# Microbial and plant-derived compounds both contribute to persistent soil organic carbon in temperate soils

Pierre Barre, Katell Quénéa, Alix Vidal, Lauric Cécillon, Bent T. Christensen, Thomas Kätterer, Andy Macdonald, Léo Petit, Alain F. Plante, Folkert van Oort, et al.

► To cite this version:

Pierre Barre, Katell Quénéa, Alix Vidal, Lauric Cécillon, Bent T. Christensen, et al. Microbial and plant-derived compounds both contribute to persistent soil organic carbon in temperate soils. Biogeochemistry, 2018, 140 (1), pp.81-92. 10.1007/s10533-018-0475-5. hal-01852142

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Microbial and plant-derived compounds both contribute to persistent soil organic carbon in temperate soils

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Pierre Barré¹, Katell Quénéa², Alix Vidal³, Lauric Cécillon⁴, Bent T. Christensen⁵, Thomas Kätterer⁶

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Kätterer⁶, Andy Macdonald⁷, Léo Petit², Alain F. Plante⁸, Folkert van Oort⁹, Claire Chenu⁹

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Affiliations:

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¹Laboratoire de Géologie de l'ENS - PSL Research University – CNRS UMR8538, 75005 Paris, France

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²Sorbonne Universités, UPMC Univ Paris 06, CNRS, EPHE, UMR 7619 Metis, 4, place Jussieu, 75252 Paris cedex 05

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³Lehrstuhl für Bodenkunde, TU München, Emil-Ramann-Strasse 2, 85356 Freising, Germany

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⁴Université Grenoble Alpes, Irstea, UR EMGR, 2 rue de la Papeterie-BP 76, F-38402 St-

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Martin-d'Hères, France

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⁵Department of Agroecology, Aarhus University, AU-Foulum, DK-8830 Tjele, Denmark

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⁶Department of Ecology, Swedish University of Agricultural Sciences, 75007 Uppsala, Sweden

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⁷Department of Sustainable Agriculture Sciences, Rothamsted Research, Harpenden,

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Hertfordshire, UK

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⁸Earth and Environmental Science, University of Pennsylvania, Philadelphia, USA

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⁹AgroParisTech - INRA, UMR 1402 ECOSYS, 78850 Thiverval Grignon, France

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*Correspondence to: Pierre Barré, e-mail:barre@geologie.ens.fr, phone: +33 1 44 32 22 94

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1 Microbial and plant-derived compounds both contribute to persistent soil organic carbon in temperate soils

4 Pierre Barré¹, Katell Quénéa², Alix Vidal³, Lauric Cécillon⁴, Bent T. Christensen⁵, Thomas Kätterer⁶

5 Kätterer⁶, Andy Macdonald⁷, Léo Petit², Alain F. Plante⁸, Folkert van Oort⁹, Claire Chenu⁹

7 Affiliations:

8 ¹Laboratoire de Géologie de l'ENS - PSL Research University – CNRS UMR8538, 75005 Paris, France

10 ²Sorbonne Universités, UPMC Univ Paris 06, CNRS, EPHE, UMR 7619 Metis, 4, place Jussieu, 75252 Paris cedex 05

12 ³Lehrstuhl für Bodenkunde, TU München, Emil-Ramann-Strasse 2, 85356 Freising, Germany

13 ⁴Université Grenoble Alpes, Irstea, UR EMGR, 2 rue de la Papeterie-BP 76, F-38402 St-Martin-d'Hères, France

15 ⁵Department of Agroecology, Aarhus University, AU-Foulum, DK-8830 Tjele, Denmark

16 ⁶Department of Ecology, Swedish University of Agricultural Sciences, 75007 Uppsala, Sweden

17 ⁷Department of Sustainable Agriculture Sciences, Rothamsted Research, Harpenden, Hertfordshire, UK

19 ⁸Earth and Environmental Science, University of Pennsylvania, Philadelphia, USA

20 ⁹AgroParisTech - INRA, UMR 1402 ECOSYS, 78850 Thiverval Grignon, France

22 *Correspondence to: Pierre Barré, e-mail:barre@geologie.ens.fr, phone: +33 1 44 32 22 94
26 **Abstract**

Our study tests the emerging paradigm that biochemical recalcitrance does not affect
substantially long-term (50 years) SOC persistence. We analyzed the molecular composition of
SOC in archived soils originating from four European long-term bare fallow experiments
(Askov, Rothamsted, Versailles and Ultuna). The soils had been collected after various periods
(up to 53 years) under bare fallow. With increasing duration of bare fallow without new organic
inputs, the relative abundance of cutin- and suberin-derived compounds declined substantially,
and the abundance of lignin-derived compounds was close to zero. Conversely, the relative
abundance of plant-derived long-chain alkanes remained almost constant or increased during
the bare fallow period. The relative abundance of N-containing compounds, considered to be
abundant in SOC derived from microbial activity, increased consistently illustrating that
microbial turnover of SOC continues even when plant inputs are stopped. The persistence of
the different families of plant-derived compounds differed markedly over the scale of half a
century, which may be ascribed to their contrasting chemical characteristics and recalcitrance,
or to differences in their interactions with the soil mineral matrix, and likely some combination
since chemical composition drives the degree of mineral association. Using soil from this
unique set of long-term bare fallow experiments, we provide direct evidence that multi-decadal
scale persistent SOC is enriched in microbe-derived compounds but also includes a substantial
fraction of plant-derived compounds.

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48 **Keywords:** TMAH-Py-GC-MS, long-term bare fallow, plant-derived compounds, soil
49 organic carbon persistence

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**Abstract**

Our study tests the emerging paradigm that biochemical recalcitrance does not affect substantially long-term (50 years) SOC persistence. We analyzed the molecular composition of SOC in archived soils originating from four European long-term bare fallow experiments (Askov, Rothamsted, Versailles and Ultuna). The soils had been collected after various periods (up to 53 years) under bare fallow. With increasing duration of bare fallow without new organic inputs, the relative abundance of cutin- and suberin-derived compounds declined substantially, and the abundance of lignin-derived compounds was close to zero. Conversely, the relative abundance of plant-derived long-chain alkanes remained almost constant or increased during the bare fallow period. The relative abundance of N-containing compounds, considered to be abundant in SOC derived from microbial activity, increased consistently illustrating that microbial turnover of SOC continues even when plant inputs are stopped. The persistence of the different families of plant-derived compounds differed markedly over the scale of half a century, which may be ascribed to their contrasting chemical characteristics and recalcitrance, or to differences in their interactions with the soil mineral matrix, and likely some combination since chemical composition drives the degree of mineral association. Using soil from this unique set of long-term bare fallow experiments, we provide direct evidence that multi-decadal scale persistent SOC is enriched in microbe-derived compounds but also includes a substantial fraction of plant-derived compounds.

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**Keywords:** TMAH-Py-GC-MS, long-term bare fallow, plant-derived compounds, soil organic carbon persistence

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## 51 Introduction

In spite of their importance for soil quality and climate regulation, the mechanisms responsible for long-term storage of soil organic carbon (SOC) remain insufficiently understood (Stockmann et al. 2013). Emerging paradigms suggest that the persistence of SOC is less defined by its chemical composition and recalcitrance than previously conceived (Schmidt et al. 2011; Lehmann and Kleber, 2015). It is envisioned that the chemical composition of organic inputs influence short-term C dynamics whereas the long-term persistence of SOC is defined mainly by its interactions with the mineral matrix and environmental constraints. Evidence supporting the reduced role of inherent biochemical recalcitrance as the primary mechanism for SOC persistence is derived from studies showing shorter residence times of specific compounds presumed to be recalcitrant compared to bulk SOC (e.g. Amelung et al. 2008; Schmidt et al. 2011), and the predominance of SOC of microbial rather than plant origin. Specifically, plant-derived compounds such as lignin show much shorter residence times than previously anticipated (e.g. Dignac et al. 2005; Amelung et al., 2008; Hofmann et al. 2009), and recent molecular level characterization suggest that microbial metabolites and necromass are important constituents of the most persistent fractions of SOC (Miltner et al. 2012; Kallenbach et al. 2016). Although biochemical recalcitrance may not play a primary and direct role in SOC stabilization, the molecular composition of SOC remains an essential feature that plays important indirect roles in its long-term persistence in soil. For example, it has been demonstrated that diverse soil minerals provide selective chemical affinities and induce chemical fractionation of SOC through differential sorption processes (e.g. Sollins et al. 2009; Mitchell et al. 2013; Coward et al. 2018).

