time intervals over a one year experiment. Moreover, these results contributed to shed new lights on the interaction between earthworms and root litter. Roots and shoots were rapidly incorporated by earthworms within casts. Two phases dominated by the incorporation of litter were detected, potentially revealing (i) the incorporation of fresh residues during the first month of the experiment and (ii) the incorporation of fresh residues potentially combined with the reingestion of older casts between the first and second months. From two months until the end of the experiment, cast molecular characteristics revealed a phase dominated by litter decomposition in the casts, the decomposition of shoot litter being more rapid than that of root litter. After one year, the casts in the mesocosm containing shoots showed similar molecular characteristics to casts used as controls, which was not the case for that containing roots. Shoot and root decomposition was monitored by aliphatic compound evolution (i.e.

decrease in long carbon chain n-alkane and n-alkanol relative abundances) and aromatic compound evolution (i.e. decrease in 4-methoxybenzoic acid methyl ester relative abundance), respectively. Non-oxidized lignin was rapidly decomposed in earthworm casts, whereas the abundance of oxidized lignin tended to increase, highlighting the need to differentiate undecomposed lignin present in fresh litter from decomposed lignin to unravel the processes of decomposition and stabilization of carbon in soils. The relative abundance of compounds with a high degree of oxidation (i.e. ω-hydroxyalkanoic, n-alkanedioic, dihydroxyhexadecanoic and trihydroxyoctadecanoic acids) was important in casts used as controls and increased upon decomposition of other casts. It can be suggested that, the more oxidized the compounds, the more preserved they are in casts, probably due to an increased adsorption onto mineral particles. Casts greatly contribute to the soil composition, as earthworms can produce approximatively 50 t.ha -1 .year -1 of casts on grassland soil surfaces [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF]. Thus, the fast incorporation of OM in cast aggregates and subsequent physical protection from decomposition should play a significant role in soil carbon stabilization. Longer term analyses are required to confirm this assumption. Figure 28 summarizes the major conclusions for this part. While no major differences between the fate of roots and shoots in casts were highlighted using bulk isotopic measurements (Chapter 3), the molecular characterization of cast samples clearly separated Cast-Control, Cast-Root and Cast-Shoot samples. Moreover, while the 'bulk' labelled root and shoot-derived carbon was equivalent after one year for Cast-Root and Cast-Shoot, respectively, the molecular characterization revealed the slower decomposition of Cast-Root compared to Cast-Shoot. As isotopic analyses, the molecular analyses also revealed, for both Cast-Root and Cast-Shoot, two key time steps for carbon dynamics in casts of this experiment: 8 and 54 weeks, symbolizing the incorporation of fresh litter and its decomposition, respectively. However, while isotopic analyses presented a phase dominated by litter incorporation until 6 months, molecular analyses revealed that decomposition was already strongly initiated after 8 weeks. The molecular characterization enabled a precise monitoring of OC in casts but did not provide any information on its localization and on the anatomical evolution of plant structures. For this purpose, innovative microscale observations were performed on Cast-Root and Cast-Shoot samples after 8 and 54 weeks of experiment (section 4.3). Prior to this analysis, an additional sample from Cast-Shoot after 24 weeks of experiment was used in order to evaluate the potential of the method (section 4.2).

The potential of combining TEM and NanoSIMS to study earthworm casts

Few studies have been using biogeochemical methods to evaluate the effect of earthworms on carbon cycling [START_REF] Hong | How do earthworms influence organic matter quantity and quality in tropical soils?[END_REF][START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF]. Artificial 13 C labelling litter has been used to follow the fate of carbon in the soil in the presence of earthworms [START_REF] Fonte | Influence of earthworm activity on aggregate-associated carbon and nitrogen dynamics differs with agroecosystem management[END_REF][START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF]. The large scale of analyses used in these studies does not give insight in the interaction between soil-plant-microorganisms. However, the heterogeneous distribution of OC in soil structures induces contrasted microbial activity areas [START_REF] Decaëns | Macroecological patterns in soil communities[END_REF][START_REF] Kuzyakov | Microbial hotspots and hot moments in soil: Concept & review[END_REF]. This distribution inside casts using in situ fine scale imaging has been little investigated, despite the high potential of this approach. In this part, the incorporation and decomposition of plant litter in earthworms cast will be investigated at the microscale using imaging techniques.

For the first time in this field of study, the innovative combination of two fine-scale imaging techniques was used, namely transmission electron microscopy (TEM) and nano-scale secondary ion mass spectrometry (NanoSIMS), to characterize the nature of OM and to locate and determine the origin of incorporated OM thanks to 13 C labelling. TEM has proven its efficiency to investigate organo-mineral associations in soil micro-aggregates [START_REF] Watteau | Root impact on the stability and types of micro-aggregates in silty soil under maize[END_REF][START_REF] Watteau | 0-20 μm aggregate typology based on the nature of aggregative organic materials in a cultivated silty topsoil[END_REF], earthworm burrows [START_REF] Pey | Structure of earthworm burrows related to organic matter of a constructed Technosol[END_REF] and casts [START_REF] Pey | Technosol composition affects Lumbricus terrestris surface cast composition and production[END_REF]. Studies using NanoSIMS to understand the role of earthworms in soil are scarce. [START_REF] Gicquel | Understanding the fate and linkage of N and S in earthworm-engineered peat soil by coupling stable isotopes and nano-scale secondary ion mass spectrometry[END_REF] have double labelled earthworms with N and S to follow their fate in the earthworm intestinal epithelium and in the burrows of a peat soil using NanoSIMS. Thus, NanoSIMS shows great potential to investigate both physical and chemical roles of earthworms in soil at the nanoscale. More details on the application of these methods in soil sciences were reported in section 1.3.4. The present section consists in an evaluation of the potential of combining TEM and NanoSIMS. In the next section (4.3), this methodology was applied to compare the incorporation and decomposition in casts root and shoot litters, at two time steps.

In order to investigate the potential of combining TEM and NanoSIMS to characterize and monitor OM in casts, two contrasted samples were analyzed: (1) artificially 13 C labelled shoot prior to its incorporation inside casts and (2) shoots incorporated inside the structurally complex earthworm casts and the microorganisms implied in their decomposition.

Methodological aspects are presented in section 2.2.3.

Results

Labelled litter characterization

Labelled litter TEM images revealed plant tissues such as thin cell walls, chlorophyll parenchyma and epidermis cells (Figure 29). Within the cells of the parenchyma, chloroplast areas containing starch grains were recognized. Similar features were identified on NanoSIMS images (Figure 30). For the isotopic image, a mask was applied to remove the resin contribution, using a defined threshold for the 12 C 14 N -signal. Black regions hence represent either a hole on the section or resin. cp, chloroplast area; cw, cell wall; ep., epidermis cell; sg, starch grain.

Earthworm cast characterization

The investigated cast contained organic and mineral particles with different sizes, either isolated or associated into aggregates (Figure 31). The different organic components in casts were identified on NanoSIMS images (Figure 32)

with a high degree of confidence thanks to their typical size and shape as observed on TEM images. In addition the high 12 C 14 N confirmed the biological origin of the observed structures.

The "laces" identified as plant cell walls on TEM images (Figure 31.a) were recognizable in NanoSIMS images (Figure 32.A), along with structures identified as bacteria, isolated or forming colonies in aggregates and few fungi (Figure 32). The organic nature of the identified structures was confirmed by the high carbon and nitrogen content evidenced by the 12 C 14 N -signal (Figure 32). The 28 Si and 12 C 14 N images clearly indicated the presence of organo-mineral associations as observed on TEM images. The δ 13 C maps revealed the presence of labelled and unlabelled OM, which were heterogenously distributed in earthworm casts (Figure 32). Both plant residues and microorganisms were labelled. Labelled cell walls from plant residues were easily recognizable (Figure 32 For the seven images obtained on the investigated casts, the mean δ 13 C was 54 ‰, varying from -5 to 148 ‰ (Table 8). A total of 38 and 66 ROI were drawn from the images, for plant residues and bacteria, respectively. Thus, we obtained the isotopic values for each selected ROI. No significant differences in δ 13 C were observed between labelled material in plant residues, bacteria and fungi. Plant residues and bacteria presented a mean δ 13 C of 238 and 334 ‰, respectively. 70 % of the labelled bacteria presented a δ 13 C between 150 and 350 ‰, whereas plant residues were more uniformly distributed between 0 and 550 ‰ (Figure 33).

Discussion

Incorporation of plant residues in casts

TEM and NanoSIMS images reveal a complex spatial organization of organic and mineral constituents in the earthworm casts. Casts are known to be rich in OM from many studies using biochemical [START_REF] Bossuyt | Rapid incorporation of carbon from fresh residues into newly formed stable microaggregates within earthworm casts[END_REF][START_REF] Bossuyt | Protection of soil carbon by microaggregates within earthworm casts[END_REF][START_REF] Pulleman | Earthworms and management affect organic matter incorporation and microaggregate formation in agricultural soils[END_REF][START_REF] Oyedele | Physicochemical properties of earthworm casts and uningested parent soil from selected sites in southwestern Nigeria[END_REF][START_REF] Fahey | Earthworm effects on the incorporation of litter C and N into soil organic matter in a sugar maple forest[END_REF] and TEM [START_REF] Pey | Technosol composition affects Lumbricus terrestris surface cast composition and production[END_REF] approaches. The association of TEM and NanoSIMS analyses enables to distinguish, on a same sample, the different constituents inside the casts and to determine whether they originated from the 13 C labelled litter deposited on the soil surface.

As expected, the observations of the image allow the visualization of the incorporation of plant residues by earthworms inside casts. Moreover, we identified plant residues in various stages of degradation (Figure 31), originating either from residues previously incorporated in the soil (δ 13 C < 0) or from the added labelled litter (Figure 32). The time of residence, the localization and the way the litter was incorporated in the soil structure could influence decomposition of the plant residues. Thus, the different stages of degradation of plant residues could result from degradation having occurred before the beginning of the experiment or from differences in residue localization on, or in, the soil structure. The various degradation levels could also result from different litter incorporation ways by earthworms. Indeed, residues can be incorporated directly or indirectly by earthworms. During the direct incorporation by ingestion, residues are highly fragmented by the grazing activity of earthworms and grinded during the transit, especially passing the gizzard where they are submitted to strong muscular contractions in the presence of mineral particles [START_REF] Shipitalo | Chemistry and micromorphology of aggregation in earthworm casts[END_REF]. The indirect incorporation consists in a pre-oral mechanism, so-called 'ploughing-in' of residues, where litter is embedded by fresh casts which initiate microbial decomposition [START_REF] Cortez | Field decomposition of leaf litters: earthwormmicroorganism interactions -the ploughing-in effect[END_REF]. The presence of hardly, partially or totally decomposed plant cells on TEM and NanoSIMS images could suggest that both mechanisms took place in the studied casts.

Abundant microorganisms in casts

Cast images obtained in this study show an important microbial population as observed in previous studies using TEM [START_REF] Barois | Transformation of the soil structure through Pontoscolex corethrurus (Oligochaeta) intestinal tract[END_REF][START_REF] Pey | Technosol composition affects Lumbricus terrestris surface cast composition and production[END_REF] or microbial analysis [START_REF] Postma-Blaauw | Earthworm species composition affects the soil bacterial community and net nitrogen mineralization[END_REF][START_REF] Frouz | Microbial properties of soil aggregates created by earthworms and other factors: spherical and prismatic soil aggregates from unreclaimed post-mining sites[END_REF]. TEM and NanoSIMS images of the earthworm casts reveal the presence of bacteria both in active and spore forms, as observed by [START_REF] Pey | Technosol composition affects Lumbricus terrestris surface cast composition and production[END_REF]. The presence of few spores may indicate that some active bacteria encountered unsuitable environmental conditions during the transit in the mobile anoxic microarea formed by the earthworm gut inducing sporulation [START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF][START_REF] Drake | As the worm turns: The earthworm gut as a transient habitat for soil microbial biomes[END_REF]. As the gut transit time is long for anecic earthworms, especially for L. terrestris (from 8 to 20 hours) [START_REF] Hartenstein | Production and carrying capacity for the earthworm Lumbricus terrestris in culture[END_REF], some microorganisms get more time for adaptation and multiplication [START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF]. Surviving cells and spores can then rapidly multiply and recover their activity in casts [START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF]. Indeed, the increased surface area of plant residues incorporated in casts and the high concentration of readily available carbon contained in mucus secreted by earthworms, favor proliferation of bacteria and their further decomposition of residues [START_REF] Parle | A Microbiological Study of Earthworm Casts[END_REF][START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF][START_REF] Amador | Microbiological characterization of the structures built by earthworms and ants in an agricultural field[END_REF][START_REF] Frouz | Microbial properties of soil aggregates created by earthworms and other factors: spherical and prismatic soil aggregates from unreclaimed post-mining sites[END_REF]. Bacteria occur in the investigated cast either isolated or in cluster inside aggregates (Figure 32.B). The presence of bacterial cluster was reported to be frequent in casts compared to bulk soil and interpreted as a consequence of higher OM content [START_REF] Kristůfek | Ultrastructural analysis of the intestinal content of earthworm Lumbricus rubellus Hoffm. (Annelida, Lumbricidae)[END_REF][START_REF] Frouz | Microbial properties of soil aggregates created by earthworms and other factors: spherical and prismatic soil aggregates from unreclaimed post-mining sites[END_REF]. We also observed fungi in the casts (Figure 31 and Figure 32). [START_REF] Tiwari | Fungal abundance and diversity in earthworm casts and in uningested soil[END_REF] have shown that fungi are more abundant and present a higher diversity in casts compared to bulk soil. More specifically, anecic earthworms generally favor the development of rapidly growing fungi that can easily assimilate materials in casts [START_REF] Moody | Selective consumption of decomposing wheat straw by earthworms[END_REF]. As in the case of bacteria, this development can be explained by the favorable growth conditions in casts [START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF].

Residue decomposition and the role of microorganisms in casts

The δ 13 C values of the plant residues observed in earthworm casts are lower than those of the initial litter (238 vs. 1004 ‰). Two explanations can be put forward to account for this difference. First, heterogeneity of labelling cannot be excluded as Nguyen [START_REF] Nguyen Tu | Variability of 13 C-labeling in plant leaves[END_REF] have demonstrated that leaves from labelled Ryegrass grown under constant 13 CO 2 -enriched atmosphere present important isotope variability, among and within leaves. In this respect, the fragments of residues incorporated by earthworms in the investigated casts would originate from poorly labelled litter fragments, as observed on the NanoSIMS image of initial litter (Figure 30). However, the isotope variability between initial litter and plant residues in casts is much larger than observed by Nguyen Tu et al. ( 2013) among and within leaves (76 % vs. 20 %). Thus, this hypothesis would at most account for a minor contribution to this difference. Secondly, selective degradation of plant residues may have resulted in 13 C depletion as non-structural compounds from litter such as sugars, which are rapidly lost as dissolved carbon, are more strongly labelled than structural compounds such as lignin [START_REF] Benner | Depletion of 13 C in lignin and its implications for stable carbon isotope studies[END_REF][START_REF] Fahey | Transport of Carbon and Nitrogen Between Litter and Soil Organic Matter in a Northern Hardwood Forest[END_REF]. Thus, degradation would result in a decrease of the δ 13 C of remaining plant residues [START_REF] Benner | Depletion of 13 C in lignin and its implications for stable carbon isotope studies[END_REF]. This hypothesis is supported by the presence of lignin-rich plant cell walls as the main recognizable labelled plant residues on NanoSIMS images of the casts (Figure 32.A), the cell constituents having been rapidly degraded. This selective degradation may take place during assimilation by microorganisms or by earthworms during ingestion. An indicator of the microbial decomposition is the labelling of microorganisms in casts (Figure 32). Indeed, microorganisms are not abundant in the initial litter and their mean δ 13 C value is of 334 ‰ in the casts, highlighting their role in residue decomposition. Furthermore, it has been demonstrated that the high microbial biomass, diversity and activity in casts lead to the recycling of plant residue constituents by microorganisms and their mineralization [START_REF] Postma-Blaauw | Earthworm species composition affects the soil bacterial community and net nitrogen mineralization[END_REF]. Labile plant constituents are the main source of microbial products [START_REF] Cotrufo | The Microbial Efficiency-Matrix Stabilization (MEMS) framework integrates plant litter decomposition with soil organic matter stabilization: do labile plant inputs form stable soil organic matter?[END_REF] because they are efficiently assimilated by microbes and litter cell contents present higher lability than cell walls [START_REF] Fahey | Transport of Carbon and Nitrogen Between Litter and Soil Organic Matter in a Northern Hardwood Forest[END_REF]. Thus, microorganisms have probably used the labelled dissolved carbon contained in plant residues. The presence of bacterial clusters in the casts (Figure 32.B) confirms that casts stands for a 'hotspot' for microbial activity. Indeed, the presence of labile substrate creates favorable conditions for microbial development and activity, as recently reviewed by [START_REF] Kuzyakov | Microbial hotspots and hot moments in soil: Concept & review[END_REF]. The selective degradation of plant residues may also occur upon ingestion by earthworms. Indeed, the latter preferentially assimilate labile carbon and generally little digest and assimilate cell wall constituents [START_REF] Brown | Functional interactions between earthworms, microorganisms, invertebrates and plants[END_REF][START_REF] Curry | The feeding ecology of earthworms -A review[END_REF].

In addition to the mean decrease in δ 13 C between initial litter and plant residues in casts, a large variability in values was observed for plant residues present in casts (from 20 to 633 ‰) (Table 8). Again, this may be explained by the labelled heterogeneity or, more likely, by the presence of residues of various decomposition stages in cast. We also observed a high variability of δ 13 C value for bacteria (from 14 to 943 ‰). [START_REF] Clode | In situ mapping of nutrient uptake in the rhizosphere using nanoscale secondary ion mass spectrometry[END_REF] interpreted this variability as the presence of different species that incorporated 13 C at different rates or by different cellular activity at the sampling time. As TEM images showed various living states of bacteria, the 'cellular activity' hypothesis is likely.

Conclusion

Earthworms are recognized as soil engineers that create organo-mineral structures influencing OC incorporation and protection in the soil. The heterogeneous distribution of OC in these structures induces areas with contrasting decomposition levels at the microscale. The combination of TEM and NanoSIMS imaging highlighted the complex microscale organization in earthworm casts. By comparing morphologies on the NanoSIMS and TEM images, the different organic components (plant residues and microorganisms) could be identified within the casts and their associations with mineral particles could be described.

This allowed us to assess the influence of earthworm activity on the OC incorporation in casts. Images also revealed that casts are populated with abundant and diverse microorganisms, which actively participate in litter decomposition, as demonstrated by their high labelling. Thanks to 13 C labelling and NanoSIMS imaging, we identified the labelled litter incorporated in casts by earthworms. We also observed a significant decrease in δ 13 C from the intact litter to plant residues incorporated in the casts, suggesting decomposition between these two contrasted samples. The various stages of plant residues decomposition and the high variability of δ 13 C values for plant residues and microorganisms highlighted the complexity of OM dynamics and the importance of microscale analyses to identify and explain this variability.

The combination of NanoSIMS and TEM showed great potential to link OM state of decomposition with their 13 C signature. This section was a first step to investigate the forms taken by the OC during the process of incorporation and decomposition, for a given time step and in one composite sample of casts. This method was further applied on cast samples containing root or shoot residues, collected at different time steps (4.3).

Monitoring the fate of roots and shoots in earthworm casts at the microscale

Soil represents a mosaic of microenvironments [START_REF] Ranjard | Quantitative and qualitative microscale distribution of bacteria in soil[END_REF] where most processes implied in OC decomposition and stabilization occur at the microscale. Indeed, microaggregates (< 250 µm) contain high concentrations of OC and enclose most of the stable soil carbon [START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF]. Thus, the study of SOM formation and dynamics requires the consideration of spatial soil heterogeneity using analytical tools at aggregate and microorganism level. Earthworms are essential soil engineers creating structures, called casts, where organic and mineral compounds are intimately associated. Cast are part of the most complex and dynamic structures in soil [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF] and were recognized as a potential sink for stable carbon in the long term ( Six et al., 2004;[START_REF] Bossuyt | Protection of soil carbon by microaggregates within earthworm casts[END_REF]. However, cast observations at the microscale are scarce and generally focus on revealing cast constituents at a given date [START_REF] Barois | Transformation of the soil structure through Pontoscolex corethrurus (Oligochaeta) intestinal tract[END_REF][START_REF] Pey | Technosol composition affects Lumbricus terrestris surface cast composition and production[END_REF][START_REF] Vidal | Incorporation of 13 C labelled shoot residues in Lumbricus terrestris casts: A combination of transmission electron microscopy and nanoscale secondary ion mass spectrometry[END_REF]. Moreover, little is known on the capacity of earthworms to ingest and transform root litter, while root-derived carbon tend to contribute more than shoot-derived carbon to the stable carbon pool in soil, [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF].

The present study aimed at (1) characterizing the state of decomposition of roots or shoots incorporated in casts, (2) identifying the microorganisms implied in this decomposition, at different time steps. To meet this objective, Cast-Shoot and Cast-Root samples collected after 8 and 54 weeks of experiment were analyzed combining TEM and NanoSIMS (section 2.2.3).

Results

Initial roots and shoots at the microscale

Prior to the investigation of Cast-Root and Cast-Shoot samples, the litter initially deposited on the soil surface was examined by TEM and NanoSIMS. Both shoot and root TEM images (Figure 34) revealed well preserved plant tissues, such as parenchyma and woody tissues.

