# Conversion of arable land to perennial bioenergy crops increases soil organic carbon stocks on the long term

Fabien Ferchaud, Sylvain Marsac, Bruno Mary

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## To cite this version:

Fabien Ferchaud, Sylvain Marsac, Bruno Mary. Conversion of arable land to perennial bioenergy crops increases soil organic carbon stocks on the long term. Agriculture, Ecosystems & Environment, 2025, 388, pp.109637. 10.1016/j.agee.2025.109637. hal-05014100

HAL Id: hal-05014100

https://hal.science/hal-05014100v1

Submitted on 31 Mar 2025

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# Conversion of arable land to perennial bioenergy crops increases soil organic carbon stocks on the long term

Fabien Ferchaud<sup>a,b,*</sup> <sup>●</sup>, Sylvain Marsac<sup>c</sup>, Bruno Mary<sup>a</sup>

## ARTICLE INFO

**Keywords:**

SOC
¹³C

Carbon sequestration

Miscanthus

Switchgrass

## ABSTRACT

Perennial C4 bioenergy crops can combine high productivity and low input requirements. However, their impact on soil organic carbon (SOC) stocks remains uncertain. The aim of this study was to assess the long-term impact of converting arable land to perennial bioenergy crops on SOC stocks for two crop species (miscanthus and switchgrass) and several crop management practices (nitrogen fertilization, soil moisture, harvest date and irrigation). We analyzed two long-term experiments located in northern and southern France. Both sites were sampled initially and after 12 or 13 years. SOC stocks were calculated at equivalent soil mass in each site and δ¹³C measurements were used to calculate changes in “new” and “old” SOC stocks. SOC stocks in the old ploughed layer increased significantly in both sites but most of the SOC storage occurred in the topsoil layer (~0–5 cm). SOC storage rate was fairly similar between miscanthus and switchgrass but was much greater in the southern site than in the northern site (0.96 vs 0.26 t C ha⁻¹ yr⁻¹). This larger storage rate was mainly explained by higher carbon inputs, as suggested by the higher accumulation rate of new SOC (1.41 vs 0.86 t C ha⁻¹ yr⁻¹). No significant effect of the management practices on the SOC change rate could be detected, but early harvest systematically reduced SOC storage compared to late harvest (by 33% for miscanthus and 12% for switchgrass). Higher carbon inputs due to late harvest or irrigated conditions were partly compensated by a higher old SOC decrease.

## 1. Introduction

Biomass is expected to be a major player toward low-carbon economies, by replacing fossil carbon for energy (IPCC, 2011), chemistry or materials (Cherubini, 2010; Ragauskas et al., 2006). The development of biomass production faces a major challenge: to produce substantial quantities while ensuring food security and preserving the environment (Tilman et al., 2009). Agriculture can be a major source of biomass (Scarlat et al., 2010) but the availability of arable crop residues is limited because of the negative impact of residue removal on soil fertility (Powlson et al., 2011). The production of dedicated bioenergy crops is therefore expected to increase significantly (Chum et al., 2011). Among these, perennial C4 bioenergy crops such as miscanthus (Miscanthus × giganteus) and switchgrass (Panicum virgatum) are of particular interest. These perennial rhizomatous grasses combine high productivity, low fertilizer or pesticide requirements and environmental benefits, for example on water quality (Gabrielle et al., 2014; Lesur et al., 2014; Somerville et al., 2010).

One of the major performance criteria for dedicated bioenergy crops is the emission of greenhouse gases during their production, which must be minimal (Don et al., 2012). The change in soil organic carbon (SOC) stock is a key term when calculating the greenhouse gas balance of bioenergy crops, because it results in either carbon dioxide emission or sequestration (Don et al., 2012). Conversion of arable land to perennial bioenergy crops is expected to increase SOC stocks but experimental results exhibit a very large variability (Knight et al., 2024; Poeplau and Don, 2014; Qin et al., 2016; Zang et al., 2018).