Nonetheless, the link between SOC persistence and molecular composition remains obscured partly because of lack of analytical procedures that allow persistent SOC to be physically separated from labile SOC. One obvious way to assure that only persistent SOC is
## 51 Introduction

In spite of their importance for soil quality and climate regulation, the mechanisms responsible for long-term storage of soil organic carbon (SOC) remain insufficiently understood (Stockmann et al. 2013). Emerging paradigms suggest that the persistence of SOC is less defined by its chemical composition and recalcitrance than previously conceived (Schmidt et al. 2011; Lehmann and Kleber, 2015). It is envisioned that the chemical composition of organic inputs influence short-term C dynamics whereas the long-term persistence of SOC is defined mainly by its interactions with the mineral matrix and environmental constraints. Evidence supporting the reduced role of inherent biochemical recalcitrance as the primary mechanism for SOC persistence is derived from studies showing shorter residence times of specific compounds presumed to be recalcitrant compared to bulk SOC (e.g. Amelung et al. 2008; Schmidt et al. 2011), and the predominance of SOC of microbial rather than plant origin. Specifically, plant-derived compounds such as lignin show much shorter residence times than previously anticipated (e.g. Dignac et al. 2005; Amelung et al., 2008; Hofmann et al. 2009), and recent molecular level characterization suggest that microbial metabolites and necromass are important constituents of the most persistent fractions of SOC (Miltner et al. 2012; Kallenbach et al. 2016). Although biochemical recalcitrance may not play a primary and direct role in SOC stabilization, the molecular composition of SOC remains an essential feature that plays important indirect roles in its long-term persistence in soil. For example, it has been demonstrated that diverse soil minerals provide selective chemical affinities and induce chemical fractionation of SOC through differential sorption processes (e.g. Sollins et al. 2009; Mitchell et al. 2013; Coward et al. 2018).

Nonetheless, the link between SOC persistence and molecular composition remains obscured partly because of lack of analytical procedures that allow persistent SOC to be physically separated from labile SOC. One obvious way to assure that only persistent SOC is
present in a soil is to cease C inputs and allow the more decomposable compounds to decompose. Long-term bare fallow experiments (LTBF) provide such conditions. LTBF experiments consist of soil plots that have been kept free of organic inputs and vegetation for decades by manual or chemical means. New C inputs to the soils have therefore been negligible during the fallow period, and the SOC remaining has consequently persisted for at least the duration of the fallow period. With regular sampling and archiving of soils, LTBF experiments now provide a unique research platform for studies of the characteristics of SOC that has persisted in soils for decades (Barré et al. 2010; 2016). It is noteworthy that the persistence over decades of a C atom in the LTBF soils does not necessarily imply that the organic molecule containing this C atom also has persisted for decades. Soil microbial activity in LTBF plots may transfer a C atom, initially present in a plant-derived molecule, into a microbe-derived molecule during the bare fallow period. Consequently, while our experimental set-up allows for studies on the persistence of C entering the soil before the fallow period, it does not reflect the persistence of pre-fallow organic matter.

This study examines changes in the molecular composition of SOC after decades of in-situ decomposition without new organic inputs. We hypothesize that the molecular composition of SOC is dominated by plant residues in the early phase of LTBF (~10-30 years), whereas plant-derived compounds contribute insignificantly to SOC that persists for half a century, and that this persistent SOC is dominated by SOC of microbial origin. We tested the hypotheses by analyzing the molecular composition of soils collected after different periods under bare fallow from four experiments located at Askov (Denmark), Rothamsted (United Kingdom), Ultuna (Sweden) and Versailles (France). The soils were analyzed using pyrolysis gas-chromatography mass-spectrometry after tetramethylammonium hydroxide derivatization (TMAH-Py-GC-MS).

Molecular characterization of SOC using Py-GC-MS is a well-established method that allows identification of plant-derived compounds such as lignin, cutin, suberin and long-chain aliphatic
present in a soil is to cease C inputs and allow the more decomposable compounds to decompose. Long-term bare fallow experiments (LTBF) provide such conditions. LTBF experiments consist of soil plots that have been kept free of organic inputs and vegetation for decades by manual or chemical means. New C inputs to the soils have therefore been negligible during the fallow period, and the SOC remaining has consequently persisted for at least the duration of the fallow period. With regular sampling and archiving of soils, LTBF experiments now provide a unique research platform for studies of the characteristics of SOC that has persisted in soils for decades (Barré et al. 2010; 2016). It is noteworthy that the persistence over decades of a C atom in the LTBF soils does not necessarily imply that the organic molecule containing this C atom also has persisted for decades. Soil microbial activity in LTBF plots may transfer a C atom, initially present in a plant-derived molecule, into a microbe-derived molecule during the bare fallow period. Consequently, while our experimental set-up allows for studies on the persistence of C entering the soil before the fallow period, it does not reflect the persistence of pre-fallow organic matter.

This study examines changes in the molecular composition of SOC after decades of in-situ decomposition without new organic inputs. We hypothesize that the molecular composition of SOC is dominated by plant residues in the early phase of LTBF (~10-30 years), whereas plant-derived compounds contribute insignificantly to SOC that persists for half a century, and that this persistent SOC is dominated by SOC of microbial origin. We tested the hypotheses by analyzing the molecular composition of soils collected after different periods under bare fallow from four experiments located at Askov (Denmark), Rothamsted (United Kingdom), Ultuna (Sweden) and Versailles (France). The soils were analyzed using pyrolysis gas-chromatography mass-spectrometry after tetramethylammonium hydroxide derivatization (TMAH-Py-GC-MS).

Molecular characterization of SOC using Py-GC-MS is a well-established method that allows identification of plant-derived compounds such as lignin, cutin, suberin and long-chain aliphatic
compounds and compounds of microbial origin such as short-chain or ramified alkanes
(Derenne & Quenea 2015). Results obtained by Py-GC-MS have been used to compare soils
from different biomes (e.g. Carr et al. 2013), and soils under contrasting land-use (e.g.
Schellekens et al. 2013; Oliveira et al. 2016) or land management practices (Buurman &
Roscoe 2011).

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## 107 Materials and Methods

Study sites and soil samples

The LTBF experiments were initiated at different times at four sites across NW Europe: (1) INRA "42 parcels" experiment initiated in 1928 and located in the grounds of the Château de Versailles in Versailles, France; (2) the long-term bare fallow initiated in 1959 and located adjacent to the Ley-Arable experiment at Rothamsted Research, Harpenden, United Kingdom; (3) the Ultuna long-term field experiment initiated in 1956 and located at the Swedish University of Agricultural Sciences, Uppsala, Sweden; and (4) the Lermarken long-term bare fallow initiated in 1956 and located at Askov Experimental Station, Denmark (Table 1). More details on the LTBF sites are available in Barré et al. (2010) and references cited therein.
Surface (up to 25 cm deep) soil samples collected just before and periodically during the bare fallow period were ground and sieved at 2 mm, air-dried and archived in the dark at ambient temperatures. In total, 26 samples were included in the study: eight from each of Rothamsted and Versailles (4 dates × 2 plots), four from Ultuna (4 dates × 1 plot) and six from Askov (3 dates × 2 plots). Sampling dates of samples included in the study are mentioned in Table 1. For the Versailles LTBF experiment, only soils sampled until 1980 (up to 51 years of bare fallow) were included in this study because molecular analyses of soils sampled after 1980 yielded compound abundances that were too low for quantification due to low SOC contents (6-8 mg C g⁻¹ soil).
compounds and compounds of microbial origin such as short-chain or ramified alkanes
(Derenne & Quenea 2015). Results obtained by Py-GC-MS have been used to compare soils
from different biomes (e.g. Carr et al. 2013), and soils under contrasting land-use (e.g.
Schellekens et al. 2013; Oliveira et al. 2016) or land management practices (Buurman &
Roscoe 2011).