Thus, the initial litter was not affected by preparative treatment. On shoot images, cells from the chlorophyll parenchyma with chloroplast areas containing starch grains were identified

Cast-Shoot and Cast-Root after 8 weeks of experiment

Cast-Shoot and Cast-Root TEM images revealed the presence of plant structures at different state of degradation (Figure 36). Similarities could be found with structures previously observed on the root and shoot images but plant structures observed in casts were at a higher degradation state (e.g.

Initial roots and shoots

The first step to monitor root and shoot incorporation and decomposition in casts at the microscale was to identify their potential anatomical specificities. TEM images revealed only few anatomical differences between initial roots and shoots:

1-The presence of chlorophyll parenchyma cells in initial shoots (Figure 34.a). Chlorophyll parenchyma cells contain the chloroplast implied in the photosynthesis achieved by plant leaves [START_REF] Waters | The making of a chloroplast[END_REF]).

2-A slightly higher abundance of woody tissues in initial roots, recognized by their thicker cell walls (Figure 34.e). It is generally admitted that roots contain higher concentrations of lignin, one of the main constituent of woody tissues, compared to shoots [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF].

3-The identification of few microorganisms in initial roots. These microorganisms have probably developed onto roots before the beginning of the experiment, as it is known that root surrounding are often enriched in microorganisms in soil [START_REF] Philippot | Going back to the roots: the microbial ecology of the rhizosphere[END_REF].

The NanoSIMS images from initial shoots (Figure 35 presented similar and high δ 13 C values (~ 700 ‰). Thus, their identification and differentiation from other soil organic sources in Cast-Root and Cast-Shoot, respectively will be ensured by their labelling, using NanoSIMS images.

Cast-Root and Cast-Shoot after 8 weeks of experiment

NanoSIMS images revealed the presence of labelled root and shoot structures in Cast-Root (Figure 37.c and d) and Cast-Shoot (Figure 37.a and b.), respectively. Thus, after 8 weeks of experiment, earthworms have incorporated both litter types in their casts. However, plant structures identified in both samples presented different degradation states, highlighting higher degradation states of shoots vs. roots, in Cast-Shoot and Cast-Root, respectively. Four hypotheses could be put forward to account for these observations:

1-Roots are expected to be less palatable than shoots for earthworms due to their increased concentration in lignin content [START_REF] Tian | Breakdown of plant residues with contrasting chemical compositions under humid tropical conditions: Effects of earthworms and millipedes[END_REF]. Thus, earthworms might have delayed the incorporation of roots compared to shoots in their casts. Observations after 54 weeks would reflect a shorter degradation period.

2-The presence of abundant intact tissues in Cast-Root might suggest that earthworms have recently favored indirect over direct incorporation of roots inside casts. Briefly, earthworms are able to initiate microbial decomposition by embedding litter into fresh casts (indirect incorporation), without ingesting any residues (direct incorporation) [START_REF] Cortez | Field decomposition of leaf litters: earthwormmicroorganism interactions -the ploughing-in effect[END_REF]. Thus, non-ingested roots would not be submitted to the grazing activity of earthworms and grinded during the transit, explaining the presence of intact residues on TEM and nanoSIMS images, as proposed by [START_REF] Vidal | Incorporation of 13 C labelled shoot residues in Lumbricus terrestris casts: A combination of transmission electron microscopy and nanoscale secondary ion mass spectrometry[END_REF] for cast observation using the same methodology.

3-Roots tend to decompose at a slower rate compared to shoots [START_REF] Balesdent | Major contribution of roots to soil carbon storage inferred from maize cultivated soils[END_REF]Gale and Cambardella, 2000;[START_REF] Puget | Short-term dynamics of root-and shoot-derived carbon from a leguminous green manure[END_REF][START_REF] Lu | Carbon dynamics of rhizodeposits, root-and shootresidues in a rice soil[END_REF].

Thus, the roots incorporated into Cast-Root might have decomposed at a slower rate compared to shoots.

4-A methodological bias also cannot be excluded due to the reduced area observed using TEM and NanoSIMS analyses, thus questioning how representative it is [START_REF] Poch | Micromorphology Techniques for Soil Organic Carbon Studies[END_REF]. However, we have studied the molecular characteristics of the same samples at a larger sampling scale (few mg) (see section 4.1). We highlighted that, after 8 weeks of experiment, the Cast-Root sample characteristics was more similar to initial roots, than Cast-Shoot to initial shoots. Moreover, the use of composite cast samples and the observation of various sections for each sample with TEM limit potential methodological biases. Thus, the contribution from fresh roots or little degraded litter in Cast-Root seems to be reliable. the microorganisms first attack cell contents which contain more labile compounds [START_REF] Fahey | Transport of Carbon and Nitrogen Between Litter and Soil Organic Matter in a Northern Hardwood Forest[END_REF], the latter representing their main source of energy and carbon [START_REF] Cotrufo | The Microbial Efficiency-Matrix Stabilization (MEMS) framework integrates plant litter decomposition with soil organic matter stabilization: do labile plant inputs form stable soil organic matter?[END_REF]. However, some bacterial clusters were also observed on plant cell walls (Figure 36.e), illustrating the diversity of microorganism metabolic capacities in casts [START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF].

The degradation of woody tissues was initiated by fungi, attacking cell walls (Figure 36.d).

Earthworms are not able to decompose lignin without the participation of microorganisms [START_REF] Neuhauser | Soil invertebrates and the degradation of vanillin, cinnamic acid, and lignins[END_REF][START_REF] Curry | The feeding ecology of earthworms -A review[END_REF]. Fungi are able to degrade more resistant tissues compared to bacteria [START_REF] Bossuyt | Influence of microbial populations and residue quality on aggregate stability[END_REF] and are particularly implied in lignin decomposition [START_REF] Tuor | Enzymes of white-rot fungi involved in lignin degradation and ecological determinants for wood decay[END_REF][START_REF] Filley | Lignin demethylation and polysaccharide decomposition in spruce sapwood degraded by brown rot fungi[END_REF][START_REF] Dignac | Carbon-13 natural abundance as a tool to study the dynamics of lignin monomers in soil: an appraisal at the Closeaux experimental field (France)[END_REF]. In Cast-Root, these processes were not emphasized on the TEM images presented, which may be explained by the reduced presence of microorganisms compared to Cast-Shoot.

Cast-Shoot TEM images also presented more organo-mineral aggregates compared to Cast-Root. These organo-mineral aggregates contained plant cell wall residues (Figure 36.b) identified as labelled on a similar zone of nanoSIMS images (Figure 37.b). Some fungi were also observed inside aggregates (Figure 36.c) and nanoSIMS images revealed that some of these microorganisms were labelled (Figure 37.a), highlighting their participation to shoot decomposition. It is well known that most microorganisms find suitable living conditions in micropores (< 10 µm in 'inner parts' of aggregates [START_REF] Hattori | Micro-organisms and soil aggregates as their microhabitats[END_REF][START_REF] Ranjard | Quantitative and qualitative microscale distribution of bacteria in soil[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF]. These aggregates illustrated the process of cast formation by earthworms, which are the result of fresh plant fragmentation, soil disruption and their further complexation with mucus during the gut transit [START_REF] Lavelle | Small-scale and large-scale effects of endogeic earthworms on soil organic matter dynamics in soils of the humid tropics[END_REF].

Thus, after 8 weeks of experiment, shoot presented a higher state of degradation in casts compared to roots, as reflected by the presence of abundant intact root structure in Cast-Root, as well as the increased organo-mineral associations and microorganism abundance in Cast-Shoot. These observations highlighted a diverging fate of roots and shoots in earthworm cast after 8 weeks of experiment. In the following, we will investigate whether this observation was still valid after 54 weeks of experiment.

Cast-Root and Cast-Shoot evolution after 54 weeks

After 54 weeks of experiment, organo-mineral associations tended to be more abundant in Cast-Shoot and Cast-Root (to a lesser extent), compared to the samples collected after 8 weeks. Thanks to the visual observation of mesocosms (see section 3.1.1, Figure 20), it was observed that, after 54 weeks, no more litter could be observed at the surface of soil mesocosms. The litter had been either mineralized and/or incorporated in casts or soil. Thus, the increased organo-mineral aggregate formation might be explained by the ageing and drying of casts, leading to an increased aggregate stability [START_REF] Shipitalo | Chemistry and micromorphology of aggregation in earthworm casts[END_REF][START_REF] Martin | Short-and long-term effects of the endogeic earthworm Millsonia anomala (Omodeo) (Megascolecidae, Oligochaeta) of tropical savannas, on soil organic matter[END_REF][START_REF] Lavelle | Small-scale and large-scale effects of endogeic earthworms on soil organic matter dynamics in soils of the humid tropics[END_REF][START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF]. This increased stability can be partly explained by: 1-The presence of minerals whose features enhance interaction with OM [START_REF] Eusterhues | Stabilisation of soil organic matter by interactions with minerals as revealed by mineral dissolution and oxidative degradation[END_REF][START_REF] Eusterhues | Organo-mineral associations in sandy acid forest soils: importance of specific surface area, iron oxides and micropores[END_REF][START_REF] Kaiser | Mineral surfaces and soil organic matter[END_REF][START_REF] Sollins | Sequential density fractionation across soils of contrasting mineralogy: evidence for both microbial-and mineral-controlled soil organic matter stabilization[END_REF]. These features include the chemical nature of soil minerals, the capacity of mineral surfaces to adsorb organic material [START_REF] Baldock | Role of the soil matrix and minerals in protecting natural organic materials against biological attack[END_REF].

2-The physical arrangement of mineral particles is related to the evolution of pore size observed in cast aggregates, which tended to be reduced from 8 to 54 weeks. Indeed as most of the decomposition processes occur within the pore space in soils, pore sizes strongly influence OM stability [START_REF] Baldock | Role of the soil matrix and minerals in protecting natural organic materials against biological attack[END_REF][START_REF] Kaiser | Mineral surfaces and soil organic matter[END_REF].

3-The presence of abundant microorganisms [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF] producing exopolymers that tend to strengthen organo-mineral bonds in casts [START_REF] Shipitalo | Chemistry and micromorphology of aggregation in earthworm casts[END_REF].

4-The production of calcite by the earthworms via the calciferous glands or derived from plant decomposition [START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF]. Indeed, calcium is a well know agent for claypolyvalent cation-OM complexes [START_REF] Sokoloff | Effect of neutral salts of sodium and calcium on carbon and nitrogen in soils[END_REF][START_REF] Baldock | Role of the soil matrix and minerals in protecting natural organic materials against biological attack[END_REF][START_REF] Clough | Physical and chemical protection of soil organic carbon in three agricultural soils with different contents of calcium carbonate[END_REF][START_REF] Wuddivira | Effects of organic matter and calcium on soil structural stability[END_REF].

Thus, in the present study, the process of stabilization of casts seemed to be initiated in Cast-Shoot, and to a lesser extend in Cast-Root, after 54 weeks of experiment.

Plant tissues were highly degraded and the remaining identified structures often corresponded to resistant ones, for both Cast-Shoot and Cast-Root. For example, in Cast-Shoot, structured woody tissues were still identifiable with an important bacterial population (Figure 38.d). It was shown that, at the early stage of degradation, woody tissues are generally first mainly attacked by fungi (as it was observed after 8 weeks in this study), which provide bacteria with intermediate decomposition products [START_REF] Romaní | Interactions of bacteria and fungi on decomposing litter: differential extracellular enzyme activities[END_REF]. Extended degradation of plant tissues was also observed in Cast-Root. For example, only the middle lamellas, and more specifically cell intersections, were still present on Figure 38.f. [START_REF] Ding | How does plant cell wall nanoscale architecture correlate with enzymatic digestibility?[END_REF] observed the degradation of dried corn stems using bright-field light and atomic force microscopy and showed that bacteria started digesting the cell walls away from the middle lamella. This could be explained by the important concentration of lignin in middle lamella compared to other cell wall structures [START_REF] Jouanin | Lignins: biosynthesis, biodegradation and bioengineering[END_REF], which explains its slower degradation.

While most microorganisms identified in samples after 8 weeks of experiment were under an intact form, samples collected after 54 weeks showed microorganisms exhibiting various states of degradation in both Cast-Shoot and Cast-Root. A chronology explaining this evolution can be proposed. After 8 weeks, fresh residues had entered casts which, coupled to the production of easily assimilated mucus by earthworms [START_REF] Drake | As the worm turns: The earthworm gut as a transient habitat for soil microbial biomes[END_REF], stimulated the growth and development of microbial communities [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF].

While microorganisms have limited capacities to move into the soil [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF], earthworms facilitate the accessibility of microorganisms to fresh substrate [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF]. After 54 weeks, the substrates started to become limiting for microorganisms, leading to the progressive starvation [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF] of microorganisms isolated from the litter. As bacteria faced unsuitable environmental conditions, some of them formed spores (Figure 38.b and h). Indeed, as discussed in section 4.2, in the present study, the sporulation might either be caused by the anoxic conditions created during the earthworm gut transit [START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF][START_REF] Drake | As the worm turns: The earthworm gut as a transient habitat for soil microbial biomes[END_REF] or by the lack of substrate (Willey et al., 2008). Other bacteria might not be able to overcome this lack of energy and carbon and thus will die and form bacterial residues (Figure 38.b and d; Figure 38.e and h) which will further fragment [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF], leaving a 'fingerprint' at their ultimate state of degradation (Figure

38.f).

Labelled structures identified on NanoSIMS images included well defined and isolated spots identified as bacteria (Figure 39.c) and fungi (Figure 39.d). Even though we cannot exclude a methodological bias due to the observation of 2D and not 3D images, these labelled microorganisms seemed to be isolated from labelled organic substrate. Two explanations can be formulated to explain this apparent decoupling between substrate and microorganisms.

First, the labelled OM assimilated by microorganisms might have been totally mineralized by microorganisms, resulting in the absence of labelled plant material. Indeed, microorganisms tend to preferentially attack easily degradable compounds, such as soluble carbon [START_REF] Fahey | Transport of Carbon and Nitrogen Between Litter and Soil Organic Matter in a Northern Hardwood Forest[END_REF]. Second, microorganisms might have been incorporated into aggregates after having assimilated organic compounds during the gut transit or before re-ingest of cast be earthworms. Indeed, a few months after cast formation, earthworms are able to re-ingest their own cast where decomposition has been initiated [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF]. Moreover, earthworms tend to favor the enclosing of organic structures such as microorganisms into aggregates [START_REF] Shan | Digestion and residue stabilization of bacterial and fungal cells, protein, peptidoglycan, and chitin by the geophagous earthworm Metaphire guillelmi[END_REF], as existing microstructures are destroyed during the gut transition and new microaggregates can be formed [START_REF] Shipitalo | Chemistry and micromorphology of aggregation in earthworm casts[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF]. Spatial inaccessibility of the organic compounds might have played an essential role in protecting OC from degradation [START_REF] Lützow | Stabilization of organic matter in temperate soils: mechanisms and their relevance under different soil conditions-a review[END_REF]. Thus, in the present study, labelled microorganisms could have been integrated or re-integrated into aggregates, where they will potentially be protected from decomposition [START_REF] Shan | Digestion and residue stabilization of bacterial and fungal cells, protein, peptidoglycan, and chitin by the geophagous earthworm Metaphire guillelmi[END_REF]. On NanoSIMS images, some 'diffused' labelling (i.e. which did not correspond to a defined organic structure) was also identified. For example, the organo-mineral aggregate identified on Figure 39.a contained, in its core, a form which was poor in 12 C 14 N, appearing as a pore, but presenting a 'diffused' labelling. Looking closer at a similar area or structure (Figure 40)

on TEM images, different organic structures could be identified in these pores such as compacted cell walls and amorphous OM forming a fuzzy veil. This veil of organic coating likely included exopolymers produced by bacteria, such as of carbohydrates, which were already observed in soil pores a long time ago [START_REF] Foster | Polysaccharides in soil fabrics[END_REF]. Contribution of plant-derived labelled dissolved OM cannot be excluded. This veil might also include bacterial cell wall residues as suggested by [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF] which described them as 'flat cell wall fragments of relatively uniform size'. These types of fragments tend to be stabilized in soil due to the complex chemical composition of microbial cell walls [START_REF] Sollins | Stabilization and destabilization of soil organic matter: mechanisms and controls[END_REF] and the spatial arrangement of soil matrix [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF]. Thus, according to [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF], the microbial necromass might highly contribute to the soil stable carbon. Even though we cannot exclude a bias created by the NanoSIMS analysis, the 'diffused' labelling identified in pores within organo-mineral aggregate might illustrate the importance of microbial residues in the longer term. However, to confirm the link between diffused labelling and microbial residues, further TEM and NanoSIMS analyses focused on microorganisms would be required.

Conclusion

The powerful combination of TEM and NanoSIMS analyses revealed a contrasted fate of root and shoot litter in earthworm casts. After two months in the presence of earthworms, plant tissues derived from shoot litter observed in casts presented an increased state of degradation compared to those derived from root litter. This was also correlated with abundant microorganisms, presenting contrasted metabolic capacities depending on their nature (fungi vs. bacteria), as well as abundant organo-mineral associations in casts containing shoot residues. Thus, the incorporation of root litter was delayed in casts and/or its decomposition was slower compared to shoots. After one year, organo-mineral aggregates were larger and more abundant in casts containing shoots, and to a lesser extent in cast containing roots, compared to the casts collected earlier. Some labelled plant residues and microorganisms were enclosed into these aggregates, which made them potentially protected from degradation. This highlighted the fast action of earthworms on aggregate formation and potential stabilization, which starts earlier in the presence of shoot compared to root litter. In both cast types, bacteria presented various state of degradation, from intact to 'fingerprint'.

Abundant bacterial residues, associated to veil of organic coatings, were identified in pores and could partly contribute to the 'diffused' labelling observed on aggregate NanoSIMS images. Thus, living or dead, microorganisms play a determinant role in casts, as degrading agents and/or potential source of carbon. Temporal microscale observations added one more straw to the haystack of mechanisms implied in cast formation and evolution depending on litter quality, highlighting their potential key contribution to soil stable OC in the longer terms. More research is needed to unravel the fate of microorganisms in earthworm casts.

One of the major conclusions from the chapter 3 was that earthworms tended to reduce the difference between the contrasted fate of root and shoot observed in soil and cast samples.

However, further analyses of casts using molecular (Section 3.1) and microscale (Section 3.2 and 3.3) analyses revealed some differences: the incorporation and/or decomposition of root is reduced in casts compared to shoot as revealed by the contrasted molecular composition and degradation state of plant structures, at the same time step. This led to an earlier enclosing of shoot vs. root derived carbon in casts. Longer term analyses are needed to investigate if these differences are maintained after few years/decades. If we consider that cast might account for 50 % of the surface soil layer [START_REF] Ponomareva | The role of earthworms in the creation of a stable structure in ley rotations[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF], these results participated to decipher the processes behind soil carbon stable pool formation.

Chapter 5.

Organic matter and microbial communities after one year of experiment

In chapter 3, we observed that earthworm casts presented high labelled litter-derived carbon content, in constant evolution along the year of experiment. Thus, we decided to dedicate chapter 4 to the molecular and microscale characterization of cast OM, at different time steps. We clearly highlighted a contrasted fate between roots and shoots in casts, as well as a high microbial contribution to organic structures, after one year of experiment. We therefore wondered whether these trends could be observed in soil samples.

In chapter 3, we observed that, along the year of experiment, labelled root-and shoot-derived carbon increased in soil samples collected at 0-20 cm. After one year, the labelled litterderived carbon was higher in mesocosms containing roots compared to those containing shoots. Moreover, the amount of carbon derived from both roots and shoots was lower in soil samples, in the presence of earthworms. Thus, in chapter 5, we decided to focus on this last key time step (54 weeks) in order to (1) characterize OM at the molecular level in soil vs. cast samples (section 5.2) and ( 2) achieve a first screening of the microbial community abundance and metabolic capacities in soil and cast samples (section 5.3). Prior to these sections, we introduce the chapter with few TEM and NanoSIMS images illustrating a first attempt to characterize OM in soil samples collected after 54 weeks of experiment (section 5.1).

Introduction -soil samples at the microscale

This introductory section aimed at briefly presenting the different organic structures identified on soil TEM and NanoSIMS images, after one year of experiment. As cast samples were described in chapter 4, the introduction focuses on soil samples.

Soil sample fractionation for microscale observations

In cast samples collected after one year of experiment, litter-derived structures were highly degraded and incorporated in aggregates; and microorganisms were abundant (section 4.3).

We observed that, the more abundant mineral particles were, the more difficult it was to localize and identify labelled organic structures. Moreover, thanks to isotopic analyses (chapter 3), we know that after one year, soil contained less labelled litter-derived carbon than cast samples. Thus, in order to increase our chances to observe organic structures on soil sample images, we decided to fractionate soil and to analyze the 0-20 µm and 20-50 µm fractions (see section 2.2.3.1 for methodological details). Indeed, it is generally admitted that smaller soil fractions are enriched in OC compared to other fractions [START_REF] Lützow | Stabilization of organic matter in temperate soils: mechanisms and their relevance under different soil conditions-a review[END_REF].