The variability in the published results remains largely unexplained but may be partly due to methodological issues. First, most studies employed a paired plot approach in which the initial SOC stock is obtained from an adjacent plot representing the former land use, sampled simultaneously to the plot converted to bioenergy cropping. This approach can lead to erroneous estimates of the SOC stock change in case of initial SOC stock differences between the two plots or if the SOC stock in the reference plot is not at equilibrium (Poeplau and Don, 2014). Secondly, SOC stocks are most often calculated at fixed depth

<footnote><sup>a</sup> BioEcoAgro Joint Research Unit, INRAE, Université de Liège, Université de Lille, Université de Picardie Jules Verne, Barenton-Bugny 02000, France</footnote>

<footnote><sup>b</sup> UMR Eco&Sols, Univ Montpellier, CIRAD, INRAE, IRD, Institut Agro Montpellier, Montpellier, France</footnote>

<footnote><sup>c</sup> Arvalis, 110 chemin de la côte vieille, Baziège 31750, France</footnote>

<footnote>* Correspondence to: INRAE, UMR Eco&Sols, 2 place Viala, Montpellier 34060, France.</footnote>

<footnote>E-mail address: fabien.ferchaud@inrae.fr (F. Ferchaud).</footnote>
instead of equivalent soil mass, i.e. without taking into account differences in soil bulk density. The fixed depth approach may overestimate SOC sequestration under bioenergy crops (Rowe et al., 2016). Finally, the age of the bioenergy crop may have an impact on the SOC stock change. For example, Hansen et al. (2004) reported a higher SOC stock under miscanthus compared to an arable plot only in a 16-year-old plot and not in a 9-year-old plantation. There is a need for diachronic studies following SOC stock evolution over time in long-term experiments. Furthermore, the use of the ¹³C signature of SOC allows to quantify the contribution of the carbon derived from C4 perennial crops to SOC stock changes (Poeplau and Don, 2014; Zang et al., 2018).

Soil and climate conditions as well as management practices may have an impact on SOC stock change under perennial bioenergy crops because of their effects on SOC turnover and/or plant carbon inputs in soil. Knight et al. (2024) analysed published SOC change rates under miscanthus and did not find any significant effect of the soil texture. In a paired site study comparing eleven arable-miscanthus and nine grassland-miscanthus land-use transitions in Great Britain, Rowe et al. (2016) found no correlation with soil texture but a negative significant correlation with the initial SOC stock. Poeplau and Don (2014) found a positive effect of the mean annual temperature on the C4 soil carbon accumulation originated from miscanthus. Few studies have investigated the effect of perennial bioenergy crop management (such as nitrogen fertilization rate and harvest date) on SOC stocks (Cattaneo et al., 2014; Follett et al., 2012). The effect of the crop species (miscanthus vs switchgrass) is also difficult to evaluate because most of the available data comes from different sites, mainly in Europe for miscanthus and in North America for switchgrass (Qin et al., 2016).

The aim of this study was to assess the long-term impacts of conversion from arable land to perennial bioenergy crops on SOC stocks for two crop species (miscanthus and switchgrass) and several crop management options (nitrogen fertilization, harvest date and irrigation). We used two long-term experiments, one located in northern France which had been previously sampled after five years (Ferchaud et al., 2016) and another located in southern France. Both sites were sampled initially and after twelve or thirteen years and SOC stocks were calculated at equivalent soil mass. We hypothesized i) that perennial bioenergy crops established on arable land would increase SOC stocks in the topsoil on the long-term and ii) that SOC changes can be affected by site, crop species and management, through differences in carbon inputs and/or SOC mineralization.

## 2. Material and methods

### 2.1. Study sites and experimental designs

#### 2.1.1. Site 1

The first site, corresponding to the long-term experiment called "Biomass & Environment" (Ferchaud et al., 2016; Ferchaud and Mary, 2016), was located at the INRAE experimental station of Estrées-Mons, northern France (Table 1, Table S1). The experiment was established in 2006 on a field cultivated with annual arable crops for many years (winter wheat was the most frequent crop) and regularly ploughed.