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## 107 Materials and Methods

Study sites and soil samples

The LTBF experiments were initiated at different times at four sites across NW Europe: (1) INRA "42 parcels" experiment initiated in 1928 and located in the grounds of the Château de Versailles in Versailles, France; (2) the long-term bare fallow initiated in 1959 and located adjacent to the Ley-Arable experiment at Rothamsted Research, Harpenden, United Kingdom; (3) the Ultuna long-term field experiment initiated in 1956 and located at the Swedish University of Agricultural Sciences, Uppsala, Sweden; and (4) the Lermarken long-term bare fallow initiated in 1956 and located at Askov Experimental Station, Denmark (Table 1). More details on the LTBF sites are available in Barré et al. (2010) and references cited therein.
Surface (up to 25 cm deep) soil samples collected just before and periodically during the bare fallow period were ground and sieved at 2 mm, air-dried and archived in the dark at ambient temperatures. In total, 26 samples were included in the study: eight from each of Rothamsted and Versailles (4 dates × 2 plots), four from Ultuna (4 dates × 1 plot) and six from Askov (3 dates × 2 plots). Sampling dates of samples included in the study are mentioned in Table 1. For the Versailles LTBF experiment, only soils sampled until 1980 (up to 51 years of bare fallow) were included in this study because molecular analyses of soils sampled after 1980 yielded compound abundances that were too low for quantification due to low SOC contents (6-8 mg C g⁻¹ soil).
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128 TMAH-Py-GC-MS TMAH analysis

129 All samples were subjected to Curie point pyrolysis in the presence of TMAH. Samples (5–10 mg, depending on OC content) were impregnated with a few drops of TMAH in 25% MeOH (1/1, w/w) prior to loading into ferromagnetic tubes. Samples were pyrolysed at a Curie temperature of 650 °C for 10 s using a pyrolysis unit (Pilodist), coupled to a gas chromatograph (GC, Trace Ultra Thermo electron) and a mass spectrometer (MS, DSQ 1 Thermo). After 10 min at 50 °C, the GC oven temperature gradually increased from 50 °C to 310 °C at 2°C min⁻¹.

135 For the sake of clarity, the carbon number of the parent compound is reported for aliphatic products since TMAH induces *in situ* methylation. Semi-quantitative information was derived from TMAH-Py-GC-MS using the Agilent ChemStation software by manually subtracting the background signal from the total ion current (TIC) of each peak of interest, and integrating each peak area. The relative abundance of each compound was calculated by dividing its peak area by the sum of all of the areas of identified pyrolysis products. Compounds corresponding to each peak were identified by MS using the NIST compound library and published data. Only results for elution times greater than 30 minutes were used because of strong co-elution and artefacts due to TMAH elution before 30 minutes. Consequently, only part of SOC (generally 50-60%) is recovered as pyrolysis effluents and characterized by Py-GC-MS (Haddix et al. 2016). Identified pyrolysis products or groups of products, and their probable origins (*i.e.*, plants, microbes or ubiquitous) are reported in Table 2 with key references.

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148 Statistical analyses

149 A principal component analysis (PCA) was performed to assess similarities and differences in molecular composition among the 26 soil samples (*i.e.*, all sites, years of LTBF duration, and
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128 TMAH-Py-GC-MS TMAH analysis

129 All samples were subjected to Curie point pyrolysis in the presence of TMAH. Samples (5–10 mg, depending on OC content) were impregnated with a few drops of TMAH in 25% MeOH (1/1, w/w) prior to loading into ferromagnetic tubes. Samples were pyrolysed at a Curie temperature of 650 °C for 10 s using a pyrolysis unit (Pilodist), coupled to a gas chromatograph (GC, Trace Ultra Thermo electron) and a mass spectrometer (MS, DSQ 1 Thermo). After 10 min at 50 °C, the GC oven temperature gradually increased from 50 °C to 310 °C at 2°C min⁻¹.

134 For the sake of clarity, the carbon number of the parent compound is reported for aliphatic products since TMAH induces *in situ* methylation. Semi-quantitative information was derived from TMAH-Py-GC-MS using the Agilent ChemStation software by manually subtracting the background signal from the total ion current (TIC) of each peak of interest, and integrating each peak area. The relative abundance of each compound was calculated by dividing its peak area by the sum of all of the areas of identified pyrolysis products. Compounds corresponding to each peak were identified by MS using the NIST compound library and published data. Only results for elution times greater than 30 minutes were used because of strong co-elution and artefacts due to TMAH elution before 30 minutes. Consequently, only part of SOC (generally 50-60%) is recovered as pyrolysis effluents and characterized by Py-GC-MS (Haddix et al. 2016). Identified pyrolysis products or groups of products, and their probable origins (*i.e.*, plants, microbes or ubiquitous) are reported in Table 2 with key references.

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148 Statistical analyses

149 A principal component analysis (PCA) was performed to assess similarities and differences in molecular composition among the 26 soil samples (*i.e.*, all sites, years of LTBF duration, and
plots) based on the 18 selected mean-centered and variance-scaled Py-GC-MS TIC relative abundances using the multivariate analysis R package Factoextra (http://www.sthda.com).

Trends in the relative abundance of each identified compound were investigated using least-squares linear regression analysis with bare fallow duration as the independent variable using the lm R statistical package.

## 157 Results and Discussion

Molecular characteristics of soils at the start of bare fallow

The thermo-chemolysates of samples collected at the start of the bare fallow periods (Figure 1) are consistent with the products commonly identified in Py-GC-MS using similar acquisition parameters (Vidal et al. 2016). At all sites, aromatic compounds are mainly represented by lignin derived compounds and to a lesser extent by phenolic compounds. Aliphatic compounds comprise *n*-alkanoic acids, *ω*-hydroxyalkanoic acids, *n*-alkanedioic acids, *n*-alkanes, *n*-alkanols, 8,16 - 9,16 and 10, 16 or 18 dihydroxyhexadecanoic acids (DiOH) and 9,10,18 trihydroxyoctadecanoic acid (TriOH). Carbohydrates and *N*-containing compounds were also observed.

The molecular composition of SOC varies substantially among experimental sites (Figure 2a) as evidenced by the lack of clustering of the year-zero samples in PC space, and conversely the clustering of samples by site and separation of sites in PC space. Differences in the molecular composition among sites are illustrated by separation along PC 1 in the scores plot (Figure 2a), where soil from Versailles and Ultuna are relatively similar but differ from those from Rothamsted and Askov. These differences between sites are maintained over the duration of the LTBF experiments, suggesting that variability in molecular composition among sites may be greater than changes over time. The relative abundance of *ω*-hydroxyalkanoic acids was greater at Rothamsted and Askov compared to Versailles and Ultuna (Figure 2,
plots) based on the 18 selected mean-centered and variance-scaled Py-GC-MS TIC relative abundances using the multivariate analysis R package Factoextra (http://www.sthda.com).

Trends in the relative abundance of each identified compound were investigated using least-squares linear regression analysis with bare fallow duration as the independent variable using the lm R statistical package.

# Results and Discussion

Molecular characteristics of soils at the start of bare fallow

The thermo-chemolysates of samples collected at the start of the bare fallow periods (Figure 1) are consistent with the products commonly identified in Py-GC-MS using similar acquisition parameters (Vidal et al. 2016). At all sites, aromatic compounds are mainly represented by lignin derived compounds and to a lesser extent by phenolic compounds. Aliphatic compounds comprise *n*-alkanoic acids, *ω*-hydroxyalkanoic acids, *n*-alkanedioic acids, *n*-alkanes, *n*-alkanols, 8,16 - 9,16 and 10, 16 or 18 dihydroxyhexadecanoic acids (DiOH) and 9,10,18 trihydroxyoctadecanoic acid (TriOH). Carbohydrates and *N*-containing compounds were also observed.

The molecular composition of SOC varies substantially among experimental sites (Figure 2a) as evidenced by the lack of clustering of the year-zero samples in PC space, and conversely the clustering of samples by site and separation of sites in PC space. Differences in the molecular composition among sites are illustrated by separation along PC 1 in the scores plot (Figure 2a), where soil from Versailles and Ultuna are relatively similar but differ from those from Rothamsted and Askov. These differences between sites are maintained over the duration of the LTBF experiments, suggesting that variability in molecular composition among sites may be greater than changes over time. The relative abundance of *ω*-hydroxyalkanoic acids was greater at Rothamsted and Askov compared to Versailles and Ultuna (Figure 2,
Supplementary Table 1). Long chain ω-hydroxyalkanoic acids (C22 and C24) are recognized as suberin-derived compounds, i.e. a typical biopolymer mainly present in plant roots (Kolattukudy 1980; Nierop 2001; Kögel-Knabner et al. 2002). Compared to other sites, Askov also shows greater relative abundances of DiOH, TriOH, and long chain *n*-alkanols. These compounds are typical plant-derived compounds, with TriOH and DiOH being commonly identified as cutin monomer-derived compounds (Riederer et al. 1993; Del Rio and Hatcher 1998; Otto and Simpson 2006). As such, Askov and to some extent also Rothamsted exhibit greater plant contributions compared to Versailles and Ultuna which in turn have greater abundances of N-containing compounds (especially Versailles) than Askov and Rothamsted. The greater proportion of N-containing compounds suggests a higher contribution of microbial products to SOM (Grandy & Neff 2007; Vancampenhout et al. 2009). The observed N-containing compounds are dominated by pyrrole and pyridine which are usually associated with microbial material whereas N-containing compounds from plant origin (for instance diketodipyrrole or diketopiperazines) were not observed. The different contribution of N-containing compounds may also be related to the different abundance of legumes in the vegetation before bare fallow implementation. However, information on plant community compositions before bare fallow implementation is not available. The addition of N fertilizers does not seem to play an important role for the relative content of N-containing compounds as a higher contribution of N-containing compounds was not observed at Askov where fertilizers were added throughout the bare fallow.

