# Ley grassland under temperate climate had a legacy effect on soil organic matter quantity, biogeochemical signature and microbial activities

A. Crème, Cornelia Rumpel, Xavier Le Roux, A. Romian, T. Lan, Abad Chabbi

► To cite this version:

A. Crème, Cornelia Rumpel, Xavier Le Roux, A. Romian, T. Lan, et al. Ley grassland under temperate climate had a legacy effect on soil organic matter quantity, biogeochemical signature and microbial activities. Soil Biology and Biochemistry, 2018, 122, pp.203-210. 10.1016/j.soilbio.2018.04.018. hal-01796515

HAL Id: hal-01796515

https://hal.science/hal-01796515

Submitted on 5 Feb 2024

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

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

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

Distributed under a Creative Commons Attribution 4.0 International License
# Ley grassland under temperate climate had a legacy effect on soil organic matter quantity, biogeochemical signature and microbial activities

A. Crème, Cornelia Rumpel, Xavier Le Roux, A. Romian, T. Lan, Abad Chabbi

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

## ► To cite this version:

A. Crème, Cornelia Rumpel, Xavier Le Roux, A. Romian, T. Lan, et al., Ley grassland under temperate climate had a legacy effect on soil organic matter quantity, biogeochemical signature and microbial activities. Soil Biology and Biochemistry, 2018, 122, pp.203-210. 10.1016/j.soilbio.2018.04.018. hal-01796515

HAL Id: hal-01796515

https://hal.science/hal-01796515v1

Submitted on 5 Feb 2024

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

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

<figure><img src="image_2.png" /></figure>
1 Ley grassland under temperate climate had a legacy effect on soil organic matter

2 quantity, biogeochemical signature and microbial activities

3

4 Crème, A¹²³, Rumpel, C.³*, Le Roux, X.⁴, Romian, A.³, Lan, T.³, Chabbi, A.¹²*

5

6 1 : INRA, UR P3F, 86600 Lusignan, France.

7 2 : INRA, ECOSYS UMR 1402 (INRA, AgroParisTech, Université Paris-Saclay), 78850

8 Thiverval-Grignon, France.

9 3 : CNRS, UPMC, IEES UMR (UPMC 113, CNRS 7618, IRD 242, INRA 1392, Université Paris 7 113, UPEC 7618), 78850 Thiverval-Grignon, France.

10

11 4 : INRA, LEM UMR 1418 (INRA, CNRS, Université Lyon 1, Université de Lyon), 69622

12 Villeurbanne, France.

13

14 *corresponding authors : abad.chabbi@inra.fr; cornelia.rumpel@inra.fr

15
1 Ley grassland under temperate climate had a legacy effect on soil organic matter

2 quantity, biogeochemical signature and microbial activities

3

4 Crème, A¹²³, Rumpel, C.³*, Le Roux, X.⁴, Romian, A.³, Lan, T.³, Chabbi, A.¹²*

5

6 1 : INRA, UR P3F, 86600 Lusignan, France.

7 2 : INRA, ECOSYS UMR 1402 (INRA, AgroParisTech, Université Paris-Saclay), 78850

8 Thiverval-Grignon, France.

9 3 : CNRS, UPMC, IEES UMR (UPMC 113, CNRS 7618, IRD 242, INRA 1392, Université Paris 7 113, UPEC 7618), 78850 Thiverval-Grignon, France.

10

11 4 : INRA, LEM UMR 1418 (INRA, CNRS, Université Lyon 1, Université de Lyon), 69622

12 Villeurbanne, France.

13

14 *corresponding authors : abad.chabbi@inra.fr; cornelia.rumpel@inra.fr

15
## Abstract

Temporary (ley) grassland introduced into cropping cycles has been advocated as being beneficial for the delivery of ecosystem services by agricultural soils. The management of these temporary grasslands has unknown effects on soil organic matter (SOM) concentrations and biogeochemical properties of the cropland soils following the grassland phase. Here, we investigated the legacy effect of differently managed temporary grasslands, i.e. change of soil properties lasting beyond three years of crop. We assessed soil organic carbon (SOC) quantity and SOM biogeochemical signature (composition of soil neutral carbohydrates and lignin), as well as microbial activities (potential C and N mineralization and denitrification). We used a long-term field experiment on Cambisol with temperate climate in western France, where temporary grassland management practices differed in terms of duration (3 or 6 years) and presence or absence of N fertilization. Topsoil (10 cm) samples were collected after a 3-yr crop rotation (maize, wheat, barley).

Our results showed that N fertilization during the grassland phase was necessary to maintain soil C and N concentrations beyond three years of crop. Temporary grassland management may affect microbial activities as indicated by contrasting polysaccharide and lignin composition. It had however, no effect on potential CO₂ and N₂O emissions during laboratory incubations. The biogeochemical signature of SOM was close to continuous grassland only in treatments with 6 yrs of fertilized temporary grassland. We thus, conclude that the legacy effects of a grassland phase on SOC quantity and properties of SOM depend on its management.

## Graphical abstract

<table><thead><tr><th></th><th colspan="2">Control</th><th colspan="3">Temporary grassland</th></tr></thead><tbody><tr><td rowspan="3">3 years<br>6 years<br>9 years</td><td>Cropland</td><td>Grassland</td><td>Cropland</td><td>Grassland</td><td>Grassland N-</td></tr><tr><td>Cropland</td><td>Grassland</td><td>Grassland</td><td>Grassland</td><td>Grassland N-</td></tr><tr><td>Cropland</td><td>Grassland</td><td>Cropland</td><td>Cropland</td><td>Cropland</td></tr><tr><td></td><td>9 yrs crop rotation<br>maize-wheat-barley</td><td>9 yrs fertilized<br>grassland</td><td colspan="2">3yrs<br>Ley Duration</td><td>6yrs<br>N+<br>Ley Fertilization</td></tr><tr><td></td><td colspan="2">Cropland SOM ≠ Grassland SOM</td><td colspan="2">Unexpected non-progressive age<br>dependent SOM effect</td><td>Positive legacy effect on SOM<br>quantity and chemical signature</td></tr><tr><td></td><td colspan="2"></td><td colspan="2">6yrs fertilized grassland introduction in crop rotation have positive<br>legacy effects on SOM content and biochemical signature</td><td></td></tr></tbody></table>
## Abstract

Temporary (ley) grassland introduced into cropping cycles has been advocated as being beneficial for the delivery of ecosystem services by agricultural soils. The management of these temporary grasslands has unknown effects on soil organic matter (SOM) concentrations and biogeochemical properties of the cropland soils following the grassland phase. Here, we investigated the legacy effect of differently managed temporary grasslands, i.e. change of soil properties lasting beyond three years of crop. We assessed soil organic carbon (SOC) quantity and SOM biogeochemical signature (composition of soil neutral carbohydrates and lignin), as well as microbial activities (potential C and N mineralization and denitrification). We used a long-term field experiment on Cambisol with temperate climate in western France, where temporary grassland management practices differed in terms of duration (3 or 6 years) and presence or absence of N fertilization. Topsoil (10 cm) samples were collected after a 3-yr crop rotation (maize, wheat, barley).

Our results showed that N fertilization during the grassland phase was necessary to maintain soil C and N concentrations beyond three years of crop. Temporary grassland management may affect microbial activities as indicated by contrasting polysaccharide and lignin composition. It had however, no effect on potential CO₂ and N₂O emissions during laboratory incubations. The biogeochemical signature of SOM was close to continuous grassland only in treatments with 6 yrs of fertilized temporary grassland. We thus, conclude that the legacy effects of a grassland phase on SOC quantity and properties of SOM depend on its management.

## Graphical abstract

<table><thead><tr><th></th><th colspan="2">Control</th><th colspan="3">Temporary grassland</th></tr></thead><tbody><tr><td rowspan="3">3 years<br>6 years<br>9 years</td><td>Cropland</td><td>Grassland</td><td>Cropland</td><td>Grassland</td><td>Grassland N-</td></tr><tr><td>Cropland</td><td>Grassland</td><td>Grassland</td><td>Grassland</td><td>Grassland N-</td></tr><tr><td>Cropland</td><td>Grassland</td><td>Cropland</td><td>Cropland</td><td>Cropland</td></tr><tr><td></td><td>9 yrs crop rotation<br>maize-wheat-barley</td><td>9 yrs fertilized<br>grassland</td><td colspan="2">3yrs<br>Ley Duration</td><td>6yrs<br>N+<br>Ley Fertilization</td></tr><tr><td></td><td colspan="2">Cropland SOM ≠ Grassland SOM</td><td colspan="2">Unexpected non-progressive age<br>dependent SOM effect</td><td>Positive legacy effect on SOM<br>quantity and chemical signature</td></tr><tr><td></td><td colspan="2"></td><td colspan="2">6yrs fertilized grassland introduction in crop rotation have positive<br>legacy effects on SOM content and biochemical signature</td><td></td></tr></tbody></table>
43

**Keywords**

44

Temporary grassland management, microbial activities, potential N₂O emission, soil organic matter biogeochemistry, long-term field experiment.

45

46

47

**Abbreviations**

48

SOM, Soil organic matter; SOC, Soil organic carbon; TG, temporary grasslands; TG3N+, temporary grassland with 3 years of fertilized ley; TG6N+, temporary grassland with 6 year of fertilized ley; TG6N-, temporary grassland with 6 years ley without fertilization; CC, permanent cropland; GG, permanent grassland; POM, particulate organic matter; IN, inorganic nitrogen; ON organic nitrogen; IP inorganic phosphorus; OP, organic phosphorus; V, vanillyl; S, syringyl; Co, *p*-coumaryl; Ac, acid; Al, aldehyde; C5, pentoses; C6, hexoses; MB-C, microbial carbon; MB-N, microbial nitrogen; CLPP, community-level physiological profiles; AWCD, average well color development; Cmin, carbon potential mineralization; Nmin, nitrogen potential mineralization; *k*, carbon mineralization constant rate; PD, potential denitrification; PCA, principal component analysis.

49

50

51

52

53

54

55

56

57

58
43

**Keywords**

44

Temporary grassland management, microbial activities, potential N₂O emission, soil organic matter biogeochemistry, long-term field experiment.

45

46

47

**Abbreviations**

48

SOM, Soil organic matter; SOC, Soil organic carbon; TG, temporary grasslands; TG3N+, temporary grassland with 3 years of fertilized ley; TG6N+, temporary grassland with 6 year of fertilized ley; TG6N-, temporary grassland with 6 years ley without fertilization; CC, permanent cropland; GG, permanent grassland; POM, particulate organic matter; IN, inorganic nitrogen; ON organic nitrogen; IP inorganic phosphorus; OP, organic phosphorus; V, vanillyl; S, syringyl; Co, *p*-coumaryl; Ac, acid; Al, aldehyde; C5, pentoses; C6, hexoses; MB-C, microbial carbon; MB-N, microbial nitrogen; CLPP, community-level physiological profiles; AWCD, average well color development; Cmin, carbon potential mineralization; Nmin, nitrogen potential mineralization; *k*, carbon mineralization constant rate; PD, potential denitrification; PCA, principal component analysis.

49

50

51

52

53

54

55

56

57

58
59

# 1. Introduction

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

Agricultural soils of the temperate regions have lost much of their organic carbon since the 1960s due to the intensification of crop production (Lal, 2004). Nowadays, rebuilding soil organic matter (SOM) stocks has become a priority because SOM greatly improves soil properties and functions. Moreover, SOM storage may be able to counteract increasing atmospheric greenhouse gas concentrations (Chabbi et al., 2017; Smith, 2016). In this context, grassland soils with high belowground C stocks can be carbon sinks (Conant et al., 2001). Therefore, the introduction of temporary or ley grassland in cropland cycles may be a suitable negative emission technology (Smith, 2016), if it increases soil C sequestration as a legacy effect (i.e. soil C lasts beyond the following cropland phase).

Temporary grasslands were shown to have several beneficial effects on soil quality related to SOM increase (Studdert et al., 1997; Franzluebbers et al., 2014). Moreover, they reduce nitrate leaching as compared to continuous cropping systems (Kunrath et al., 2015). Recent work also showed that introduction of temporary mowed grassland in the cropping cycle led to higher contribution of SOM in macro-aggregates as compared to permanent arable land (Panettieri et al., 2017). This effect was observed as a legacy effect after 3 yrs of continuous cropping, and explained by higher amounts of root litter input during the grassland phase (Armas-Herrera et al., 2016; Attard et al., 2016). However, in order to secure the C sink function of grasslands, judicious management is necessary (Smith et al., 2015). Indeed, management of temporary grasslands in terms of cutting or grazing, fertilisation and duration, influences the decomposition and stabilisation processes of SOM through its impact on C, N and P coupling and decoupling (Acharya et al., 2012; Attard et al., 2016; Conant et al., 2001; Crème et al., 2016; Klumpp et al., 2009; Lemaire et al., 2015; Rumpel et al., 2015). Beneficial effects of temporary grasslands may depend on the interplay between environmental constraints, soil microorganisms and SOM characteristics. To date the effect of temporary grassland management on these variables is still unknown.

In this study, we investigated the legacy effect (i.e. changes observable after 3 years of crop) of differently managed temporary grasslands on SOM and microbial properties. The aim of the study was to better apprehend the impact of temporary grassland management on SOM biogeochemical composition, nutrient forms as well as potential microbial activities. We analysed SOM molecular signatures of soil neutral carbohydrates and lignin, susceptible to be affected by grassland management (Crème et al., 2017). These two macromolecules were chosen because their nature and fate in soil are contrasting (Kögel-Knabner and Rumpel, 2018). As biomarkers, their quantity and composition may give valuable information on
59

# 1. Introduction

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

Agricultural soils of the temperate regions have lost much of their organic carbon since the 1960s due to the intensification of crop production (Lal, 2004). Nowadays, rebuilding soil organic matter (SOM) stocks has become a priority because SOM greatly improves soil properties and functions. Moreover, SOM storage may be able to counteract increasing atmospheric greenhouse gas concentrations (Chabbi et al., 2017; Smith, 2016). In this context, grassland soils with high belowground C stocks can be carbon sinks (Conant et al., 2001). Therefore, the introduction of temporary or ley grassland in cropland cycles may be a suitable negative emission technology (Smith, 2016), if it increases soil C sequestration as a legacy effect (i.e. soil C lasts beyond the following cropland phase).

Temporary grasslands were shown to have several beneficial effects on soil quality related to SOM increase (Studdert et al., 1997; Franzluebbers et al., 2014). Moreover, they reduce nitrate leaching as compared to continuous cropping systems (Kunrath et al., 2015). Recent work also showed that introduction of temporary mowed grassland in the cropping cycle led to higher contribution of SOM in macro-aggregates as compared to permanent arable land (Panettieri et al., 2017). This effect was observed as a legacy effect after 3 yrs of continuous cropping, and explained by higher amounts of root litter input during the grassland phase (Armas-Herrera et al., 2016; Attard et al., 2016). However, in order to secure the C sink function of grasslands, judicious management is necessary (Smith et al., 2015). Indeed, management of temporary grasslands in terms of cutting or grazing, fertilisation and duration, influences the decomposition and stabilisation processes of SOM through its impact on C, N and P coupling and decoupling (Acharya et al., 2012; Attard et al., 2016; Conant et al., 2001; Crème et al., 2016; Klumpp et al., 2009; Lemaire et al., 2015; Rumpel et al., 2015). Beneficial effects of temporary grasslands may depend on the interplay between environmental constraints, soil microorganisms and SOM characteristics. To date the effect of temporary grassland management on these variables is still unknown.