The experiment included two perennial bioenergy crops (Table 2): miscanthus (Miscanthus × giganteus Greef & Deuter) and switchgrass (Panicum virgatum cv. Kanlow). These crops were harvested either early in autumn (October) or late in late winter (February of March). Two levels of mineral nitrogen (N) fertilization were also tested: 0 (N-) and 120 (N+) kg N ha⁻¹ yr⁻¹. The experiment was a split-block design (three blocks) with the crop species and harvest dates in the main plots (Miscanthus Early, Miscanthus Late, Switchgrass Early, Switchgrass Late) and nitrogen fertilization rates (N- and N+) in the subplots (Figure S1).

The whole experimental plot was mouldboard ploughed in December 2005 at a depth of ca. 25 cm. Miscanthus was planted in April 2006 (1.5 rhizomes m⁻²) and switchgrass was sown in June 2006 (seed rate = 15 kg ha⁻¹). The crops were not harvested during the first year: the aboveground biomass was chopped in January 2007 and left on the soil surface. In the N+ treatments, nitrogen fertilization was applied every year from 2007 as urea ammonium nitrate solution, with a single application in April.

#### 2.1.2. Site 2

The second site was located in Montgaillard-Lauragais, southern France (Table 1, Table S2). The experiment was also set up in 2006, on a plot with a long-term arable crop history. The crop rotation was durum wheat-sunflower during at least ten years before 2006 and the plot was regularly ploughed.

The experiment included the same crops and cultivars as site 1. It was divided into two parts separated by ca. 50 m (Figure S2): one part focusing on harvest date and the other part focusing on water availability (Table 2).

In the first part ("harvest date" part), early harvest (end of September to mid-October) and late harvest (mid-March to end of April) were compared for each crop. The crops were established in two separated zones: one zone with three plots of miscanthus and another zone with

<table><caption>**Table 1**

Main characteristics of the two experimental sites.</caption><thead><tr><th></th><th>Site 1</th><th>Site 2</th></tr></thead><tbody><tr><td>Location</td><td>Estrées-Mons (France)</td><td>Montgaillard-Lauragais (France)</td></tr><tr><td>Latitude (WGS84)</td><td>79.872</td><td>43.433</td></tr><tr><td>Longitude (WGS84)</td><td>3.013</td><td>1.679</td></tr><tr><td>Former land use</td><td>Arable crops</td><td>Arable crops</td></tr><tr><td>Starting year</td><td>2006</td><td>2006</td></tr><tr><td>SOC sampling years</td><td>2006 and 2018</td><td>2006 and 2019</td></tr><tr><td>Mean air temperature (°C)<sup>a</sup></td><td>10.7</td><td>13.7</td></tr><tr><td>Cumulative precipitation (mm yr<sup>-1</sup>)<sup>a</sup></td><td>649</td><td>658</td></tr><tr><td>Cumulative Penman potential evapotranspiration (mm yr<sup>-1</sup>)<sup>a</sup></td><td>713</td><td>915</td></tr><tr><td></td><td colspan="2" style="text-align: center;">Harvest date part</td></tr><tr><td></td><td>Haplic Luvisol</td><td>Haplic Calcisol</td><td>Haplic Calcisol</td></tr><tr><td>Soil type<sup>b</sup></td><td></td><td></td><td></td></tr><tr><td>Clay 0–30 cm (g kg<sup>-1</sup>)</td><td>180</td><td>253</td><td>288</td></tr><tr><td>Silt 0–30 cm (g kg<sup>-1</sup>)</td><td>766</td><td>366</td><td>371</td></tr><tr><td>Sand 0–30 cm (g kg<sup>-1</sup>)</td><td>52</td><td>348</td><td>339</td></tr><tr><td>CaCO<sub>3</sub> 0–30 cm (g kg<sup>-1</sup>)</td><td>2</td><td>33</td><td>2</td></tr><tr><td>Initial pH in water 0–30 cm</td><td>7.8</td><td>8.3</td><td>7.7</td></tr><tr><td>Initial C:N ratio 0–30 cm</td><td>9.0</td><td>7.5</td><td>8.3</td></tr></tbody></table>

<footnote>a Between 2006 and 2017 in site 1 and between 2006 and 2018 in site 2.</footnote>

<footnote>b IUSS Working Group WRB (2022)</footnote>
three plots of switchgrass. Each plot was divided into two subplots, one in early harvest and one in late harvest. In fact, the early treatment was harvested early during six years, from 2008 to 2013, and harvested late after 2013. The mineral nitrogen fertilization was similar for the two treatments.