A number of edaphic factors may explain the observed differences among sites in molecular composition, including climate (temperature, precipitation), management history, pre-fallow vegetation and productivity, and soil properties such as texture, mineralogy and pH. The SOC associated with fine mineral fractions has been ascribed to microbial origin (Nierop et al. 2005; Grandy & Neff 2008), partly because of the affinity of reactive mineral surfaces for
Supplementary Table 1). Long chain ω-hydroxyalkanoic acids (C22 and C24) are recognized as suberin-derived compounds, i.e. a typical biopolymer mainly present in plant roots (Kolattukudy 1980; Nierop 2001; Kögel-Knabner et al. 2002). Compared to other sites, Askov also shows greater relative abundances of DiOH, TriOH, and long chain *n*-alkanols. These compounds are typical plant-derived compounds, with TriOH and DiOH being commonly identified as cutin monomer-derived compounds (Riederer et al. 1993; Del Rio and Hatcher 1998; Otto and Simpson 2006). As such, Askov and to some extent also Rothamsted exhibit greater plant contributions compared to Versailles and Ultuna which in turn have greater abundances of N-containing compounds (especially Versailles) than Askov and Rothamsted. The greater proportion of N-containing compounds suggests a higher contribution of microbial products to SOM (Grandy & Neff 2007; Vancampenhout et al. 2009). The observed N-containing compounds are dominated by pyrrole and pyridine which are usually associated with microbial material whereas N-containing compounds from plant origin (for instance diketodipyrrole or diketopiperazines) were not observed. The different contribution of N-containing compounds may also be related to the different abundance of legumes in the vegetation before bare fallow implementation. However, information on plant community compositions before bare fallow implementation is not available. The addition of N fertilizers does not seem to play an important role for the relative content of N-containing compounds as a higher contribution of N-containing compounds was not observed at Askov where fertilizers were added throughout the bare fallow.

A number of edaphic factors may explain the observed differences among sites in molecular composition, including climate (temperature, precipitation), management history, pre-fallow vegetation and productivity, and soil properties such as texture, mineralogy and pH. The SOC associated with fine mineral fractions has been ascribed to microbial origin (Nierop et al. 2005; Grandy & Neff 2008), partly because of the affinity of reactive mineral surfaces for
N-containing functional groups. The long agricultural history and sandy texture at Askov compared to the three other sites (Table 1), may explain why compounds usually associated with fine fractions (i.e., microbe-derived N-containing compounds) are recovered in smaller relative proportions at Askov. Conversely, high proportions of fine particles in Versailles and Ultuna soils may explain the greater proportions of microbial products. Despite the high proportion of fine particles in soils at Rothamsted, results indicate a large plant-derived contribution. Before being converted to fallow, the Rothamsted site had a long history of permanent grassland and probably a greater accumulation of plant inputs than the other sites, as reflected by the larger initial soil C contents (Table 1). The larger pre-fallow plant inputs at Rothamsted might explain the greater relative proportion of plant-derived compounds in the fallowed soil.

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Evolution of SOC molecular composition during the bare fallow period

The degree to which samples from the same site cluster in PCA scores reflects the degree to which SOC molecular composition changed relative to other sites. Tight clustering of samples from the same site but different bare fallow durations would indicate less change in chemical composition compared to more scattered samples (Figure 2a). As such, changes in SOC composition appears to be greatest at Rothamsted, followed by Versailles, Ultuna, and Askov.

PCA loadings (Figure 2b) indicate that most plant-derived compounds (ω-hydroxyalkanoic acids, long chain *n*-alkanedioic acids, dihydroxyhexadecanoic acids, trihydroxyoctadecanoic acid and long chain *n*-alkanoic acids) contribute negatively to PC 1 whereas microbial-derived compounds (FAmicro) have a positive contribution to PC 1. N-containing compounds are considered to be mainly of microbial origin and have a positive contribution to PC 1, whereas ubiquitous compounds considered to be mostly derived from plants such as short chain diacids (C₈, C₉) contribute negatively to PC 1. The changes in SOC molecular composition with time

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N-containing functional groups. The long agricultural history and sandy texture at Askov compared to the three other sites (Table 1), may explain why compounds usually associated with fine fractions (i.e., microbe-derived N-containing compounds) are recovered in smaller relative proportions at Askov. Conversely, high proportions of fine particles in Versailles and Ultuna soils may explain the greater proportions of microbial products. Despite the high proportion of fine particles in soils at Rothamsted, results indicate a large plant-derived contribution. Before being converted to fallow, the Rothamsted site had a long history of permanent grassland and probably a greater accumulation of plant inputs than the other sites, as reflected by the larger initial soil C contents (Table 1). The larger pre-fallow plant inputs at Rothamsted might explain the greater relative proportion of plant-derived compounds in the fallowed soil.

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Evolution of SOC molecular composition during the bare fallow period

The degree to which samples from the same site cluster in PCA scores reflects the degree to which SOC molecular composition changed relative to other sites. Tight clustering of samples from the same site but different bare fallow durations would indicate less change in chemical composition compared to more scattered samples (Figure 2a). As such, changes in SOC composition appears to be greatest at Rothamsted, followed by Versailles, Ultuna, and Askov.

PCA loadings (Figure 2b) indicate that most plant-derived compounds (ω-hydroxyalkanoic acids, long chain *n*-alkanedioic acids, dihydroxyhexadecanoic acids, trihydroxyoctadecanoic acid and long chain *n*-alkanoic acids) contribute negatively to PC 1 whereas microbial-derived compounds (FAmicro) have a positive contribution to PC 1. N-containing compounds are considered to be mainly of microbial origin and have a positive contribution to PC 1, whereas ubiquitous compounds considered to be mostly derived from plants such as short chain diacids (C8, C9) contribute negatively to PC 1. The changes in SOC molecular composition with time

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under bare fallow for samples from Versailles and Ultuna are illustrated by PCA trends toward more positive PC 1 and more negative PC 2 scores (i.e., towards the bottom-right quadrant; Figure 2a). For samples from Rothamsted, only the trend towards more negative PC2 scores is observed. Lack of divergence in PC scores for samples within sites during the first 25 years of bare fallow suggests that changes in the chemical composition of SOM were limited for all sites except Rothamsted. This site showed the greatest loss of SOC during the first 25 years (from 30 mg C g⁻¹ soil to 15 mg C g⁻¹ soil). Conversely, no temporal evolution is observed in PCA scores for Askov samples.

Temporal trends in the relative abundances of pyrolysis products were compound specific (Figures 2a and 3; supplementary Table 1). Lignin-derived compounds decreased with bare fallow duration at all sites, though the trend is not statistically significant for Askov (Figure 3; supplementary Table 1). Lignin-derived compounds are not observed after 50 years of bare fallow at Versailles, Ultuna and Rothamsted, suggesting that the downward trend might have been more pronounced at Askov after a longer period of bare fallow. These results are consistent with previous results showing a low persistence of lignin compounds in soils (Dignac et al. 2005; Kelleher et al. 2006; Hofmann et al. 2009). Cutin- and suberin-derived compounds (ωOH, diacids long chain, TriOH and DiOH) contributed similarly to the PCA axis (Figure 2b). As for lignin-derived compounds, the sum of cutin- and suberin-derived compounds during the bare fallow period showed that their relative contributions decreased with duration of bare fallow, except at Askov with the shortest bare-fallow period (Figure 3; Supplementary Table 1). The decrease in cutin+suberin derived compounds is statistically significant for Rothamsted and Versailles, but not for Ultuna (Supplementary Table 2), likely because of the smaller number of samples retrieved at this site. A rapid decay of cutin- and suberin-derived compounds (water repellant wax-like substances from the cuticle) has previously been reported (Armas-Herrera et al. 2016). Conversely, the relative abundance of long chain alkanes, also of plant
under bare fallow for samples from Versailles and Ultuna are illustrated by PCA trends toward more positive PC 1 and more negative PC 2 scores (i.e., towards the bottom-right quadrant; Figure 2a). For samples from Rothamsted, only the trend towards more negative PC2 scores is observed. Lack of divergence in PC scores for samples within sites during the first 25 years of bare fallow suggests that changes in the chemical composition of SOM were limited for all sites except Rothamsted. This site showed the greatest loss of SOC during the first 25 years (from 30 mg C g⁻¹ soil to 15 mg C g⁻¹ soil). Conversely, no temporal evolution is observed in PCA scores for Askov samples.