Organic structures observed in soil samples

Despite soil fractionation, the differentiation between soil samples from different mesocosms was complex and required further analyzes. We succeeded in obtaining TEM and NanoSIMS images from both 0-20 and 20-50 µm fractions from the soil collected in the mesocosm Shoot-E, at 0-20 cm depth. Thus, this sample was chosen to illustrate the diversity of structures identified in soil samples and to introduce this chapter on soil and cast samples collected after 54 weeks of experiment. Logically, the 0-20 µm soil fraction contained microaggregates (mainly between 5-10 µm) (Figure 41), while the 20-50 µm fraction was mainly represented by larger aggregates (> 20 µm) (Figure 42). Microorganisms were identified in both 0-20 µm and 20-50 µm fractions, mainly integrated into aggregates (Figure 41 and Figure 42).

Labelled structures identified on NanoSIMS images of soil samples were scarce (Figure 43).

The few labelled structures were integrated into aggregates and associated with mineral This introductory section illustrated that the labelled litter-derived OC in soil samples could either be in the form of plant residues (dissolved or highly degraded) and/or microorganisms (dead or alive). However, at this fine scale, we were not able to investigate satisfactorily, the impact of litter quality and earthworms on OC cycling and microbial communities. Thus, section 5.2 aimed at characterizing root-vs. shoot-derived OC in soil and casts at the molecular level, after one year of experiment. Section 5.3 investigated the impact of litter quality and earthworms on the microbial community abundance and metabolic potentials.

Molecular characterization of SOM in soil and casts

In order to investigate the impact of litter quality and earthworms on OC cycling, the present section aimed at characterizing the root-and shoot-derived SOM at the molecular level, in soil and cast samples, after one year of experiment. As presented in section 4.1, cast collected in mesocosms incubated with roots or shoots presented significant differences at the molecular level. This clear differentiation was made possible thanks to the highly labelled litter-derived carbon in casts. The proportion of carbon derived from labelled litter is lower in soil samples and heterogeneously distributed, as revealed by TEM and NanoSIMS images (section 5.1).

Moreover, a large proportion of OM in soil samples should be derived from past vegetation.

Thus, in order to maximize the chances to differentiate cast and soil samples collected in different mesocosms, we decided to extract lipids from samples and to analyze separately lipid-free samples with Py-GC-MS (section 5.2.1) and lipid extract with GC-MS (section 5.2.2) (see section 2.2.2 for methodological details). Indeed, [START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF] recommended the separated comparison of free lipids and ester-bound lipid to obtain complementary information on past vegetation, soil processed and lipid transformations.

Pyrolysis on lipid-free samples

Initial root and shoot litter

The aim of the present section is to characterize OM in lipid-free soil and cast samples. For this purpose, TMAH-Py-GC-MS was first achieved on initial lipid-free shoot and root litter in order to identify specific indicators of roots and shoots that could be researched in soil and cast samples. Indeed, the monomers derived from suberin and cutin, typical biopolymers present in roots and shoots [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF], are used to trace root-and shoot-derived OM in soil samples [START_REF] Del Río | Analysis of aliphatic biopolymers using thermochemolysis with tetramethylammonium hydroxide (TMAH) and gas chromatography-mass spectrometry[END_REF][START_REF] Nierop | Temporal and vertical organic matter differentiation along a vegetation succession as revealed by pyrolysis and thermally assisted hydrolysis and methylation[END_REF][START_REF] Otto | Sources and composition of hydrolysable aliphatic lipids and phenols in soils from western Canada[END_REF][START_REF] Mendez-Millan | Can cutin and suberin biomarkers be used to trace shoot and root-derived organic matter? A molecular and isotopic approach[END_REF]. Overall, lipid-free root and shoot litter chromatograms presented the same aromatic compounds as described in section 4.1 on root and shoot litter analyzed without lipid extraction (Appendix 4.A to D). Both aromatic and aliphatic compounds could be identified. Briefly, aromatic compounds encompassed mainly lignin-and/or suberinderived compounds from p-coumaryl (H), guaiacyl (G) and syringyl (S), as well as more ubiquitous phenols (P). The assignement of these compounds is available in Table 6 (Section 4.1). As observed in section 4.1, the relative abundance of aromatic compounds with respect to aliphatic compounds was higher in roots compared to shoots. Moreover 4-methoxybenzoic acid methyl ester (P3) appeared to be the most suitable aromatic product to distinguish root and shoot litter. Thus, as concluded in section 4.1.1, the relative abundance of the aromatic compounds and specific pyrolysis products especially abundant in roots, such as P3, were used below to identify root contribution in soil and cast samples.

As lipids contain high concentrations of aliphatic compounds [START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF], their relative abundance was logically reduced in lipid-free samples (Appendix 4.A to C), compared to samples without lipid extraction (Appendix 4.B to D). For example, long chain n-alkanoic acids, n-alkanes and n-alkanols were virtually absent from the lipid-free shoot sample. On the contrary, dihydroxy (DiOH) and trihydroxy (TriOH) acids relative abundances were high, while these compounds were not observed on pyro-chromatograms from the samples without lipid extraction, likely due to the overprinting of other aliphatics.

These compounds are typical cutin-derived compounds [START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF][START_REF] Otto | Sources and composition of hydrolysable aliphatic lipids and phenols in soils from western Canada[END_REF][START_REF] Mendez-Millan | Can cutin and suberin biomarkers be used to trace shoot and root-derived organic matter? A molecular and isotopic approach[END_REF]. Long chain ω-hydroxyalkanoic acids, as well as mono unsaturated ω-hydroxyalkanoic acids and n-alkanedioic acid C 18 , were identified in root litter. These compounds are attributed to suberin [START_REF] Nierop | Rapid molecular assessment of the bioturbation extent in sandy soil horizons under pine using ester-bound lipids by on-line thermally assisted hydrolysis and methylation-gas chromatography/mass spectrometry[END_REF][START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF][START_REF] Otto | Sources and composition of hydrolysable aliphatic lipids and phenols in soils from western Canada[END_REF][START_REF] Mendez-Millan | Can cutin and suberin biomarkers be used to trace shoot and root-derived organic matter? A molecular and isotopic approach[END_REF][START_REF] Angst | The fate of cutin and suberin of decaying leaves, needles and roots -Inferences from the initial decomposition of bound fatty acids[END_REF]. Thus, the relative abundance of DiOH and TriOH could be potentially used as an indicator of shootderived OM in soil and cast samples. Root-derived OM could be potentially recognized thanks to the relative abundance of long chain ω-hydroxyalkanoic acids, as well as mono unsaturated ω-hydroxyalkanoic acids and n-alkanedioic acid C 18 ,

Soil and cast control samples

The analysis of pyrolysis products in the control soil sample (Control-NE) enabled the assessment of the contribution of the SOM, independently from the incorporation of shoot or root constituents. The analysis of pyrolysis products in the control cast sample (Cast-Control)

and its comparison to Control-NE allowed the assessment of a potential earthworm impact on SOM molecular characteristics, independently from the incorporation of shoot or root constituents. Control-NE and Cast-Control contained the same pyrolysis products with similar relative abundances (Figure 44). In the present study, earthworm did not impact significantly the molecular characteristics of SOM, in the absence of litter, after one year. Thus, pyrolysis products identified on cast and soil control samples were mainly attributed to OM initially present in the soil. On the contrary, [START_REF] Hong | How do earthworms influence organic matter quantity and quality in tropical soils?[END_REF] showed that dry casts and bulk soil could be differentiated at the molecular level, when focusing on aggregates < 2000 µm. These differences were mainly attributed to the relative abundance of polysaccharides and N-containing compounds, which were not included in the present study.

Indeed, in the present study, we only used compounds that could clearly be identified on pyrochromatograms in order to dertimine their origin. This was not the case for polysaccharides and N-containing compounds. Moreover, [START_REF] Hong | How do earthworms influence organic matter quantity and quality in tropical soils?[END_REF] interpreted cast and soil differences partly by the incorporation of plant material in casts. However, in the present study, earthworms in the mesocosms corresponding to the Cast-Control sample were deprived from fresh litter during the whole year of experiment. Thus, they did not have the opportunity to incorporate fresh plants into their casts. [START_REF] Otto | Sources and composition of hydrolysable aliphatic lipids and phenols in soils from western Canada[END_REF][START_REF] Mendez-Millan | Can cutin and suberin biomarkers be used to trace shoot and root-derived organic matter? A molecular and isotopic approach[END_REF]. The important relative abundance of P3, long chain ω-hydroxyalkanoic acids, as well as monounsaturated ω-hydroxyalkanoic acids and nalkanedioic acid C 18 (Figure 44) highlighted the important contribution of root-derived carbon in both control soil and casts. This is consistent with the dominant root contribution which has often been observed in temperate forest soil [START_REF] Chefetz | Characterization of Organic Matter in Soils by Thermochemolysis Using Tetramethylammonium Hydroxide (TMAH)[END_REF][START_REF] Rumpel | Vertical distribution, age, and chemical composition of organic carbon in two forest soils of different pedogenesis[END_REF][START_REF] Nierop | Organic matter formation in sandy subsurface horizons of Dutch coastal dunes in relation to soil acidification[END_REF][START_REF] Otto | Sources and composition of hydrolysable aliphatic lipids and phenols in soils from western Canada[END_REF][START_REF] Feng | The distribution and degradation of biomarkers in Alberta grassland soil profiles[END_REF]Mueller et al., 2012).

Root-and shoot-derived compounds in soil and casts

As soil and casts presented the same pyrolysis products, a PCA was carried out in order to highlight potential differences between the soil and cast samples collected in the different mesocosms, after 54 weeks of experiment. Almost the same pyrolysis products as those presented in Table 6 (Chapter 4) were selected for the PCA. The same aromatic compounds were chosen, while few changes were operated for aliphatic compounds. The 11 aromatic products were used as 11 separated variables. In total, 31 aliphatic compounds were selected for the PCA, gathered either according to their common origin or considered as individual variable, leading to 12 aliphatic variables. Due to the high number of variables compared to the number of samples, the PCA will be used only to describe general trends. The few changes operated for aliphatic compound compared to the previous PCA realized in section 4.1 (Table 6) were listed below:

 As mono unsaturated ω-hydroxyalkanoic acids and n-alkanedioic acid C 18 (ωOH18uns and Dia18uns, respectively) are important suberin-derived compounds, they were used as individual variables.

 Even carbon numbered C 26 -30 n-alkanols were used for the OH indicator (C 26 -34 in and C 17i,a in Table 6)

Figure 45. PCA carried out with the 23 variables (see Table 6 for the main description) of the 9 soil and cast samples, with root and shoot litter used as illustrative samples. A, distribution of variables on the correlation circle and B, distribution of the soil and cast samples. This was particularly emphasized in Cast-Root and Cast-Shoot (Figure 46.A) . The three classes of compounds were almost absent from root and shoot chromatograms. Thus, these results might either illustrate (1) the increase in the relative abundance of other compounds in cast samples and/or (2) the decomposition of compounds originally present in the soil, during cast formation and ageing. This decomposition might reflect a priming effect caused by the incorpation of fresh plants [START_REF] Fontaine | Stability of organic carbon in deep soil layers controlled by fresh carbon supply[END_REF][START_REF] Fontaine | Fungi mediate long term sequestration of carbon and nitrogen in soil through their priming effect[END_REF][START_REF] Kuzyakov | Priming effects: Interactions between living and dead organic matter[END_REF] and/or by the presence of mucus in casts [START_REF] Lu | Carbon dynamics of rhizodeposits, root-and shootresidues in a rice soil[END_REF][START_REF] Coq | Earthworm activity affects soil aggregation and organic matter dynamics according to the quality and localization of crop residues-An experimental study (Madagascar)[END_REF]. Indeed, the presence of such easily available compounds might have stimulated the microbial activity and the decomposition of 'old' SOM.

Root contribution in soil and cast samples

The PCA cleary highlighted an important contribution of root-derived compounds in soil and casts incubated with roots. Root-NE, Root-E and Cast-Root were characterized either by high relative abundance of aromatic compounds (e.g. P3 and G5) or long chain ω-hydroxyalkanoic acids (ωOH), as well as ωOH18uns and Dia18uns. These compounds were described as the most representative compounds in root litter (Appendix 4.A). This was confirmed by the representation of the relative abundance of P3 and the sum of ωOH, ωOH18uns and Dia18uns (Figure 46.A and B). Cast-Root and Root-NE were clearly distinct from the other samples. This is in agreement with the isotopic results, revealing a high contribution from labelled litter-derived carbon, as

Cast-Root and Root-NE presented δ 13 C values of 127 and 126 ‰, respectively (Table 9).

However, while Cast-Root was similar to root litter molecular characteristics, Root-NE was mainly represented by ωOH, ωOH18uns and Dia18uns (Figure 45 and Figure 46.A andB).

This could result from the preferential incorporation of ωOH, ωOH18uns and Dia18uns compared to aromatic compounds in the soil. Indeed, as root litter was deposited on the soil surface, most of the labelled derived compounds have probably been incorporated under soluble forms. It was observed that, in the initial phase of decomposition and without any physical transfer of litter into the soil, lignin-derived compounds tended to accumulate in litter, while non-structural compounds were preferentially lost from litter and transferred to the soil [START_REF] Cotrufo | Formation of soil organic matter via biochemical and physical pathways of litter mass loss[END_REF]. This could explain why lignin-derived compounds in roots were slightly less transferred to the soil, compared to casts, where lignin can be transferred via the physical action of earthworms. Root-E was less separated from the other samples, which is consistent with isotopic results (δ 13 C value: -25.3‰) (Table 9). These observations are consistent with the tendency of earthworms to homogeneize root and shoot characteristics in soil, as proposed in chapter 3. After one year, cast and soil samples collected in mesocosms incubated with roots presented different molecular characteristics from other samples. On the contrary, samples collected in mesocosms incubated with shoots presented molecular characteristics closer to control samples.

While

Various hypotheses can be put forward to explain these results:

1-Roots incorporated in either cast or soil presented lower decomposition rates compared to shoots. This could be partly explained by the higher relative abundance of aromatic compounds in root litter, which tends to be more resistant to decomposition [START_REF] Balesdent | Major contribution of roots to soil carbon storage inferred from maize cultivated soils[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF]. Thus, while most of the shoot-derived compounds have been degraded after one year, root-derived compounds might be preferentially preserved in soil and cast samples.

2-While root litter molecular characteristics were highly different from control samples, shoot litter were closer to control sample characteristics. This could partly explain the difficulty to differentiate samples collected in mesocosms incubated with shoots from control samples. However, pyrolysis analyses were realized on lipid-free samples and it was shown that shoots tend to contain more lipids compared to roots [START_REF] Bianchi | Surface lipid composition of C3 and C4 plants[END_REF][START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF]. The molecules responsible for the differentiation between shoot and control samples might be present in the fraction containing lipids, which was extracted from cast and soil samples.

In order to test these hypotheses, the free lipid fractions of these samples were analyzed (see section 2.2.2.1 for methodological details).

Lipids in cast and soil samples

Initial root and shoot litter

The aim of the present section is to characterize the OM in soil and cast free lipid samples, after 54 weeks of experiment. Lipids were proportionally more abundant in shoots vs. roots (7.1 vs. 1.6 % of the total litter mass). This can be explained by the presence of a layer of epicuticular waxes made of lipids, covering the surface of leaves [START_REF] Bianchi | Surface lipid composition of C3 and C4 plants[END_REF].

Five major families of compounds were identified in both root and shoot litter: n-alkanes, nalkanoic acids, n-alkanedioic acids, n-alkanols and sterols (Figure 47.A and B). These compounds were described as typical plant-derived lipids in the literature [START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments-IV. Preliminary results from a study of the effect of soil pH on organic matter decay[END_REF][START_REF] Bull | Organic geochemical studies of soils from the Rothamsted classical experiments-V. The fate of lipids in different long-term experiments[END_REF][START_REF] Wiesenberg | Source determination of lipids in bulk soil and soil density fractions after four years of wheat cropping[END_REF].

Few aromatic compounds were identified in root litter lipids (Figure 47.A): mainly ligninderived compounds from p-coumaryl (H1), guaiacyl (G3, G4 and G5) and syringyl (S2), as well as phenol (P3). Their relative abundance was low, except for P3. [START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF] also observed the presence of low molecular weight phenolic compounds in extractable lipid chromatograms from Corsican pine roots. This result is in agreement with the higher concentrations of lignin in roots compared to shoots [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF].

Despite root and shoot lipids comprised similar families, their distribution according to carbon chain length and the relative abundance of molecules of each family diverged. In roots (Figure 47.A), C 16 n-alkanoic acid presented the highest relative abundance, followed by the unsaturated C 18:1-2 counterparts and sterols. C 20 to C 26 n-alkanoic acids with even carbon numbered chain were also observed in roots (Figure 47.A), whereas they were not detected in shoot sample lipids (Figure 47.B). Long chain n-alkanoic acids (> 20) had previously been identified in C3 plant roots [START_REF] Wiesenberg | Source and turnover of organic matter in agricultural soils derived from n-alkane/n-carboxylic acid compositions and C-isotope signatures[END_REF][START_REF] Wiesenberg | Source determination of lipids in bulk soil and soil density fractions after four years of wheat cropping[END_REF] and were also observed in suberin-derived compounds [START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. Shoot lipids were mainly represented by unsaturated C 18:3 n-alkanoic acid and C 26 n-alkanol, whereas they were virtually absent or in low relative abundance, respectively, in the root sample (Figure 47.A).

Unsaturated n-alkanoic acids, such as C 18:3, are typical compounds from vegetal biomass which decompose rapidly in soils [START_REF] Wiesenberg | Source determination of lipids in bulk soil and soil density fractions after four years of wheat cropping[END_REF]Quenea et al., 2012). This dominant C 26 n-alkanol peak was already reported by several authors as a typical grass plant constituent [START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments-I. Total lipid extracts, solvent insoluble residues and humic acids from Broadbalk Wilderness[END_REF][START_REF] Jansen | The straightchain lipid biomarker composition of plant species responsible for the dominant biomass production along two altitudinal transects in the Ecuadorian Andes[END_REF]. [START_REF] Jansen | The straightchain lipid biomarker composition of plant species responsible for the dominant biomass production along two altitudinal transects in the Ecuadorian Andes[END_REF], who analyzed lipid extracts from roots and shoots from 19 plant species from Ecuadorian Andes, observed that the quantity of n-alkanols extracted from roots was much lower compared to shoots. This is in agreement with the low n-alkanol relative abundance observed in roots. C 29 and C 31 n-alkanes were identified in both root and shoot samples. These n-alkanes are typical constituents of superior plant waxes and were identified in shoots from C3 plants [START_REF] Wiesenberg | Source and turnover of organic matter in agricultural soils derived from n-alkane/n-carboxylic acid compositions and C-isotope signatures[END_REF] and roots [START_REF] Jansen | The straightchain lipid biomarker composition of plant species responsible for the dominant biomass production along two altitudinal transects in the Ecuadorian Andes[END_REF]. Three main sterols were identified in both root and shoot extracts: cholesterol, stigmasterol and sitosterol. [START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF] found sitosterol to be one of the dominant compounds in root and needle extracts from Corsican pine.

Thus, the main differences between root and shoot litter lipids relied on the presence of aromatic compounds, as well as the higher relative abundance of long chain n-alkanoic acids and sterols in root litter, while shoot litter presented a higher relative abundance of n-alkanol

C 26 .

The soil and cast control

In the control soil extract (Figure 48.A), n-alkanoic acids ranged from C 9 to C 32 , with a strong even-over-odd preference and a maximum at C 16 , as frequently reported in the literature [START_REF] Jandl | Contribution of primary organic matter to the fatty acid pool in agricultural soils[END_REF][START_REF] Wiesenberg | Source determination of lipids in bulk soil and soil density fractions after four years of wheat cropping[END_REF]. While C 16 and C 18 n-alkanoic acids are ubiquitous in living organisms, C 20 -C 26 n-alkanoic acids were described as good indicators of vegetal biomass incorporation in soils [START_REF] Wiesenberg | Source and turnover of organic matter in agricultural soils derived from n-alkane/n-carboxylic acid compositions and C-isotope signatures[END_REF] and they can also be derived from the oxidation of n-alkanols [START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments-IV. Preliminary results from a study of the effect of soil pH on organic matter decay[END_REF]. The presence of iso and anteiso C 15 n-alkanoic acids highlighted a bacterial contribution [START_REF] Wannigama | A comparison of lipid components of the fresh and dead leaves and pneumatophores of the mangrove Avicennia marina[END_REF]Quenea et al., 2012). C 16 to C 33 n-alkanes were identified in soil, with a strong odd-over-even preference, an increased relative abundance of odd chain C 29 to C 33 and a maximum at C 31 , as frequently observed in the literature [START_REF] Bull | Organic geochemical studies of soils from the Rothamsted classical experiments-V. The fate of lipids in different long-term experiments[END_REF][START_REF] Lichtfouse | A novel pathway of soil organic matter formation by selective preservation of resistant straight-chain biopolymers: chemical and isotope evidence[END_REF][START_REF] Wiesenberg | Source determination of lipids in bulk soil and soil density fractions after four years of wheat cropping[END_REF]. C 12 to C 32 n-alkanols were observed with dominant even-over-odd chain and a maximum at C 26 , also representing the dominant compounds of the entire chromatogram.