In this study, we investigated the legacy effect (i.e. changes observable after 3 years of crop) of differently managed temporary grasslands on SOM and microbial properties. The aim of the study was to better apprehend the impact of temporary grassland management on SOM biogeochemical composition, nutrient forms as well as potential microbial activities. We analysed SOM molecular signatures of soil neutral carbohydrates and lignin, susceptible to be affected by grassland management (Crème et al., 2017). These two macromolecules were chosen because their nature and fate in soil are contrasting (Kögel-Knabner and Rumpel, 2018). As biomarkers, their quantity and composition may give valuable information on
degradation and stabilisation processes of SOM. Non-cellulosic carbohydrates have a low degree of polymerization and may be plant or microbial derived (Murayama, 1984; Kögel-Knabner, 2017, 2002; Kuzyakov et al., 2000; Rumpel and Dignac, 2006). In contrast, comparatively more recalcitrant compounds such as aromatic lignins are exclusively plant-derived. They are mineralizable only by a restricted number of decomposers (white-rot fungi) (Frei, 2013; Kögel-Knabner, 2017, 2002). In addition, we determined SOM C:N:P stoichiometry, which may change in response to grassland management (Crème et al., 2016). We also quantified the amount of particulate organic matter and potential microbial activities involved in CO2 and N2O emissions and nutrient release. We hypothesised (i) that temporary grassland has legacy effects on SOM quantity, molecular composition and microbial activities, observable after a 3 year crop rotation following the end of the grassland phase; (ii) that the magnitude of the legacy effect would depend on the length of the grassland phase (6 versus 3 years); and (iii) that the magnitude of the legacy effect would be sensitive to nitrogen fertilization applied during the grassland phase.

## 2. Materials and methods

### 2.1. Site description

The experimental site is the long term observatory on environmental research called "agrosystems biogeochemical cycles and biodiversity" (SOERE ACBB) situated at the INRA research station in Lusignan (46°25′12.91" N; 0°07′29.35" E), western France.

The mean annual temperature is 12°C and the mean annual precipitation is around 750 mm. The experiment was established in 2005 on a Cambisol with a silty-loam texture and a bulk density of 1.43 kg m⁻³ (Chabbi et al., 2009; Moni et al., 2010). The profile can be divided into two main domains: three red-brown upper horizons characterised by a loamy texture overlying three lower clayey rubefied horizons rich in kaolinite and iron oxides (Ducloux and Chesseron, 1988). The lower rubefied horizons were classified as Paleo-Ferralsol from the late Mesozoic period, while the upper horizons were classified as Cambisol formed from Ferralsol horizons under temperate climate (Ducloux and Chesseron, 1988).

At this site, in 2005, a long-term field experiment was established consisting of ley grassland introduction in the cropping cycle with 5 different treatments in terms of fertilization and duration of the grassland phase (Table 1): TG3N+ corresponds to 3 yrs of fertilized ley, TG6N+ to 6 yrs of fertilized ley and TG6N- is a 6 yrs ley without fertilization. In addition, two control treatments with permanent cropland (CC) consisting of maize-wheat-barley rotation and with permanent grassland (GG) were established. Treatments were
degradation and stabilisation processes of SOM. Non-cellulosic carbohydrates have a low degree of polymerization and may be plant or microbial derived (Murayama, 1984; Kögel-Knabner, 2017, 2002; Kuzyakov et al., 2000; Rumpel and Dignac, 2006). In contrast, comparatively more recalcitrant compounds such as aromatic lignins are exclusively plant-derived. They are mineralizable only by a restricted number of decomposers (white-rot fungi) (Frei, 2013; Kögel-Knabner, 2017, 2002). In addition, we determined SOM C:N:P stoichiometry, which may change in response to grassland management (Crème et al., 2016). We also quantified the amount of particulate organic matter and potential microbial activities involved in CO2 and N2O emissions and nutrient release. We hypothesised (i) that temporary grassland has legacy effects on SOM quantity, molecular composition and microbial activities, observable after a 3 year crop rotation following the end of the grassland phase; (ii) that the magnitude of the legacy effect would depend on the length of the grassland phase (6 versus 3 years); and (iii) that the magnitude of the legacy effect would be sensitive to nitrogen fertilization applied during the grassland phase.

## 2. Materials and methods

### 2.1. Site description

The experimental site is the long term observatory on environmental research called "agrosystems biogeochemical cycles and biodiversity" (SOERE ACBB) situated at the INRA research station in Lusignan (46°25′12.91" N; 0°07′29.35" E), western France.

The mean annual temperature is 12°C and the mean annual precipitation is around 750 mm. The experiment was established in 2005 on a Cambisol with a silty-loam texture and a bulk density of 1.43 kg m⁻³ (Chabbi et al., 2009; Moni et al., 2010). The profile can be divided into two main domains: three red-brown upper horizons characterised by a loamy texture overlying three lower clayey rubefied horizons rich in kaolinite and iron oxides (Ducloux and Chesseron, 1988). The lower rubefied horizons were classified as Paleo-Ferralsol from the late Mesozoic period, while the upper horizons were classified as Cambisol formed from Ferralsol horizons under temperate climate (Ducloux and Chesseron, 1988).

At this site, in 2005, a long-term field experiment was established consisting of ley grassland introduction in the cropping cycle with 5 different treatments in terms of fertilization and duration of the grassland phase (Table 1): TG3N+ corresponds to 3 yrs of fertilized ley, TG6N+ to 6 yrs of fertilized ley and TG6N- is a 6 yrs ley without fertilization. In addition, two control treatments with permanent cropland (CC) consisting of maize-wheat-barley rotation and with permanent grassland (GG) were established. Treatments were
129

replicated 4 times in 4 separated blocks. Before the beginning of the experiment, the whole surface had been homogenized by 3 years of cropping (barley, barley and wheat).

130

131

The species sown for CC in April 2005, was *Zea mays* L. (maize) with a density of 8.5 seeds m⁻². In October 2005, *Triticum aestivum* L. (wheat) was sown with density of 150 seeds m⁻². In October 2006, *Hordeum vulgare* L. (barley) was sown with density of 165 seeds m⁻². This crop rotation of 3 yrs was repeated when the soil was under permanent cropland (CC). For ley grassland, this crop rotation was alternated with a grassland phase (TG). To establish grassland, a species mixture containing *Dactylis glomerata* L. (cocksfoot), *Festuca arundinacea* Schreb (tall fescue) and *Lolium perenne* L. (rye-grass) was sown.

132

133

134

135

136

137

138

The grasslands were harvested 3-4 times a year depending on their productivity. The biomass was removed. Nitrogen was the only element added through mineral fertilizer application. The N-fertilizer amount needed to account for biomass removal was determined each year by soil analyses. In consequence, fertilization varied with years and ranged between 36 and 160 kg N ha⁻¹ year⁻¹ for cropland, and between 170 and 380 kg N ha⁻¹ year⁻¹ for fertilized grassland phases according to the residual soil N amount determined at the end of the previous growing season. It is important to note that in unfertilised temporary grassland (TG6N-) differed from the other treatments only with regards to fertilisation during the grassland phase.

139

140

141

142

143

144

145

146

147

148

## 2.2. Soil sampling

149

The top 10 cm soil was sampled in March 2014 for each plot (i.e. 5 treatments × 4 blocks), nine years after implantation of the experiment. All soils except GG soils had been under cropland rotation for the last three years (year 7 to 9, Table 1). Two soil cores (8 cm diameter) were randomly taken from each plot, and mixed to yield one composite sample per plot. Fresh sub-samples were used for biological analysis. The remaining soil was air-dried, and aliquots were ground for chemical analysis.

150

151

152

153

154

155

156

## 2.3. Soil elemental composition

157

Total carbon (C) and total nitrogen (N) concentrations were determined using an elemental analyzer (CHN NA 150, Carlo Erba). No carbonate was present in the soil, therefore soil C is considered to be exclusively organic.

158

159

160

Nitrate-N (NO₃⁻) and ammonium-N (NH₄⁺) were determined colorimetrically after extraction of soil with a 0.5 M KCl solution (1:5 weight/volume) (Page et al., 1982).

161

162

Total soil phosphorus (P) was determined with the ignition method (Walker and Adams, 1958) using the modifications introduced by Ivanoff et al., (1998). Briefly 0.5 g of

163
129

replicated 4 times in 4 separated blocks. Before the beginning of the experiment, the whole surface had been homogenized by 3 years of cropping (barley, barley and wheat).

130

131

The species sown for CC in April 2005, was *Zea mays* L. (maize) with a density of 8.5 seeds m⁻². In October 2005, *Triticum aestivum* L. (wheat) was sown with density of 150 seeds m⁻². In October 2006, *Hordeum vulgare* L. (barley) was sown with density of 165 seeds m⁻². This crop rotation of 3 yrs was repeated when the soil was under permanent cropland (CC). For ley grassland, this crop rotation was alternated with a grassland phase (TG). To establish grassland, a species mixture containing *Dactylis glomerata* L. (cocksfoot), *Festuca arundinacea* Schreb (tall fescue) and *Lolium perenne* L. (rye-grass) was sown.

132

133

134

135

136

137

138

The grasslands were harvested 3-4 times a year depending on their productivity. The biomass was removed. Nitrogen was the only element added through mineral fertilizer application. The N-fertilizer amount needed to account for biomass removal was determined each year by soil analyses. In consequence, fertilization varied with years and ranged between 36 and 160 kg N ha⁻¹ year⁻¹ for cropland, and between 170 and 380 kg N ha⁻¹ year⁻¹ for fertilized grassland phases according to the residual soil N amount determined at the end of the previous growing season. It is important to note that in unfertilised temporary grassland (TG6N-) differed from the other treatments only with regards to fertilisation during the grassland phase.

139

140

141

142

143

144

145

146

147

148

## 2.2. Soil sampling

149

The top 10 cm soil was sampled in March 2014 for each plot (i.e. 5 treatments × 4 blocks), nine years after implantation of the experiment. All soils except GG soils had been under cropland rotation for the last three years (year 7 to 9, Table 1). Two soil cores (8 cm diameter) were randomly taken from each plot, and mixed to yield one composite sample per plot. Fresh sub-samples were used for biological analysis. The remaining soil was air-dried, and aliquots were ground for chemical analysis.

150

151

152

153

154

155

156

## 2.3. Soil elemental composition

157

Total carbon (C) and total nitrogen (N) concentrations were determined using an elemental analyzer (CHN NA 150, Carlo Erba). No carbonate was present in the soil, therefore soil C is considered to be exclusively organic.

158

159

160

Nitrate-N (NO₃⁻) and ammonium-N (NH₄⁺) were determined colorimetrically after extraction of soil with a 0.5 M KCl solution (1:5 weight/volume) (Page et al., 1982).

161

162

Total soil phosphorus (P) was determined with the ignition method (Walker and Adams, 1958) using the modifications introduced by Ivanoff et al., (1998). Briefly 0.5 g of

163
dry soil was ignited for 1 h at 550°C before extraction during 24 h by shaking with 1 M
sulphuric acid (H₂SO₄) for total P. A second (unignited) replicate of 0.5 g dry soil was
extracted with 30 mL of 1 M H₂SO₄ for 24 h for inorganic phosphorus (IP) determination.
The suspensions were centrifuged at 17000 g for 10 min and the supernatants analyzed. The
orthophosphate content in each extract was measured as acid molybdenum complex
colorimetrically with detection at 880 nm (Murphy and Riley, 1962). The organic phosphorus
(OP) content was determined by difference.

171

172

## 2.4. Particulate organic matter

Particulate organic matter (POM) was separated from soil by physical fractionation
(Zimmermann, 2007 modified by Poeplau et al. 2013). Briefly, 30 g of soil were dispersed in
150 mL of deionized water using an ultrasonic probe with a calibrated energy output of 22 J
mL⁻¹ to break down macro-aggregates. The soil suspension was sieved over a 63 µm sieve
with a minimum of 2 L of deionized water. The fraction > 63 µm was freeze-dried and
separated by density fractionation into POM and sand and stable aggregate. Thirty mL of
sodium polytungstate solution with a density of 1.8 kg m⁻³ was added and stirred 1 h followed
by centrifugation for 15 minutes at 10000 RPM. The floating POM fraction and the
submerged sand and stable aggregate fraction were separated and recovered, after vacuum
filtration using glass fiber filters (Whatman GF/C 0.45 µm). They were rinsed with deionized
water, freeze-dried, ground and analyzed for their C and N concentrations (see above).

173

174

175

176

177

178

179

180

181

182

183

184

185

## 2.5. Lignin

Soil lignin content was determined after alkaline cupric oxide oxidation (Hedges and
Ertel, 1982; Koegel and Bochter, 1985) and quantification of the released monomers by gas
chromatography. Briefly, oxidation was carried out with CuO at high temperature (172°C)
under alkaline conditions (2 M NaOH) using 500 mg of soil, 250 mg of CuO, 50 mg of
ammonium ferrous hexahydrate and 50 mg of glucose. After cooling the samples, 0.5 mL of
the recovery standard ethylvanillin (60 mg L⁻¹) was added to assess losses during the
purification procedure. Thereafter, samples were acidified with 5 M HCl before removal of
humic acids by centrifugation (10 minutes at 17000 RPM) and solid-phase extraction of the
phenolic oxidation products using C18 reversed phase columns. The phenols were derivatised
with BSTFA and quantified as trimethylsilyl derivatives by gas chromatography with an HP
gas chromatograph (HP GC 6890) equipped with a flame ionization detector and a SGE BPX-
5 column (50 m length, 0.25 mm inner diameter, 0.32 µm coating). Samples were injected in
split mode (1:10). The GC oven temperature was programmed at 100°C during 2 min, then

186

187

188

189

190

191

192

193

194

195

196

197

198
dry soil was ignited for 1 h at 550°C before extraction during 24 h by shaking with 1 M
sulphuric acid (H₂SO₄) for total P. A second (unignited) replicate of 0.5 g dry soil was
extracted with 30 mL of 1 M H₂SO₄ for 24 h for inorganic phosphorus (IP) determination.
The suspensions were centrifuged at 17000 g for 10 min and the supernatants analyzed. The
orthophosphate content in each extract was measured as acid molybdenum complex
colorimetrically with detection at 880 nm (Murphy and Riley, 1962). The organic phosphorus
(OP) content was determined by difference.

171

172

## 2.4. Particulate organic matter

Particulate organic matter (POM) was separated from soil by physical fractionation
(Zimmermann, 2007 modified by Poeplau et al. 2013). Briefly, 30 g of soil were dispersed in
150 mL of deionized water using an ultrasonic probe with a calibrated energy output of 22 J
mL⁻¹ to break down macro-aggregates. The soil suspension was sieved over a 63 µm sieve
with a minimum of 2 L of deionized water. The fraction > 63 µm was freeze-dried and
separated by density fractionation into POM and sand and stable aggregate. Thirty mL of
sodium polytungstate solution with a density of 1.8 kg m⁻³ was added and stirred 1 h followed
by centrifugation for 15 minutes at 10000 RPM. The floating POM fraction and the
submerged sand and stable aggregate fraction were separated and recovered, after vacuum
filtration using glass fiber filters (Whatman GF/C 0.45 µm). They were rinsed with deionized
water, freeze-dried, ground and analyzed for their C and N concentrations (see above).

173

174

175

176

177

178

179

180

181

182

183

184

185

## 2.5. Lignin

Soil lignin content was determined after alkaline cupric oxide oxidation (Hedges and
Ertel, 1982; Koegel and Bochter, 1985) and quantification of the released monomers by gas
chromatography. Briefly, oxidation was carried out with CuO at high temperature (172°C)
under alkaline conditions (2 M NaOH) using 500 mg of soil, 250 mg of CuO, 50 mg of
ammonium ferrous hexahydrate and 50 mg of glucose. After cooling the samples, 0.5 mL of
the recovery standard ethylvanillin (60 mg L⁻¹) was added to assess losses during the
purification procedure. Thereafter, samples were acidified with 5 M HCl before removal of
humic acids by centrifugation (10 minutes at 17000 RPM) and solid-phase extraction of the
phenolic oxidation products using C18 reversed phase columns. The phenols were derivatised
with BSTFA and quantified as trimethylsilyl derivatives by gas chromatography with an HP
gas chromatograph (HP GC 6890) equipped with a flame ionization detector and a SGE BPX-
5 column (50 m length, 0.25 mm inner diameter, 0.32 µm coating). Samples were injected in
split mode (1:10). The GC oven temperature was programmed at 100°C during 2 min, then

186

187

188

189

190

191

192

193

194

195

196

197

198
from 100°C to 172°C with a heating rate of 8°C min⁻¹, from 172 to 184°C at 4°C min⁻¹, and
from 184 to 300°C at a rate of 10°C min⁻¹.