In the second part ("water supply" part), two treatments were compared for each crop: without irrigation (Rainfed) or irrigated at a rate corresponding to maximal evapotranspiration (Irrigated). The crops were also established in two separated zones: one zone with six plots of miscanthus and another zone with six plots of switchgrass. Each treatment was randomly affected to three plots of miscanthus and three plots of switchgrass. In the irrigated treatment, irrigation was applied during seven years (2008–2014), at the rate of 367 ± 149 mm yr⁻¹ for miscanthus and 299 ± 157 mm yr⁻¹ for switchgrass (mean ± standard deviation between years). The mineral nitrogen fertilization was similar in the two treatments.

In both parts of the experiment, miscanthus was planted in late April 2006 (1.5 rhizomes m⁻²) and switchgrass was sown in early June 2006 (seed rate = 10 kg ha⁻¹). The crops were not harvested during the first year. Around 20% of the miscanthus plants died during the first year and were replaced in spring 2007 by plants coming from an adjacent plot also planted in 2006, without any impact on the stand variability. Three switchgrass plots which were badly established after the first year (S6, S7 and S8) were sown again in 2007. Except in 2006 and 2007, nitrogen fertilization was applied every year as ammonium nitrate with either one application of 80 kg N ha⁻¹ (April) or two applications of 100 kg N ha⁻¹ (April and May).

## 2.2. Soil sampling and bulk density measurements

### 2.2.1. Initial measurements

The two sites were initially sampled in 2006. At Estrées-Mons (site 1), two soil cores of 8-cm diameter were taken in each plot (one north and one south) in May 2006, down to 40 cm depth, using a powered soil corer (Humax soil sampler, Switzerland). Each core was divided into three layers: 0–20, 20–Y and Y–40 cm. Y refers to the old ploughing depth which was identified by detecting a change in soil color. It was on average equal to 33.9 cm. Bulk density was measured in April 2006 (every 5 cm from 0 to 40 cm) in six areas spread over the experimental plot using a gamma densitometer (LCP-INRA, Angers, France). At

Mongaillard-Lauragais (site 2), six soil cores of 2 cm diameter were collected in each plot in August 2006 down to 60 cm depth using a hydraulic coring device and divided into two layers (0–30 cm and 30–60 cm). The 30 cm depth corresponds to the old ploughing depth. For each layer and plot, the six samples were pooled to obtain a composite soil sample. The bulk density was measured with steel cylinders, in three different areas and four depths.

Soil samples taken in each site were oven dried at 35 °C for 96 h and crushed to pass through a 2 mm sieve. A subsample was then finely ground with a ball mill (PM 400, Retsch, Germany) and kept for analysis.

### 2.2.2. Final measurements

The final sampling took place in April 2018 at Estrées-Mons (site 1) and April 2019 at Montgaillard-Lauragais (site 2). Soil sampling and processing were achieved by the same team and with the same methods on both sites, to ensure homogeneity between sites.

Twenty-four plots were sampled in each site (8 treatments × 3 replicates). Six soil cores were taken down to 60 cm depth in each plot, using a powered soil corer of 8 cm diameter (Humax soil sampler, Switzerland). The six cores were located inside a micro-plot (size 2–3.8 m²) and were taken in intra-row and inter-row zones to ensure a representative sampling. The micro-plot of site 1 was located north of each plot, close to the previous sampling achieved in 2011 (Ferchaud et al., 2016).

Soil cores were divided into six layers: 0–5, 5–20, 20–30, 30–35, 35–40 and 40–60 cm. Coarse residues (> 2 mm), roots and rhizomes were then carefully separated from the soil by handpicking. Soil samples were oven dried at 35 °C for 96 h. Samples were pooled by two for a given layer, except for layers 0–5 and 5–20 cm, and then crushed to pass through a 2 mm sieve. A total of 576 soil samples was obtained for each site. Finally, a subsample was finely ground with a ball mill (PM 400, Retsch, Germany) before analysis.