Long chain n-alkanols (> 20) were recognized as typically derived from higher shoot plant waxes [START_REF] Van Bergen | Organic geochemical studies of soils from the Rothamsted Classical Experiments-IV. Preliminary results from a study of the effect of soil pH on organic matter decay[END_REF][START_REF] Bull | Organic geochemical studies of soils from the Rothamsted classical experiments-V. The fate of lipids in different long-term experiments[END_REF][START_REF] Quenea | Variation in lipid relative abundance and composition among different particle size fractions of a forest soil[END_REF]Quenea et al., , 2006;;[START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF] and were dominant constituants (C 26 ) of shoot litter extract (Figure 47.B). n-Alkanols are essential indicators of past plant types [START_REF] Jansen | Characteristic straight-chain lipid ratios as a quick method to assess past forestpáramo transitions in the Ecuadorian Andes[END_REF] and can represent an important contribution to stabilized OM (Mueller et al., 2012). For example, [START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF] found n-alkanol C 26 to be, by far, the dominant compound from soil lipid extracts collected in a pine forest. As pine biomass did not contain this n-alkanol, authors suggested that the soil was probably covered by grass vegetation before pine trees were implanted. C 22 , C 24 and C 26 ωhydroxyalkanoic acids were also identified in soil. These acids are generally described as derived products from the hydrolyses of cutins and suberins (Quenea et al., 2012). Not surprisingly, most compounds identified in the soil control were derived from plant biomass and more specifically from grass plant. This is in agreement with the > 50 year permanent pasture land use of the soil collected for the experiment.

The cast control extract was almost identical to the soil control extract (Figure 48.B), with the exception of two isomers of long chain unsaturated n-alkanoic acid: the arachidonic acid (C 20:4 ) (Figure 48.B and Appendix 5). Arachidonic acid is a polyunsaturated n-alkanoic acid derived from eukaryotic organisms [START_REF] Packter | Lipids in plants and microbes: By J L Harwood and N J Russel[END_REF]. Studies illustrating the link between this acid and earthworms are scarce. [START_REF] Schaefer | Effects of Lumbricus terrestris, Allolobophora chlorotica and Eisenia fetida on microbial community dynamics in oil-contaminated soil[END_REF] highlighted its significant increase in the presence of earthworms in an oil-contaminated soil. It was also identified in earthworm tissues [START_REF] Albro | Lipids of the earthworm Lumbricus terrestris[END_REF] or in its gut content [START_REF] Sampedro | Changes in the fatty acid profiles through the digestive tract of the earthworm Lumbricus terrestris L[END_REF]. Thus, in the present study, the arachidonic acid identified in earthworm casts can be originated from (1) earthworms itself (i.e. their tissue or gut content) and/or ( 2) microorganisms (e.g. fungi)

stimulated by the earthworm.

A probable strong legacy from past vegetation

As for pyrolysis products, as free lipids identified in controls of soil and casts were almost identical, a PCA was realized on 64 compounds gathered in 15 variables presented in Appendix 6.C. The PCA generated two factors explaining 67 % of the variance (Appendix 6.

A and B). F1 separated cast from soil samples, but no clear distinction could be made according to the litter quality (i.e. roots vs. shoots). The main variable responsible for cast and soil sample separation was the presence of arachidonic acid (FA20uns) in cast samples as described above for the Cast-Control. However, differences observed in free lipids of roots and shoots were not reflected in cast and soil samples. Important differences observed in lipid composition of different plant species and organs are not necessarily encountered in the soil [START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF]Mueller et al., 2012). Many factors can explain this distortion between plant and soil lipid composition (1) legacy from other plant inputs in the soil [START_REF] Nierop | The complementarity of extractable and ester-bound lipids in a soil profile under pine[END_REF], ( 2) diverging affinity of lipids from different origins with soil mineral phase [START_REF] Lützow | Stabilization of organic matter in temperate soils: mechanisms and their relevance under different soil conditions-a review[END_REF] (3) soil organism community abundance and composition (Mueller et al., 2012). Moreover, lipids might also be transformed in other compound classes over time.

For example, n-alkanols and n-alkanes might be oxidized into n-alkanoic acids during decomposition [START_REF] Amblès | Incorporation of natural monoacids from plant residues into an hydromorphic forest podzol[END_REF]. The description of Cast-Control and Control-NE chromatograms highlighted a strong contribution from plant-derived compounds in the lipid extracts. After 54 weeks of experiment, the legacy from past plant inputs seemed to mask the potential differences between free lipid samples collected in mesocosms containing different litter quality. Moreover, as plants originally present on the soil collected for the experiment, as well as those used for the experiment were both grass vegetation, the differentiation is even more complicated.

Conclusion

After one year of experiment, root-derived compounds (e.g. 4-methoxybenzoic acid methyl ester, ω-hydroxyalkanoic acids) were clearly identified in cast and soil samples collected in mesocosms incubated with root litter. On the contrary, samples collected in mesocoms incubated with shoot litter could not be differentiated from control samples neither in lipidfree samples or free lipid extracts. This probably highlighted a slower decomposition/transformation of root-derived compounds in soil and cast samples. However, samples collected in mesocosms containing shoot litter, especially Cast-Shoot, presented a significant signal of carbon derived from labelled litter (Table 9). Why was it impossible to clearly separate, by molecular analyses, samples collected in the mesocosm incubated with shoots from the control samples?

In section 4.1, we observed that, from 2 to 24 weeks of experiment, casts in mesocosms containing shoots were clearly differentiated from the control samples, particularly thanks to the higher relative abundance of n-alkanols and n-alkanes. However, after 54 weeks of experiment, casts from the mesocosm containing shoots showed similar molecular characteristics to casts used as controls. Thus, n-alkanols and n-alkanes coming from fresh litter were probably identified in casts because earthworms incorporated high concentration of fresh litter at the beginning of the experiment. However, these two series of compounds have probably been degraded in casts from 24 to 54 weeks.

Similarly, in section 5.2, n-alkanols and n-alkanes were identified in free lipid shoot sample.

However, these compounds could not be used as indicators of shoot-derived compounds after 54 weeks of experiment, in soil and cast free lipid samples. This is consistent with observations from section 4.1. n-Alkanol and n-alkane degradation might have been achieved by microorganisms, which could be identified on TEM and NanoSIMS images from soil samples, in section 5.1. The microbial community seems also to play a key role in cast samples, after 54 weeks, as underlined by the presence of arachidonic acid in section 5.2 and the abundant microorganisms identified on TEM and nanoSIMS images in section 4.3. Thus, in section 5.3, we achieved a first screening of the microbial community abundance and diversity in soil and casts samples, after 54 weeks of experiment.

Microbial characterization in soil and cast

Microorganisms, represented at 90 % by bacteria and fungi, are the primary decomposers in soil [START_REF] Six | Bacterial and fungal contributions to carbon sequestration in agroecosystems[END_REF]. Indeed, as described in section 1.2.5.1, soil microorganisms have a direct role on litter decomposition by mediating the processes of organic compound mineralization and transformation in soil. Inputs of fresh litter in soil provide microorganisms with easily available substrate, which accelerates the decomposition of OM originally present in the soil [START_REF] Kuzyakov | Microbial hotspots and hot moments in soil: Concept & review[END_REF]. It can be questioned, how the input of different litter quality (i.e. roots vs. shoot, in the present study), would influence microbial community functions and abundance in soil and casts?

Soil fauna, especially earthworms in temperate ecosystems, can greatly impact microorganism activity and abundance [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF]. The direction of this impact is still debated: do earthworms stimulate or depress soil microbial communities [START_REF] Aira | Eisenia fetida (Oligochaeta: Lumbricidae) modifies the structure and physiological capabilities of microbial communities improving carbon mineralization during vermicomposting of pig manure[END_REF]? Most studies investigating the link between earthworms and soil microbial communities have been mainly

used biochemical parameters such as enzymatic activities, fatty acid profiles, microbial biomass and microbial respiration [START_REF] Tiunov | Microbial biomass, biovolume and respiration in Lumbricus terrestris L. cast material of different age[END_REF][START_REF] Sampedro | Changes in the fatty acid profiles through the digestive tract of the earthworm Lumbricus terrestris L[END_REF].

Real-time Polymerase Chain Reaction (Q-PCR) is commonly used to quantify taxonomic (e.g. bacteria and fungi) gene markers in environmental samples [START_REF] Smith | Advantages and limitations of quantitative PCR (Q-PCR)based approaches in microbial ecology[END_REF].

More recently, a method investigating microbial community function and structure has been applied to soil science, the Biolog® method. Biolog® is a culture-dependent method evaluating the community-level physiological profiles of microbial community, based on the capacity to use different carbon sources [START_REF] Hill | Methods for assessing the composition and diversity of soil microbial communities[END_REF]. One of the great advantages of Biolog® method is that it is easy and rapid [START_REF] Preston-Mafham | Analysis of microbial community functional diversity using sole-carbon-source utilisation profiles-a critique[END_REF]. However, studies using Biolog® method to investigate the impact of earthworms on microbial communities are still scarce [START_REF] Aira | Eisenia fetida (Oligochaeta: Lumbricidae) modifies the structure and physiological capabilities of microbial communities improving carbon mineralization during vermicomposting of pig manure[END_REF][START_REF] Amador | Microbiological characterization of the structures built by earthworms and ants in an agricultural field[END_REF][START_REF] Sheehan | The effects of earthworm functional diversity on microbial biomass and the microbial community level physiological profile of soils[END_REF][START_REF] Sen | Do earthworms affect dynamics of functional response and genetic structure of microbial community in a lab-scale composting system?[END_REF]. For example, Sheenan et al., (2008) showed that the impact on the metabolic potential of the soil microbial community depended on the diversity and identity of earthworms from different ecological categories. [START_REF] Sen | Do earthworms affect dynamics of functional response and genetic structure of microbial community in a lab-scale composting system?[END_REF] highlight different microbial community functions and diversity in a compost of sugar industry waste incubated during 60 days, with or without earthworms.

The aim of the present section was to obtain a preliminary appreciation of the link between litter quality, earthworms and microbial community in soil and casts. Complex community-level microbial interactions occurring in soil requires the use of both quantitative and qualitative methods to investigate the impact of soil factors on microbial communities [START_REF] Hill | Methods for assessing the composition and diversity of soil microbial communities[END_REF]. In order to access a first screening of the microbial abundance and functional diversity, Q-PCR and Biolog® Ecoplates TM were carried out on cast and soil samples, at two depths (0-20 cm and 40-60 cm), after 54 weeks of experiment (see section 2.2.4 for methodological details).

Results

Quantification of microbial communities by real-time PCR

Real-time PCR was applied in order to obtain the abundance of fungi and bacteria in soil and cast samples.

Soil samples collected at 40-60 cm presented a lower bacterial abundance compared to cast and soil collected at 0-20 cm (Figure 49.A), whatever the mesocosm. However, differences were rarely statistically significant due to the high variability of data. They were no clear difference between cast and soil samples collected at 0-20 cm, whatever the litter quality (root or shoot), although bacterial abundance in Cast-Shoot tended to be slightly higher compared to soil. Thus, the bacterial abundance in casts with litter ≥ soil 0-20 cm >> soil 40-60 cm.

Regarding the fungal abundance, the difference between surface (cast and soil 0-20 cm) and deeper samples was not as clear as for bacteria (Figure 49.B). The fungal abundance was lower in deeper soil samples compared to surface ones in Shoot-E, Shoot-NE and Control-NE (to a lesser extend). Although not statistically significant, Cast-Root and Cast-Shoot tended to present a slightly higher fungal abundance, compared to other samples, especially the one collected in Control-E and Root-E, whatever the depth. Thus, the fungal abundance in cast with litter ≥ soil without earthworms > soil with earthworms (with the exception of Shoot-E 0-20 cm).

Catabolic profiles of microbial communities by Biolog® EcoPlate™

Biolog® method was applied in order to obtain the diversity and metabolic potential of the microbial community in soil and cast samples.

The average well color development (AWCD) reveals the mean carbon substrate use efficiency of the cultivable microbial community of a given sample. Thus, the higher the AWCD, the more efficient is the microbial community to use Biolog® subtrates, in average.

AWCD was approximatively twice higher in Cast-Root, Cast-Shoot, Root-NE 0-20 cm and Shoot-NE 0-20 cm (to a lesser extent), compared to other samples (Figure 50.A). The functional richness (FR) expresses the number of substrates used by the microbial community. Thus, the higher the FR, the higher diversity of functions the microbial community can achieve. The same trend as for AWCD, although not statistically significant, was observed for FR, but Shoot-NE 0-20 cm was not different from the other samples (Figure

50.B).

Thus, the number of substrates used by the microbial community (FR), as well as the substrate use efficiency (AWCD) were higher in cast with litter = Root-NE 0-20 cm > other cast and soil samples.

The metabolic potential and functional differences in the microbial community in terms of capacities to degrade different carbon substrates were represented using polygons (Figure 51). Each summit of the polycon regroups several carbon substrates in one family, which In order to specify the metabolic potential of the microbial community of a given sample, a PCA was carried out on cast and soil samples. Each sample was expressed as using each the optical density (blank corrected) divided by the AWCD, for each substrate. Each substrate was used as one variable. Thus, the preliminary PCA included 31 variables (detailed in Appendix 1) and 15 samples. In order to simplify the representation and interpretation, only substrates presenting a contribution to factor 1 or 2 higher than 5 % were selected and the PCA was carried out again, including 12 variables. The PCA generated two factors explaining 55 % of the variance (Figure 52

Discussion

The abundance of bacteria and fungi in soil and casts

Bacterial abundance was lower in soil collected at 40-60 cm compared to the other samples.

In chapter 3, we observed that virtually no carbon derived from labelled litter could be detected at this depth. Thus, the amount of fresh litter-derived compounds reaching 40-60 cm was probably very low after 54 weeks of experiment. Most bacteria depend on the availability of carbon to develop and decompose SOM (Kuzyakov et al., 2000;[START_REF] Kuzyakov | Priming effects: Interactions between living and dead organic matter[END_REF]. Thus, the lack of fresh substrate at 40-60 cm has probably limited the development of bacteria, explaining their lower abundance at this depth. Bacterial abundance tended to be slightly higher in casts collected in mesocosms incubated with either roots or shoots, although not significant, compared to other samples (Figure 49.A). This is agreement with the abundant microbial community (dead or alive) observed on TEM cast images at this time step (section 4. 3.3.3). This could be explained by the presence of fresh substrate, rich in easily available carbon, in casts derived from (1) the fresh roots and shoots incorporated in casts [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF] and/or (2) the mucus produced by earthworms [START_REF] Drake | As the worm turns: The earthworm gut as a transient habitat for soil microbial biomes[END_REF]. However, after one year, bacterial abundance differences between casts and soil at 0-20 cm are reduced. This might be explained by the fact that, after one year, casts are ageing and drying, and litter incorporated into casts presented high degradation state, as illustrated on TEM and NanoSIMS images (section 4. 3.3.3). Thus, after one year, the bacterial community has probably started to decrease, explaining the slight difference with soil samples. To be confirmed, microbiological analyses should be performed at previous time steps.

Contrary to bacterial abundance results, less fungal abundance differences were observed between 0-20 cm and 40-60 cm soil samples (Figure 49.B). This is unexpected regarding the admitted resource exploitation strategy of fungi. Indeed, fungi are generally mainly restricted to the soil surface (< 20 cm) as they need oxygen to decompose complex substrates such as phenolic compounds [START_REF] Ekschmitt | Soil-carbon preservation through habitat constraints and biological limitations on decomposer activity[END_REF]. The abundance of fungi tended to be slightly reduced in soils in the presence of earthworms, compared to casts and soil without earthworms. It might be hypothesized that earthworms have feed on fungi in soils, as reported in the literature [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF][START_REF] Shan | Digestion and residue stabilization of bacterial and fungal cells, protein, peptidoglycan, and chitin by the geophagous earthworm Metaphire guillelmi[END_REF]. However, in that case, why would the fungal abundance not be reduced in casts? Some studies observed that some microorganisms could be affected by the anoxic conditions encountered during the transit into the earthworm gut [START_REF] Brown | How do earthworms affect microfloral and faunal community diversity?[END_REF][START_REF] Drake | As the worm turns: The earthworm gut as a transient habitat for soil microbial biomes[END_REF]. However, the favorable conditions encountered in casts (i.e. fresh substrate) make fungi develop rapidly after cast excretion [START_REF] Moody | Selective consumption of decomposing wheat straw by earthworms[END_REF]. Thus, it can be proposed that earthworms feed on fungi in soil, explaining the lower abundance in the presence of earthworms, but that earthworms enhance the development of fungi in casts after their excretion. It is important to note, that the two genes selected for quantification do not necessarily reflect the whole microbial community. Indeed, the DNA quantification before real-time PCR analyses (Appendix 7) reflected a significantly higher quantity of DNA in the presence of earthworms, in cast and soil incubated with litter, in the presence of both roots and shoots. Thus, other microorganisms than bacteria and fungi might be enhanced in the presence of earthworms.

Microbial community metabolic profiles in control samples

Although the abundance of bacteria and fungi did not highlighted marked differences between samples, functional diversity revealed some. Microbial communities from most control samples could be differentiated from others by using simple substrates, such as carbohydrates (i-Erythritol, β-Methyl-D-glucoside and α-D-Lactose) and one carboxylic acid (α-Ketobutyric acid) (Figure 52). Gomez-Brandon et al. (2010) studied the impact of epigeic earthworms on microbial community, in the presence of grape marc, after 15 days of incubation. They found that microbial communities from control samples had higher capacities to degrade i-Erythritol, compared to samples influenced by earthworms. [START_REF] Nunan | Metabolising old soil carbon: Simply a matter of simple organic matter?[END_REF] obtained similar results comparing the microbial communities from bare-fallow plots to cultivated counterparts. They showed that microbial communities which were deprived from fresh plant inputs (bare-fallow soil) tended to use substrates that required low investment, with low energetic return. In the present study, communities from control samples might have used the same strategy.

A B

Cast-Control and control soil samples could not be differentiated in the present study, while [START_REF] Amador | Microbiological characterization of the structures built by earthworms and ants in an agricultural field[END_REF], who used Biolog® to compare structures formed by ant and anecic earthworms in USA, observed strong divergence in substrate use between casts and bulk soil. However, these authors analyzed casts formed less than 24 hours after their formation, while casts analyzed in the present study had already start ageing and drying. This could also explained why [START_REF] Amador | Microbiological characterization of the structures built by earthworms and ants in an agricultural field[END_REF] showed that microorganisms in casts had higher capacities to grow on α-Ketobutyric acid and carbohydrates, compared to bulk soil, contrary to the present study. Indeed, fresh casts generally reveal a high bacterial community using simple substrates [START_REF] Wolters | Invertebrate control of soil organic matter stability[END_REF], as they contain important concentrations of mucus, made of simple substrates which can be easily assimilated by microorganisms [START_REF] Martin | Les mucus intestinaux de ver de terre moteur de leurs interactions avec la microflore[END_REF].

Link between microbial communities and litter inputs

Microbial communities from Cast-Root and Cast-Shoot, as well as Root-NE collected at 0-20 cm, had the capacity to use a high number of substrates (FR) (Figure 50.B), compared to the other samples. The efficiency to use these substrates (AWCD) was also higher (Figure 50.A).

Microbial communities from these samples present higher capacities to use almost all substrate families (Figure 51.A and B). The PCA on individual substrates revealed that their capacity to use α-Cyclodextrin, Glycogen L-Phenylalanin differentiated them most from the other samples. α-Cyclodextrin and Glycogen are two polymers which are mainly representative of plant inputs [START_REF] Nunan | Metabolising old soil carbon: Simply a matter of simple organic matter?[END_REF] and L-Phenylalanin is an amino acid. Gomez-Brandon et al. (2010) also found an increase in the capacity to degrade polymers and amino acids after 15 days. Thus, the microbial communities from these samples have been exposed recently to plant inputs. This is in agreement with results from Chapter 3, which highlighted the higher proportion of carbon derived from labeled litter in these samples. Thus, after 54 weeks, microbial communities from these samples present a great potential to degrade plant inputs. This is consistent with the hypothesis that roots incorporated into the soil exhibit a lower state of degradation compared to shoots, as observed on TEM and NanoSIMS images (section 4.3.3.3). Although Cast-Root and Cast-Shoot were not separated on the PCA, the metabolic potential regrouped by families (Figure 52) presented that communities from Cast-Shoot tended to be more able to degrade phenolic compounds compared to Cast-Root. This could appear at variance with the fact that roots contain more lignin-derived compounds, which are phenolic compounds, compared to shoots. However, it could also be hypothesized that microorganisms present in Cast-Root still have access to more easily degradable compounds, while microorganisms present in Cast-Shoot have already used simple compounds and are attacking more complex material such as phenolic ones.

It has to be noted that the Biolog® method describes the metabolic potential of cultivable microorganisms, capable of growing on high nutrient and substrate concentrations, as well as oxygen availability [START_REF] Amador | Microbiological characterization of the structures built by earthworms and ants in an agricultural field[END_REF]. It is estimated that less than 1 % of soil microorganisms can be recovered on a culture media [START_REF] Hill | Methods for assessing the composition and diversity of soil microbial communities[END_REF][START_REF] Kirk | Methods of studying soil microbial diversity[END_REF] and many fungi cannot grow on culture media [START_REF] Van Elsas | Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis[END_REF]. Thus, these results do not reflect the impact of litter quality and earthworms on the whole microbial community, but only on a restricted fraction.