The content and state of decomposition of lignin were characterized by this method.
The total lignin concentration of the sample is represented by the sum of phenolic oxidation
products vanillyl (V), syringyl (S) and p-coumaryl (Co) with their acid (Ac), aldehyde (Al)
and ketone side chains. Thus, the concentration of V phenols corresponded to the sum of the
concentrations of vanillin, acetovanillone and vanillic acid. S phenol concentrations were
calculated as the sum of syringaldehyde, acetosyringone and syringic acid. The Co phenol
concentrations were estimated as the sum of ferulic and p-coumaric acid. The ratios between
the acid and aldehyde forms of the phenol monomers V and S can be used to infer the state of
lignin degradation. Usually (Ac/Al)~V~ and (Ac/Al)~S~ are increasing with increasing lignin
degradation (Koegel and Bochter, 1985). Likewise, the ratios Co/V and S/V are decreasing
with increasing lignin degradation because of the preferential degradation of S and Co as
compared to V units (Bahri et al., 2006).

213

214

## 2.6. *Sugar*

The concentrations of neutral non-cellulosic carbohydrates were determined after
trifluoroacetic acid (TFA) hydrolysis and reduction-acetylation of the monomers released by
gas chromatography (Rumpel and Dignac 2006; modified by Eder et al 2010). The analysis
was performed using 500 mg of air-dried and ground soil samples. Hydrolysis was carried out
at 105°C for 4 h with 5 mL of 4 M TFA. After cooling the samples, 0.5 mL of the recovery
standard methylglucose (3 g L⁻¹) was added and the soil was removed by filtration through
glass fiber filters (Whatman GF/C 0.45 µm). After evaporation of TFA using a vacuum
speedvac (Thermo scientific, USA), the sugars were re-dissolved in 0.9 µL Na₂-EDTA and
the internal standard myo-inositol (1.75 g L⁻¹) was added. Reduction of aldoses to alditols was
performed at 40°C for 1 h 30 after addition of 1 mL of sodium borohydride (NaBH₄) in
dimethylsulfoxide (20 g L⁻¹). After cooling, acetylation was carried out by addition of 0.2 mL
acetic acid. Thereafter, 2 ml of acetic anhydride were added and methylimidazole (0.2 mL)
was used as a catalyst of the reaction. After 10 min, the reaction was stopped by addition of
10 mL ice-cold distilled water. The derivatised monosaccharide units were extracted by
liquid-liquid extraction using a vortex mixer following addition of 1 mL dichloromethane.
The gas chromatographic measurements of the monosaccharides in dichloromethane were
performed with an HP GC 6890 gas chromatograph equipped with a flame ionization detector
operating in split mode (1:10). Separation of the monosaccharide units was achieved with a
60 m fused silica capillary column (SGE BPX 70, 0.32 mm internal diameter, 0.25 mm film
from 100°C to 172°C with a heating rate of 8°C min⁻¹, from 172 to 184°C at 4°C min⁻¹, and
from 184 to 300°C at a rate of 10°C min⁻¹.

The content and state of decomposition of lignin were characterized by this method.
The total lignin concentration of the sample is represented by the sum of phenolic oxidation
products vanillyl (V), syringyl (S) and p-coumaryl (Co) with their acid (Ac), aldehyde (Al)
and ketone side chains. Thus, the concentration of V phenols corresponded to the sum of the
concentrations of vanillin, acetovanillone and vanillic acid. S phenol concentrations were
calculated as the sum of syringaldehyde, acetosyringone and syringic acid. The Co phenol
concentrations were estimated as the sum of ferulic and p-coumaric acid. The ratios between
the acid and aldehyde forms of the phenol monomers V and S can be used to infer the state of
lignin degradation. Usually (Ac/Al)~v~ and (Ac/Al)~s~ are increasing with increasing lignin
degradation (Koegel and Bochter, 1985). Likewise, the ratios Co/V and S/V are decreasing
with increasing lignin degradation because of the preferential degradation of S and Co as
compared to V units (Bahri et al., 2006).

213

214

## 2.6. *Sugar*

The concentrations of neutral non-cellulosic carbohydrates were determined after
trifluoroacetic acid (TFA) hydrolysis and reduction-acetylation of the monomers released by
gas chromatography (Rumpel and Dignac 2006; modified by Eder et al 2010). The analysis
was performed using 500 mg of air-dried and ground soil samples. Hydrolysis was carried out
at 105°C for 4 h with 5 mL of 4 M TFA. After cooling the samples, 0.5 mL of the recovery
standard methylglucose (3 g L⁻¹) was added and the soil was removed by filtration through
glass fiber filters (Whatman GF/C 0.45 µm). After evaporation of TFA using a vacuum
speedvac (Thermo scientific, USA), the sugars were re-dissolved in 0.9 µL Na₂-EDTA and
the internal standard myo-inositol (1.75 g L⁻¹) was added. Reduction of aldoses to alditols was
performed at 40°C for 1 h 30 after addition of 1 mL of sodium borohydride (NaBH₄) in
dimethylsulfoxide (20 g L⁻¹). After cooling, acetylation was carried out by addition of 0.2 mL
acetic acid. Thereafter, 2 ml of acetic anhydride were added and methylimidazole (0.2 mL)
was used as a catalyst of the reaction. After 10 min, the reaction was stopped by addition of
10 mL ice-cold distilled water. The derivatised monosaccharide units were extracted by
liquid-liquid extraction using a vortex mixer following addition of 1 mL dichloromethane.
The gas chromatographic measurements of the monosaccharides in dichloromethane were
performed with an HP GC 6890 gas chromatograph equipped with a flame ionization detector
operating in split mode (1:10). Separation of the monosaccharide units was achieved with a
60 m fused silica capillary column (SGE BPX 70, 0.32 mm internal diameter, 0.25 mm film
thickness) and the following temperature program: from 200 to 250°C at 8 °C min⁻¹ and 15 min isothermal at 250°C. Carrier gas was helium with a flow rate of 1.0 mL min⁻¹. The injector was kept at 250°C and the detector at 260°C. The concentrations of individual sugars were calculated based on the total ion current of the recovery standard methylglucose and the internal standard myo-inositol. The non-cellulosic sugar content of the samples was determined as sum of monosaccharides released: pentoses (xylose, ribose and arabinose), hexoses (glucose, galactose and mannose), and desoxyhexoses (fucose and rhamnose) (Koegel-Knabner, 2002). To determine the plant and microbial origin of soil carbohydrates, the ratios C6/C5 and desoxyC6/C5 were calculated. The decay degree of plant-derived sugars was determined by the ratio of xylose/mannose (Oades, 1984).

## 2.7. Microbial biomass

Microbial biomass was determined on fresh soil samples by the chloroform fumigation-extraction method (Vance *et al.*, 1987). Briefly, the fumigation-extraction technique is based on fumigation of soil with chloroform (CHCl₃) for 16 hours, which allows lysis of microbial cells. For each plot, dissolved C and N were extracted from a fumigated soil sample and an unfumigated control with a 0.03 M K₂SO₄ solution (1:4 weight/volume). The extracts were filtered at 0.7 µm and freeze-dried. C and N of the dry extracts were determined with an elemental analyzer (CHN NA 1500, Carlo Erba). Microbial C (MB-C) and N (MB-N) were calculated as differences between paired fumigated and non fumigated soils, using a multiplication factor of 2.22 for C and N (Jenkinsen *et al.*, 2004).

## 2.8. Potential mineralization of C and N

The potential C and N mineralization (Cmin and Nmin) of soils was determined under controlled conditions. The fresh soil sieved at 4 mm was incubated at field moisture in three replicates of 30 g and one of 50 g for the last step (around 140 g in total) in jars of 1 liter at 20°C for 56 days. All samples were pre-incubated for 2 days under the same condition and the atmosphere was flush with CO₂-free atmosphere at the beginning of the experiment. To avoid anaerobic conditions, the atmosphere was flushed regularly with CO₂-free synthetic air.

The accumulated CO₂ concentration was measured at day 0, 1, 3, 7, 10, 14, 21, 28, 42 and 56 with a micro gas chromatograph (Agilent 590). C mineralization was modeled with R (Studio Version 0.98.1062) using a first order exponential model with one C pool. This model gave the best fit after model evaluation. We used the following equation:

$$C(t) = C_0(1 - e^{(-kt)})$$
thickness) and the following temperature program: from 200 to 250°C at 8 °C min⁻¹ and 15 min isothermal at 250°C. Carrier gas was helium with a flow rate of 1.0 mL min⁻¹. The injector was kept at 250°C and the detector at 260°C. The concentrations of individual sugars were calculated based on the total ion current of the recovery standard methylglucose and the internal standard myo-inositol. The non-cellulosic sugar content of the samples was determined as sum of monosaccharides released: pentoses (xylose, ribose and arabinose), hexoses (glucose, galactose and mannose), and desoxyhexoses (fucose and rhamnose) (Koegel-Knabner, 2002). To determine the plant and microbial origin of soil carbohydrates, the ratios C6/C5 and desoxyC6/C5 were calculated. The decay degree of plant-derived sugars was determined by the ratio of xylose/mannose (Oades, 1984).

## 2.7. Microbial biomass

Microbial biomass was determined on fresh soil samples by the chloroform fumigation-extraction method (Vance *et al.*, 1987). Briefly, the fumigation-extraction technique is based on fumigation of soil with chloroform (CHCl₃) for 16 hours, which allows lysis of microbial cells. For each plot, dissolved C and N were extracted from a fumigated soil sample and an unfumigated control with a 0.03 M K₂SO₄ solution (1:4 weight/volume). The extracts were filtered at 0.7 µm and freeze-dried. C and N of the dry extracts were determined with an elemental analyzer (CHN NA 1500, Carlo Erba). Microbial C (MB-C) and N (MB-N) were calculated as differences between paired fumigated and non fumigated soils, using a multiplication factor of 2.22 for C and N (Jenkinsen *et al.*, 2004).

## 2.8. Potential mineralization of C and N

The potential C and N mineralization (Cmin and Nmin) of soils was determined under controlled conditions. The fresh soil sieved at 4 mm was incubated at field moisture in three replicates of 30 g and one of 50 g for the last step (around 140 g in total) in jars of 1 liter at 20°C for 56 days. All samples were pre-incubated for 2 days under the same condition and the atmosphere was flush with CO₂-free atmosphere at the beginning of the experiment. To avoid anaerobic conditions, the atmosphere was flushed regularly with CO₂-free synthetic air.

The accumulated CO₂ concentration was measured at day 0, 1, 3, 7, 10, 14, 21, 28, 42 and 56 with a micro gas chromatograph (Agilent 590). C mineralization was modeled with R (Studio Version 0.98.1062) using a first order exponential model with one C pool. This model gave the best fit after model evaluation. We used the following equation:

$$C(t) = C_0(1 - e^{(-kt)})$$
268

269

270

Where C(t) is the cumulative amount of C mineralized after time t (% C₀), C₀ is the initial amount of organic carbon, t is the incubation time (day) and k is the C mineralization rate constant.

271

272

273

The soil was collected at day 7, 14, 28 and 56 to follow the N mineralization. The concentration of inorganic N of each sample was determined on fresh soil as described above (1.3).

274

275

## 2.9. Potential denitrification

276

277

278

279

280

281

282

Potential gross rate of N₂O production, i.e. potential denitrification (PD), was measured as the N₂O production rate from soil samples amended with NO₃⁻ and labile C, headspace atmosphere being replaced by a He:C₂H₂ mixture (90:10) to ensure anaerobic conditions and inhibition of N₂O reductase (Patra et al., 2005). Soil samples were incubated at 28°C for 8h. N₂O concentration was measured after 2h, 4h, 6h and 8h with a gas chromatograph. A linear rate of N₂O production was always observed during the denitrification assays.

283

284

## 2.10. Statistical analysis

285

286

287

288

A two-way ANOVA was used followed by Tukey HSD post hoc test. For sugar and lignin data, the first factor accounted for different treatments, while the second factor was the analytical series, which impacted significantly the results. For all other data, the first factor was the treatments and the second factors the field plot.

289

290

291

Principal component analysis (PCA) was carried out for soil parameters with package FactoMineR. The PCA function of the FactoMineR package automatically transforms data to a standardized normal distribution.

292

293

# 3. Results

294

295

## 3.1. Soil C, N and P concentrations and forms

296

297

298

299

300

301

302

The C concentrations ranged between 9.1 and 16.8 mg g⁻¹, and those of N between 1.01 and 1.65 mg g⁻¹ (Table 2). Lowest concentrations of both elements were observed for unfertilised temporary grassland soil (TG6N-), and highest for continuous grassland (GG). Both elements followed a similar pattern and C/N ratios were thus unaffected by treatments. Temporary grassland duration had no linear effect on soil C and N as demonstrated by the significant increase of C and N concentrations for TG3N+ as compared to continuous cropping (CC), while no such an effect was observed for TG6N+.
268

269

270

Where C(t) is the cumulative amount of C mineralized after time t (% C₀), C₀ is the initial amount of organic carbon, t is the incubation time (day) and k is the C mineralization rate constant.

271

272

273

The soil was collected at day 7, 14, 28 and 56 to follow the N mineralization. The concentration of inorganic N of each sample was determined on fresh soil as described above (1.3).

274

275

## 2.9. Potential denitrification

276

277

278

279

280

281

282

Potential gross rate of N₂O production, i.e. potential denitrification (PD), was measured as the N₂O production rate from soil samples amended with NO₃⁻ and labile C, headspace atmosphere being replaced by a He:C₂H₂ mixture (90:10) to ensure anaerobic conditions and inhibition of N₂O reductase (Patra et al., 2005). Soil samples were incubated at 28°C for 8h. N₂O concentration was measured after 2h, 4h, 6h and 8h with a gas chromatograph. A linear rate of N₂O production was always observed during the denitrification assays.

283

284

## 2.10. Statistical analysis

285

286

287

288

A two-way ANOVA was used followed by Tukey HSD post hoc test. For sugar and lignin data, the first factor accounted for different treatments, while the second factor was the analytical series, which impacted significantly the results. For all other data, the first factor was the treatments and the second factors the field plot.

289

290

291

Principal component analysis (PCA) was carried out for soil parameters with package FactoMineR. The PCA function of the FactoMineR package automatically transforms data to a standardized normal distribution.

292

293

# 3. Results

294

295

## 3.1. Soil C, N and P concentrations and forms

296

297

298

299

300

301

302

The C concentrations ranged between 9.1 and 16.8 mg g⁻¹, and those of N between 1.01 and 1.65 mg g⁻¹ (Table 2). Lowest concentrations of both elements were observed for unfertilised temporary grassland soil (TG6N-), and highest for continuous grassland (GG). Both elements followed a similar pattern and C/N ratios were thus unaffected by treatments. Temporary grassland duration had no linear effect on soil C and N as demonstrated by the significant increase of C and N concentrations for TG3N+ as compared to continuous cropping (CC), while no such an effect was observed for TG6N+.
Total soil phosphorus (P) concentrations did not vary significantly among treatments (Table 2). Significant differences were noted for the C/P ratio, which increased following fertilisation and was highest in GG (Table 2). Continuous grassland also showed the highest proportion of C in the POM fraction and the highest NH4+/NO3- ratio (Table 2).