Temporal trends in the relative abundances of pyrolysis products were compound specific (Figures 2a and 3; supplementary Table 1). Lignin-derived compounds decreased with bare fallow duration at all sites, though the trend is not statistically significant for Askov (Figure 3; supplementary Table 1). Lignin-derived compounds are not observed after 50 years of bare fallow at Versailles, Ultuna and Rothamsted, suggesting that the downward trend might have been more pronounced at Askov after a longer period of bare fallow. These results are consistent with previous results showing a low persistence of lignin compounds in soils (Dignac et al. 2005; Kelleher et al. 2006; Hofmann et al. 2009). Cutin- and suberin-derived compounds (ωOH, diacids long chain, TriOH and DiOH) contributed similarly to the PCA axis (Figure 2b). As for lignin-derived compounds, the sum of cutin- and suberin-derived compounds during the bare fallow period showed that their relative contributions decreased with duration of bare fallow, except at Askov with the shortest bare-fallow period (Figure 3; Supplementary Table 1). The decrease in cutin+suberin derived compounds is statistically significant for Rothamsted and Versailles, but not for Ultuna (Supplementary Table 2), likely because of the smaller number of samples retrieved at this site. A rapid decay of cutin- and suberin-derived compounds (water repellant wax-like substances from the cuticle) has previously been reported (Armas-Herrera et al. 2016). Conversely, the relative abundance of long chain alkanes, also of plant
origin, showed no trends with bare fallow duration, with the exception of Versailles where a statistically significant increase was observed. However, the significance of this trend relies on the last sampling date (Figure 3; Supplementary Table 2). The different slopes of the regressions models for the various groups of plant-derived compounds (Supplementary Table 2) suggest that alkanes have a greater persistence than cutin and suberin, which in turn are more persistent than lignin. Thus, the chemistry of these plant-derived compounds affects their persistence in soil for at least 50 years.

The relative abundance of N-containing compounds tended to increase with bare fallow duration at all sites except Askov (Figure 3), the increases being statistically significant for Rothamsted and Versailles (Supplementary Table 2). This is consistent with Carr et al. (2013), who observed a relative decrease in lignin-derived compounds and a relative increase in N-containing compounds in soils with degraded SOM retrieved from arid and semi-arid regions in South Africa. For all other compounds, the trends with duration of bare fallow were minor, not statistically significant or not consistent across sites (supplementary Table 2).

Plant and microbial contributions to persistent SOC

Differences in the relative abundance of plant-derived, microbial-derived, and N-containing compounds between the start of the bare fallow period and the last sampling varied by compound group and site (Figure 4). Except at Askov, the relative abundances of plant-derived compounds decreased and microbial products and N-containing compounds increased after decades of bare fallow. This illustrates that the microbial turnover of SOC continues even when plant inputs are stopped and may promote an ongoing relative increase of microbial products in the bare fallow soils compared to plant-derived compounds. However, compounds of plant origin are still recovered in substantial quantities after 50 years without new organic inputs. At Rothamsted plant-derived compounds represent about 50% of relative peak areas while they
origin, showed no trends with bare fallow duration, with the exception of Versailles where a statistically significant increase was observed. However, the significance of this trend relies on the last sampling date (Figure 3; Supplementary Table 2). The different slopes of the regressions models for the various groups of plant-derived compounds (Supplementary Table 2) suggest that alkanes have a greater persistence than cutin and suberin, which in turn are more persistent than lignin. Thus, the chemistry of these plant-derived compounds affects their persistence in soil for at least 50 years.

The relative abundance of N-containing compounds tended to increase with bare fallow duration at all sites except Askov (Figure 3), the increases being statistically significant for Rothamsted and Versailles (Supplementary Table 2). This is consistent with Carr et al. (2013), who observed a relative decrease in lignin-derived compounds and a relative increase in N-containing compounds in soils with degraded SOM retrieved from arid and semi-arid regions in South Africa. For all other compounds, the trends with duration of bare fallow were minor, not statistically significant or not consistent across sites (supplementary Table 2).

Plant and microbial contributions to persistent SOC

Differences in the relative abundance of plant-derived, microbial-derived, and N-containing compounds between the start of the bare fallow period and the last sampling varied by compound group and site (Figure 4). Except at Askov, the relative abundances of plant-derived compounds decreased and microbial products and N-containing compounds increased after decades of bare fallow. This illustrates that the microbial turnover of SOC continues even when plant inputs are stopped and may promote an ongoing relative increase of microbial products in the bare fallow soils compared to plant-derived compounds. However, compounds of plant origin are still recovered in substantial quantities after 50 years without new organic inputs. At Rothamsted plant-derived compounds represent about 50% of relative peak areas while they
accounted for 25% at Versailles and Ultuna at the last sampling. The presence of plant-derived compounds after 50 years of bare fallow substantiates the influence of initial plant chemistry on the resulting SOC composition. Contrary to our hypothesis, this demonstrates that persistent SOC is not composed exclusively of compounds derived from microbial activity but that plant-derived compounds contribute to SOC even after several decades of degradation. Our findings contribute to the current debate on the composition of persistent SOC and the importance of substrate quality for the chemical composition of SOC that is formed during consecutive cycles of microbial turnover (Kallenbach et al. 2016; Liang et al. 2017). Thus, biochemical characteristics remain a mechanism to consider for long-term (>50 years) persistence of SOC. Chemically complex plant components undergo substantial alteration during microbial decomposition, and decomposition products, as well as compounds originating from the microbial population, are retained in organo-mineral associations (Christensen 2001). Indeed, previous studies demonstrated negligible amounts of non-mineral associated particulate OM (Balabane & Plante 2004).

Wickings et al. (2012) proposed three hypotheses to describe changes in the chemical composition of litter during decomposition: (1) the chemical convergence hypothesis (“all litters will converge eventually towards a common chemistry”); (2) the initial litter quality hypothesis (“the initial litter quality has persistent effects on litter chemical composition”); (3) the decomposer control hypothesis (“changes of litter chemistry during decomposition do not simply reflect the effects of time or initial litter quality, but are also strongly influenced by the functional attributes of distinct decomposer communities”). Our dataset allows for testing these hypotheses of organic matter decomposition in soil. Previous results from spectroscopic analyses (MIRS and NEXAFS) of LTBF soils failed to provide clear evidence of chemical trends and convergence in SOC composition over time (Barré et al. 2016). Conversely, the Py-GC-MS data reported here demonstrate that, despite contrasting initial molecular
accounted for 25% at Versailles and Ultuna at the last sampling. The presence of plant-derived compounds after 50 years of bare fallow substantiates the influence of initial plant chemistry on the resulting SOC composition. Contrary to our hypothesis, this demonstrates that persistent SOC is not composed exclusively of compounds derived from microbial activity but that plant-derived compounds contribute to SOC even after several decades of degradation. Our findings contribute to the current debate on the composition of persistent SOC and the importance of substrate quality for the chemical composition of SOC that is formed during consecutive cycles of microbial turnover (Kallenbach et al. 2016; Liang et al. 2017). Thus, biochemical characteristics remain a mechanism to consider for long-term (>50 years) persistence of SOC. Chemically complex plant components undergo substantial alteration during microbial decomposition, and decomposition products, as well as compounds originating from the microbial population, are retained in organo-mineral associations (Christensen 2001). Indeed, previous studies demonstrated negligible amounts of non-mineral associated particulate OM (Balabane & Plante 2004).

Wickings et al. (2012) proposed three hypotheses to describe changes in the chemical composition of litter during decomposition: (1) the chemical convergence hypothesis (“all litters will converge eventually towards a common chemistry”); (2) the initial litter quality hypothesis (“the initial litter quality has persistent effects on litter chemical composition”); (3) the decomposer control hypothesis (“changes of litter chemistry during decomposition do not simply reflect the effects of time or initial litter quality, but are also strongly influenced by the functional attributes of distinct decomposer communities”). Our dataset allows for testing these hypotheses of organic matter decomposition in soil. Previous results from spectroscopic analyses (MIRS and NEXAFS) of LTBF soils failed to provide clear evidence of chemical trends and convergence in SOC composition over time (Barré et al. 2016). Conversely, the Py-GC-MS data reported here demonstrate that, despite contrasting initial molecular
characteristics, SOC in soils subject to more than 25 years of bare fallow had similar chemical trajectories during degradation. The trajectory was characterized by a decrease in lignin-, cutin- and suberin-derived compounds and a relative increase in N-containing compounds and alkanes (Figure 3). However, initial differences in SOC composition among the sites were reflected even after 50 years of decomposition, and these differences among sites appear to be greater than the changes undergone during long-term decomposition. The degradation of SOC may therefore be explained by a combination of the chemical convergence and the initial chemical quality hypotheses proposed by Wickings et al. (2012). Our focus on the evolution of the chemical and molecular composition of SOC during decomposition under long-term bare fallow does not address the structure of the decomposer communities. Consequently, the decomposer control hypothesis cannot be rules out.