Conclusion

Fungal and bacterial abundance did not seem to be influenced by the litter quality. However, bacterial abundance depended on the depth of the soil sample in the mesocosm (i.e. fewer bacteria in deeper layers). Fungal abundance tended to be less abundant in the presence of earthworms in soil samples, but their abundance was slightly higher in casts. This suggested a paradoxical impact of earthworms on the fungal community. Despite Biolog® enabled the investigation of a small portion of the microbial community, some differences of metabolic potential could be observed between samples. In casts, the presence of litter (roots or shoots) favored microbial communities able to degrade a higher diversity of substrates, especially two polymers (Cyclodextrin and Glycogen) and an amino acid (L-Phenylalanin). The same observation was made for the soil taken at 0-20 cm and incubated with roots. Thus, in the present study, even though the quantity of microorganisms was not particularly dependent on litter inputs, their metabolic potential was related to litter availability. After one year of experiment, the amount of litter available for microbial decomposition and its degradation stage seems to regulate the microbial community functions, more than the initial chemical composition. Indeed, the microbial communities have the highest substrate use efficiency in samples presenting the highest percentage of carbon derived from labelled litter (i.e. Cast-Root, Cast-Shoot and Root-NE) as observed in Chapter 3.

In section 5.1 and chapter 4, microscale observations using TEM and NanoSIMS enabled the identification of organic structures in soil and casts, respectively. However, we were not able to investigate satisfactorily the impact of litter quality and earthworms in soil samples, at the microscale. Thus, we applied molecular analyses in order to characterize OM. Lipid-free analysis using Py-GC-MS highlighted a significant contribution of root-derived compounds in soil collected in mesocosms incubated with roots, after one year of experiment, illustrating the impact of litter quality on SOM composition (section 5.2.1). Analysis of lipids enabled the clear differentiation between cast and soil samples, revealing the presence of arachidonic acids in casts, thus illustrating the contribution of earthworms and/or microorganisms to SOM composition (section 5.2.2). In order to clarify the role of microorganisms on SOM composition and decomposition in the present study, we carried out a first screening of the microbial community abundance and functions (section 5.3). Microbial community metabolic capacities depended on the litter availability, more than the litter chemistry. The combination of different microscopic (i.e. TEM and NanoSIMS) molecular (i.e. Py-GC-MS and GC-MS) and microbiological (i.e. Q-PCR and Biolog®) analyses, as well as the combination of different disciplines (i.e. biogeochemistry and microbiology), gave complementary information on the impact of litter quality and earthworms on SOM characteristics and microorganisms. The present section illustrates the potential of multidisciplinary approaches to decifer processes of SOM formation, decomposition and stabilization.

Conclusions and perspectives

Soils are fundamental to all civilization by underpinning the delivery of a wide range of ecosystem goods and services, including climate and carbon regulation. They are therefore major players in the terrestrial carbon cycle and represent an important carbon sink. Carbon inputs and outputs are mainly regulated by biotic and abiotic factors. The present study focused on two essential biotic factors: litter inputs, which represent the main source of SOM; and earthworms, which are key soil engineers in temperate regions. We aimed at investigating the fate of 13 C labelled root and shoot in the presence or absence of earthworms, based on a one year mesocosm experiment. For this purpose, we used a multidisciplinary approach at different spatio-temporal scales, in order to quantify, localize and characterize the OC incorporated in soil and earthworms casts. Figure 53 summarizes the main conclusions and perspectives detailed below.

First, a general appreciation on the localization and quantification of carbon derived from roots and shoots was carried out in casts and soil at two depths (0-20 cm and 40-60 cm) (Chapter 3). Carbon derived from litter continuously increased in soil at 0-20 cm depth, whereas no significant increase of litter-derived carbon could be noted at 40-60 cm. Roots incorporated in soil 0-20 cm layer tended to decompose at a slower rate compared to shoots.

Earthworms accelerated litter decomposition and minimized the difference between root-and shoot-derived carbon after 54 weeks of experiment, in soil and casts.

Earthworm casts contained high concentrations of carbon derived from labelled litter, which evolved constantly and rapidly during the year of experiment. Thus, a focus was made on casts, to investigate, at the molecular and microscopic level, root-and shoot-derived carbon incorporation and decomposition in these complex organo-mineral structures (Chapter 4). Contrary to 'bulk isotopic' analyses, molecular and microscopic analyses revealed important differences between casts in the mesocosms incubated with roots or shoots, along the year of experiment. At the molecular level, roots and shoots could be clearly followed in casts thanks to the monitoring of aliphatic (mainly n-alkanols and n-alkanes) and aromatic (mainly 4-methoxybenzoic acid, methyl ester) compounds, respectively. After one year, while cast collected in the mesocosm with shoots presented similar molecular characteristics to control samples, this was not the case for cast collected in the mesocosm incubated with roots (Section 4.1). Thus, roots were incorporated and/or decomposed at slower rate in casts compared to shoots. This was confirmed at the microscopic scale as shoots tended to be integrated more rapidly in cast organo-mineral aggregates compared to roots, in the presence of abundant microorganisms involved in their decomposition (Section

and 4.3).

The few labelled spots identified on soil sample fractions, after 54 weeks of experiment could be assigned to past or present microbial activity. Thus, the last chapter of this study was dedicated to a first screening of potential molecular and microbial differences in cast and soil samples collected after 54 weeks of experiment (Chapter 5). In soil and cast lipidfree samples, the addition of root litter was clearly reflected at the molecular level, mainly due to the presence of 4-methoxybenzoic acid methyl ester and ω-hydroxyalkanoic acids. On the contrary, samples collected in mesocoms incubated with shoot litter could not be differentiated from control samples neither in soil pyrolysate or lipid extract. This confirmed the lower decomposition state of roots incorporated in soil and casts samples after one year, as proposed in Chapter 3. The main difference in lipid extracts between soil and cast samples lies on the presence of arachidonic acids in casts, which could be of microbial origin (Section 5.1). Despite relatively similar bacterial and fungal abundance between all cast and soil (0-20 cm) samples, microbial communities presented diverging metabolic potentials. Communities from casts collected in mesocosms incubated with litter, as well as soil incubated with roots but no earthworms, presented the potential to use an increased diversity of substrate and more intensively, compared to communities from other samples. More specifically, these communities were particularly able to use substrates derived from plants, such as polymers and amino acids, reflecting microorganism dependence on substrate availability (Section 5.2).

Root and shoot in soil and casts -chemical composition and physical protection

As mentioned in Chapter 1, it is widely recognized that roots tend to decompose at a slower rate compared to shoots, related to the debated contribution from three factors: root chemical recalcitrance, as well as physico-chemical and physical protection in soil. In this study, both roots and shoots were deposited on the soil surface. Thus, the physical protection of carbon potentially induced by the localization of root vs. shoot in the soil was not investigated.

Initially labelled roots and shoots used in this experiment had similar labelling, diverging molecular composition and hardly differentiable structures at the microscale. After one year of experiment, in soil at 0-20 cm without earthworms and incubated with roots, the 'bulk' isotopic signal and the relative abundance of litter-derived molecules were higher compared to other samples. This root contribution in soil was probably responsible for the increased metabolic potential of the microbial communities, particularly in using polymers and amino acids. Thus, the chemical composition of roots and shoots had a significant impact on the fate of carbon in soil, with an increased contribution from root derived carbon in soil after one year. One of the main limits of this study was the inability to obtain a carbon balance due to the size and limited number of mesocosms, which constrained the sampling design. Indeed, mesocosms were not used as destructive samples and only small soil quantities could be collected for each time step. As mentioned in Chapter 1, litter deposited at the soil surface can either be mineralized, releasing CO 2 to the atmosphere, or incorporated into soil in the form of organic compounds. After incorporation, these compounds can be mineralized, transferred in deeper layers and/or stored in soil. Thus, one of the perspectives of this experiment could be to use destructive samples for each time step and factor, in order to monitor and quantify the different pools of carbon along the experiment by:  Quantifying the CO 2 and 13 CO 2 released to the atmosphere  Weighting the litter remaining at the soil surface and quantifying/characterizing its carbon  Weighting soil and casts and quantifying/characterizing the carbon incorporated This carbon balance would facilitate the differentiation between the mineralized carbon, from that remaining in the litter or incorporated into the soil.

As earthworm casts evolved rapidly and not necessarily homogeneously during the experiment, processes explaining the fate of roots and shoots were more complex compared to those occurring in soils. After one year of experiment, casts presented similar 'bulk' isotopic signals from roots and shoots (Chapter 3) and microbial community metabolic potentials (Section 5.2), but different molecular contributions from roots and shoots (Section 4.1) with slightly different aspects at the microscale (Section 4.2). Thus, in the present study, the chemical composition tended to influence the litter palatability for earthworms. Root litter was probably less palatable for earthworms, which might have delayed the incorporation of roots, compared to shoots into casts. Along the year of experiment, physical interactions might also have played an important role in cast formation and evolution, as organomineral associations tended to increase during drying and ageing of casts (Section 4.3). The main limit encountered to study casts in the present study was the inability to differentiate incorporation from decomposition. Indeed, casts are very dynamic structures that can be formed, destroyed and re-ingested by earthworms. For example, when root residues where observed to be less degraded in casts after 8 weeks, it was difficult to know if it highlighted an increased or delayed incorporation of roots compared to shoots and/or if roots incorporated in casts decomposed slower. Despite the partial response thanks to mesocosm visual observations, some perspective could be proposed to improve the understanding of cast formation and evolution in the presence of roots or shoots. Roots and shoots could be incubated in the presence of earthworms and soils, in microcosms. In the first stage of the experiment, cast formation would be strictly followed (e.g. using cameras) in order to distinguish direct from indirect incorporation of litter by earthworms. At repeated time steps, casts could be quantified. One part of cast samples could be used for characterization, while the other could be incubated for several weeks/months, in the absence of earthworms, in order to highlight cast evolution, without re-ingestion and incorporation of fresh litter. Thus, it would be possible to distinguish if the enclosing of litter-derived compounds in casts would mainly be related to the repeated mechanical action of earthworms or to the drying and ageing of cast inducing stabilization.

Thus, in the short term, chemical composition of roots vs. shoots tended to play an important role on (1) incorporation of litter by earthworms as roots seemed to be less palatable for earthworms compared to roots, (2) incorporation and/or decomposition of roots in soil, as root were either incorporated later in soil compared to shoots or decomposed slower in soil compared to shoots. In the longer term, as observed in casts where processes of decomposition are accelerated, the physical protection of OM within aggregates, highlighted the crucial role of mineral properties on soil interactions. Moreover, the encapsulation of OM, in the form of amorphous OM -including plant residues, microorganisms, their products or residues -highlighted the potential of cast as a source of stable OC. This is in agreement with [START_REF] Kögel-Knabner | Organo-mineral associations in temperate soils: Integrating biology, mineralogy, and organic matter chemistry[END_REF] who concluded their review as followed: 'Recalcitrance and accessibility/aggregation seem to determine the turnover dynamics in fast and intermediate cycling OM pools, but for long-term OC preservation the interactions with mineral surfaces (…) are a major control in all soils investigated here'. However, the experiment was not long enough to confirm these potential stabilization processes at longer terms. Thus, we assume this study fulfills the objective of combining methods and communities to study SOM cycling, as suggested by [START_REF] Schmidt | Persistence of soil organic matter as an ecosystem property[END_REF]. However, this effort should be applied for longer term experiment at both laboratory and field scale.

The paradoxical role of earthworms on the fate of litter in casts and soil

In the present study, earthworms tended to concentrate an important proportion of labelled litter in casts. As described in Chapter 1, the litter is submitted to several mechanical, physical and chemical attacks during its incorporation into casts. Thus, molecular and microscale analyses highlighted an accelerated incorporation and decomposition in these structures (Chapter 4) compared to the soil (Chapter 3 and 5). After one year, while the litter-derived carbon tended to increase in soil, it was drastically decreasing in casts. This was mainly illustrated by the decrease in root lignin-derived compounds and their increased oxidation, as well as the decrease in shoot-derived n-alkanes and n-alkanols. At the microscale this was highlighted by the increased degradation state of plant structures and their progressive enclosing in microaggregates, associated with the important contribution of microorganisms. Thus, as described in Chapter 1, in the longer term, earthworms favor the physical protection of organic compounds in casts. Although this was already reported in the literature [START_REF] Bossuyt | Protection of soil carbon by microaggregates within earthworm casts[END_REF][START_REF] Pulleman | Earthworms and management affect organic matter incorporation and microaggregate formation in agricultural soils[END_REF], the microbial nature (i.e. living or dead microorganisms and microbial products) of these protected organic structures was rarely described. Further multidisciplinary analyses of microbial contribution in casts in the short and longer terms would fill this gap (see issue n°4).

Despite the differentiation between root and shoot-derived compounds in cast at the molecular level, in soil, earthworms tended to minimize the difference between roots and shoots at the isotopic, microbial and molecular scale. Thus, the higher contribution of root-derived carbon in soils compared with shoot-derived carbon should be questioned in the presence of earthworms. However, in the present study, we did not left the choice for earthworms to choose between roots and shoots and litter was deposited on the soil surface, which is not the case in natural conditions. Thus, these observations should be verified using in situ or field experiments.

Compounds at trace level in complex samples

The distinction between root-and shoot-derived compounds was possible at molecular level and microscale in casts because litter had been highly incorporated by earthworms. However, the proportion of carbon derived from labelled litter was much lower in soil samples. Indeed, at the microscale, few labelled spots were detected (Chapter 5, introduction) and at the molecular level, the strong molecular background derived from the past vegetation probably masked most of the differences between soil samples (Section 5.1). Moreover, one of the efforts of this study was to investigate two soil depths. However, no significant increase in litter-derived carbon was observed in 40-60 cm and it was decided to focus on casts and soil at 0-20 cm in the present study. It has to be noted that the slight increase in the 40-60 cm layer, after one year of experiment in the presence of earthworms (Chapter 3), might have a strong impact in the longer term. Indeed, the slow turnover of organo-mineral structures in subsoils makes the small amount of OM transferred to the subsoil (in the form of dissolved OM, for example) more preserved compared with the important amount of organic in the topsoil [START_REF] Kaiser | Mineral surfaces and soil organic matter[END_REF]. This could also be related to the lower bacterial abundance and metabolic potential observed in soil samples collected at 40-60 cm (Section 5.2). Indeed, if microorganisms are less abundant and active in the subsoil, the mineralization and decomposition of litter-derived compounds should be limited. It was also reviewed that the stable carbon in subsoil could be mainly derived from microbial derived-compounds (Kögel-Knabner and Amelung, 2014). Thus, one of the limits of this study was the difficulty to identify and characterize small amounts of carbon derived from labelled litter.

To partially solve this problem, the roots and shoots originally present in the field could be analyzed in order to identify the molecular characteristics derived from the past vegetation in soil samples. However, as both past vegetation and labelled litter are grassland plants, the distinction between the two sources in soil samples would probably be difficult. Another more direct and precise possibility would be to apply compound specific stable isotope analyses on 0-20 cm and 40-60 cm samples. These analyses allow 'the isotopic analysis of individual substances occurring at trace levels in very complex mixture' [START_REF] Amelung | Combining biomarker with stable isotope analyses for assessing the transformation and turnover of soil organic matter[END_REF].

Moreover, as plant litters were deposited at the soil surface, an important proportion of OC has probably been transferred in soil under dissolved form (DOC). Thus, the DOC might contain a large proportion of labelled litter-derived carbon. It was shown that shoots tented to contain higher concentration of water soluble carbon compared to roots [START_REF] Beuch | Effect of the organic residues of Miscanthus giganteus on the soil organic matter level of arable soils[END_REF][START_REF] Bird | Fine Roots vs. Needles: A Comparison of 13 C and 15 N Dynamics in a Ponderosa Pine Forest Soil[END_REF][START_REF] Shi | Decomposition of roots and shoots of perennial grasses and annual barley-separately or in two residue mixes[END_REF]. DOC monitoring on litter and soil samples along the year of experiment would probably bring new elements to explain the fate of roots and shoots in soils.

Moreover, soil samples from the 20-40 cm layer were conditioned for storage, at every time steps and could be analyzed in order to obtain a full appreciation of labelled compound distribution along the soil 'profile'.

The microbial contribution to carbon cycling

One of the important results of the present study was the essential role of microorganisms in decomposing litter (Chapter 4 and 5). Thanks to microscale observations using the innovative combination of TEM and nanoSIMS (Chapter 4), it was also observed that, in the presence of earthworms, microorganisms could be enclosed in microaggregates which might lead to their protection and participate to the preserved carbon pool in soils. However, microbial analyses were realized only on the ultimate time step and used as a first screening of the microbial community abundance and diversity. Bacterial and fungal quantification was carried out by using two genes which did not necessarily represented the entire DNA contribution. Moreover, the metabolic potential was analyzed thanks to Biolog® on a very small proportion of the soil microbial community (< 1%). Further experiment should be realized in order to precise the impact of litter quality on microbial community at different time step. For example, the microbial respiration could be estimated by measuring CO 2 and 13 CO 2 . Microbial community profiles could be obtained using Biolog® and/or MicroResp TM which was described as more sensitive compared to Biolog® [START_REF] Campbell | A rapid microtiter plate method to measure carbon dioxide evolved from carbon substrate amendments so as to determine the physiological profiles of soil microbial communities by using whole soil[END_REF]. However, MicroResp TM analysis requires a higher quantity of soil, as well as soils containing the minimum of carbonates. The microbial community characterization and its implication in labelled litter decomposition could also be specified using Phospholipid. Fatty Acid -Stable Isotope Probing (PLFA-SIP). Indeed, PLFA-SIP can be used to trace labelled compounds within the microbial community [START_REF] Neufeld | Methodological considerations for the use of stable isotope probing in microbial ecology[END_REF][START_REF] Adam | Degradation of 13 C-labeled pyrene in soilcompost mixtures and fertilized soil[END_REF]. Finally, combining TEM and NanoSIMS might show great potential to 1 µm bacteria Bacterial degradation confirm the process of microbial cell wall fragmentation and stabilization in soil proposed by [START_REF] Miltner | SOM genesis: microbial biomass as a significant source[END_REF]. This potential can be illustrated on casts in the TEM image presented below (Figure 54), illustrating the different states of bacterial degradation within a bacterial aggregate. Microscale analyses should be combined to microbial analyses in order to obtain a general appreciation of the microbial abundance and activity.

However, microscale analyses also presented some drawbacks in the present study:

 The limited number of samples and images observation, which made the generalization of results complicated.

 The difficulty in identifying exactly the same structures on TEM and nanoSIMS images. This could be improved by trying analyzing exactly same sections for nanoSIMS and TEM.

However, this is complicated by the particularly fragile structure on the sections that rapidly degrade.

 The difficulty to obtain reliable δ 13 C values due to the potential bias caused by the presence of resin containing carbon, as well as the presence of punctual artefacts potentially created by boron oxides. The contamination by boron oxides can be addressed by increasing the sputtering time.

 The fact that TEM and nanoSIMS analyses are carried out at the 2D level. Thus, an apparently isolated labelled compound might be in contact with other labelled structures in another dimension.

Soil as a carbon sink

This study fits in a context of increasing interest for soil sciences, particularly for the soil as a carbon sink. Despite the short term considered in this study compared to the scale of carbon stabilization, it provides some key issues of reflection on factors controlling soil carbon stabilization. The fate of roots is different from shoots in soil and casts, under the same condition of experiment, illustrating the crucial role of litter quality on carbon cycling.

However, this observation is questioned while making the system more complex by adding earthworms. Earthworms incorporate rapidly roots and shoots in casts and in soils, to a lesser extent: they enhance physical protection of organic compounds in microaggregates. These processes lead to a relative homogenization of root-and shoot-derived compounds in the presence of earthworms, after one year of experiment. Thus, while studying soil carbon stabilization, the role of soil invertebrates should be particularly considered. Moreover, structures formed by earthworms contain a significant proportion of living and dead microorganisms enclosed in microaggregates. Microorganisms isolated from substrates would not be able to participate in organic compound mineralization and would not release carbon in the atmosphere. In the longer terms, microorganisms would die and their derived compounds might highly contribute to the stable carbon pool. Thus, microorganisms incorporated in casts would both participate to reduce carbon emission in the atmosphere and increase the soil stable carbon pool. Back in natural conditions, if we consider that casts might account for half of the surface soil layer [START_REF] Ponomareva | The role of earthworms in the creation of a stable structure in ley rotations[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF], what would be the microbial contribution to stable carbon? [START_REF] Oades | The retention of organic matter in soils[END_REF][START_REF] Cortez | Field decomposition of leaf litters: relationships between decomposition rates and soil moisture, soil temperature and earthworm activity[END_REF]. Le transfert de litière dans le sol et sa décomposition passe par l'action de la faune et des microorganismes du sol. Dans cette thèse, nous nous intéressons particulièrement aux vers de terre, considérés comme étant les ingénieurs du sol dominants dans les régions tempérées [START_REF] Lee | Earthworms: their ecology and relationships with soils and land use[END_REF]. Ces invertébrés ont un rôle clé dans la structuration du sol et le cycle du carbone [START_REF] Blouin | A review of earthworm impact on soil function and ecosystem services[END_REF]. Ils sont capables d'ingérer des composés organiques (restes de plantes et microorganismes) et minéraux (particules de sol), en différentes proportions en fonction de leur catégorie écologique. Pendant l'ingestion, le sol et les restes de plantes sont mélangés, associés au mucus produit par le ver de terre, puis rejetés le long des galeries ou sous forme de turricules déposés à la surface du sol [START_REF] Guggenberger | Soil organic matter within earthworm casts of an anecic-endogeic tropical pasture community, Colombia[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF]) (Figure 12). Les turricules sont des structures organo-minérales complexes où une partie du CO est rapidement minéralisée à court terme, alors qu'une autre partie est stabilisée à plus long terme [START_REF] Brown | Regulation of soil organic matter dynamics and microbial activityin the drilosphere and the role of interactionswith other edaphic functional domains[END_REF]. Il est reconnu que le comportement alimentaire des vers de terre dépend de la qualité de la litière et de sa palatabilité. Cependant, l'impact des vers de terre sur l'incorporation et la décomposition des racines dans le sol est peu connue [START_REF] Curry | The feeding ecology of earthworms -A review[END_REF][START_REF] Zangerlé | Do earthworms and roots cooperate to build soil macroaggregates? A microcosm experiment[END_REF]. L'objectif de cette thèse est d'étudier le devenir de racines et de parties aériennes dans les sols, en présence, ou en absence de vers de terre. Une expérimentation en mésocosmes a été mise en place afin de suivre l'incorporation de parties racinaires et aériennes de ray-grass marquées au carbone 13, en présence de vers de terre épi-anéciques (Lumbricus terrestris L.).