## 3.2. Soil carbohydrate signature

Total soil carbohydrate concentrations ranged between 3.9 and 5.9 mg g⁻¹ soil, and decreased in the order GG > TG6N+ > TG3N+ > CC > TG6N- (Table 3). The carbohydrate content expressed on a total C basis was with 43% highest for TG6N- and similar for all other treatments (Table 3). A C6/C5 > 2 was recorded for the soils of the treatments CC and TG (Table 3). This indicated a primarily microbial origin of the carbohydrates. Both ratios indicating microbial sugar contribution (C6/C5 and desoxyC6/C5) were lowest for GG and highest for CC, with temporary grasslands being in an intermediate position (Table 3).

## 3.3. Lignin signature

Total soil lignin concentrations varied between 0.20 and 0.35 mg g⁻¹ soil, and were not different for GG and CC, in contrast to TG6 and TG3, which showed significantly lower values (Table 4). Lignin contents were comprised between 1.9 and 2.4 % of total soil C without any differences among treatments (Table 4). The Ac/Al ratios of vanillyl and syringyl indicated similar lignin degradation for GG, CC and TG, except for the ratio (Ac/Al)₅, which differentiated TG3+ and TG6+ (Table 4). The ratio Co/V was significantly higher for CC than GG, indicating a lower degree of soil lignin degradation. TG6N+ and TG6N- showed intermediate values, while the highest Co/V and S/V ratios were recorded for TG3N+ (Table 4).

## 3.4. Microbial biomass

The C and N concentrations of soil microbial biomass (MB-C and MB-N) are presented in Table 5. MB-N did not vary significantly among treatments. In contrast, MB-C with decreased in the order GG > TG6N+ > TG3N+ > CC > TG6N- (Table 5) and the MB-C/MB-N ratio consequently showed highest values for GG (Table 5).

## 3.5. Potential microbial activities

Treatment effects on C mineralisation during 56 days of incubation are presented in Fig. 1. At the end of incubation, between 3.8 and 10.5 % of initial C were mineralised, highest mineralisation rate constant *k* being recorded for soils of the treatment GG (Fig. 1).
Total soil phosphorus (P) concentrations did not vary significantly among treatments (Table 2). Significant differences were noted for the C/P ratio, which increased following fertilisation and was highest in GG (Table 2). Continuous grassland also showed the highest proportion of C in the POM fraction and the highest NH4+/NO3- ratio (Table 2).

## 3.2. Soil carbohydrate signature

Total soil carbohydrate concentrations ranged between 3.9 and 5.9 mg g⁻¹ soil, and decreased in the order GG > TG6N+ > TG3N+ > CC > TG6N- (Table 3). The carbohydrate content expressed on a total C basis was with 43% highest for TG6N- and similar for all other treatments (Table 3). A C6/C5 > 2 was recorded for the soils of the treatments CC and TG (Table 3). This indicated a primarily microbial origin of the carbohydrates. Both ratios indicating microbial sugar contribution (C6/C5 and desoxyC6/C5) were lowest for GG and highest for CC, with temporary grasslands being in an intermediate position (Table 3).

## 3.3. Lignin signature

Total soil lignin concentrations varied between 0.20 and 0.35 mg g⁻¹ soil, and were not different for GG and CC, in contrast to TG6 and TG3, which showed significantly lower values (Table 4). Lignin contents were comprised between 1.9 and 2.4 % of total soil C without any differences among treatments (Table 4). The Ac/Al ratios of vanillyl and syringyl indicated similar lignin degradation for GG, CC and TG, except for the ratio (Ac/Al)₃S, which differentiated TG3+ and TG6+ (Table 4). The ratio Co/V was significantly higher for CC than GG, indicating a lower degree of soil lignin degradation. TG6N+ and TG6N- showed intermediate values, while the highest Co/V and S/V ratios were recorded for TG3N+ (Table 4).

## 3.4. Microbial biomass

The C and N concentrations of soil microbial biomass (MB-C and MB-N) are presented in Table 5. MB-N did not vary significantly among treatments. In contrast, MB-C with decreased in the order GG > TG6N+ > TG3N+ > CC > TG6N- (Table 5) and the MB-C/MB-N ratio consequently showed highest values for GG (Table 5).

## 3.5. Potential microbial activities

Treatment effects on C mineralisation during 56 days of incubation are presented in Fig. 1. At the end of incubation, between 3.8 and 10.5 % of initial C were mineralised, highest mineralisation rate constant *k* being recorded for soils of the treatment GG (Fig. 1).
N mineralisation dynamics during the incubation (for N-NH₄⁺ and N-NO₃⁻) are shown in Fig. 2. During the first 15 days of incubation, a decrease of soil ammonium concentration was observed (Fig. 2A). Thereafter, the N-NH₄⁺ concentrations increased again to reach highest values for GG and TG6N+. The other three treatments showed similar NH₄⁺ concentrations at the end of the incubation, with TG3N+ and TG6N- presenting significantly lower values than GG. Nitrate (NO₃⁻) concentrations increased during the incubation in all treatments (Fig. 2B). GG showed significantly higher nitrate accumulation than CC. Temporary grasslands were in an intermediate position (Fig. 2B).

Potential denitrification ranged between 0.72 and 1.13 µgN-N₂O g⁻¹ soil h⁻¹, and did not reveal any differences among treatments (Table 5).

### 3.6. *Principal component analyses*

To investigate the parameters most affected by land use legacy, we carried out a PCA (Fig. 3). Based on Pearson correlation coefficients among variables of the dataset, it appeared that C, N, MB-C, lignin, sugar concentration and *k* were positively correlated (all *r* > 0.57 and *p* < 0.001) and all were negatively correlated to C6/C5 sugar ratio (*p* < 0.01). Soil P was positively correlated only with Co/V (*r* = 0.61) and S/V (*r* = 0.51) lignin ratios; S/V itself correlated negatively with sugar (*r* = -0.53) and POM (*r* = -0.64). PD was positively correlated with the S/V lignin ratio (*r* = 0.54; *p* = 0.05).

The first two dimensions of the PCA explained 73.17 % of the variability. The first dimension, representing 52.15% of the variability, was strongly and positively (*r* > 0.78) correlated with sugar and lignin concentrations, soil C and N concentrations, and the C mineralization rate constant *k*, and negatively with the C6/C5 sugar ratio (Fig. 3B). The second dimension, which represented 21.02 % of the variability, was strongly and positively (*r* > 0.8) correlated to the concentration of soil P and Co/V and S/V lignin ratios and more weakly with PD (*r* = 0.51). It was negatively correlated to POM (*r* = -0.45) (Fig. 3B).

On the PCA plan (Fig. 3A), GG was separated in the first dimension from all other treatments that had been under crop rotation for the last three years. However, grassland management before the cropland phase affected the location of the treatments within the PCA plan. While six-year fertilised grassland TG6N+ was in an intermediate position between CC and GG, TG6N- is located next to CC in the opposite direction (Fig. 3 A). The treatment TG3N+ overlaps with CC.

## 4. Discussion
N mineralisation dynamics during the incubation (for N-NH₄⁺ and N-NO₃⁻) are shown in Fig. 2. During the first 15 days of incubation, a decrease of soil ammonium concentration was observed (Fig. 2A). Thereafter, the N-NH₄⁺ concentrations increased again to reach highest values for GG and TG6N+. The other three treatments showed similar NH₄⁺ concentrations at the end of the incubation, with TG3N+ and TG6N- presenting significantly lower values than GG. Nitrate (NO₃⁻) concentrations increased during the incubation in all treatments (Fig. 2B). GG showed significantly higher nitrate accumulation than CC. Temporary grasslands were in an intermediate position (Fig. 2B).

Potential denitrification ranged between 0.72 and 1.13 µgN-N₂O g⁻¹ soil h⁻¹, and did not reveal any differences among treatments (Table 5).

### 3.6. *Principal component analyses*

To investigate the parameters most affected by land use legacy, we carried out a PCA (Fig. 3). Based on Pearson correlation coefficients among variables of the dataset, it appeared that C, N, MB-C, lignin, sugar concentration and *k* were positively correlated (all *r* > 0.57 and *p* < 0.001) and all were negatively correlated to C6/C5 sugar ratio (*p* < 0.01). Soil P was positively correlated only with Co/V (*r* = 0.61) and S/V (*r* = 0.51) lignin ratios; S/V itself correlated negatively with sugar (*r* = -0.53) and POM (*r* = -0.64). PD was positively correlated with the S/V lignin ratio (*r* = 0.54; *p* = 0.05).

The first two dimensions of the PCA explained 73.17 % of the variability. The first dimension, representing 52.15% of the variability, was strongly and positively (*r* > 0.78) correlated with sugar and lignin concentrations, soil C and N concentrations, and the C mineralization rate constant *k*, and negatively with the C6/C5 sugar ratio (Fig. 3B). The second dimension, which represented 21.02 % of the variability, was strongly and positively (*r* > 0.8) correlated to the concentration of soil P and Co/V and S/V lignin ratios and more weakly with PD (*r* = 0.51). It was negatively correlated to POM (*r* = -0.45) (Fig. 3B).

On the PCA plan (Fig. 3A), GG was separated in the first dimension from all other treatments that had been under crop rotation for the last three years. However, grassland management before the cropland phase affected the location of the treatments within the PCA plan. While six-year fertilised grassland TG6N+ was in an intermediate position between CC and GG, TG6N- is located next to CC in the opposite direction (Fig. 3 A). The treatment TG3N+ overlaps with CC.

## 4. Discussion
4.1. Grassland soil and arable soil – two different systems with contrasting SOM characteristics and microbial activities

Nine years of continuous grassland (GG) led to higher soil C and N concentrations than 9 yrs of continuous crop (CC), in accordance with literature results (Guggenberger et al., 1994; INRA, 2002; Lal, 2004; Soussana et al., 2004). The PCA clearly showed differentiation of GG and CC along the first dimension, which correlated to C, N, MB-C, lignin, sugar, *k* and the C6/C5 sugar ratio. Soils under both land use systems can thus be distinguished not only according to their C and N concentrations but also according to the biochemical nature of their SOM (Rumpel et al., 2009; Rumpel and Chabbi, 2010) and potential microbial activities (Attard et al., 2016).

Highest concentrations of particulate organic matter, indicating the presence of fresh litter, were found for GG. This may be explained by (1) lower mineralisation of SOM in soil permanently covered by vegetation and absence of tillage (Soussana et al., 2004) and/or (2) higher constant inputs of fresh plant material originating from grass roots (Acharya et al., 2012; Post and Kwon, 2000). High inputs of litter and rhizodeposition under grassland may also sustain a more active microbial community (Attard et al., 2016; Carter et al., 1998; Murphy et al., 2007). This is in agreement with the higher potential C mineralisation and microbial biomass (MB-C) in GG as compared to CC.

Lignin concentrations and contents were similar for CC and GG. These results are in contrast with those recorded by other authors (Guggenberger et al., 1994; Lobe et al., 2002; Rumpel and Chabbi, 2010). In the present study, differences in the lignin signature between the two systems concerned the Co/V ratio. The lower Co/V ratio for CC indicates a more degraded lignin signature as compared to GG, in accordance with observations made by other authors (Lobe et al., 2002). This is supported by a higher contribution of microbial sugars in CC as compared to GG, suggesting more processed SOM in soil under continuous cropping. Moreover, the C/P ratio of 11.6 for CC as compared to 18.4 for GG could indicate a more stabilized SOM under continuous crop (Kirkby et al., 2013).

Overall, nine years after the establishment of mowed and fertilised grassland, soils showed clear differences as compared to cropland concerning the nature of their SOM, nutrient concentrations as well as amount and activity of microbial biomass. According to our data, it seems that while long-term grasslands are beneficial for increasing SOM concentrations,
4.1. Grassland soil and arable soil – two different systems with contrasting SOM characteristics and microbial activities

Nine years of continuous grassland (GG) led to higher soil C and N concentrations than 9 yrs of continuous crop (CC), in accordance with literature results (Guggenberger et al., 1994; INRA, 2002; Lal, 2004; Soussana et al., 2004). The PCA clearly showed differentiation of GG and CC along the first dimension, which correlated to C, N, MB-C, lignin, sugar, *k* and the C6/C5 sugar ratio. Soils under both land use systems can thus be distinguished not only according to their C and N concentrations but also according to the biochemical nature of their SOM (Rumpel et al., 2009; Rumpel and Chabbi, 2010) and potential microbial activities (Attard et al., 2016).

Highest concentrations of particulate organic matter, indicating the presence of fresh litter, were found for GG. This may be explained by (1) lower mineralisation of SOM in soil permanently covered by vegetation and absence of tillage (Soussana et al., 2004) and/or (2) higher constant inputs of fresh plant material originating from grass roots (Acharya et al., 2012; Post and Kwon, 2000). High inputs of litter and rhizodeposition under grassland may also sustain a more active microbial community (Attard et al., 2016; Carter et al., 1998; Murphy et al., 2007). This is in agreement with the higher potential C mineralisation and microbial biomass (MB-C) in GG as compared to CC.

Lignin concentrations and contents were similar for CC and GG. These results are in contrast with those recorded by other authors (Guggenberger et al., 1994; Lobe et al., 2002; Rumpel and Chabbi, 2010). In the present study, differences in the lignin signature between the two systems concerned the Co/V ratio. The lower Co/V ratio for CC indicates a more degraded lignin signature as compared to GG, in accordance with observations made by other authors (Lobe et al., 2002). This is supported by a higher contribution of microbial sugars in CC as compared to GG, suggesting more processed SOM in soil under continuous cropping. Moreover, the C/P ratio of 11.6 for CC as compared to 18.4 for GG could indicate a more stabilized SOM under continuous crop (Kirkby et al., 2013).

Overall, nine years after the establishment of mowed and fertilised grassland, soils showed clear differences as compared to cropland concerning the nature of their SOM, nutrient concentrations as well as amount and activity of microbial biomass. According to our data, it seems that while long-term grasslands are beneficial for increasing SOM concentrations,
408

at the same time they contain less processed SOM, which may increase the potential for CO2 emissions but on the other hand is beneficial in terms of nutrient retention, as illustrated for example by the higher NH4+/NO3- ratio.

409

410

411

412

## 4.2. Legacy effect of N fertilisation during 6 yrs of temporary grassland

413

414

Our data showed that the unfertilised 6 yrs temporary grassland (TG6N-) presented the lowest soil C and N concentrations of all treatments. This may be explained by biomass export following mowing leading to nutrient removal and thus loss of productivity during the grassland phase. Although soils were fertilised during the following three years of cropland, C and N concentrations were significantly lower for TG6N- than TG6N+. Moreover, absence of fertilisation during 6 yrs of temporary grassland induced a legacy effect concerning SOM stoichiometric ratios, with the lowest C/N and C/P ratios of all treatments. These data suggest low amounts of plant derived matter, and a high proportion of soil C being composed of microbial derived material in TG6N- (Kirkby et al., 2013). This is supported by the high proportions of microbial sugars present in soil of TG6N-. In contrast, the lignin concentrations on a soil or C basis were not impacted by fertilisation during the grassland phase, even if lignin degradation is generally affected by N availability (Kiem and Kögel-Knabner, 2003; Knicker, 2011).

415

416

417

418

419

420

421

422

423

424

425

426

427

428

The strong legacy effect of absence of fertilisation during the grassland phase on soil parameters is illustrated by PCA analyses, as the soils of TG6N- were distinguished from all treatments along the first dimension. This shows that N fertilisation of mowed grasslands is necessary to allow grassland soil to acquire a specific grassland SOM signature and maintain important soil functions (Conant et al., 2001; Lal, 2004; Rumpel et al., 2015). Indeed, N fertilisation is a limiting factor for biological productivity, plant biomass formation and SOM input (Knicker, 2011). Thus, beneficial effects of temporary grasslands related to soil C are dependent on N fertilization.

429

430

431

432

433

434

435

436

437

## 4.3. Legacy effects of grassland duration

438

439

To investigate legacy effects of temporary grassland duration, we compared the effects of 3 and 6 yrs fertilised mowed temporary grassland (TG3N+ and TG6N+). Our data show that 3 yrs of temporary grassland increased concentrations of major elements as compared to continuous crop and even as compared to TG6N+. The biogeochemical signature of SOM is

440

441

442
at the same time they contain less processed SOM, which may increase the potential for CO2 emissions but on the other hand is beneficial in terms of nutrient retention, as illustrated for example by the higher NH4+/NO3- ratio.