312

313 Acknowledgements

The INSU EC2CO program is acknowledged for financial support (CARACAS project). We thank Rothamsted Research and the Lawes Agricultural Trust for access to archived samples and the BBSRC for support under the Institute National Capabilities program grant. Related information and data can be found in the electronic Rothamsted Archive (era.rothamsted.ac.uk).

The Danish contribution was financially supported by The Ministry of Environment and Food.

The Swedish contribution was supported by the Faculty of Natural Resources and Agriculture at the Swedish University of Agricultural Sciences. We thank the two reviewers for their constructive comments on the manuscript.

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characteristics, SOC in soils subject to more than 25 years of bare fallow had similar chemical trajectories during degradation. The trajectory was characterized by a decrease in lignin-, cutin- and suberin-derived compounds and a relative increase in N-containing compounds and alkanes (Figure 3). However, initial differences in SOC composition among the sites were reflected even after 50 years of decomposition, and these differences among sites appear to be greater than the changes undergone during long-term decomposition. The degradation of SOC may therefore be explained by a combination of the chemical convergence and the initial chemical quality hypotheses proposed by Wickings et al. (2012). Our focus on the evolution of the chemical and molecular composition of SOC during decomposition under long-term bare fallow does not address the structure of the decomposer communities. Consequently, the decomposer control hypothesis cannot be rules out.

312

313 Acknowledgements

The INSU EC2CO program is acknowledged for financial support (CARACAS project). We thank Rothamsted Research and the Lawes Agricultural Trust for access to archived samples and the BBSRC for support under the Institute National Capabilities program grant. Related information and data can be found in the electronic Rothamsted Archive (era.rothamsted.ac.uk).

The Danish contribution was financially supported by The Ministry of Environment and Food.

The Swedish contribution was supported by the Faculty of Natural Resources and Agriculture at the Swedish University of Agricultural Sciences. We thank the two reviewers for their constructive comments on the manuscript.

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Quépéa K, Mathieu J, Derenne S (2012) Soil lipids from accelerated solvent extraction:

419

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45-52
421 Riederer M, Matzke K, Ziegler F, Kögel-Knabner I (1993) Occurrence, distribution and fate of
422 the lipid plant biopolymers cutin and suberin in temperate forest soils. Organic
423 Geochem., 20: 1063-1076

424 Rieley G, Collier RJ, Jones DM, Eglinton G, Eakin PA, Fallick AE (1991) Sources of
425 sedimentary lipids deduced from stable carbon-isotope analyses of individual
426 compounds. Nature, 352(6334), 425.

427 Schellekens J, Barberá GG, Buurman P, Pérez-Jordà G, Martínez-Cortizas A (2013) Soil
428 organic matter dynamics in Mediterranean A-horizons — the use of analytical pyrolysis
429 to ascertain land-use history. J. Anal. Appl. Pyrolysis, 104: 287-298

430 Schmidt MWI, Torn MS, Abiven S, Dittmar T, Guggenberger G, Janssens IA, Kleber M,
431 Kogel-Knabner I, Lehmann J, Manning DAC, Nannipieri P, Rasse DP, Weiner S,
432 Trumbore SE (2011) Persistence of soil organic matter. Nature, 478: 49-56

433 Sollins P, Kramer MG, Swanston C, Lajtha K, Filley T, Aufdenkampe AK, Wagai R, Bowden
434 RD (2009) Sequential density fractionation across soils of contrasted mineralogy:
435 evidence for both microbial- and mineral-controlled soil organic matter stabilization.
436 Biogeochem., 96: 209-231

437 Stockmann U, Adams MA, Crawford JW, Field DJ, et al. (2013) The knowns, known
438 unknowns and unknowns of sequestration of soil organic carbon. Agriculture
439 Ecosystems & Environment, 164: 80-99

440 Vancampenhout K, Wouters K, De Vos B, Buurman P, Swennen R, Deckers J
441 (2009) Differences in chemical composition of soil organic matter in natural ecosystems
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443 568-579

444 Vidal A, Quenea K, Alexis M, Derenne S (2016) Molecular fate of root and shoot litter on
445 incorporation and decomposition in earthworm casts. Organic Geochem., 101: 1-10
421 Riederer M, Matzke K, Ziegler F, Kögel-Knabner I (1993) Occurrence, distribution and fate of
422 the lipid plant biopolymers cutin and suberin in temperate forest soils. Organic
423 Geochem., 20: 1063-1076

424 Rieley G, Collier RJ, Jones DM, Eglinton G, Eakin PA, Fallick AE (1991) Sources of
425 sedimentary lipids deduced from stable carbon-isotope analyses of individual
426 compounds. Nature, 352(6334), 425.

427 Schellekens J, Barberá GG, Buurman P, Pérez-Jordà G, Martínez-Cortizas A (2013) Soil
428 organic matter dynamics in Mediterranean A-horizons — the use of analytical pyrolysis
429 to ascertain land-use history. J. Anal. Appl. Pyrolysis, 104: 287-298

430 Schmidt MWI, Torn MS, Abiven S, Dittmar T, Guggenberger G, Janssens IA, Kleber M,
431 Kogel-Knabner I, Lehmann J, Manning DAC, Nannipieri P, Rasse DP, Weiner S,
432 Trumbore SE (2011) Persistence of soil organic matter. Nature, 478: 49-56

433 Sollins P, Kramer MG, Swanston C, Lajtha K, Filley T, Aufdenkampe AK, Wagai R, Bowden
434 RD (2009) Sequential density fractionation across soils of contrasted mineralogy:
435 evidence for both microbial- and mineral-controlled soil organic matter stabilization.
436 Biogeochem., 96: 209-231

437 Stockmann U, Adams MA, Crawford JW, Field DJ, et al. (2013) The knowns, known
438 unknowns and unknowns of sequestration of soil organic carbon. Agriculture
439 Ecosystems & Environment, 164: 80-99

440 Vancampenhout K, Wouters K, De Vos B, Buurman P, Swennen R, Deckers J
441 (2009) Differences in chemical composition of soil organic matter in natural ecosystems
442 from different climatic regions – a pyrolysis-GC/MS study. Soil Biol. Biochem., 41:
443 568-579

444 Vidal A, Quenea K, Alexis M, Derenne S (2016) Molecular fate of root and shoot litter on
445 incorporation and decomposition in earthworm casts. Organic Geochem., 101: 1-10
446

Wickings K, Grandy SG, Reed SC, Cleveland CC (2012) The origin of litter chemical

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Wannigama GP, Volkman JK, Gillan FT, Nichols PD, Johns RB (1981) A comparison of lipid

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Organic Geochem., 41: 1049-1055

454

455
446

Wickings K, Grandy SG, Reed SC, Cleveland CC (2012) The origin of litter chemical

447

complexity during decomposition. Ecol. Let., 15: 1180-1188

448

Wannigama GP, Volkman JK, Gillan FT, Nichols PD, Johns RB (1981) A comparison of lipid

449

components of the fresh and dead leaves and pneumatophores of the mangrove

450

Avicennia marina. Phytochemistry, 20: 659-666

451

Wiesenfeld GLB, Gocke M, Kuzyakov Y (2010) Fast incorporation of root-derived lipids and

452

fatty acids into soil – evidence from a short term multiple pulse labelling experiment.