Le marquage artificiel de la litière permet de suivre son incorporation dans le sol et de différencier la matière organique (MO) ajoutée de celle initialement présente dans le sol [START_REF] Thompson | Pulse-labelling a cover crop with 13 C to follow its decomposition in soil under field conditions[END_REF][START_REF] Klumpp | Long-term steady state 13 C labelling to investigate soil carbon turnover in grasslands[END_REF][START_REF] Brüggemann | Carbon allocation and carbon isotope fluxes in the plantsoil-atmosphere continuum: a review[END_REF][START_REF] Comeau | Carbon input from 13C-labeled crops in four soil organic matter fractions[END_REF]Fayer et al., 2013). Il est important de noter que les racines et les parties aériennes ont été déposées à la surface du sol. Ce travail se concentre donc spécifiquement sur l'impact des caractéristiques chimiques de la litière sur le cycle du carbone du sol [START_REF] Rasse | Is soil carbon mostly root carbon? Mechanisms for a specific stabilisation[END_REF]. La les structures formées par les vers de terre contiennent de nombreux microorganismes, vivants ou morts, inclus dans les agrégats. Les microorganismes morts pourraient contribuer de manière importante au stock de carbone stable dans le sol à long terme. Si l'on considère que les turricules pourraient représenter la moitié du la surface du sol en condition naturelle [START_REF] Ponomareva | The role of earthworms in the creation of a stable structure in ley rotations[END_REF][START_REF] Six | A history of research on the link between (micro)aggregates, soil biota, and soil organic matter dynamics[END_REF], quelle serait alors la contribution des microorganismes au carbone stable ? [START_REF] Adler | Lignin chemistry-past, present and future[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF] [START_REF] Dinel | Soil lipids: origin, nature, content, decomposition, and effect on soil physical properties[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF] For the isotopic image, a mask was applied to remove the resin contribution, using a defined threshold for the 12 C 14 N -signal. Black regions hence represent either a hole on the section or resin. cp, chloroplast area; cw, cell wall; ep., epidermis cell; sg, starch grain. .

List of figures

List of tables

Table 1. Aliphatic lipids identified in soil samples and their principal sources (Otto and Simpson, 2005) 2. Capacity of soil microorganisms to decompose complex molecular structures [START_REF] Ekschmitt | Strategies used by soil biota to overcome soil organic matter stability-why is dead organic matter left over in the soil?[END_REF] The fate of 13 C labelled root and shoot litter in soil and earthworm casts: a multidisciplinary approach based on a mesocosm experiment Soil represents an important carbon sink, which inputs and outputs are governed by numerous biotic and abiotic factors. In the present study, we focused on two essential biotic factors: litter inputs, which account for the main source of SOM; and earthworms, which are key soil engineers in temperate regions. We aimed at investigating the fate of 13C-labelled Ryegrass root and shoot litter, in the soil and epi-anecic earthworm casts (Lumbricus terrestris), based on a one year mesocosm experiment. Soil samples were collected at two depths, as well as surface casts, at the beginning and regularly during the 54 weeks of experiment. In order to integrate SOM complexity and heterogeneous composition, we used a multidisciplinary approach at different spatio-temporal scales to quantify, localize and characterize the organic carbon incorporated in soil and earthworm casts. The chemical composition of roots and shoots had a significant impact on the fate of carbon in soil, with an increased contribution from root-derived carbon in soil and casts after one year. The chemical composition also tended to influence the litter palatability for earthworms. As organo-mineral associations tended to increase during drying and ageing of casts, physical interactions might have also played an important role in cast formation and evolution. We highlighted that earthworms tended to minimize the diverging fate of root and shoot residues on both soil and cast carbon, after the year of experiment. Thus, while studying soil carbon incorporation, decomposition and stabilization, the role of soil invertebrates should be particularly considered. We also observed the crucial role of microorganisms in decomposing litter and their potentially high participation to the preservation of carbon pool in soil and particularly in casts.

Key words -Soil organic carbon, litter, roots vs. shoots, earthworms, casts, isotopic analyses, molecular characterization, microscopic observations, microbiological analyses. Key words -Carbone organique du sol, litière, racines vs. parties aériennes, turricules, vers de terre, analyse isotopique, caractérisation moléculaire, observation microscopiques, analyses microbiologique.

  pour les nombreuses heures passées à observer mes échantillons et pour nos échanges scientifiques. Merci pour tes rires, ton soutien, ta délicatesse et nos envolées rêveuses chargées d'imaginaire. J'ai eu également la chance de collaborer avec Laurent Remusat que je remercie pour m'avoir fait confiance et m'avoir formée au NanoSIMS. Merci d'être resté avec moi de longues soirées au laboratoire, toujours dans la bonne humeur! Je tiens également à remercier Marie Alexis et Thanh Thuy Nguyen Tu pour les analyses complémentaires à celles de la thèse, ainsi que les discussions scientifiques et sur l'enseignement. Et merci à Thanh pour les chocolats de Pâques cachés dans les bureaux et les corrections de CV! Merci à Jérôme Mathieu pour son regard d'expert sur les vers de terre. Merci à Véronique Vaury pour les analyses isotopiques et ses desserts, aussi. Enfin, je tiens à remercier tout particulièrement Christelle Anquetil pour avoir participé à l'ensemble des analyses à l'échelle moléculaire. Merci Christelle pour ton humour débordant, ton soutien exemplaire et infaillible au quotidien, ainsi que le magnifique dessin sur ma porte! Je remercie également Céline Roose-Amsaleg, Laurence Millot-Cornette, Thomas Lerch, ainsi que Julie Leloup pour les analyses microbiologiques et l'aide à l'interprétation de mes résultats. Je remercie également les membres de mon comité de pilotage Isabelle Basile-Doelsch, Naoise Nunan, Yvan Capowiez, ainsi que François Baudin pour les discussions scientifiques que nous avons eues au sujet de ma thèse. Merci à Appoline Auclerc pour ses conseils sur les vers de terre et à Jean Christophe Lata pour le choqueur à vers de terre. Merci à Patrick Dumont, le jardinier de la serre qui avait toujours un oeil sur mes mésocosmes et m'a offert de nombreuses plantes que je garde précieusement. Sans oublier les jardiniers de l'UPMC qui m'ont aidé à vider les mésocosmes à la fin de l'expérimentation. Un grand merci également à tous les membres du couloir 56-66 dont je n'ai pas encore parlé qui m'ont permis de passer trois années dans une ambiance particulièrement sympathique et détendue: Maryse (que je n'embêterai plus avec mes vers de terre et mes chansons débiles), Arnaud (son poisson HDR et feu Bubulle), Agnès, Véro, Jean-Robert (pour les nombreuses fois où il m'a aidé dans les démarches administratives), Emmanuel (pour les analyses TOC), Sylvain. Merci aux stagiaires encadrés ou co-encadrés Anne-Sophie, Lucas et Elise qui ont fait des analyses sur mes échantillons. Merci à Valérie, Nadine et Nora pour le soutien administratif.

Figure 1 .

 1 Figure 1. Atmospheric concentrations of carbon dioxide (CO2) from Mauna Loa (red) and South Pole (black) since 1958 (IPCC, 2013).

Figure 2 .

 2 Figure 2. Overall balance of the global carbon cycle between 2004 and 2013 (Le Quéré et al., 2015).

Figure 3 .

 3 Figure 3. Simplified representation of a soil aggregate.

Figure 4 .

 4 Figure 4. (1) Historical and (2) emerging views of organic matter formation and preservation in the soil[START_REF] Schmidt | Persistence of soil organic matter as an ecosystem property[END_REF].

Figure 6 .

 6 Figure 6. Chemical structure of lignin precursors: (1) coumaryl/hydroxyphenyl alcohol, (2) coniferyl/guaiacyl alcohol and (3) sinapyl/syringyl alcohol (Kögel-Knabner, 2002).

Figure 12 .

 12 Figure 12. How do earthworm casts form? 1) Soil aggregates and plant residues are ingested by the earthworm; 2) In the gizzard, most aggregates are disrupted and plant residues are fragmented, with the aid of sand particles; 3) The presence of microbial activity, digestive enzymes and mucus favors the decomposition of organic compounds; 4) Organic and mineral compounds are rearranged with mucus and polysaccharides produced by microorganisms to form casts dejected by the earthworm.

Figure 15 .

 15 Figure 15. General objectives of the PhD thesis and the analytical tools used to fulfill this objective. EA-IRMS, elemental analysis -isotope ratio mass spectrometry; TEM, transmission electron microscopy; NanoSIMS, nano-scale secondary ion mass spectrometry; GC-MS, gas chromatograph-mass spectrometry; Py-GC-MS, pyrolysis-gas chromatographmass spectrometry; Q-PCR, real time Polymerase Chain Reaction; Biolog, Catabolic profiles of microbial communities by Biolog® EcoPlate™. The chapters referring to each approach are indicated under each analytical method.

Figure 18 .

 18 Figure 18. Samples collected at each time step and for each mesocosm.

  PowerSoil DNA Isolation Kit (Mo Bio, USA). The DNA concentration was quantified using Quant-iTTM dsDNA High-Sensitivity Kit (Invitrogen, Canada). DNA extracts were then diluted to 0.5 ng.µL -1 . Real time PCR was achieved, targeting the 16S rRNA gene for bacteria and the internal transcribed spacer (ITS) gene for fungi using a StepOne TM Real-Rime PCR (Applied BioSystem, USA). Primers were used to carry out the reaction: 341F 50for ITS. In a total reaction volume of 20 µL, each sample contained: 1 ng of DNA template, 7.5 µL of Power SYBR® Green PCR Master Mix (Applied Biosystem, USA) and 0.1 µM of each primer. The PCR program included an initial denaturation process which was achieved at 94°C for 10 s, annealing at 60°C and 55 °C (for 16S rDNA and ITS, respectively) for 30 s, extension at 72 °C for 10 s and 1 cycle and 30 s at 80°C. The melting curve was obtained by gradual heating from 65 to 95 °C at 0.1 °C/s, with fluorescence acquisition at 0.1 °C intervals. Copy number standard curves were obtained using 16S rRNA genes from Pseudomonas aeruginosa (strain 01), contained in PGEM-T easy (Promega) and linearized by SalI. Results were analyzed thanks to StepOne V 2.2.2 software.

Figure 19 .

 19 Figure 19. Schematic representation of a BiologTM microplate with the different carbon sources used as substrate in each well. For details, see Appendix 1.Plates were incubated at 30°C and absorbance at 590 nm was measured daily for 11 days using the SPECTROstar Nano (BMG LABTECH) microplate reader. The heterotrophic utilization of carbon sources resulted in the reduction of tetrazolium to purple formazan. The data from 5 days were used (Appendix 2) to determine the average well-color development (AWCD), calculated for each well following equation 4.

  available for this time step. No more residues were observed at the soil surface in the mesocosm containing earthworms. Mesocosm without earthworms and controls were visually similar to the previous time step.

Figure 20 .

 20 Figure 20. Visual appreciation of mesocosm evolution along the year of experiment.

  ) C lab in Root-E and NE tended to be higher than Shoot-E and NE, respectively. Contrary to other mesocosms, C lab in Shoot-E little varied from 8 th week until the end of experiment, with values of 1.13, 0.85 and 0.96 % after 8, 24 and 54 weeks, respectively. There was no significant difference of C lab between mesocosms in the 40-60 cm soil layer during the year of experiment (Figure21.B). However, at 54 weeks C lab reached 0.2 % in Root-E and Shoot-E whereas it remained lower than 0.05 % without earthworms.

Figure 21 .

 21 Figure 21. Proportion of carbon derived from labelled litter (C lab ) measured in the soil samples at the 0-20 cm (A) and 40-60 cm (B) layer, for the seven time steps, in mesocosms with labelled residues (n=3).The letters above histogram bars represent statistical results of Kruskal-Wallis test; different letters indicate significant difference between mesocosms, for a given step time. E: with earthworms, NE: without earthworms.

Figure 22 .

 22 Figure 22. Proportion of carbon derived from labelled litter (Clab) in casts sampled in mesocosms containing root (Cast-Root) or shoot residues (Cast-Shoot) (n=3). Letters above histogram bars represent statistical results of Kruskal-Wallis test, different letters indicate significant difference between mesocosms, for a given time step.

  example, after 24 weeks, the RC org was 1.1 and 0.89 for Root-NE and Shoot-NE, respectively. RC org in Control-E was generally lower than 1 during the whole experiment. The increase of RC org observed for Control-NE after 24 weeks can be explained by the development of mosses in this mesocosm from 24 weeks until the end of the experiment, potentially increasing the OC concentration in the soil 0-20 cm layer.

Figure 23 .

 23 Figure 23. Organic carbon content relative to the soil organic carbon content at the beginning of the experiment (RC org ) (n=3), for the 0-20 cm soil layer. The letters above histogram bars represent statistical results of Kruskal-Wallis test; different letters indicate significant difference between mesocosms, for a given time step.

Figure 24 .

 24 Figure 24. Organic carbon relative to the soil organic carbon at the beginning of the experiment (RCorg) measured in casts sampled in the mesocosms containing no litter (Cast-Control), root (Cast-Root) or shoot residues (Cast-Shoot) (n=3).The letters above histogram bars represent statistical results of Kruskal-Wallis test; different letters indicate significant differences between mesocosms, for a given time step.

Part 1

 1 was published in Organic Geochemistry. 101 (2016), 1-10 Molecular fate of root and shoot litter upon incorporation and decomposition in earthworm casts A. Vidal, K. Quenea, M. Alexis, S. Derenne Part 2 was published in Soil Biology and Biochemistry. 93 (2016), 8-16 Incorporation of 13 C labelled shoot residues in Lumbricus terrestris casts: A combination of Transmission Electron Microscopy and Nanoscale Secondary Ion Mass Spectrometry A.Vidal, L. Remusat, F. Watteau, S. Derenne, K. Quenea

  The litter and soil ingested by earthworms are transferred into casts which contain high concentration of OC compared to the soil (Chapter 1). It was shown in chapter 3 that earthworms were able to ingest roots and minimize the diverging fate of root and shoot residues in soil and casts. At the bulk isotopic scale, Cast-Root and Cast-Shoot presented approximately the same root and shoot contributions, respectively, along the year of experiment. One can wonder whether these trends are observed at the molecular scale?

Figure 25 .

 25 Figure 25. TIC trace of 650 °C Curie point pyrolysate of A, root litter; B, shoot litter and C, Cast-Control sample, after two weeks.Some compounds are illustrated with their structures. Aromatic compounds (red symbols) including compounds derived from: G for guiacyl ( ), S for syringyl ( )and H for coumaryl ( ), as well as other phenolic compounds ( ); aliphatic compounds (blue symbols) including methyl esters of n-alkanoic acids ( ), ωhydroxyalkanoic acids ( ), n-alkanedioic acids ( ), n-alkanes ( ), n-alkanols ( ), 8,16 -9,16 and 10, 16 or 18 dimethoxyhexadecanoic acids (DiOH), 9,10,18 trimethoxyoctadecanoic acid (TriOH) and contaminants (+). Numbers indicate chain length.Retention time(min)

  .A and B). F1 clearly separated Cast-Root from Cast-Control and Cast-Shoot samples. F2 clearly separated Cast-Control and Cast-Shoot samples (Figure 26.B). On the PCA, Cast-Control samples were represented mainly by the higher relative abundance of n-alkanoic acids (FAeven), ω-hydroxyacids (ωOH), n-alkanedioic acids (Dia) and C 16 dihydroxyacids (DiOH16) and was poorly represented by 3-(3,4-dimethoxyphenyl) 2-propenoic acid, methyl ester (G5).The differentiation between Cast-Shoot and Cast-Root reflected the main differences in compound relative abundances highlighted in the root and shoot chromatograms (Figure 26.A and B). Cast-Root samples were characterized by a high relative contribution of aromatic compounds and with a similar composition to root litter products (Figure 26.B). Aliphatic compounds were relatively more abundant in Cast-Control and Cast-Shoot samples. The latter could be further distinguished through the higher relative abundance of the long chain n-alkanes (Alk) and n-alkanols (OH) (Figure 26.B). The shoot litter sample was slightly distant from Cast-Shoot samples and was characterized as being opposite to Cast-Control samples. As a result, Cast-Shoot samples exhibit intermediate molecular characteristics between shoot litter and Cast-Control samples. Altogether, these observations revealed a clear contribution from root and shoot litter to the respective Cast-Root and Cast-Shoot samples. This was confirmed by the δ 13 C values of Cast-Root and Cast-Shoot compared with Cast-Control samples after two weeks (668, 550 and -26‰, respectively; Table

Figure 26 .

 26 Figure 26. PCA carried out with the 22 variables (see Table6for description) of the 15 cast samples, with root and shoot litter used as illustrative samples. A, distribution of variables on the correlation circle and B, distribution of the cast samples, with root and shoot litter illustrative samples. Numbers indicated next to samples correspond to the number of weeks after the beginning of the experiment.

(

  ωOH) were relatively more abundant in Cast-Control samples than other samples, the sum of these compounds was used to represent Cast-Control sample dynamics (Dia + ωOH) (Figure 27.A). As 4-methoxybenzoic acid methyl ester (P3) was the most abundant lignin-derived compound from root litter, its evolution along the year of experiment was used to follow the root contribution to Cast-Root samples (Figure 27.B). Finally, Cast-Shoot samples were characterized by higher n-alkane (Alk) and n-alkanol (OH) relative abundances than other samples, and the sum of these compounds was used to represent Cast-Shoot dynamics (Alk + OH) (Figure 27.C). For Control-Cast samples, the molecular composition barely evolved from 2 to 54 weeks of the experiment as shown by the homogenous group formed by the samples on the PCA (Figure 26.B) and the rather constant Dia + ωOH relative abundance between 4 and 54 weeks (Figure 27.A). However, between 2 and 4 weeks, the Dia + ωOH relative abundance slightly decreased. This trend was also observed for TriOH, FAeven and OH (Appendix3.A).

  according to the date of sampling (Figure 26.B): after (1) 2, 4 and 24 weeks, (2) 8 weeks and (3) 54 weeks. Cast-Root and Cast-Shoot samples collected after 8 weeks were molecularly closer to the root and shoot litter (to a lesser extent), respectively, than the samples from the first group. This observation was confirmed by the evolution along the year of P3 (Figure 27.B) and Alk + OH (Figure 27.C) relative abundance from Cast-Root and Cast-Shoot, respectively. Indeed, P3 and Alk + OH relative abundance increased between 4 and 8 weeks,

  Figure 27. Relative abundances of A,the sum of C 20 , C 22 , C 24 and C 26 ω-hydroxyalkanoic acids and C 22 and C 24 n-alkanedioc acids (Dia + ωOH); B, 4-methoxybenzoic acid methyl ester (P3) and C, The sum of C 25-33 n-alkanes and C 26-34 n-alkanols (Alk + OH) from Cast-Control, Cast-Root and Cast-Shoot samples.

  isotopic and OC content values after 54 weeks (Table

  relative abundance of Dia + ωOH increased during decomposition in both Cast-Root and Cast-Shoot samples (Figure 26.B and Figure 27.A), becoming closer to the Cast-Control characteristics. The same trend was observed for dihydroxyhexadecanoic acids (DiOH16) and trihydroxyoctadecanoic acid (TriOH), but to a lesser extent compared to Dia + ωOH (Appendix 3.B). This probably reflected the preservation of these compounds in earthworm casts along the year of the experiment, even though their production during degradation of root and shoot litter in Cast-Root and Cast-Shoot samples, respectively, cannot be excluded.

Figure 28 .

 28 Figure 28. The molecular fate of roots and shoots in earthworms casts.

  The mean δ 13 C value for plant tissue was 1004 ‰. Cell walls and chloroplasts presented different mean δ 13 C values, with 804 (standard deviation: 120) and 1041 (standard deviation: 57) ‰, respectively.

Figure 29 .

 29 Figure 29. TEM micrographs of initial shoots of Lolium multiflorum. cp, chloroplast area; cw, cell wall; ep, epidermis; sg, starch grain.

Figure 30 .