411

412

## 4.2. Legacy effect of N fertilisation during 6 yrs of temporary grassland

413

Our data showed that the unfertilised 6 yrs temporary grassland (TG6N-) presented the lowest soil C and N concentrations of all treatments. This may be explained by biomass export following mowing leading to nutrient removal and thus loss of productivity during the grassland phase. Although soils were fertilised during the following three years of cropland, C and N concentrations were significantly lower for TG6N- than TG6N+. Moreover, absence of fertilisation during 6 yrs of temporary grassland induced a legacy effect concerning SOM stoichiometric ratios, with the lowest C/N and C/P ratios of all treatments. These data suggest low amounts of plant derived matter, and a high proportion of soil C being composed of microbial derived material in TG6N- (Kirkby et al., 2013). This is supported by the high proportions of microbial sugars present in soil of TG6N-. In contrast, the lignin concentrations on a soil or C basis were not impacted by fertilisation during the grassland phase, even if lignin degradation is generally affected by N availability (Kiem and Kögel-Knabner, 2003; Knicker, 2011).

427

The strong legacy effect of absence of fertilisation during the grassland phase on soil parameters is illustrated by PCA analyses, as the soils of TG6N- were distinguished from all treatments along the first dimension. This shows that N fertilisation of mowed grasslands is necessary to allow grassland soil to acquire a specific grassland SOM signature and maintain important soil functions (Conant et al., 2001; Lal, 2004; Rumpel et al., 2015). Indeed, N fertilisation is a limiting factor for biological productivity, plant biomass formation and SOM input (Knicker, 2011). Thus, beneficial effects of temporary grasslands related to soil C are dependent on N fertilization.

428

429

430

431

432

433

434

435

436

437

## 4.3. Legacy effects of grassland duration

438

439

To investigate legacy effects of temporary grassland duration, we compared the effects of 3 and 6 yrs fertilised mowed temporary grassland (TG3N+ and TG6N+). Our data show that 3 yrs of temporary grassland increased concentrations of major elements as compared to continuous crop and even as compared to TG6N+. The biogeochemical signature of SOM is

440

441

442
distinct for TG3N+ as illustrated by lower lignin degradation state for TG3N+ than TG6N+. Parameters related to the sugar signature are intermediate between CC and TG6N+, suggesting an age dependent legacy effect. It is interesting to note that comparatively labile compounds like carbohydrates, which control the initial phase of plant litter degradation, showed an age dependent (non-significant) legacy effect of duration, while for lignin, a recalcitrant compound, the legacy effect was not age dependent and significant.

449

In the PCA plan, TG6N+ was placed along the first dimension between CC and GG, whereas TG3N+ was not different from CC and differentiated from TG6N+ along the second dimension characterised by P, Co/V and S/V lignin ratios, PD and POM. It was interesting to see an unexpected non-linear response of these parameters to TG age. Non age-dependent effects for grasslands of short duration have already been observed by other authors for root biomass (Acharya et al., 2012) and for C and N fluxes (Attard et al., 2016). These data indicate that after 3 yrs of grassland, the nature of SOM was still in a transitional state and did not yet acquire grassland characteristics, for example a typical biogeochemical grassland soil signature with high lignin concentration. In our system, grasslands of longer duration were needed to be able to differentiate temperate grassland from continuously cropped soils beyond the three-year cropland phase.

461

## 5. Conclusion

In this study, we investigated the biogeochemical nature of SOM in 0-10 cm of cropland and grassland soils as well as the legacy effect (i.e. changes in parameters lasting beyond three years of crop) of differently managed temporary grasslands inserted into crop rotations. Our results show that even if no effect on potential microbial activity was detected, C concentrations of arable soil may be increased through the introduction of temporary grasslands with specific management in terms of fertilisation and duration. Strong legacy effects were related to N-fertilisation, which was necessary to preserve a typical grassland biogeochemical signature and SOM concentrations beyond three years of crop. Grassland duration showed no age-dependent legacy effects concerning C concentrations and other soil properties. However, overall it seems that only after temporary grassland duration of 6 years, soils acquired biogeochemical characteristics close to continuous grassland. In contrast, soils, which supported 3 years of temporary grassland were similar to those supporting continuous crop after the end of the cropland phase.

463

464

465

466

467

468

469

470

471

472

473

474

475

476

## Acknowledgements
distinct for TG3N+ as illustrated by lower lignin degradation state for TG3N+ than TG6N+. Parameters related to the sugar signature are intermediate between CC and TG6N+, suggesting an age dependent legacy effect. It is interesting to note that comparatively labile compounds like carbohydrates, which control the initial phase of plant litter degradation, showed an age dependent (non-significant) legacy effect of duration, while for lignin, a recalcitrant compound, the legacy effect was not age dependent and significant.

449

In the PCA plan, TG6N+ was placed along the first dimension between CC and GG, whereas TG3N+ was not different from CC and differentiated from TG6N+ along the second dimension characterised by P, Co/V and S/V lignin ratios, PD and POM. It was interesting to see an unexpected non-linear response of these parameters to TG age. Non age-dependent effects for grasslands of short duration have already been observed by other authors for root biomass (Acharya et al., 2012) and for C and N fluxes (Attard et al., 2016). These data indicate that after 3 yrs of grassland, the nature of SOM was still in a transitional state and did not yet acquire grassland characteristics, for example a typical biogeochemical grassland soil signature with high lignin concentration. In our system, grasslands of longer duration were needed to be able to differentiate temperate grassland from continuously cropped soils beyond the three-year cropland phase.

461

## 5. Conclusion

In this study, we investigated the biogeochemical nature of SOM in 0-10 cm of cropland and grassland soils as well as the legacy effect (i.e. changes in parameters lasting beyond three years of crop) of differently managed temporary grasslands inserted into crop rotations. Our results show that even if no effect on potential microbial activity was detected, C concentrations of arable soil may be increased through the introduction of temporary grasslands with specific management in terms of fertilisation and duration. Strong legacy effects were related to N-fertilisation, which was necessary to preserve a typical grassland biogeochemical signature and SOM concentrations beyond three years of crop. Grassland duration showed no age-dependent legacy effects concerning C concentrations and other soil properties. However, overall it seems that only after temporary grassland duration of 6 years, soils acquired biogeochemical characteristics close to continuous grassland. In contrast, soils, which supported 3 years of temporary grassland were similar to those supporting continuous crop after the end of the cropland phase.

463

464

465

466

467

468

469

470

471

472

473

474

475

476

## Acknowledgements
478 This study was carried out under the framework of the AEGES project funded by ADEME.

479 A.C. is grateful to the European commission (EXPEER project [grant number 262060]) and

480 the region Poitou-Charente for joint funding of a PhD grant. Xavier Charrier and the SOERE

481 ACBB are acknowledged for providing access to an excellent field experiment. Potential

482 denitrification assays were performed on the AME plateform (LEM, UMR1418).

483

484 References

485

486 Acharya, B.S., Rasmussen, J., Eriksen, J., 2012. Grassland carbon sequestration and

487 emissions following cultivation in a mixed crop rotation. Agriculture, Ecosystems &

488 Environment 153, 33–39.

489 Amelung, W., Brodowski, S., Sandhage-Hofmann, A., Bol, R., 2008. Combining biomarker

490 with stable isotope analyses for assessing the transformation and turnover of soil

491 organic matter. Advances in Agronomy. 100, 155–250.

492 Ammann, C., Flechard, C.R., Leifeld, J., Neftel, A., Fuhrer, J., 2007. The carbon budget of

493 newly established temperate grassland depends on management intensity. Agriculture,

494 Ecosystems & Environment, 121, 5–20.

495 Armas-Herrera, C.M., Dignac, M.-F., Rumpel, C., Arbelo, C.D., Chabbi, A., 2016.

496 Management effects on composition and dynamics of cutin and suberin in topsoil

497 under agricultural use. European Journal of Soil Science 67, 360–373.

498 Attard, E., Le Roux, X., Charrier, X., Delfosse, O., Guillaumaud, N., Lemaire, G., Recous, S.,

499 2016. Delayed and asymmetric responses of soil C pools and N fluxes to

500 grassland/cropland conversions. Soil Biology & Biochemistry 97, 31–39.

501 Attard, E., Recous, S., Chabbi, A., De Berranger, C., Guillaumaud, N., Labreuche, J.,

502 Philippot, L., Schmid, B., Le Roux, X., 2011. Soil environmental conditions rather

503 than denitrifier abundance and diversity drive potential denitrification after changes in

504 land uses. Global Change Biology 17, 1975–1989.

505 Bahri, H., Dignac, M.-F., Rumpel, C., Rasse, D.P., Chenu, C., Mariotti, A., 2006. Lignin

506 turnover kinetics in an agricultural soil is monomer specific. Soil Biology &

507 Biochemistry 38, 1977–1988.

508 Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985. Chloroform fumigation and

509 the release of soil nitrogen: A rapid direct extraction method to measure microbial

510 biomass nitrogen in soil. Soil Biology & Biochemistry 17, 837–842.

511 Carter, M., Gregorich, E., Angers, D., Donald, R., Bolinder, M., 1998. Organic C and N

512 storage, and organic C fractions, in adjacent cultivated and forested soils of eastern

513 Canada. Soil & Tillage Research 47, 253–261.

514 Chabbi, A., Kögel-Knabner, I., Rumpel, C., 2009. Stabilised carbon in subsoil horizons is

515 located in spatially distinct parts of the soil profile. Soil Biology & Biochemistry 41,

516 Chabbi, A., Lehmann, J., Ciais, P., Loescher, H.W., Cotrufo, M.F., Don, A., SanClements,

517 M., Schipper, L., Six, J., Smith, P., Rumpel, C., 2017. Aligning agriculture and

518 climate policy. Nature Climate Change 7, 307–309.

519 Christensen, B.T., Rasmussen, J., Eriksen, J., Hansen, E.M., 2009. Soil carbon storage and

520 yields of spring barley following grass leys of different age. European Journal of

521 Agronomy 31, 29–35.

522 Conant, R.T., Paustian, K., Elliott, E.T., 2001. Grassland Management and Conversion into

523 Grassland: Effects on Soil Carbon. Ecological Applications 11, 343–355.

524 Cotrufo, M.F., Wallenstein, M.D., Boot, C.M., Denef, K., Paul, E., 2013. The Microbial

525 Efficiency-Matrix Stabilization (MEMS) framework integrates plant litter
478 This study was carried out under the framework of the AEGES project funded by ADEME.

479 A.C. is grateful to the European commission (EXPEER project [grant number 262060]) and

480 the region Poitou-Charente for joint funding of a PhD grant. Xavier Charrier and the SOERE

481 ACBB are acknowledged for providing access to an excellent field experiment. Potential

482 denitrification assays were performed on the AME plateform (LEM, UMR1418).

483

484 References

485

486 Acharya, B.S., Rasmussen, J., Eriksen, J., 2012. Grassland carbon sequestration and

487 emissions following cultivation in a mixed crop rotation. Agriculture, Ecosystems &

488 Environment 153, 33–39.

489 Amelung, W., Brodowski, S., Sandhage-Hofmann, A., Bol, R., 2008. Combining biomarker

490 with stable isotope analyses for assessing the transformation and turnover of soil

491 organic matter. Advances in Agronomy. 100, 155–250.

492 Ammann, C., Flechard, C.R., Leifeld, J., Neftel, A., Fuhrer, J., 2007. The carbon budget of

493 newly established temperate grassland depends on management intensity. Agriculture,

494 Ecosystems & Environment, 121, 5–20.

495 Armas-Herrera, C.M., Dignac, M.-F., Rumpel, C., Arbelo, C.D., Chabbi, A., 2016.

496 Management effects on composition and dynamics of cutin and suberin in topsoil

497 under agricultural use. European Journal of Soil Science 67, 360–373.

498 Attard, E., Le Roux, X., Charrier, X., Delfosse, O., Guillaumaud, N., Lemaire, G., Recous, S.,

499 2016. Delayed and asymmetric responses of soil C pools and N fluxes to

500 grassland/cropland conversions. Soil Biology & Biochemistry 97, 31–39.

501 Attard, E., Recous, S., Chabbi, A., De Berranger, C., Guillaumaud, N., Labreuche, J.,

502 Philippot, L., Schmid, B., Le Roux, X., 2011. Soil environmental conditions rather

503 than denitrifier abundance and diversity drive potential denitrification after changes in

504 land uses. Global Change Biology 17, 1975–1989.

505 Bahri, H., Dignac, M.-F., Rumpel, C., Rasse, D.P., Chenu, C., Mariotti, A., 2006. Lignin

506 turnover kinetics in an agricultural soil is monomer specific. Soil Biology &

507 Biochemistry 38, 1977–1988.

508 Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D.S., 1985. Chloroform fumigation and

509 the release of soil nitrogen: A rapid direct extraction method to measure microbial

510 biomass nitrogen in soil. Soil Biology & Biochemistry 17, 837–842.

511 Carter, M., Gregorich, E., Angers, D., Donald, R., Bolinder, M., 1998. Organic C and N

512 storage, and organic C fractions, in adjacent cultivated and forested soils of eastern

513 Canada. Soil & Tillage Research 47, 253–261.

514 Chabbi, A., Kögel-Knabner, I., Rumpel, C., 2009. Stabilised carbon in subsoil horizons is

515 located in spatially distinct parts of the soil profile. Soil Biology & Biochemistry 41,

516 Chabbi, A., Lehmann, J., Ciais, P., Loescher, H.W., Cotrufo, M.F., Don, A., SanClements,

517 M., Schipper, L., Six, J., Smith, P., Rumpel, C., 2017. Aligning agriculture and

518 climate policy. Nature Climate Change 7, 307–309.

519 Christensen, B.T., Rasmussen, J., Eriksen, J., Hansen, E.M., 2009. Soil carbon storage and

520 yields of spring barley following grass leys of different age. European Journal of

521 Agronomy 31, 29–35.

522 Conant, R.T., Paustian, K., Elliott, E.T., 2001. Grassland Management and Conversion into

523 Grassland: Effects on Soil Carbon. Ecological Applications 11, 343–355.

524 Cotrufo, M.F., Wallenstein, M.D., Boot, C.M., Denef, K., Paul, E., 2013. The Microbial

525 Efficiency-Matrix Stabilization (MEMS) framework integrates plant litter
526 decomposition with soil organic matter stabilization: do labile plant inputs form stable
527 soil organic matter? Global Change Biology 19, 988-995.

528 Crème, A., Chabbi, A., Gastal, F., Rumpel, C., 2017. Biogeochemical nature of grassland soil
529 organic matter under plant communities with two nitrogen sources. Plant and Soil 415,
530 189-201.

531 Crème, A., Rumpel, C., Gastal, F., Gil, M. de la L.M., Chabbi, A., 2016. Effects of grasses
532 and a legume grown in monoculture or mixture on soil organic matter and phosphorus
533 forms. Plant and Soil 402, 117-128.

534 Ducloux, J. and Chesseron, C., 1988. Les terres rouges à chataigniers de l'ouest de la France.
535 Ann. Soc. Sci. nat. Charente-Maritime, 7(7): 853-868.

536 Eder, E., Spielvogel, S., Kölbl, A., Albert, G., Kögel-Knabner, I., 2010. Analysis of
537 hydrolysable neutral sugars in mineral soils: Improvement of alditol acetylation for
538 gas chromatographic separation and measurement. Organic Geochemistry 41, 580-
539 585.

540 Franzluebbers, A.J., Sawchik, J., Taboada, M.A., 2014. Agronomic and environmental
541 impacts of pasture-crop rotations in temperate North and South America. Agriculture,
542 Ecosystems & Environment, 190, 18-26.