453

Organic Geochem., 41: 1049-1055

454

455
<figure><img src="image_1.png" /><figcaption>**Fig. 1** Total ion concentration (TIC) traces of 650°C Curie point pyrolysate of initial samples of each site. Compound families are identified using various symbols. **C** refers to “carbohydrates”, **N** to “N-containing compounds”, **●** to “lignin-derived” compounds and **★** to other phenolic compounds. Aliphatic compounds were identified as follows: methyl esters of microbe-derived fatty acids (◇), long-chain methyl esters of n-alkanoic acids (◆), ω-hydroxyalkanoic acids (■), n-alkanedioic acids (CAL), n-alkanes (Θ), n-alkanols (♥), 8,16-9,16 and 10, 16 or 18 dihydroxyhexadecanoic acids (DiOH16), 9,10,18 trihydroxyoctadecanoic acid (TriOH). + corresponds to a contaminant and ? to an unidentified compound.</figcaption></figure>
<figure><img src="image_2.png" /><figcaption>**Fig. 1** Total ion concentration (TIC) traces of 650°C Curie point pyrolysate of initial samples of each site. Compound families are identified using various symbols. **C** refers to “carbohydrates”, **N** to “N-containing compounds”, **●** to “lignin-derived” compounds and **★** to other phenolic compounds. Aliphatic compounds were identified as follows: methyl esters of microbe-derived fatty acids (◇), long-chain methyl esters of n-alkanoic acids (◆), ω-hydroxyalkanoic acids (■), n-alkanedioic acids (CAL), n-alkanes (Θ), n-alkanols (♥), 8,16 - 9,16 and 10, 16 or 18 dihydroxyhexadecanoic acids (DiOH16), 9,10,18 trihydroxyoctadecanoic acid (TriOH). + corresponds to a contaminant and ? to an unidentified compound.</figcaption></figure>
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /><figcaption>**Fig. 2** Scores (a) and loadings (b) of principal component analysis carried out with the relative abundance of the 18 compounds (see Table 2 for description) of soil samples from different durations of long-term bare fallow at four sites. Arrows on Figure 2a were added from visual interpretation. Variables in black and grey with solid lines correspond to plant-derived and microbe-derived compounds respectively. Variables in black with dashed lines correspond to ubiquitous compounds.</figcaption></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2** Scores (a) and loadings (b) of principal component analysis carried out with the relative abundance of the 18 compounds (see Table 2 for description) of soil samples from different durations of long-term bare fallow at four sites. Arrows on Figure 2a were added from visual interpretation. Variables in black and grey with solid lines correspond to plant-derived and microbe-derived compounds respectively. Variables in black with dashed lines correspond to ubiquitous compounds.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 3** Evolution with bare fallow duration of the relative abundances of lignin, cutin+suberin (sum of ωOH, diacids long chain, TriOH and DiOH), long-chain alkanes and N-containing compounds at four long-term bare fallow sites. As cutin- and suberin-derived products (ω-hydroxyalkanoic acids, n-alkanedioic acids, dihydroxyhexadecanoic acids, trihydroxyoctadecanoic acid) had a similar contribution to the PCA axis (Figure 2b), their relative abundances were combined in this figure. Dotted lines refer to statistically significant (p<0.05) relationships.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3** Evolution with bare fallow duration of the relative abundances of lignin, cutin+suberin (sum of ωOH, diacids long chain, TriOH and DiOH), long-chain alkanes and N-containing compounds at four long-term bare fallow sites. As cutin- and suberin-derived products (ω-hydroxyalkanoic acids, n-alkanedioic acids, dihydroxyhexadecanoic acids, trihydroxyoctadecanoic acid) had a similar contribution to the PCA axis (Figure 2b), their relative abundances were combined in this figure. Dotted lines refer to statistically significant (p<0.05) relationships.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 4** Differences in the relative abundances of plant-derived, microbe-derived and N-compounds between initial and final soil samples from four long-term bare fallow sites. All compounds within the plant and microbial origins groups were summed. For Rothamsted, Versailles and Askov, where two replicates were analyzed, the two values were averaged. Differences were obtained by subtracting the averaged relative abundances in the final samples from the averaged values in initial samples.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 4** Differences in the relative abundances of plant-derived, microbe-derived and N-compounds between initial and final soil samples from four long-term bare fallow sites. All compounds within the plant and microbial origins groups were summed. For Rothamsted, Versailles and Askov, where two replicates were analyzed, the two values were averaged. Differences were obtained by subtracting the averaged relative abundances in the final samples from the averaged values in initial samples.</figcaption></figure>
<table><thead><tr><th></th><th>Versailles (FR)</th><th>Rothamsted (UK)</th><th>Ultuna (SE)</th><th>Askov (DK)</th></tr></thead><tbody><tr><td>Location</td><td>48°48' N<br>2°08' E</td><td>51°82' N<br>0°35' E</td><td>59°49' N<br>17°38' E</td><td>55°28' N<br>9°07' E</td></tr><tr><td>Mean annual<br>temperature (°C)</td><td>10.7</td><td>9.5</td><td>5.5</td><td>7.8</td></tr><tr><td>Mean annual<br>precipitation<br>(mm)</td><td>628</td><td>712</td><td>533</td><td>862</td></tr><tr><td>FAO soil<br>classification</td><td>Luvisol</td><td>Chromic Luvisol</td><td>Eutric<br>Cambisol</td><td>Orthic Luvisol</td></tr><tr><td>Previous land-use</td><td>Unmanaged<br>grassland</td><td>Managed<br>grassland</td><td>Arable</td><td>Arable</td></tr><tr><td>Soil textural class<br>(% sand/ silt/clay)</td><td>Silty loam<br>(26/57/17)</td><td>Flinty silty clay<br>loam (13/62/25)</td><td>Clay loam<br>(23/41/36)</td><td>Coarse sandy<br>loam (79/11/10)</td></tr><tr><td>Sampling depth<br>(cm)</td><td>25</td><td>23</td><td>20</td><td>20</td></tr><tr><td>Experiment<br>initiation year</td><td>1928</td><td>1959</td><td>1956</td><td>1956</td></tr><tr><td>Number of field<br>replicate plots</td><td>2</td><td>2</td><td>1</td><td>2</td></tr><tr><td>Sampling year<br>(OC concentration<br>mgC g<sup>-1</sup> soil)</td><td>1929<br>(18.7 ; 16.2)<br>1939<br>(13.2 ; 11.9)<br>1950<br>(11.9 ; 11.3)<br>1980<br>(7.5 ; 7.4)</td><td>1959<br>(30.5 ; 30.1)<br>1971<br>(18.2 ; 18.1)<br>1987<br>(12.5 ; 13.9)<br>2008<br>(9.7 ; 10.3)</td><td>1956<br>(15.0)<br>1967<br>(13.3)<br>1985<br>(10.6)<br>2009<br>(9.1)</td><td>1956<br>(17.5 ; 16.9)<br>1968<br>(11.8 ; 14.1)<br>1983<br>(11.4 ; 11.2)</td></tr></tbody></table>

509

**Table 1** Selected site characteristics of four long-term bare fallow field experiments, and the identification of archived soil samples used for characterization of the molecular composition of soil organic matter.

510

511

512

513

514

515

516
<table><thead><tr><th></th><th>Versailles (FR)</th><th>Rothamsted (UK)</th><th>Ultuna (SE)</th><th>Askov (DK)</th></tr></thead><tbody><tr><td>Location</td><td>48°48' N<br>2°08' E</td><td>51°82' N<br>0°35' E</td><td>59°49' N<br>17°38' E</td><td>55°28' N<br>9°07' E</td></tr><tr><td>Mean annual temperature (°C)</td><td>10.7</td><td>9.5</td><td>5.5</td><td>7.8</td></tr><tr><td>Mean annual precipitation (mm)</td><td>628</td><td>712</td><td>533</td><td>862</td></tr><tr><td>FAO soil classification</td><td>Luvisol</td><td>Chromic Luvisol</td><td>Eutric Cambisol</td><td>Orthic Luvisol</td></tr><tr><td>Previous land-use</td><td>Unmanaged grassland</td><td>Managed grassland</td><td>Arable</td><td>Arable</td></tr><tr><td>Soil textural class (% sand/ silt/clay)</td><td>Silty loam (26/57/17)</td><td>Flinty silty clay loam (13/62/25)</td><td>Clay loam (23/41/36)</td><td>Coarse sandy loam (79/11/10)</td></tr><tr><td>Sampling depth (cm)</td><td>25</td><td>23</td><td>20</td><td>20</td></tr><tr><td>Experiment initiation year</td><td>1928</td><td>1959</td><td>1956</td><td>1956</td></tr><tr><td>Number of field replicate plots</td><td>2</td><td>2</td><td>1</td><td>2</td></tr><tr><td>Sampling year (OC concentration mgC g<sup>-1</sup> soil)</td><td>1929 (18.7 ; 16.2)<br>1939 (13.2 ; 11.9)<br>1950 (11.9 ; 11.3)<br>1980 (7.5 ; 7.4)</td><td>1959 (30.5 ; 30.1)<br>1971 (18.2 ; 18.1)<br>1987 (12.5 ; 13.9)<br>2008 (9.7 ; 10.3)</td><td>1956 (15.0)<br>1967 (13.3)<br>1985 (10.6)<br>2009 (9.1)</td><td>1956 (17.5 ; 16.9)<br>1968 (11.8 ; 14.1)<br>1983 (11.4 ; 11.2)</td></tr></tbody></table>

509

**Table 1** Selected site characteristics of four long-term bare fallow field experiments, and the identification of archived soil samples used for characterization of the molecular composition of soil organic matter.