 30 Figure 30. NanoSIMS images of the 12 C 14 N -and δ 13 C in initial shoots of Lolium multiflorum.For the isotopic image, a mask was applied to remove the resin contribution, using a defined threshold for the 12 C 14 N -signal. Black regions hence represent either a hole on the section or resin. cp, chloroplast area; cw, cell wall; ep., epidermis cell; sg, starch grain.

Figure 31 .

 31 Figure 31. TEM micrographs of casts of Lumbricus terrestris. (a) Differently-sized organic and mineral particles, isolated or associated; (b) Packed plant cells; (c) Degraded cell walls; (d) Organomineral aggregate; (e) Organo-mineral and bacterial aggregates and (f) Fungi. aom, amorphous organic matter; b, bacteria; ba, bacterial aggregate; br, bacterial residue; cw, cell wall; f, fungi; fr, fungal residue; h, hole; m, mineral; s, bacterial spore.

  Aggregates showed variable proportion of OM -plant residues and microorganisms -and minerals. Organic constituents presented a large diversity of nature. Plant tissues could still be identified and they exhibited various degradation stages as a function of their fragmentation and degradation level (Figure 31.b Vegetal tissues were still recognizable even though cells were packed (Figure 31.b) or cell walls degraded (Figure 31.c). Many long and twisting 'laces' (Figure 31.a) ressembling to thin cell walls of initial litter (Figure 29), were therefore recognized as cell wall residues. Cell wall residues were either isolated (Figure 31.a) or included in aggregates with quartz or clays (Figure 31.a and d). Minerals were also present as isolated particles (Figure 31.a and d). Microorganisms were also frequently observed. As plant tissues were not completely degraded, bacteria colonized these residues (Figure 31.c). Bacteria could also be observed either isolated or forming colonies (Figure 31.a) and/or associated within aggregates (Figure 31.a and d). Amorphous OM, likely including exopolymers produced by bacteria was associated with minerals to form typical bacterial aggregates (Figure 31.e and f). Most of these bacteria still appeared intact, but some residues were present as bacterial cell walls (Figure 31.a and f) and spores, recognized for their dark core and coat (Willey et al., 2008) (Figure 31.c and e). Some fungi, intact or as residues were identified, based on their size ( > bacteria) and/or their thick cell wall as in extant organisms (Figure 31.f).

  .A). Structures identified as labelled microorganisms were mainly represented by individual bacteria in aggregates (Figure 32.A and B) or gathered as colonies (Figure 32.B). Labelled fungi were also observed (Figure 32.B). For exemple, on the figure 4, the δ 13 C for plant residues, bacteria and fungi reached 279, 304 and 191 ‰, respectively.

Figure 32 .

 32 Figure32. NanoSIMS maps of 16 O -, 28 Si -, 12 C 14 N -and δ 13 C illustrating the organic matter diversity (A) and highlighting the presence of individual bacteria and bacterial aggregate (B) in casts of Lumbricus terrestris. For the isotopic image, a mask was applied to remove the epoxy resin contribution, using a defined threshold for the 12 C 14 N -element. b, bacteria; ba, bacterial aggregate; cw, cell wall; f, fungi.

(

  Figure 34.a). Few microorganisms were present on roots (Figure 34.f) whereas none could be recognized on shoots. The same features were observed on NanoSIMS images, as reflected in Figure 35, for shoots and roots. The mean δ 13 C value, observed for plant tissues on roots or shoots NanoSIMS images, was 700‰ for the 3 images presented in Figure 35.

Figure 34 .

 34 Figure 34. TEM micrographs of initial shoots and roots. (a) chlorophyll parenchyma and epidermis cells; (b) and (e) woody tissue; (c) woody and parenchyma tissue; (d) parenchyma tissue and (f) microorganisms present in initial roots. b, bacteria; cp, chloroplast area; cw, cell wall; edc, electron dense compounds; ep, epidermis; f, fungus; sg, starch grain.

Figure 35 .Figure 36 .

 3536 Figure 35. NanoSIMS images of the 12 C 14 N -and δ 13 C in initial (a) shoots and (b), (c) roots of Lolium multiflorum. cw, cell wall.

  Figure 34.c preserved structures vs. Figure 36.a -partially degraded parenchyma cells and well preserved woody tissues). Both Cast-Shoot and Cast-Root images revealed the presence of preserved plant tissues (Figure 36.a and g), long and thin laces identified as parenchyma cell wall residues (Figure 36.b and f), organo-mineral aggregates (Figure 36.b and i) and microorganisms (Figure 36.c, d, e and g). However, intact/barely degraded plant structures were more abundant in Cast-Root compared to Cast-Shoot, while Cast-Shoot was more represented by organo-mineral associations, compared to Cast-Root. On Cast-Root images, mineral particles were either isolated (Figure 36.g), regrouped in mineraldominated aggregates (Figure 36.i) and/or rarely associated with plant structure. Microorganisms, mainly fungi and bacteria, were more frequently observed on Cast-Shoot images compared to Cast-Root ones. Fungi were identified in woody tissues, attacking cell walls (Figure 36.d) and inside organo-mineral aggregates (Figure 36.c). Bacteria were either isolated, at the center of parenchyma cells (Figure 36.a) or inside organo-mineral aggregates (Figure 36.c). Colonies of bacteria were observed on plant cell walls (Figure 36.e). Some microorganisms were also revealed in Cast-Root (Figure 36.h and g), but to a lesser extent compared to Cast-Shoot. Similar areas identified on cast TEM images could be recognized on cast NanoSIMS images. For Cast-Shoot, Figure 36.c and b were comparable to Figure 37.a and b, respectively. For Cast-Root, Figure 36.g and h were comparable to Figure 37.c and d, respectively. NanoSIMS Cast-Shoot images confirmed that labelled microorganisms and degraded plant structures were integrated into organo-mineral aggregates (Figure 37.a and b, respectively). Labelled plant structures observed on NanoSIMS Cast-Root images presented a lower degree of degradation and interaction with mineral particles (Figure 37.c and d).

Figure 37 .

 37 Figure 37. NanoSIMS maps of 28 Si-, 12 C 14 N -and δ 13 C illustrating contrasted degradation states of labelled plant tissues and microorganisms implied in their decomposition, after 8 weeks of experiment, for both Cast-Shoot (a and b) and Cast-Root (c and d). aom, amorphous organic matter; b, bacteria; cw, cell wall; f, fungus.

Figure 38 .

 38 Figure 38. TEM micrographs of Cast-Shoot and Cast-Root samples after 54 weeks of experiment. (a), (b), (c) and (g) organo-mineral aggregates; (d) partially degraded woody tissues; (e) cell walls residues; (f) highly degraded cells with the cell intersection left (g) microorganisms. aom, amorphous organic matter; b, bacteria; br, bacterial residue; cw, cell wall; ci, cell intersection; f, fungus; fi, fingerprint of past bacterial activity; s, spore forming bacteria. A focus of (a) and (b) images, corresponding to the red scare, is available in the discussion (Figure 40).

Figure 39 .

 39 Figure 39. NanoSIMS maps of 28 Si -, 12 C 14 N -and δ 13 C illustrating the integration of labelled organic structures (both plant tissues and microorganisms) inside organo-mineral aggregates, after 54 weeks of experiment, for both Cast-Shoot (a and b) and Cast-Root (c and d). b, bacteria; cw, cell wall; d, 'diffused' labelling; f, fungus; mi, microorganism (bacteria or fungi).

  .a) and roots (Figure 35.b and c)

  Figure 37.a). Parenchyma cells were submitted to an important degradation, while woody tissues remained often intact (Figure 36.a). Moreover, as observed on Figure 36.a, most of

Figure 40 .

 40 Figure 40. (a) Focus on fig. 38 a and (b) fig. 38 b, illustrating the composition of organic residues appearing as pores on 12 C 14 N images of fig. a. aom, amorphous organic matter; br, bacterial residues; cw, cell wall.

Figure 41 .

 41 Figure 41. Example of TEM micrographs of the 0-20 µm fraction of a soil sample collected after 54 weeks of experiment (in the mesocosm Shoot-E) illustrating the presence of small aggregates (a and c), as well as the microbial presence in soil samples (b). aom, amorphous organic matter; b, bacteria; mi, microorganisms.

  for cast samples collected after 54 weeks, the labelling was either 'diffused' (Figure 43.a and b) or forming spots (Figure 43.c) which could be identified as microbial clusters as observed on TEM images (Figure 42). As proposed in section 4.3, this 'diffused' labelling might either be attributed to (1) dissolved and/or highly degraded OM, (2) microbial products and/or (3) dead microbial residues (even though we cannot exclude a methodological bias). As concluded in the Chapter 4, these observations highlighted the importance of living or dead microorganisms on carbon cycling and potential carbon stabilization in soils.

Figure 42 .

 42 Figure 42. Example of TEM micrographs of the 20-50 µm fraction of a soil sample collected after 54 weeks of experiment (in the mesocosm Shoot-E).This image corresponds to a mosaic of 4 images. b, bacteria; ci, cell intersection; fi, fingerprint of past bacterial activity.

Figure 43 .

 43 Figure 43. NanoSIMS maps of 28 Si -, 12 C 14 N -and δ 13 C illustrating the labelled organic structures inside organo-mineral aggregates of soil 0-20 µm (A) and 20-50 µm (B and C) samples, after 54 weeks of experiment. d, diffused labelled; b, possible bacterial cluster; e, possible edge effect.

Figure 44 .

 44 Figure 44. TIC trace of 650 °C Curie point pyrolysate of A, Control-NE; B, Cast-Control, after 54 weeks of experiment.Some compounds are illustrated with their structures. Aromatic compounds (red symbols) including compounds derived from: G for guiacyl ( ), S for syringyl ( ) and H for coumaryl ( ), as well as other phenolic compounds ( );aliphatic compounds (blue symbols) including methyl esters of n-alkanoic acids ( ),ω-hydroxyalkanoic acids( ),n-alkanedioic acids ( ), n-alkanols ( ), 8,16 -9,16 and 10, 16 or 18 dimethoxyhexadecanoic acids (DiOH) and 9,10,18 trimethoxyoctadecanoic acid (TriOH) Numbers indicate chain length.

  two factors explaining 58 % of the variance (Figure 45.A and B). F1 separated casts from soil samples. F2 separated samples collected in mesocosms incubated with roots (Root-NE, Root-E and Cast-Root) from samples either collected in mesocosms incubated with shoots (Shoot-NE, Shoot-E and Cast-Shoot) or without litter (Control-NE, Cantrol-E and Cast-Control) (Figure 45.B). Cast vs. soil samples Control-NE and Cast-Control, presented the same pyrolysis products, with similar relative abundances. However, the PCA clearly separated cast from soil samples. Compared to the soil, cast samples were characterized by a slightly lower relative abundance of n-alkanols (OH), n-alkanoic acids (FAeven) and n-alkanedioc acids (Dia) (Figure 45 and Figure 46.A).

Figure 46 .

 46 Figure 46. Relative abundances of A, the sum of C 20 to C 30 even chain n-alkanoic acids, C 26 to C 30 even chain n-alkanols, as well as C 22 to C 26 n-alkanedioic acids; B, 4-methoxybenzoic acid methyl ester (P3) and C, the sum of C 20 , C 22 , C 24 and C 26 ω-hydroxyalkanoic acids, as well as mono unsaturated ω-hydroxyalkanoic acids and n-alkanedioc acids C 18 (ωOH + ωOH18uns + Dia18uns) from cast and soil samples, after 54 weeks of experiment.

  P3 and ωOH + ωOH18uns + Dia18uns clearly separated samples containing rootderived compounds compared to others, DiOH and TriOH did not enable the separation between samples containing shoot-derived compounds and others, contrary to what was anticipated in section 5.2.1.1.

Figure 47 .

 47 Figure 47. TIC trace for lipid extracts of A, initial root litter and B, initial shoot litter. Aromatic compounds (red symbols) including compounds derived from guiacyl ( ), 3,4,5trimethoxy benzaldehyde ( )and 3-(4-methoxyphenyl) 2-propenoic acid, methyl ester ( ), and 4-methoxybenzoic acid ( );aliphatic compounds: n-alkanes ( ); n-alkanoic acids ( ); nalkanedioic acids ( ); n-alkanols ( ); Sterols: Ch, cholesterol ( ); St, stigmasterol ( ); Si, sitosterol ( ). Numbers indicate chain length.

Figure 48 .

 48 Figure 48. TIC trace for lipid extracts of A, Control-NE and B, Cast-Control, after 54 weeks of experiment. 4-methoxybenzoic acid ( ); n-alkanes ( ); methyl ester aliphatic compounds: n-alkanoic acids ( ); ω-hydroxyalkanoic acids ( ); n-alkanols ( ); Sterols: Ch, cholesterol ( ); St, stigmasterol ( ); Si, sitosterol ( ). Numbers indicate chain length. A focus was made on the only qualitative difference observed between Control-NE and Cast-Control. As both samples were very similar, only the dominant molecules are indicated on the chromatogram of Cast-Control.

Figure 49 .

 49 Figure 49. Abundance of bacteria and fungi obtained by Q-PCR analyses (n=3).The standard error is represented. Letters above histogram bars represent statistical results of Kruskal-Wallis test; different letters indicating significant difference (p-value < 0.05).

composition is described in Appendix 1 .

 1 The position on each of the six axes indicates the mean potential for the degradation of a given family of substrate by the cultivable microbial community. As observed using AWCD (Figure 50.A), the microbial community from Cast-Root and Cast-Shoot present a significantly higher capacity to use carbon substrates, whatever the family of substrate, compared to Cast-Control (Figure 51.A). Microbial communities from Cast-Root were generally less adapted to use phenolic compounds and carboxylic acids (to a lesser extent) and slightly better able to use polymers, compared to Cast-Shoot. The metabolic potential of microbial communities was highly variable across soil samples collected in different mesocosms, at 0-20 cm (Figure 51.B). Communities from Root-NE presented almost identical metabolic potentials compared to Cast-Root. Microbial communities from Root-E were particularly less adapted to use polymers, phenolic compounds and carboxylic acids, compared to Root-NE. Communities from Shoot-NE, presented better capacities to use most substrates (except carbohydrates and polymers), compared to Shoot-E. Finally, Shoot-E communities were hardly differentiated from control 0observed using AWDC, microbial communities from Root-NE and Shoot-NE collected at 40-60 cm presented lower metabolic capacities compared to those collected at 0-20 cm (Figure 51.C). Microbial communities from Root-NE presented higher capacities to use phenolic compounds compared to the other samples. Communities from Root-E collected at 40-60 cm presented the same metabolic profile but in lower intensity, compared to those collected at 0-20 cm.

Figure 50 .Figure 51 .

 5051 Figure 50. A, Average well color development (AWCD) and B, functional richness (FR) obtained for cast and soil samples, after 54 weeks of experiment (n=3).The standard error is represented. Letters above histogram bars represent statistical results of Kruskal-Wallis test; different letters indicating significant difference (p-value < 0.05). No statistically significant differences were observed for FR.

  .A and B). Communities from all control samples, with the exception of Control-E collected at 40-60 cm, were particularly adapted to use i-Erythritol, β-Methyl-D-glucoside, α-D-Lactose and α-Ketobutyric acid. Communities from Cast-Root, Cast-Shoot and Root-NE tended to be more adapted to use α-Cyclodextrin, Glycogen and L-Phenylalanine compared to other samples. Finally, communities from Control-E, Root-E and Shoot-E collected at 40-60 cm were globally less capable to use most of the substrates used in the PCA.

Figure 52 .

 52 Figure 52. PCA carried out with the 12 variables of the 15 soil and cast samples. Each variable represents the metabolic potential of microbial communities to use a given carbon substrate. A, distribution of variables on the correlation circle and B, distribution of the soil and cast samples.

Figure 53 .

 53 Figure 53. Simplified summary of main conclusions and perspectives (in blue -perspective, numbers indicate the issue number where a specific point is discussed).

Figure 54 .

 54 Figure 54. Potential of TEM images to illustrate the different state of bacterial degradation within bacterial aggregate.

  Dans cette thèse nous nous concentrons sur deux facteurs clés dans la régulation du cycle du carbone des sols -la qualité de la litière (ex. caractéristiques physico-chimiques et structure anatomique) et la faune du sol.La source principale de la MOS provient de la litière. La qualité de la litière détermine : combien de carbone sera incorporé au sol, combien de temps prendra la décomposition de cette litière et quel sera son impact final sur le stock de carbone stable du sol. Les parties aériennes de la plante ont longtemps été considérées comme la source principale de la MOS.Pourtant, les racines sont aujourd'hui reconnues comme étant des collaboratrices indispensables à la formation de carbone stable dans le sol. La contribution relative des racines et des parties aériennes à la MOS a fait l'objet de nombreuses études ces dix dernières années. Il est maintenant reconnu que les racines se décomposent plus lentement que les parties aériennes. Cependant, les raisons permettant d'expliquer la décomposition plus lente des racines et leur plus forte contribution au carbone stable du sol restent largement débattues dans la communauté scientifique. Ce phénomène est-il lié à la composition chimique, à la protection physique et/ou à la protection physico-chimique des racines dans le sol(Rasse et al., 2005) (Figure9).

Figure 9 .

 9 Figure 9. Hypothèses proposées pour expliquer la forte contribution du carbone dérivé des racines dans le sol (Rasse et al., 2005).

Figure 12 .

 12 Figure 12. Comment se forment les turricules de vers de terre? 1) Le sol et les restes de plantes sont ingérés par le ver de terre; 2) Dans le gésier, les pluparts de agrégats du sol sont détruits et les restes de plantes sont fragmentés, à l'aide des particules de sable; 3) La présence de microorganismes, d'enzymes digestives et de mucus favorisent la décomposition des composés organiques; 4) Les composés organiques et minéraux sont associés avec le mucus et les exsudats produits par les microorganismes (ex. polysaccharides), puis rejetés sous forme de turricules par le ver de terre.

  Figure 15. Objectifs généraux de la thèse et méthodes employées pour répondre à ces objectifs. EA-IRMS, analyseur élémentaire couplé à un spectromètre de masse ; TEM, microscope électronique à transmission; NanoSIMS, Nano Secondary Ion Mass Spectrometer ; GC-MS, chromatographie gazeuse couplée à un spectromètre de masse ; Py-GC-MS, pyrolyse couplée à la chromatographie gazeuse et à un spectromètre de masse ; q-PCR, réaction en chaîne par polymérase en temps réel ; Biolog, profils cataboliques de la communauté microbienne par Biolog® Ecoplate™. Les chapitres où les méthodes analytiques sont utilisées sur les échantillons sont indiqués en dessous de chaque encadré.

Figure 21 .

 21 Figure 21. Pourcentage de carbone issu de la litière marquée (C lab ) calculé à partir du δ 13 C (analyses AE-IRMS) pour les échantillons de sol, prélevé à 0-20 cm. Root, partie racinaire ; Shoot, partie aérienne ; E, avec vers de terre ; NE, Sans vers de terre.

Figure 22 .

 22 Figure 22. Pourcentage de carbone issu de la litière marquée (C lab ) calculé à partir du δ 13 C (analyses AE-IRMS) pour les échantillons de turricules. Cast-Root, turricules prélevés dans le mésocosme contenant des parties racinaires ; Cast-Shoot turricules prélevés dans le mésocosme contenant des parties aériennes.

Figure 26 .BFigure 37 . 3 .

 26373 Figure 26. Analyse en composante principale obtenue à partir de 22 variables (cf. Tableau 6 pour la description de toutes les variables) sur 15 échantillons de turricules. Les parties racinaires et aériennes sont utilisées comme individus illustratifs. A, distribution des variables sur le cercle de corrélation et B, distribution des échantillons. Les nombres indiquent les semaines de prélèvement des échantillons après le début de l'expérimentation.

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 12345 Figure 1. Atmospheric concentrations of carbon dioxide (CO2) from Mauna Loa (red) and South Pole (black) since 1958 (IPCC, 2013). .......................................................................... 20 Figure 2. Overall balance of the global carbon cycle between 2004 and 2013 (Le Quéré et al., 2015). ........................................................................................................................................ 20 Figure 3. Simplified representation of a soil aggregate........................................................... 22 Figure 4. (1) Historical and (2) emerging views of organic matter formation and preservation in the soil (Schmidt et al., 2011). ............................................................................................. 25 Figure 5. Model of spruce lignin[START_REF] Adler | Lignin chemistry-past, present and future[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. ................................ 29

Figure 6 .Figure 7 .Figure 8 .

 678 Figure5. Model of spruce lignin[START_REF] Adler | Lignin chemistry-past, present and future[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. ................................ 29 Figure 6. Chemical structure of lignin precursors: (1) coumaryl/hydroxyphenyl alcohol, (2) coniferyl/guaiacyl alcohol and (3) sinapyl/syringyl alcohol (Kögel-Knabner, 2002). ............ 29 Figure 7. Proposed models and monomer structures of (1) cutin and (2) suberin (Kögel-Knabner, 2002; Kolattukudy, 1980). ........................................................................................ 31 Figure 8. Major components of lipids in plant and microorganisms identified in soils[START_REF] Dinel | Soil lipids: origin, nature, content, decomposition, and effect on soil physical properties[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. ......................................................................................... 33

Figure 9 .Figure 10 .Figure 11 .Figure 12 .Figure 13 .Figure 14 .Figure 15 .Figure 17 .Figure 18 .Figure 19 .Figure 20 .Figure 21 .Figure 22 .Figure 23 .Figure 24 .Figure 25 .Figure 26 .Figure 27 .Figure 28 .Figure 29 .Figure 30 .