543 Frei, M., 2013. Lignin: Characterization of a Multifaceted Crop Component. The Scientific
544 World Journal 2013, 1-25.

545 Garland, J.L., Mills, A.L., 1991. Classification and characterization of heterotrophic microbial
546 communities on the basis of patterns of community-level sole-carbon-source
547 utilization. Applied and Environmental Microbiology 57, 2351-2359.

548 Guggenberger, G., Christensen, B. t., Zech, W., 1994. Land-use effects on the composition of
549 organic matter in particle-size separates of soil: I. Lignin and carbohydrate signature.
550 European Journal of Soil Science 45, 449-458.

551 Hedges, J.I., Ertel, J.R., 1982. Characterization of lignin by gas capillary chromatography of
552 cupric oxide oxidation products. Analytical Chemistry 54, 174-178.

553 Hedley, M.J., Stewart, J.W.B., Chauhan, B.S., 1982. Changes in inorganic and organic soil
554 phosphorus fractions induced by cultivation practices and by laboratory incubations.
555 Soil Science Society of America Journal 46, 970.

556 INRA, 2002. Stocker du carbone dans les sols agricoles de France ? Special report.

557 Ivanoff, D.B., Reddy, K.R., Robinson, S., 1998. chemical fractionation of organic phosphorus
558 in selected histosols1. Soil Science 163, 36-45.

559 Jenkinson, D.S., Brookes, P.C., Powlson, D.S., 2004. Measuring soil microbial biomass. Soil
560 Biology and Biochemistry, 36, 5-7.

561 Kiem, R., Kögel-Knabner, I., 2003. Contribution of lignin and polysaccharides to the
562 refractory carbon pool in C-depleted arable soils. Soil Biology & Biochemistry 35,
563 101-118.

564 Kirkby, C.A., Richardson, A.E., Wade, L.J., Batten, G.D., Blanchard, C., Kirkegaard, J.A.,
565 2013. Carbon-nutrient stoichiometry to increase soil carbon sequestration. Soil
566 Biology & Biochemistry 60, 77-86.

567 Klumpp, K., Fontaine, S., Attard, E., Le Roux, X., Gleixner, G., Soussana, J.-F., 2009.
568 Grazing triggers soil carbon loss by altering plant roots and their control on soil
569 microbial community. Journal of Ecology 97, 876-885.

570 Knicker, H., 2011. Soil organic N - An under-rated player for C sequestration in soils? Soil
571 Biology & Biochemistry 43, 1118-1129.

572 Koegel, I., 1986. Estimation and decomposition pattern of the lignin component in forest
573 humus layers. Soil Biology & Biochemistry 18, 589-594.

574 Koegel, I., Bochter, R., 1985. Characterization of lignin in forest humus layers by high-
575 performance liquid chromatography of cupric oxide oxidation products. Soil Biology
576 & Biochemistry 17, 637-640.
526 decomposition with soil organic matter stabilization: do labile plant inputs form stable
527 soil organic matter? Global Change Biology 19, 988-995.

528 Crème, A., Chabbi, A., Gastal, F., Rumpel, C., 2017. Biogeochemical nature of grassland soil
529 organic matter under plant communities with two nitrogen sources. Plant and Soil 415,
530 189-201.

531 Crème, A., Rumpel, C., Gastal, F., Gil, M. de la L.M., Chabbi, A., 2016. Effects of grasses
532 and a legume grown in monoculture or mixture on soil organic matter and phosphorus
533 forms. Plant and Soil 402, 117-128.

534 Ducloux, J. and Chesseron, C., 1988. Les terres rouges à chataigniers de l'ouest de la France.
535 Ann. Soc. Sci. nat. Charente-Maritime, 7(7): 853-868.

536 Eder, E., Spielvogel, S., Kölbl, A., Albert, G., Kögel-Knabner, I., 2010. Analysis of
537 hydrolysable neutral sugars in mineral soils: Improvement of alditol acetylation for
538 gas chromatographic separation and measurement. Organic Geochemistry 41, 580-
539 585.

540 Franzluebbers, A.J., Sawchik, J., Taboada, M.A., 2014. Agronomic and environmental
541 impacts of pasture-crop rotations in temperate North and South America. Agriculture,
542 Ecosystems & Environment, 190, 18-26.

543 Frei, M., 2013. Lignin: Characterization of a Multifaceted Crop Component. The Scientific
544 World Journal 2013, 1-25.

545 Garland, J.L., Mills, A.L., 1991. Classification and characterization of heterotrophic microbial
546 communities on the basis of patterns of community-level sole-carbon-source
547 utilization. Applied and Environmental Microbiology 57, 2351-2359.

548 Guggenberger, G., Christensen, B. t., Zech, W., 1994. Land-use effects on the composition of
549 organic matter in particle-size separates of soil: I. Lignin and carbohydrate signature.
550 European Journal of Soil Science 45, 449-458.

551 Hedges, J.I., Ertel, J.R., 1982. Characterization of lignin by gas capillary chromatography of
552 cupric oxide oxidation products. Analytical Chemistry 54, 174-178.

553 Hedley, M.J., Stewart, J.W.B., Chauhan, B.S., 1982. Changes in inorganic and organic soil
554 phosphorus fractions induced by cultivation practices and by laboratory incubations.
555 Soil Science Society of America Journal 46, 970.

556 INRA, 2002. Stocker du carbone dans les sols agricoles de France ? Special report.

557 Ivanoff, D.B., Reddy, K.R., Robinson, S., 1998. chemical fractionation of organic phosphorus
558 in selected histosols1. Soil Science 163, 36-45.

559 Jenkinson, D.S., Brookes, P.C., Powlson, D.S., 2004. Measuring soil microbial biomass. Soil
560 Biology and Biochemistry, 36, 5-7.

561 Kiem, R., Kögel-Knabner, I., 2003. Contribution of lignin and polysaccharides to the
562 refractory carbon pool in C-depleted arable soils. Soil Biology & Biochemistry 35,
563 101-118.

564 Kirkby, C.A., Richardson, A.E., Wade, L.J., Batten, G.D., Blanchard, C., Kirkegaard, J.A.,
565 2013. Carbon-nutrient stoichiometry to increase soil carbon sequestration. Soil
566 Biology & Biochemistry 60, 77-86.

567 Klumpp, K., Fontaine, S., Attard, E., Le Roux, X., Gleixner, G., Soussana, J.-F., 2009.
568 Grazing triggers soil carbon loss by altering plant roots and their control on soil
569 microbial community. Journal of Ecology 97, 876-885.

570 Knicker, H., 2011. Soil organic N - An under-rated player for C sequestration in soils? Soil
571 Biology & Biochemistry 43, 1118-1129.

572 Koegel, I., 1986. Estimation and decomposition pattern of the lignin component in forest
573 humus layers. Soil Biology & Biochemistry 18, 589-594.

574 Koegel, I., Bochter, R., 1985. Characterization of lignin in forest humus layers by high-
575 performance liquid chromatography of cupric oxide oxidation products. Soil Biology
576 & Biochemistry 17, 637-640.
577 Koegel-Knabner, I., 2017. The macromolecular organic composition of plant and microbial
578 residues as inputs to soil organic matter: Fourteen years on. Soil Biology &
579 Biochemistry 105, A3-A8.

580 Koegel-Knabner, I., 2002. The macromolecular organic composition of plant and microbial
581 residues as inputs to soil organic matter. Soil Biology & Biochemistry 34, 139-162.

582 Koegel-Knabner, I., Rumpel, C., 2018. Advances in molecular approaches for understanding
583 soil organic matter composition, origin and turnover – a historical overview. Advances
584 in Agronomy 148, in press.

585 Kunrath, T.R., de Berranger, C., Charrier, X., Gastal, F., de Faccio Carvalho, P.C., Lemaire,
586 G., Emile, J.-C., Durand, J.-L., 2015. How much do sod-based rotations reduce nitrate
587 leaching in a cereal cropping system? Agricultural Water Management 150, 46-56.

588 Kuzyakov, Y., Domanski, G., others, 2000. Carbon input by plants into the soil. Review.
589 Journal of Plant Nutrition and Soil Science 163, 421-431.

590 Lal, R., 2004. Soil Carbon Sequestration Impacts on global climate change and food Security.
591 Science 304, 1623-1627.

592 Lemaire, G., Gastal, F., Franzluebbers, A., Chabbi, A., 2015. Grassland-cropping rotations:
593 an avenue for agricultural diversification to reconcile high production with
594 environmental quality. Environmental Management 56, 1065-1077.

595 Lobe, I., Du Preez, C.C., Amelung, W., 2002. Influence of prolonged arable cropping on
596 lignin compounds in sandy soils of the South African Highveld. European Journal of
597 Soil Science 53, 553-562.

598 Moni, C., Rumpel, C., Virto, I., Chabbi, A., Chenu, C., 2010. Relative importance of sorption
599 versus aggregation for organic matter storage in subsoil horizons of two contrasting
600 soils. European Journal of Soil Science 61, 958-969.

601 Murayama, S., 1984. Changes in the monosaccharide composition during the decomposition
602 of straws under field conditions. Soil Science and Plant Nutrition, 30, 367-381.

603 Murphy, D.V., Stockdale, E.A., Poulton, P.R., Willison, T.W., Goulding, K.W.T., 2007.
604 Seasonal dynamics of carbon and nitrogen pools and fluxes under continuous arable
605 and ley-arable rotations in a temperate environment. European Journal of Soil Science
606 58, 1410-1424.

607 Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination of
608 phosphate in natural waters. Analytica Chimica Acta 27, 31-36.

609 Oades, J.M., 1984. Soil organic matter and structural stability: mechanisms and implications
610 for management. Plant and Soil 76, 319-337.

611 Page, A. L., R. H. Miller and D. R. Keeney (Ed., 1982): Methods of soil analysis; 2. Chemical
612 and microbiological properties, 2. Aufl. 1184 S., American Soc. of Agronomy (Publ.),
613 Madison, Wisconsin.

614 Panettieri, M., Rumpel, C., Dignac, M.-F., Chabbi, A., 2017. Does grassland introduction into
615 cropping cycles affect carbon dynamics through changes of allocation of soil organic
616 matter within aggregate fractions? Science of The Total Environment 576, 251-263.

617 Patra, A.K., Abbadie, L., Clays-Josserand, A., Degrange, V., Grayston, S.J., Loiseau, P.,
618 Louault, F., Mahmood, S., Nazaret, S., Philippot, L., Poly, F., Prosser, J.I., Richaume,
619 A., Le Roux, X., 2005. Effects of grazing on microbial functional groups involved in
620 soil N dynamics. Ecological Monographs 75, 65-80.

621 Poeplau, C., Don, A., Dondini, M., Leifeld, J., Nemo, R., Schumacher, J., Senapati, N.,
622 Wiesmeier, M., 2013. Reproducibility of a soil organic carbon fractionation method to
derive RothC carbon pools. European Journal of Soil Science 64, 735-746.

624 Post, W.M., Kwon, K.C., 2000. Soil carbon sequestration and land-use change: processes and
625 potential. Global Change Biology 6, 317-327.

626 Rumpel, C., Chabbi, A., 2010. Response of bulk chemical composition, lignin and
627 carbohydrate signature to grassland conversion in a ley-arable cropping system.
628 Nutrient Cycling in Agroecosystems 88, 173-182.
577 Koegel-Knabner, I., 2017. The macromolecular organic composition of plant and microbial
578 residues as inputs to soil organic matter: Fourteen years on. Soil Biology &
579 Biochemistry 105, A3-A8.

580 Koegel-Knabner, I., 2002. The macromolecular organic composition of plant and microbial
581 residues as inputs to soil organic matter. Soil Biology & Biochemistry 34, 139-162.

582 Koegel-Knabner, I., Rumpel, C., 2018. Advances in molecular approaches for understanding
583 soil organic matter composition, origin and turnover – a historical overview. Advances
584 in Agronomy 148, in press.

585 Kunrath, T.R., de Berranger, C., Charrier, X., Gastal, F., de Faccio Carvalho, P.C., Lemaire, G., Emile, J.-C., Durand, J.-L., 2015. How much do sod-based rotations reduce nitrate leaching in a cereal cropping system? Agricultural Water Management 150, 46-56.

588 Kuzyakov, Y., Domanski, G., others, 2000. Carbon input by plants into the soil. Review.
589 Journal of Plant Nutrition and Soil Science 163, 421-431.

590 Lal, R., 2004. Soil Carbon Sequestration Impacts on global climate change and food Security.
591 Science 304, 1623-1627.

592 Lemaire, G., Gastal, F., Franzluebbers, A., Chabbi, A., 2015. Grassland-cropping rotations: an avenue for agricultural diversification to reconcile high production with environmental quality. Environmental Management 56, 1065-1077.

595 Lobe, I., Du Preez, C.C., Amelung, W., 2002. Influence of prolonged arable cropping on lignin compounds in sandy soils of the South African Highveld. European Journal of Soil Science 53, 553-562.

598 Moni, C., Rumpel, C., Virto, I., Chabbi, A., Chenu, C., 2010. Relative importance of sorption versus aggregation for organic matter storage in subsoil horizons of two contrasting soils. European Journal of Soil Science 61, 958-969.

601 Murayama, S., 1984. Changes in the monosaccharide composition during the decomposition
602 of straws under field conditions. Soil Science and Plant Nutrition, 30, 367-381.

603 Murphy, D.V., Stockdale, E.A., Poulton, P.R., Willison, T.W., Goulding, K.W.T., 2007.
604 Seasonal dynamics of carbon and nitrogen pools and fluxes under continuous arable
605 and ley-arable rotations in a temperate environment. European Journal of Soil Science
606 58, 1410-1424.

607 Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27, 31-36.

609 Oades, J.M., 1984. Soil organic matter and structural stability: mechanisms and implications
610 for management. Plant and Soil 76, 319-337.

611 Page, A. L., R. H. Miller and D. R. Keeney (Ed., 1982): Methods of soil analysis; 2. Chemical
612 and microbiological properties, 2. Aufl. 1184 S., American Soc. of Agronomy (Publ.),
613 Madison, Wisconsin.

614 Panettieri, M., Rumpel, C., Dignac, M.-F., Chabbi, A., 2017. Does grassland introduction into cropping cycles affect carbon dynamics through changes of allocation of soil organic matter within aggregate fractions? Science of The Total Environment 576, 251-263.

617 Patra, A.K., Abbadie, L., Clays-Josserand, A., Degrange, V., Grayston, S.J., Loiseau, P., Louault, F., Mahmood, S., Nazaret, S., Philippot, L., Poly, F., Prosser, J.I., Richaume, A., Le Roux, X., 2005. Effects of grazing on microbial functional groups involved in soil N dynamics. Ecological Monographs 75, 65-80.

621 Poeplau, C., Don, A., Dondini, M., Leifeld, J., Nemo, R., Schumacher, J., Senapati, N.,
622 Wiesmeier, M., 2013. Reproducibility of a soil organic carbon fractionation method to
derive RothC carbon pools. European Journal of Soil Science 64, 735-746.

624 Post, W.M., Kwon, K.C., 2000. Soil carbon sequestration and land-use change: processes and potential. Global Change Biology 6, 317-327.

626 Rumpel, C., Chabbi, A., 2010. Response of bulk chemical composition, lignin and carbohydrate signature to grassland conversion in a ley-arable cropping system. *Nutrient Cycling in Agroecosystems* 88, 173-182.
629 Rumpel, C., Chabbi, A., Nunan, N., Dignac, M.-F., 2009. Impact of landuse change on the
630 molecular composition of soil organic matter. Journal of Analytical and Applied
631 Pyrolysis 85, 431-434.

632 Rumpel, C., Crème, A., Ngo, P.T., Velásquez, G., Mora, M.L., Chabbi, A., 2015. The impact
633 of grassland management on biogeochemical cycles involving carbon, nitrogen and
634 phosphorus. Journal of Soil Science and Plant Nutrition 15, 353-371.