510

511

512

513

514

515

516
<table><caption>**Table 2** Identified pyrolysis products, the probable origins of each product or group of products, and associated key references</caption><thead><tr><th>Code (Figure 2)</th><th>Symbol (Figure 1)</th><th>Compound</th><th>Origin</th><th>Key references</th></tr></thead><tbody><tr><td>Lignin</td><td>●</td><td>Lignin derived aromatic compounds</td><td>Higher plants</td><td>Clifford et al. (1995), Chefetz et al. (2000), Nierop (2001) and Otto et al. (2005)</td></tr><tr><td colspan="5"><u>Other phenolic compounds</u></td></tr><tr><td>P1</td><td>★</td><td>1,4-Dimethoxy benzene</td><td>Higher plants</td><td>Chefetz et al. (2000)</td></tr><tr><td>P2</td><td>★</td><td>1,2,4-Trimethoxy benzene (4-methoxy catechol)</td><td>Higher plants</td><td>Fabbri & Helleur (1999)</td></tr><tr><td colspan="5"><u>Aliphatic compounds</u></td></tr><tr><td>FAmicro</td><td>◇</td><td>C14, C14i, C15i,a and C17i,a n-alkanoic acids</td><td>Microorganisms</td><td>Wannigama et al. (1981), Quénéa et al. (2012)</td></tr><tr><td>FA_lc</td><td>◆</td><td>C20-32 even chain n-alkanoic acid</td><td>Higher plants</td><td>Chefetz et al. (2000), Wannigama et al. (1981)</td></tr><tr><td>FA16</td><td>FA16</td><td>C16 n-alkanoic acid</td><td>Ubiquitous</td><td>Nierop (2001)</td></tr><tr><td>FA18</td><td>FA18</td><td>C18 n-alkanoic acid</td><td>Ubiquitous</td><td>Wiesenberga et al. (2010)</td></tr><tr><td>FAuns</td><td>C16:1<br>C18:1</td><td>C16:1 and C18:1 n-alkanoic acid</td><td>Ubiquitous</td><td>Wiesenberga et al. (2010)</td></tr><tr><td>Alk_lc</td><td>◎</td><td>C25-33 n-alkanes</td><td>Higher plants</td><td>Rieley et al. (1991), Wiesenberga et al. (2010)</td></tr><tr><td>Alk_ram_sc</td><td>◎</td><td>C13-15 n-alkanes + ramified short chain alkanes</td><td>Microorganisms</td><td>Han and Calvin (1969)</td></tr><tr><td>OH</td><td>♥</td><td>C18-30 n-alkanols</td><td>Higher plants</td><td>Kolattukudy (1980), Quénéa et al. (2006)</td></tr><tr><td>ωOH</td><td>▲</td><td>C16-26 even chain x-hydroxyalkanoic acids</td><td>Higher plants (Cutin and Suberin)</td><td>Kolattukudy (1980) and Nierop (2001)</td></tr><tr><td>Dia_sc</td><td>●</td><td>C8, C9 n-alkanedioic acids</td><td>Ubiquitous</td><td>Kolattukudy (1980) and Nierop (2001), Grasset & Amblès (1998)</td></tr><tr><td>Dia_lc</td><td>●</td><td>C18, C22 and C24 n-alkanedioic acids</td><td>Higher plants (Cutin and Suberin)</td><td>Kolattukudy (1980) and Nierop (2001)</td></tr><tr><td>DiOH</td><td>DiOH16</td><td>8,16 - 9,16 and 10, 16 or 18 dihydroxyhexadecanoic acids</td><td>Higher plants (Cutin)</td><td>Riederer et al. (1993) and Del Río & Hatcher (1998)</td></tr><tr><td>TriOH</td><td>TriOH</td><td>9,10,18 trihydroxyoctadecanoic acid</td><td>Higher plants (Cutin)</td><td>Otto & Simpson (2006)</td></tr><tr><td colspan="5"><u>Other compounds</u></td></tr><tr><td>Ch</td><td>C</td><td>Carbohydrates</td><td>Ubiquitous</td><td></td></tr><tr><td>N-compounds</td><td>N</td><td>N-containing compounds</td><td>Ubiquitous</td><td></td></tr></tbody></table>
<table><caption>**Table 2** Identified pyrolysis products, the probable origins of each product or group of products, and associated key references</caption><thead><tr><th>Code (Figure 2)</th><th>Symbol (Figure 1)</th><th>Compound</th><th>Origin</th><th>Key references</th></tr></thead><tbody><tr><td>Lignin</td><td>●</td><td>Lignin derived aromatic compounds</td><td>Higher plants</td><td>Clifford et al. (1995), Chefetz et al. (2000), Nierop (2001) and Otto et al. (2005)</td></tr><tr><td colspan="5"><u>Other phenolic compounds</u></td></tr><tr><td>P1</td><td>★</td><td>1,4-Dimethoxy benzene</td><td>Higher plants</td><td>Chefetz et al. (2000)</td></tr><tr><td>P2</td><td>★</td><td>1,2,4-Trimethoxy benzene (4-methoxy catechol)</td><td>Higher plants</td><td>Fabbri & Helleur (1999)</td></tr><tr><td colspan="5"><u>Aliphatic compounds</u></td></tr><tr><td>FAmicro</td><td>◇</td><td>C14, C14i, C15i,a and C17i,a n-alkanoic acids</td><td>Microorganisms</td><td>Wannigama et al. (1981), Quénéa et al. (2012)</td></tr><tr><td>FA_lc</td><td>◆</td><td>C20-32 even chain n-alkanoic acid</td><td>Higher plants</td><td>Chefetz et al. (2000), Wannigama et al. (1981)</td></tr><tr><td>FA16</td><td>FA16</td><td>C16 n-alkanoic acid</td><td>Ubiquitous</td><td>Nierop (2001)</td></tr><tr><td>FA18</td><td>FA18</td><td>C18 n-alkanoic acid</td><td>Ubiquitous</td><td>Wiesenberga et al. (2010)</td></tr><tr><td>FAuns</td><td>C16:1<br>C18:1</td><td>C16:1 and C18:1 n-alkanoic acid</td><td>Ubiquitous</td><td>Wiesenberga et al. (2010)</td></tr><tr><td>Alk_lc</td><td>◎</td><td>C25-33 n-alkanes</td><td>Higher plants</td><td>Rieley et al. (1991), Wiesenberga et al. (2010)</td></tr><tr><td>Alk_ram_sc</td><td>◎</td><td>C13-15 n-alkanes + ramified short chain alkanes</td><td>Microorganisms</td><td>Han and Calvin (1969)</td></tr><tr><td>OH</td><td>♥</td><td>C18-30 n-alkanols</td><td>Higher plants</td><td>Kolattukudy (1980), Quénéa et al. (2006)</td></tr><tr><td>ωOH</td><td>~</td><td>C16-26 even chain x-hydroxyalkanoic acids</td><td>Higher plants (Cutin and Suberin)</td><td>Kolattukudy (1980) and Nierop (2001)</td></tr><tr><td>Dia_sc</td><td>●</td><td>C8, C9 n-alkanedioic acids</td><td>Ubiquitous</td><td>Kolattukudy (1980) and Nierop (2001), Grasset & Amblès (1998)</td></tr><tr><td>Dia_lc</td><td>●</td><td>C18, C22 and C24 n-alkanedioic acids</td><td>Higher plants (Cutin and Suberin)</td><td>Kolattukudy (1980) and Nierop (2001)</td></tr><tr><td>DiOH</td><td>DiOH16</td><td>8,16 - 9,16 and 10, 16 or 18 dihydroxyhexadecanoic acids</td><td>Higher plants (Cutin)</td><td>Riederer et al. (1993) and Del Río & Hatcher (1998)</td></tr><tr><td>TriOH</td><td>TriOH</td><td>9,10,18 trihydroxyoctadecanoic acid</td><td>Higher plants (Cutin)</td><td>Otto & Simpson (2006)</td></tr><tr><td colspan="5"><u>Other compounds</u></td></tr><tr><td>Ch</td><td>C</td><td>Carbohydrates</td><td>Ubiquitous</td><td></td></tr><tr><td>N-compounds</td><td>N</td><td>N-containing compounds</td><td>Ubiquitous</td><td></td></tr></tbody></table>