 91011121314151718192021222324252627282930 Figure 8. Major components of lipids in plant and microorganisms identified in soils[START_REF] Dinel | Soil lipids: origin, nature, content, decomposition, and effect on soil physical properties[END_REF][START_REF] Kögel-Knabner | The macromolecular organic composition of plant and microbial residues as inputs to soil organic matter[END_REF]. ......................................................................................... 33 Figure 9. Processes explaining the protection of root derived carbon in the soil (Rasse et al., 2005). ........................................................................................................................................ 36 Figure 10. The size spectrum of soil constituents (Totsche et al., 2010). ............................... 36 Figure 11. Formation of non-living SOM from microbial necromass (Miltner et al., 2012). . 41 Figure 12. How do earthworm casts form? 1) Soil aggregates and plant residues are ingested by the earthworm; 2) In the gizzard, most aggregates are disrupted and plant residues are fragmented, with the aid of sand particles; 3) The presence of microbial activity, digestive enzymes and mucus favors the decomposition of organic compounds; 4) Organic and mineral compounds are rearranged with mucus and polysaccharides produced by microorganisms to form casts dejected by the earthworm. ..................................................................................... 43 Figure 13. Approximate organic carbon (t.ha-1.an-1) balance during its ingestion, assimilation and dejection by earthworms (in blue: the fate of organic carbon after cast formation) (Adapted from Bouché, 2014). ............................................................................... 44 Figure 14. Simplified representation of the effect of earthworms on the soil organic carbon (Corg) at different time and spatial scale (Adapted from Brown et al., 2000). ........................ 45 Figure 15. General objectives of the PhD thesis and the analytical tools used to fulfill this objective. EA-IRMS, elemental analysis -isotope ratio mass spectrometry; TEM, transmission electron microscopy; NanoSIMS, nano-scale secondary ion mass spectrometry; GC-MS, gas chromatograph-mass spectrometry; Py-GC-MS, pyrolysis-gas chromatographmass spectrometry; Q-PCR, real time Polymerase Chain Reaction; Biolog, Catabolic profiles of microbial communities by Biolog® EcoPlate™. The chapters referring to each approach are indicated under each analytical tool. .................................................................................. 49 Figure 16. A. Experimental design. E: with earthworms, NE: without earthworms; B. Illustration of mesocosms used in the experiment. .................................................................. 53

Figure 31 .Figure 32 .Figure 34 .Figure 35 .Figure 36 .Figure 37 .Figure 38 .Figure 39 .Figure 41 .Figure 42 .Figure 43 .Figure 44 .Figure 45 .Figure 46 .Figure 47 .Figure 48 .Figure 49 .Figure 51 .Figure 53 .Figure 54 .

 3132343536373839414243444546474849515354 Figure30. NanoSIMS images of the 12 C 14 N -and δ 13 C in initial shoots of Lolium multiflorum. For the isotopic image, a mask was applied to remove the resin contribution, using a defined threshold for the 12 C 14 N -signal. Black regions hence represent either a hole on the section or resin. cp, chloroplast area; cw, cell wall; ep., epidermis cell; sg, starch grain. ........................ 95 Figure 31. TEM micrographs of casts of Lumbricus terrestris. (a) Differently-sized organic and mineral particles, isolated or associated; (b) Packed plant cells; (c) Degraded cell walls; (d) Organo-mineral aggregate; (e) Organo-mineral and bacterial aggregates and (f) Fungi. aom, amorphous organic matter; b, bacteria; ba, bacterial aggregate; br, bacterial residue; cw, cell wall; f, fungi; fr, fungal residue; h, hole; m, mineral; s, bacterial spore. .......................... 96 Figure 32. NanoSIMS maps of 16 O -, 28 Si -, 12 C 14 N -and δ 13 C illustrating the organic matter diversity (A) and highlighting the presence of individual bacteria and bacterial aggregate (B) in casts of Lumbricus terrestris. For the isotopic image, a mask was applied to remove the epoxy resin contribution, using a defined threshold for the 12 C 14 N -element. b, bacteria; ba, bacterial aggregate; cw, cell wall; f, fungi. .............................................................................. 98 Figure 33. δ 13 C histograms for bacteria and plant residues, with a 50 ‰ interval. The number of manually drawn ROI was 38 and 66, for plant residues and bacteria, respectively. ........... 99 Figure 34. TEM micrographs of initial shoots and roots. (a) chlorophyll parenchyma and epidermis cells; (b) and (e) woody tissue; (c) woody and parenchyma tissue; (d) parenchyma tissue and (f) microorganisms present in initial roots. b, bacteria; cp, chloroplast area; cw, cell wall; edc, electron dense compounds; ep, epidermis; f, fungus; sg, starch grain. ................. 105 Figure 35. NanoSIMS images of the 12 C 14 N -and δ 13 C in initial (a) shoots and (b), (c) roots of Lolium multiflorum. cw, cell wall. ........................................................................................ 106 Figure 36. TEM micrographs of Cast-Shoot and Cast-Root samples after 8 weeks of experiment.(a) Partially degraded parenchyma cells and well-preserved woody tissues; (b) organo-mineral aggregate; (c) fungus in an organo-mineral aggregate; (d) fungi attacking woody tissues; (e) bacteria inside cell walls; (f) and (h) compacted cell walls; (g) intact cell walls and (i) organo-mineral aggregate dominated by mineral particles. aom, amorphous organic matter; b, bacteria; cw, cell wall; ci, cell intersection; f, fungus. .............................. 107 Figure 37. NanoSIMS maps of 28 Si -, 12 C 14 N -and δ 13 C illustrating contrasted degradation states of labelled plant tissues and microorganisms implied in their decomposition, after 8 weeks of experiment, for both Cast-Shoot (a and b) and Cast-Root (c and d). aom, amorphous organic matter; b, bacteria; cw, cell wall; f, fungus. .............................................................. 109 Figure 38. TEM micrographs of Cast-Shoot and Cast-Root samples after 54 weeks of experiment. (a), (b), (c) and (g) organo-mineral aggregates; (d) partially degraded woody tissues; (e) cell walls residues; (f) highly degraded cells with the cell intersection left (g) microorganisms. aom, amorphous organic matter; b, bacteria; br, bacterial residue; cw, cell wall; ci, cell intersection; f, fungus; fi, fingerprint of past bacterial activity; s, spore forming bacteria. A focus of (a) and (b) images, corresponding to the red scare, is available in the discussion (Figure 40). .......................................................................................................... 111 Figure 39. NanoSIMS maps of 28 Si -, 12 C 14 N -and δ 13 C illustrating the integration of labelled organic structures (both plant tissues and microorganisms) inside organo-mineral aggregates,

  . ........................................................................................................................ 33 Table

Table 3 .

 3 . ........................................................................................................... 39 Initial composition of the soil used in the experiment. ............................................. 52 Table 4. Initial litter characterization (n=13). Numbers in parentheses indicate the standard deviation. .................................................................................................................................. 53 Table 5. Summary of analyses performed and discussed in this manuscript.Colors indicate the chapter concerned by a given analysis (pink for chapter 3, brown for chapter 4 and yellow for chapter 5). Asterisks (*) indicate analyses performed on the 3 replicates of soil. For other analyses, soil replicates were pooled to form a composite sample. ......................................... 55 Table 6. Pyrolysis products used for the PCA.Most of the common names for the nonmethylated form of aromatic compounds are indicated between brackets; the probable origins and/or significance of each product or group of products, with key references are also mentioned. ................................................................................................................................ 85 Table 7. δ 13 C values and OC content for Cast-Control, Cast-Root and Cast-Shoot samples over time (numbers in parentheses indicate the standard deviation, n = 3). ............................ 87 Table 8. δ 13 C values for the total NanoSIMS image, labelled plant residues, bacteria and fungi.The total δ 13 C values of NanoSIMS images were obtained using a same threshold defined thanks to the 12 C 14 N -distribution. For each category; -n-: number of manually drawn ROI used to obtain the mean δ 13 C values, the minimum and maximum of the δ 13 C values are also indicated. The images 5 and 6 correspond to Figs. 4 and 5, respectively. ........................ 99 Table 9. δ 13 C values and organic carbon (OC) content for cast and soil samples after 54 weeks of experiment (numbers in parentheses indicate the standard deviation, n = 3). ........ 133 VIDAL Alix -Thèse de doctorat -2016

  Devenir de litières de parties racinaires et aériennes marquées au 13 C dans le sol et les turricules de vers de terre: une approche pluridisciplinaire basée sur une expérimentation en mésocosmes. Le sol représente un puits de carbone important, dont les entrées et les sorties sont gouvernées par de nombreux facteurs biotiques et abiotiques. Dans cette étude, nous nous sommes intéressés à deux facteurs biotiques essentiels : la qualité de la litière, principale source des MOS ; et les vers de terre, ingénieurs clés des sols tempérés. Nous avons étudié le devenir de parties racinaires et aériennes, marquées au carbone 13, dans le sol et dans des turricules de vers de terre épi-anéciques (Lumbricus terrestris), au cours d'une expérimentation en mésocosmes d'une année. Des échantillons de sol ont été prélevés à deux profondeurs, ainsi que des turricules de surface, au début et pendant l'expérience qui a été menée pendant 54 semaines. Afin d'appréhender la complexité et la composition hétérogène des SOM, nous avons utilisé une approche pluridisciplinaire à différentes échelles spatio-temporelles pour quantifier, localiser et caractériser le carbone organique incorporé dans le sol et les turricules. La composition chimique des parties racinaires et aériennes a un impact significatif sur le devenir du carbone dans le sol. La contribution des racines, par rapport aux parties aériennes est plus importante dans le sol et les turricules, après une année d'expérience. La composition chimique semble également influencer la palatabilité de la litière pour les vers de terre. Les associations organo-minérales ont tendance à augmenter au cours du séchage et du vieillissement des turricules. Après une année, d'expérience, les interactions physiques semblent donc également jouer un rôle important dans la formation et l'évolution des turricules. Nous avons mis en évidence que les vers de terre avaient tendance à minimiser l'effet contrasté des parties racinaires et aériennes sur le carbone du sol et des turricules, après un an d'expérience. Il est donc important de prendre en compte les invertébrés du sol lorsqu'on étudie les processus d'incorporation, de décomposition et de stabilisation du carbone dans le sol. Nous avons aussi observé le role essentiel des microorganismes dans la décomposition de la litière, ainsi que leur participation potentiellement importante à la préservation du carbone dans le sol, et ce particulièrement dans les turricules.

Main effect of earthworms on soil OC

		Mineralization enhanced	Stabilization enhanced	Transport of OC in the subsurface
		Earthworm gut Fresh casts and burrows	Drying and ageing casts and burrows	Soil profil
	Time scale	Hours	Days and weeks	Months and years

E NE Root Control Shoot contained

  Two mesocosms contained 250 g of shoots, two others 250 g of roots and the latest two were used as control. Ground roots and shoots were deposited on the soil surface.

	Characteristics	Units	Values
	Clay (< 2 μm)	g.kg -1	189
	Loam (2-50 μm)	g.kg -1	248
	Sand (50-2000 μm)	g.kg -1	563
	Total carbonates (CaCO 3 ) g.kg -1	19.0
	CEC Metson	cmol.kg -1 9.90
	Organic Carbon	g.kg -1	12.1
	Nitrogen	g.kg -1	1.30

Figure 16. A. Experimental design. E: with earthworms, NE: without earthworms; B. Illustration of mesocosms used in the experiment.

2.1.2. Root-shoot residues and earthworms

	Plants of Italian Ryegrass (Lolium multiflorum Lam.) were artificially labelled with 13 C at the
	Atomic Energy and Alternative Energies Commission (CEA) in Cadarache (France). In order
	to obtain homogeneous material, plants were grown under a controlled and constant 13 CO 2
	enriched atmosphere. Root and shoot were separated and dried. They were chopped during
	40 seconds with a laboratory blender (Waring Commercial) in order to obtain residues of few
	millimeters. The mean δ 13 C isotopic values were 1324 ‰ (± 42) and 1632 ‰ (± 16) for roots
	and shoots, respectively (Table 4). The method used to obtain δ 13 C, carbon and nitrogen is
	described in section 2.2.1.
	Table 4. Initial litter characterization (n=13). Numbers in parentheses indicate the standard
	deviation.
		δ 13 C	C	N	C:N
	Units	‰	g.kg -1	g.kg -1	-
	Roots	1323 (42)	402 (40)	13.1 (1.2) 30.7 (3.1)
	Shoots	1632 (16)	408 (6.3)	27.7 (2.3) 14.8 (1.1)

Table 5 .

 5 Summary of analyses performed and discussed in this manuscript. Colors indicate the chapter concerned by a given analysis (pink for

chapter 3, brown for chapter 4 and yellow for chapter 5). Asterisks

			(*) indicate analyses performed on the 3 replicates of soil.
	For other analyses, soil replicates were pooled to form a composite sample.
	Analysis type	Method	Casts	Soil 0-

20 cm Soil 40-60 cm Time step(s) concerned

			All 8, 24, 54 54 All	54	All	54
	Isotopic	EA-IRMS	*		*
	Molecular	Py-GC-MS GC-MS (lipids)
	Microscopic	TEM NanoSIMS
	Microbiologic	Biolog Q-PCR		* *	* *
	2

.2.1. δ 13 C analyses and organic carbon

Table 7 .

 7 δ 13 C values and OC content for Cast-Control, Cast-Root and Cast-Shoot samples over time (numbers in parentheses indicate the standard deviation, n = 3).

			δ 13 C (‰)		Organic carbon (C) (g.kg -1 )
	Weeks	Cast-Control	Cast-Root	Cast-Shoot	Cast-Control	Cast-Root	Cast-Shoot
	2	-25.9 (0.9)	668 (7.5)	550 (8.9)	17.9 (1.2)	25.7 (1.0)	23.9 (0.5)
	4	-24.5 (1.5)	351 (3.7)	505 (11)	17.3 (0.5)	26.6 (0.2)	24.7 (1.8)
	8	-25.3 (1.3)	673 (25)	938 (3.0)	19.0 (2.2)	55.0 (0.8)	34.9 (1.2)
	24	-27.5 (0.2)	671 (1.3)	627 (12)	18.8 (0.1)	42.7 (1.6)	29.8 (1.4)
	54	-28.6 (0.1)	127 (8.2)	168 (1.9)	16.8 (2.5)	22.2 (0.6)	20.6 (1.6)

Table 8 .

 8 δ 13 C values for the total NanoSIMS image, labelled plant residues, bacteria and fungi.The total δ 13 C values of NanoSIMS images were obtained using a same threshold defined thanks to the 12 C 14 N -distribution. For each category; -n-: number of manually drawn ROI used to obtain the mean δ 13 C values, the minimum and maximum of the δ 13 C values are also indicated. The images 5 and 6 correspond to Figs.4 and 5, respectively.

	Images id Total Plant residues	Bacteria			Fungi
	Units	‰		‰				(min ; max) ‰			(min ; max) ‰
	1	72		250 (n=4)	(22 ; 401)	186 (n=13) (14 ; 497)	NA
	2	104		253 (n=12) (20 ; 509)	395 (n=4) (122 ; 597) NA
	3	-5		129 (n=1)	-	408 (n=7) (109 ; 943) NA
	4	47		176 (n=6)	(45 ; 326)	483 (n=2) (258 ; 707) NA
	5	10		279 (n=3)	(201 ; 358) 304 (n=17) (109 ; 723) 191 (n=1)
	6	3			158 (n=4)	(31 ; 270)	236 (n=12) (144 ; 489) NA
	7	148		421 (n=8)	(281 ; 633) 326 (n=11) (115 ; 665) NA
		12
		10
	Number of ROI	4 6 8									Bacteria Plant residues
		2
		0
		50 0-	100 50-	150 100-	200 150-	250 200-	300 250-	350 δ13C intervals (‰) 400 450 500 550 600 300-350-400-450-500-550-	650 600-	700 650-	750 700-	800 750-	850 800-	900 850-	950 900-	1000 950-

Figure 33. δ 13 C histograms for bacteria and plant residues, with a 50 ‰ interval. The number of manually drawn ROI was 38 and 66, for plant residues and bacteria, respectively.

Table 6 )

 6  Even carbon numbered C 20 -30 n-alkanoic acids were used for the FAeven indicator (C 20 -34 in

Table 6 )

 6  Dihydroxyoctadecanoic acid (DiOH18) was added to the DiOH indicator (only C 16 in

Table 6 )

 6  FAmicro indicator included n-alkanoic acids C 15i,a , C 17i,a and C 16:1 (C 14 , C 14i , C 15i,a , C 16:1

Table 9 .

 9 δ 13 C values and organic carbon (OC) content for cast and soil samples after 54 weeks of experiment (numbers in parentheses indicate the standard deviation, n = 3).

	Samples	δ 13 C (‰) OC (g.kg -1 )
	Cast-Control	-28.6 (0.1)	16.8 (2.5)
	Cast-Root	127 (8.2)	22.2 (0.6)
	Cast-Shoot	168 (1.9)	20.6 (1.6)
	Control-E	-28.0 (0.1)	13.6 (0.8)
	Root-E	-25.3 (29)	15.0 (1.1)
	Shoot-E	-12.1 (7.2)	12.8 (1.4)
	Control-NE	-28.1 (0.0)	11.4 (0.7)
	Root-NE	125.7

(9.6)

12.0 (1.7)

12.8 (1.6)

  Quantity of DNA extracted from soil and cast samples before real-time PCR analyses.La société du 21 ème siècle devra faire face à de nombreux défis. La concentration en CO 2 dans l'atmosphère augmente et le changement climatique est devenu un problème mondial de grande ampleur. En effet, le dernier rapport du GIEC a annoncé que le réchauffement climatique devrait atteindre au moins 1,5°C d'ici la fin du 21 ème siècle. De plus, la population Les sols sont indispensables à une multitude de services écosystémiques comme les cycles des nutriments, l'approvisionnement en aliments, la régulation du climat et le stockage du carbone. Le sol représente ainsi un réservoir de 2500 GtC, soit quatre fois le réservoir biotique et trois fois le réservoir atmosphérique[START_REF] Lal | Soil carbon sequestration impacts on global climate change and food security[END_REF]. La dégradation de la matière organique des sols (MOS) par minéralisation participe à l'émission de CO 2 dans

	Code FAshort FAodd Appendix 7. mondiale devrait dépasser les 9 milliard d'ici 2050, ce qui engendrerai une augmentation de Compound n-alkanoic acids Short carbon chain C 9 , C 10, C 12 , C 14 , C 15 , C 15i,a , C 16 :1 C Long odd carbon chain C 21 -C 29 FAeven Long even carbon chain C 20 -C 34 FA16 C 16 FA18 C 18 FA18uns C 18:1 70% de la demande en approvisionnement alimentaire mondial. En parallèle, depuis la soil) révolution industrielle, l'agriculture est un des grands responsables des émissions de gaz à DNA (µg.g -1 effet de serre (IPCC, 2013).
	FA20uns	C 20:4 (Arachidonic acid)
		n-alkanes
	Alkshort	Short carbon chain C 16 -C 19
	Alkodd	Long odd carbon chain C 21 -C 33
	Alkeven	Long even carbon chain C 20 -C 28
		n-alkanols
	OHshort	Short carbon chain C 12, C 16 andC 18
	OHodd	Long odd carbon chain C 21 -C 29
	OHeven	Long even carbon chain C 20 -C 32
		ω-hydroxyalkanoic acids
	OmeOH	Long even carbon chain C 22 , C 24 , C 26
		Sterols
	Sterol	Cholesterol, stigmaterol, sitosterol
	La formation de la MOS dépend principalement des apports fournis par les plantes. Ces
	apports peuvent provenir des parties aériennes (feuilles, tiges) et/ou des parties racinaires
	(racines et rhizodéposition). La stabilisation de la MOS repose sur de nombreux facteurs
	abiotiques (ex. climat et caractéristiques physico-chimiques du sol) et biotiques (ex. qualité de
	la litière, activité de la faune et des microorganismes du sol)

l'atmosphère. A l'inverse, l'apport et la stabilisation de MOS favorise le stockage de carbone dans le sol. Limiter les émissions de CO 2 et/ou de favoriser la stabilisation de la MOS grâce à la modification des pratiques agricoles demande moins d'investissement que les autres techniques de stockage du carbone

[START_REF] Hoffert | Advanced technology paths to global climate stability: energy for a greenhouse planet[END_REF][START_REF] Kell | Large-scale sequestration of atmospheric carbon via plant roots in natural and agricultural ecosystems: why and how[END_REF]

. Cette idée gagne de plus en plus de crédibilité scientifique, économique et politique. Par exemple, en 2015, le gouvernement français a annoncé son souhait de développer des pratiques agricoles visant à augmenter les stocks de MOS de 4‰ par an. Il est donc essentiel de mieux connaitre les processus de formation et de stockage de la MOS.

Remerciements

Peer Reviewed Articles

Vidal, A., Remusat, L., Watteau, F., Derenne, S., Quenea, K., 2016. Incorporation of 13 C 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.

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

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

Oral communications

1-Seminar at the Technische Universität München (TUM), 2016. Frieising, Germany.

The fate of 13 C labelled roots and shoots in soil in the presence of earthworms -a multidisciplinary approach. Vidal, A.