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

638 Smith, P., 2016. Soil carbon sequestration and biochar as negative emission technologies.
639 Global Change Biology 22, 1315-1324.

640 Smith, P., Cotrufo, M.F., Rumpel, C., Paustian, K., Kuikman, P.J., Elliott, J.A., McDowell,
641 R., Griffiths, R.I., Asakawa, S., Bustamante, M., House, J.I., Sobocká, J., Harper, R.,
642 Pan, G., West, P.C., Gerber, J.S., Clark, J.M., Adhya, T., Scholes, R.J., Scholes, M.C.,
643 2015. Biogeochemical cycles and biodiversity as key drivers of ecosystem services
644 provided by soils. Soil 1, 665-685.

645 Sollins, P., Homann, P., Caldwell, B.A., 1996. Stabilization and destabilization of soil organic
646 matter: mechanisms and controls. Geoderma 74, 65-105.

647 Soussana, J.-F., Soussana, J.-F., Loiseau, P., Vuichard, N., Ceschia, E., Balesdent, J.,
648 Chevallier, T., Arrouays, D., 2004. Carbon cycling and sequestration opportunities in
649 temperate grasslands. Soil Use and Management 20, 219-230.

650 Stefanowicz, A., 2006. The Biolog plates technique as a tool in ecological studies of
651 microbial communities. Polish Journal of Environmental Studies 15, 669.

652 Studdert, G.A., Echeverría, H.E., Casanovas, E.M., 1997. Crop-Pasture Rotation for
653 Sustaining the Quality and Productivity of a Typic Argiudoll. Soil Science Society of
654 America Journal 61, 1466-1472.

655 Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil
656 microbial biomass C. Soil Biology & Biochemistry 19, 703-707.

657 Walker, T.W., Adams, A.F.R., 1958. Studies on soil organic matter: i. Influence of
658 phosphorus content of parent materials on accumulations of carbon, nitrogen, sulfur,
659 and organic phosphorus in grassland soils. Soil Science 85, 307-318.

660 Zimmermann, M., Leifeld, J., Schmidt, M.W.I., Smith, P., Fuhrer, J., 2007. Measured soil
661 organic matter fractions can be related to pools in the RothC model. European Journal
662 of Soil Science 58, 658-667.

663
664
629 Rumpel, C., Chabbi, A., Nunan, N., Dignac, M.-F., 2009. Impact of landuse change on the
630 molecular composition of soil organic matter. Journal of Analytical and Applied
631 Pyrolysis 85, 431-434.

632 Rumpel, C., Crème, A., Ngo, P.T., Velásquez, G., Mora, M.L., Chabbi, A., 2015. The impact
633 of grassland management on biogeochemical cycles involving carbon, nitrogen and
634 phosphorus. Journal of Soil Science and Plant Nutrition 15, 353-371.

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

638 Smith, P., 2016. Soil carbon sequestration and biochar as negative emission technologies.
639 Global Change Biology 22, 1315-1324.

640 Smith, P., Cotrufo, M.F., Rumpel, C., Paustian, K., Kuikman, P.J., Elliott, J.A., McDowell,
641 R., Griffiths, R.I., Asakawa, S., Bustamante, M., House, J.I., Sobocká, J., Harper, R.,
642 Pan, G., West, P.C., Gerber, J.S., Clark, J.M., Adhya, T., Scholes, R.J., Scholes, M.C.,
643 2015. Biogeochemical cycles and biodiversity as key drivers of ecosystem services
644 provided by soils. Soil 1, 665-685.

645 Sollins, P., Homann, P., Caldwell, B.A., 1996. Stabilization and destabilization of soil organic
646 matter: mechanisms and controls. Geoderma 74, 65-105.

647 Soussana, J.-F., Soussana, J.-F., Loiseau, P., Vuichard, N., Ceschia, E., Balesdent, J.,
648 Chevallier, T., Arrouays, D., 2004. Carbon cycling and sequestration opportunities in
649 temperate grasslands. Soil Use and Management 20, 219-230.

650 Stefanowicz, A., 2006. The Biolog plates technique as a tool in ecological studies of
651 microbial communities. Polish Journal of Environmental Studies 15, 669.

652 Studdert, G.A., Echeverría, H.E., Casanovas, E.M., 1997. Crop-Pasture Rotation for
653 Sustaining the Quality and Productivity of a Typic Argiudoll. Soil Science Society of
654 America Journal 61, 1466-1472.

655 Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction method for measuring soil
656 microbial biomass C. Soil Biology & Biochemistry 19, 703-707.

657 Walker, T.W., Adams, A.F.R., 1958. Studies on soil organic matter: i. Influence of
658 phosphorus content of parent materials on accumulations of carbon, nitrogen, sulfur,
659 and organic phosphorus in grassland soils. Soil Science 85, 307-318.

660 Zimmermann, M., Leifeld, J., Schmidt, M.W.I., Smith, P., Fuhrer, J., 2007. Measured soil
661 organic matter fractions can be related to pools in the RothC model. European Journal
662 of Soil Science 58, 658-667.

663
664
<table><caption>**Table 1.** Experimental design of the study site (SOERE ACBB)* with the 5 treatments**. The vertical bold line indicates the date of sampling (March 2014)</caption><thead><tr><th></th><th></th><th>2005</th><th>2006</th><th>2007</th><th>2008</th><th>2009</th><th>2010</th><th>2011</th><th>2012</th><th>2013</th><th>2014</th></tr></thead><tbody><tr><td>CC</td><td>Continuous cropping</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td></tr><tr><td>TG3N+</td><td>TG fertilized, 3 yrs</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Grassland</td><td>N +</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td></tr><tr><td>TG6N+</td><td>TG fertilized, 6 yrs</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Grassland</td><td>N +</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td></tr><tr><td>TG6N-</td><td>TG unfertilized, 6 yrs</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Grassland</td><td>N -</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td></tr><tr><td>GG</td><td>Continuous grassland</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Grassland</td><td>N +</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td></tr></tbody></table>

* Long-term observatory on environmental research called “agrosystems biogeochemical cycles and biodiversity”

** permanent cropland (CC), 3 yrs fertilised temporary grassland (TG3N+), 6 yrs fertilised temporary grassland (TG6N+), 6 yrs unfertilised temporary grassland (TG6N-) and 9 yrs permanent grassland (GG).
<table><caption>**Table 1.** Experimental design of the study site (SOERE ACBB)* with the 5 treatments**. The vertical bold line indicates the date of sampling (March 2014)</caption><thead><tr><th></th><th></th><th>2005</th><th>2006</th><th>2007</th><th>2008</th><th>2009</th><th>2010</th><th>2011</th><th>2012</th><th>2013</th><th>2014</th></tr></thead><tbody><tr><td>CC</td><td>Continuous cropping</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td></tr><tr><td>TG3N+</td><td>TG fertilized, 3 yrs</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Grassland</td><td>N +</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td></tr><tr><td>TG6N+</td><td>TG fertilized, 6 yrs</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Grassland</td><td>N +</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td></tr><tr><td>TG6N-</td><td>TG unfertilized, 6 yrs</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Grassland</td><td>N -</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td></tr><tr><td>GG</td><td>Continuous grassland</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Grassland</td><td>N +</td><td>Maize</td><td>Wheat</td><td>Barley</td><td>Maize</td><td>Wheat</td></tr></tbody></table>

* Long-term observatory on environmental research called “agrosystems biogeochemical cycles and biodiversity”

** permanent cropland (CC), 3 yrs fertilised temporary grassland (TG3N+), 6 yrs fertilised temporary grassland (TG6N+), 6 yrs unfertilised temporary grassland (TG6N-) and 9 yrs permanent grassland (GG).
<table><caption>**Table 2.** Soil characteristics* under the 5 treatments**. Data are presented as mean of 4 replicates. For each column, different letters indicate significant differences (p < 0.05).</caption><thead><tr><th></th><th>C</th><th>N</th><th>P</th><th>C/N</th><th>C/P</th><th>POM</th><th>NH<sub>4</sub><sup>+</sup>/NO<sub>3</sub><sup>-</sup></th><th>IP</th><th>OP</th></tr><tr><th></th><th>mg g<sup>-1</sup></th><th>mg g<sup>-1</sup></th><th>µg g<sup>-1</sup></th><th></th><th></th><th>% C</th><th>µg g<sup>-1</sup></th><th>µg g<sup>-1</sup></th><th>µg g<sup>-1</sup></th></tr></thead><tbody><tr><td>CC</td><td>11.33<sup>c</sup></td><td>1.22<sup>c</sup></td><td>979.9<sup>ab</sup></td><td>9.3<sup>a</sup></td><td>11.6<sup>bc</sup></td><td>11.5<sup>b</sup></td><td>0.22<sup>b</sup></td><td>539.7<sup>ab</sup></td><td>440.2<sup>a</sup></td></tr><tr><td>TG3N+</td><td>12.97<sup>b</sup></td><td>1.39<sup>b</sup></td><td>1135.8<sup>a</sup></td><td>9.4<sup>a</sup></td><td>11.7<sup>bc</sup></td><td>9.7<sup>b</sup></td><td>0.13<sup>ab</sup></td><td>605.7<sup>a</sup></td><td>530.1<sup>a</sup></td></tr><tr><td>TG6N+</td><td>12.25<sup>bc</sup></td><td>1.27<sup>c</sup></td><td>915.6<sup>ab</sup></td><td>9.8<sup>a</sup></td><td>13.5<sup>b</sup></td><td>14.3<sup>b</sup></td><td>0.16<sup>b</sup></td><td>430.4<sup>ab</sup></td><td>485.3<sup>a</sup></td></tr><tr><td>TG6N-</td><td>9.14<sup>d</sup></td><td>1.01<sup>d</sup></td><td>827.3<sup>b</sup></td><td>9.0<sup>a</sup></td><td>11.1<sup>c</sup></td><td>14.1<sup>b</sup></td><td>0.16<sup>ab</sup></td><td>472.3<sup>ab</sup></td><td>354.9<sup>a</sup></td></tr><tr><td>GG</td><td>16.83<sup>a</sup></td><td>1.65<sup>a</sup></td><td>926.1<sup>ab</sup></td><td>10.2<sup>a</sup></td><td>18.4<sup>a</sup></td><td>19.9<sup>a</sup></td><td>0.74<sup>a</sup></td><td>392.8<sup>b</sup></td><td>533.3<sup>a</sup></td></tr><tr><td>F-value</td><td>187.6</td><td>91.4</td><td>4.2</td><td>3.3</td><td>41.5</td><td>45.5</td><td>10.3</td><td>3.7</td><td>0.9</td></tr></tbody></table>

671 *POM, particulate organic matter; IN, inorganic nitrogen; IP, inorganic phosphorus; OP, organic phosphorus.

672 ** permanent cropland (CC), 3 yrs fertilised temporary grassland (TG3N+), 6 yrs fertilised temporary grassland (TG6N+), 6 yrs unfertilised temporary grassland (TG6N-) and 9 yrs permanent grassland (GG).
<table><caption>**Table 2.** Soil characteristics* under the 5 treatments**. Data are presented as mean of 4 replicates. For each column, different letters indicate significant differences (*p* < 0.05).</caption><thead><tr><th></th><th>C</th><th>N</th><th>P</th><th>C/N</th><th>C/P</th><th>POM</th><th>NH<sub>4</sub><sup>+</sup>/NO<sub>3</sub><sup>-</sup></th><th>IP</th><th>OP</th></tr><tr><th></th><th>mg g<sup>-1</sup></th><th>mg g<sup>-1</sup></th><th>µg g<sup>-1</sup></th><th></th><th></th><th>% C</th><th>µg g<sup>-1</sup></th><th>µg g<sup>-1</sup></th><th>µg g<sup>-1</sup></th></tr></thead><tbody><tr><td>CC</td><td>11.33<sup>c</sup></td><td>1.22<sup>c</sup></td><td>979.9<sup>ab</sup></td><td>9.3<sup>a</sup></td><td>11.6<sup>bc</sup></td><td>11.5<sup>b</sup></td><td>0.22<sup>b</sup></td><td>539.7<sup>ab</sup></td><td>440.2<sup>a</sup></td></tr><tr><td>TG3N+</td><td>12.97<sup>b</sup></td><td>1.39<sup>b</sup></td><td>1135.8<sup>a</sup></td><td>9.4<sup>a</sup></td><td>11.7<sup>bc</sup></td><td>9.7<sup>b</sup></td><td>0.13<sup>ab</sup></td><td>605.7<sup>a</sup></td><td>530.1<sup>a</sup></td></tr><tr><td>TG6N+</td><td>12.25<sup>bc</sup></td><td>1.27<sup>c</sup></td><td>915.6<sup>ab</sup></td><td>9.8<sup>a</sup></td><td>13.5<sup>b</sup></td><td>14.3<sup>b</sup></td><td>0.16<sup>b</sup></td><td>430.4<sup>ab</sup></td><td>485.3<sup>a</sup></td></tr><tr><td>TG6N-</td><td>9.14<sup>d</sup></td><td>1.01<sup>d</sup></td><td>827.3<sup>b</sup></td><td>9.0<sup>a</sup></td><td>11.1<sup>c</sup></td><td>14.1<sup>b</sup></td><td>0.16<sup>ab</sup></td><td>472.3<sup>ab</sup></td><td>354.9<sup>a</sup></td></tr><tr><td>GG</td><td>16.83<sup>a</sup></td><td>1.65<sup>a</sup></td><td>926.1<sup>ab</sup></td><td>10.2<sup>a</sup></td><td>18.4<sup>a</sup></td><td>19.9<sup>a</sup></td><td>0.74<sup>a</sup></td><td>392.8<sup>b</sup></td><td>533.3<sup>a</sup></td></tr><tr><td>F-value</td><td>187.6</td><td>91.4</td><td>4.2</td><td>3.3</td><td>41.5</td><td>45.5</td><td>10.3</td><td>3.7</td><td>0.9</td></tr></tbody></table>

671 *POM, particulate organic matter; IN, inorganic nitrogen; IP, inorganic phosphorus; OP, organic phosphorus.

672 ** permanent cropland (CC), 3 yrs fertilised temporary grassland (TG3N+), 6 yrs fertilised temporary grassland (TG6N+), 6 yrs unfertilised temporary grassland (TG6N-) and 9 yrs permanent grassland (GG).
<table><caption>**Table 3.** Sugar signature* of soil under the five treatments**. Data are presented as mean of 4 replicates. For each column, different letters indicate significant differences (*p* < 0.05).</caption><thead><tr><th></th><th>Content<br>% C</th><th>Concentration<br>mg g<sup>-1</sup> soil</th><th>C6/C5</th><th>DesoxyC6/C5</th><th>Xylose<br>/mannose</th></tr></thead><tbody><tr><td>CC</td><td>38.3<sup>b</sup></td><td>4.29<sup>bc</sup></td><td>2.92<sup>a</sup></td><td>0.37<sup>a</sup></td><td>0.23<sup>c</sup></td></tr><tr><td>TG3N+</td><td>34.8<sup>c</sup></td><td>4.50<sup>b</sup></td><td>2.61<sup>ab</sup></td><td>0.30<sup>b</sup></td><td>0.26<sup>bc</sup></td></tr><tr><td>TG6N+</td><td>37.6<sup>bc</sup></td><td>4.61<sup>b</sup></td><td>2.37<sup>b</sup></td><td>0.33<sup>b</sup></td><td>0.31<sup>b</sup></td></tr><tr><td>TG6N-</td><td>43.1<sup>a</sup></td><td>3.94<sup>c</sup></td><td>2.90<sup>a</sup></td><td>0.29<sup>b</sup></td><td>0.23<sup>c</sup></td></tr><tr><td>GG</td><td>35.5<sup>bc</sup></td><td>5.98<sup>a</sup></td><td>1.51<sup>c</sup></td><td>0.22<sup>c</sup></td><td>0.60<sup>a</sup></td></tr><tr><td>F-value</td><td>21.7</td><td>85.6</td><td>60.6</td><td>33.8</td><td>131.8</td></tr></tbody></table>

676 *C6/C5, hexose / pentose ratio; desoxyC6/C5, desoxyhexose / pentose ratio.

677 ** permanent cropland (CC), 3 yrs fertilized temporary grassland (TG3N+), 6 yrs fertilized

678 temporary grassland (TG6N+), 6 yrs unfertilized temporary grassland (TG6N-) and

679 permanent grassland (GG).
<table><caption>**Table 3.** Sugar signature* of soil under the five treatments**. Data are presented as mean of 4 replicates. For each column, different letters indicate significant differences (*p* < 0.05).</caption><thead><tr><th></th><th>Content<br>% C</th><th>Concentration<br>mg g<sup>-1</sup> soil</th><th>C6/C5</th><th>DesoxyC6/C5</th><th>Xylose<br>/mannose</th></tr></thead><tbody><tr><td>CC</td><td>38.3<sup>b</sup></td><td>4.29<sup>bc</sup></td><td>2.92<sup>a</sup></td><td>0.37<sup>a</sup></td><td>0.23<sup>c</sup></td></tr><tr><td>TG3N+</td><td>34.8<sup>c</sup></td><td>4.50<sup>b</sup></td><td>2.61<sup>ab</sup></td><td>0.30<sup>b</sup></td><td>0.26<sup>bc</sup></td></tr><tr><td>TG6N+</td><td>37.6<sup>bc</sup></td><td>4.61<sup>b</sup></td><td>2.37<sup>b</sup></td><td>0.33<sup>b</sup></td><td>0.31<sup>b</sup></td></tr><tr><td>TG6N-</td><td>43.1<sup>a</sup></td><td>3.94<sup>c</sup></td><td>2.90<sup>a</sup></td><td>0.29<sup>b</sup></td><td>0.23<sup>c</sup></td></tr><tr><td>GG</td><td>35.5<sup>bc</sup></td><td>5.98<sup>a</sup></td><td>1.51<sup>c</sup></td><td>0.22<sup>c</sup></td><td>0.60<sup>a</sup></td></tr><tr><td>F-value</td><td>21.7</td><td>85.6</td><td>60.6</td><td>33.8</td><td>131.8</td></tr></tbody></table>

676 *C6/C5, hexose / pentose ratio; desoxyC6/C5, desoxyhexose / pentose ratio.

677 ** permanent cropland (CC), 3 yrs fertilized temporary grassland (TG3N+), 6 yrs fertilized

678 temporary grassland (TG6N+), 6 yrs unfertilized temporary grassland (TG6N-) and

679 permanent grassland (GG).
<table><caption>**Table 4.** Lignin signature* of soil under the five treatments. Data are presented as mean of 4 replicates. For each column, different letters indicate significant differences (*p* < 0.05).</caption><thead><tr><th></th><th>Content<br>% C</th><th>Concentration<br>mg g<sup>-1</sup> soil</th><th>(Ac/Al)v</th><th>(Ac/Al)s</th><th>Co/V</th><th>S/V</th></tr></thead><tbody><tr><td>CC</td><td>2.4<sup>a</sup></td><td>0.27<sup>ab</sup></td><td>0.45<sup>a</sup></td><td>0.54<sup>ab</sup></td><td>0.46<sup>b</sup></td><td>1.40<sup>b</sup></td></tr><tr><td>TG3N+</td><td>1.9<sup>a</sup></td><td>0.25<sup>b</sup></td><td>0.39<sup>a</sup></td><td>0.51<sup>b</sup></td><td>0.66<sup>a</sup></td><td>1.60<sup>a</sup></td></tr><tr><td>TG6N+</td><td>2.0<sup>a</sup></td><td>0.25<sup>b</sup></td><td>0.48<sup>a</sup></td><td>0.56<sup>a</sup></td><td>0.52<sup>ab</sup></td><td>1.40<sup>b</sup></td></tr><tr><td>TG6N-</td><td>2.2<sup>a</sup></td><td>0.20<sup>b</sup></td><td>0.46<sup>a</sup></td><td>0.52<sup>ab</sup></td><td>0.50<sup>ab</sup></td><td>1.43<sup>ab</sup></td></tr><tr><td>GG</td><td>2.1<sup>a</sup></td><td>0.35<sup>a</sup></td><td>0.47<sup>a</sup></td><td>0.52<sup>ab</sup></td><td>0.66<sup>a</sup></td><td>1.29<sup>b</sup></td></tr><tr><td>F-value</td><td>2.7</td><td>6.9</td><td>0.9</td><td>5.2</td><td>7.0</td><td>9.0</td></tr></tbody></table>

*(Ac/Al)V, (acid/aldehyde)vanillyl ratio ;(Ac/Al)S, (acid/aldehyde)syringyl ratio ;Co/V, p-coumaryl/vanillyl ratio ;S/V, syringyl/vanillyl ratio.

** permanent cropland (Cr), 3 yrs fertilized temporary grassland (TG3N+), 6 yrs fertilized temporary grassland (TG6N+), 6 yrs unfertilized temporary grassland (TG6N-) and permanent grassland (GG).
<table><caption>**Table 4.** Lignin signature* of soil under the five treatments. Data are presented as mean of 4 replicates. For each column, different letters indicate significant differences (p < 0.05).</caption><thead><tr><th></th><th>Content<br>% C</th><th>Concentration<br>mg g<sup>-1</sup> soil</th><th>(Ac/Al)v</th><th>(Ac/Al)s</th><th>Co/V</th><th>S/V</th></tr></thead><tbody><tr><td>CC</td><td>2.4<sup>a</sup></td><td>0.27<sup>ab</sup></td><td>0.45<sup>a</sup></td><td>0.54<sup>ab</sup></td><td>0.46<sup>b</sup></td><td>1.40<sup>b</sup></td></tr><tr><td>TG3N+</td><td>1.9<sup>a</sup></td><td>0.25<sup>b</sup></td><td>0.39<sup>a</sup></td><td>0.51<sup>b</sup></td><td>0.66<sup>a</sup></td><td>1.60<sup>a</sup></td></tr><tr><td>TG6N+</td><td>2.0<sup>a</sup></td><td>0.25<sup>b</sup></td><td>0.48<sup>a</sup></td><td>0.56<sup>a</sup></td><td>0.52<sup>ab</sup></td><td>1.40<sup>b</sup></td></tr><tr><td>TG6N-</td><td>2.2<sup>a</sup></td><td>0.20<sup>b</sup></td><td>0.46<sup>a</sup></td><td>0.52<sup>ab</sup></td><td>0.50<sup>ab</sup></td><td>1.43<sup>ab</sup></td></tr><tr><td>GG</td><td>2.1<sup>a</sup></td><td>0.35<sup>a</sup></td><td>0.47<sup>a</sup></td><td>0.52<sup>ab</sup></td><td>0.66<sup>a</sup></td><td>1.29<sup>b</sup></td></tr><tr><td>F-value</td><td>2.7</td><td>6.9</td><td>0.9</td><td>5.2</td><td>7.0</td><td>9.0</td></tr></tbody></table>

*(Ac/Al)V, (acid/aldehyde)vanillyl ratio ;(Ac/Al)S, (acid/aldehyde)syringyl ratio ;Co/V, p-coumaryl/vanillyl ratio ;S/V, syringyl/vanillyl ratio.

** permanent cropland (Cr), 3 yrs fertilized temporary grassland (TG3N+), 6 yrs fertilized temporary grassland (TG6N+), 6 yrs unfertilized temporary grassland (TG6N-) and permanent grassland (GG).
<table><caption>**Table 5.** Microbial parameters* for soil under the five treatments**. Data are presented as mean of 4 replicates. For each column, different letters indicate significant differences (p < 0.05).</caption><thead><tr><th></th><th>MB-C</th><th>MB-N</th><th>MB-C/MB-N</th><th>Potential denitrification</th></tr><tr><th></th><th>µg g<sup>-1</sup></th><th>µg g<sup>-1</sup></th><th></th><th>µgN-N<sub>2</sub>O g<sup>-1</sup> h<sup>-1</sup></th></tr></thead><tbody><tr><td>CC</td><td>143.6<sup>b</sup></td><td>24.1<sup>a</sup></td><td>5.9<sup>b</sup></td><td>1.09<sup>a</sup></td></tr><tr><td>TG3N+</td><td>180.9<sup>ab</sup></td><td>30.8<sup>a</sup></td><td>5.8<sup>b</sup></td><td>1.13<sup>a</sup></td></tr><tr><td>TG6N+</td><td>204.4<sup>ab</sup></td><td>26.7<sup>a</sup></td><td>7.6<sup>a</sup></td><td>1.05<sup>a</sup></td></tr><tr><td>TG6N-</td><td>135.2<sup>b</sup></td><td>25.2<sup>a</sup></td><td>5.3<sup>b</sup></td><td>0.93<sup>a</sup></td></tr><tr><td>GG</td><td>231.3<sup>a</sup></td><td>33.8<sup>a</sup></td><td>6.8<sup>ab</sup></td><td>0.72<sup>a</sup></td></tr><tr><td>F-value</td><td>6.3</td><td>2.4</td><td>7.3</td><td>1.5</td></tr></tbody></table>

685 * MB-C, microbial carbon; MB-N, microbial nitrogen; AWCD, average well color development.

686 ** permanent cropland (CC), 3 yrs fertilized temporary grassland (TG3N+), 6 yrs fertilized temporary grassland (TG6N+), 6 yrs unfertilized temporary grassland (TG6N-) and permanent grassland (GG).
<table><caption>**Table 5.** Microbial parameters* for soil under the five treatments**. Data are presented as mean of 4 replicates. For each column, different letters indicate significant differences (p < 0.05).</caption><thead><tr><th></th><th>MB-C</th><th>MB-N</th><th>MB-C/MB-N</th><th>Potential denitrification</th></tr><tr><th></th><th>µg g<sup>-1</sup></th><th>µg g<sup>-1</sup></th><th></th><th>µgN-N<sub>2</sub>O g<sup>-1</sup> h<sup>-1</sup></th></tr></thead><tbody><tr><td>CC</td><td>143.6<sup>b</sup></td><td>24.1<sup>a</sup></td><td>5.9<sup>b</sup></td><td>1.09<sup>a</sup></td></tr><tr><td>TG3N+</td><td>180.9<sup>ab</sup></td><td>30.8<sup>a</sup></td><td>5.8<sup>b</sup></td><td>1.13<sup>a</sup></td></tr><tr><td>TG6N+</td><td>204.4<sup>ab</sup></td><td>26.7<sup>a</sup></td><td>7.6<sup>a</sup></td><td>1.05<sup>a</sup></td></tr><tr><td>TG6N-</td><td>135.2<sup>b</sup></td><td>25.2<sup>a</sup></td><td>5.3<sup>b</sup></td><td>0.93<sup>a</sup></td></tr><tr><td>GG</td><td>231.3<sup>a</sup></td><td>33.8<sup>a</sup></td><td>6.8<sup>ab</sup></td><td>0.72<sup>a</sup></td></tr><tr><td>F-value</td><td>6.3</td><td>2.4</td><td>7.3</td><td>1.5</td></tr></tbody></table>

685 * MB-C, microbial carbon; MB-N, microbial nitrogen; AWCD, average well color development.

686 ** permanent cropland (CC), 3 yrs fertilized temporary grassland (TG3N+), 6 yrs fertilized temporary grassland (TG6N+), 6 yrs unfertilized temporary grassland (TG6N-) and permanent grassland (GG).
<table><caption>**Table 6.** Pearson's regression correlation of variables and treatments with dimension 1 and 2 of PCA for analysis of soil under permanent cropland (CC), 3 yrs fertilized temporary grassland (TG3N+), 6 yrs fertilized temporary grassland (TG6N+), 6 yrs unfertilized temporary grassland (TG6N-) and permanent grassland (GG).</caption><thead><tr><th colspan="2">Dimension 1 (52.15%)</th><th colspan="2">Dimension 2 (21.02%)</th></tr><tr><th>treatment</th><th>estimate (p-value)</th><th>treatment</th><th>estimate (p-value)</th></tr></thead><tbody><tr><td>GG</td><td>5.11 (2.32e-08)</td><td>TG3N+</td><td>2.22 (0.00129)</td></tr><tr><td>TG6N-</td><td>-2.56 (4.25e-07)</td><td></td><td></td></tr></tbody></table>
<table><caption>**Table 6.** Pearson's regression correlation of variables and treatments with dimension 1 and 2 of PCA for analysis for soil under permanent cropland (CC), 3 yrs fertilized temporary grassland (TG3N+), 6 yrs fertilized temporary grassland (TG6N+), 6 yrs unfertilized temporary grassland (TG6N-) and permanent grassland (GG).</caption><thead><tr><th colspan="2">Dimension 1 (52.15%)</th><th colspan="2">Dimension 2 (21.02%)</th></tr><tr><th>treatment</th><th>estimate (p-value)</th><th>treatment</th><th>estimate (p-value)</th></tr></thead><tbody><tr><td>GG</td><td>5.11 (2.32e<sup>-08</sup>)</td><td>TG3N+</td><td>2.22 (0.00129)</td></tr><tr><td>TG6N-</td><td>-2.56 (4.25e<sup>-07</sup>)</td><td></td><td></td></tr></tbody></table>
<figure><img src="image_2.png" /></figure>

**Fig. 1.** Potential mineralization of soil carbon with observed (point) and simulated data (line) of soil under permanent cropland (CC), 3 yrs fertilized temporary grassland (TG3N+), 6 yrs fertilized temporary grassland (TG6N+), 6 yrs unfertilized temporary grassland (TG6N-) and permanent grassland (GG). *k* is the C mineralization constant rate used for simulation (C(t) = C₀(1 - e^(-kt)). Different letters indicate significant differences (*p* < 0.5) between treatments. Data are presented as mean of 4 replicates.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Potential mineralization of soil carbon with observed (point) and simulated data (line) of soil under permanent cropland (CC), 3 yrs fertilized temporary grassland (TG3N+), 6 yrs fertilized temporary grassland (TG6N+), 6 yrs unfertilized temporary grassland (TG6N-) and permanent grassland (GG). *k* is the C mineralization constant rate used for simulation (C(t) = C₀(1 - e^(-kt)). Different letters indicate significant differences (*p* < 0.5) between treatments. Data are presented as mean of 4 replicates.</figcaption></figure>
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

**Fig. 2.** Potential mineralization and organization of soil inorganic nitrogen for (A) ammonium and (B) nitrate of soil under permanent cropland (CC), 3 years fertilized temporary grassland (TG3N+), 6 years fertilized temporary grassland (TG6N+), 6 years unfertilized temporary grassland (TG6N-) and permanent grassland (GG). * indicates significant differences (p < 0.5) for Anova 2 ways and Tukey HSD post hoc test results between treatments per day. Data are presented as mean of 4 replicates.
A

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>

**Fig. 2.** Potential mineralization and organization of soil inorganic nitrogen for (A) ammonium and (B) nitrate of soil under permanent cropland (CC), 3 years fertilized temporary grassland (TG3N+), 6 years fertilized temporary grassland (TG6N+), 6 years unfertilized temporary grassland (TG6N-) and permanent grassland (GG). \* indicates significant differences (p < 0.5) for Anova 2 ways and Tukey HSD post hoc test results between treatments per day. Data are presented as mean of 4 replicates.
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Principal component analysis of soil under permanent cropland (CC), 3 years fertilized temporary grassland (TG3N+), 6 years fertilized temporary grassland (TG6N+), 6 years unfertilized temporary grassland (TG6N-) and permanent grassland (GG). (A) Variable factor map and (B) Individual factor map.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Principal component analysis of soil under permanent cropland (CC), 3 years fertilized temporary grassland (TG3N+), 6 years fertilized temporary grassland (TG6N+), 6 years unfertilized temporary grassland (TG6N-) and permanent grassland (GG). (A) Variable factor map and (B) Individual factor map.</figcaption></figure>