Bulk density was determined in the same micro-plots of each site just before soil sampling. It was measured in the upper soil layer (0–5 cm) using steel cylinders of 96 cm³ (diameter 5 cm, length 5 cm) which were pushed into the soil (four replicates per plots). The soil was dried out 48 h at 105 °C and weighed. A gamma densitometer (LCP-INRA, Angers, France) was used in the layers between 5 and 40 cm (every 5 cm), with two replicates per plot in site 1 and one replicate per plot in site 2. For the 40–60 cm layer, we assumed that the bulk density did not vary with

<table><caption>**Table 2**
Experimental treatments tested in site 1 and site 2.</caption><thead><tr><th>Treatment name</th><th>Crop</th><th>Harvest date</th><th>Mineral nitrogen fertilisation<br>(kg N ha<sup>-1</sup> yr<sup>-1</sup>)<sup>a</sup></th><th>Irrigation<br>(mm yr<sup>-1</sup>)<sup>a</sup></th></tr></thead><tbody><tr><td colspan="5"><strong>Site 1</strong></td></tr><tr><td>Miscanthus Early N-</td><td>Miscanthus × giganteus</td><td>Early</td><td>0</td><td>0</td></tr><tr><td>Miscanthus Early N+</td><td>Miscanthus × giganteus</td><td>Early</td><td>110</td><td>0</td></tr><tr><td>Miscanthus Late N-</td><td>Miscanthus × giganteus</td><td>Late</td><td>0</td><td>0</td></tr><tr><td>Miscanthus Late N+</td><td>Miscanthus × giganteus</td><td>Late</td><td>110</td><td>0</td></tr><tr><td>Switchgrass Early N-</td><td>Panicum virgartum</td><td>Early</td><td>0</td><td>0</td></tr><tr><td>Switchgrass Early N+</td><td>Panicum virgartum</td><td>Early</td><td>110</td><td>0</td></tr><tr><td>Switchgrass Late N-</td><td>Panicum virgartum</td><td>Late</td><td>0</td><td>0</td></tr><tr><td>Switchgrass Late N+</td><td>Panicum virgartum</td><td>Late</td><td>110</td><td>0</td></tr><tr><td colspan="5"><strong>Site 2: Harvest date part</strong></td></tr><tr><td>Miscanthus Early</td><td>Miscanthus × giganteus</td><td>Early<sup>b</sup></td><td>68</td><td>12<sup>c</sup></td></tr><tr><td>Miscanthus Late</td><td>Miscanthus × giganteus</td><td>Late</td><td>68</td><td>12<sup>c</sup></td></tr><tr><td>Switchgrass Early</td><td>Panicum virgartum</td><td>Early<sup>b</sup></td><td>68</td><td>12<sup>c</sup></td></tr><tr><td>Switchgrass Late</td><td>Panicum virgartum</td><td>Late</td><td>68</td><td>12<sup>c</sup></td></tr><tr><td colspan="5"><strong>Site 2: Water supply part</strong></td></tr><tr><td>Miscanthus Rainfed</td><td>Miscanthus × giganteus</td><td>Late</td><td>132</td><td>12<sup>c</sup></td></tr><tr><td>Miscanthus Irrigated</td><td>Miscanthus × giganteus</td><td>Late</td><td>132</td><td>209<sup>d</sup></td></tr><tr><td>Switchgrass Rainfed</td><td>Panicum virgartum</td><td>Late</td><td>132</td><td>12<sup>c</sup></td></tr><tr><td>Switchgrass Irrigated</td><td>Panicum virgartum</td><td>Late</td><td>132</td><td>173<sup>d</sup></td></tr></tbody></table>

<footnote>a Mean value for 2006–2017 in Site 1 and 2006–2018 in Site 2.</footnote>

<footnote>b Early harvest from 2008 to 2013 and late harvest after 2013.</footnote>

<footnote>c 90 and 60 mm applied in 2006 and 2007 respectively (to ensure a good crop establishment) and no irrigation after 2007.</footnote>

<footnote>d Irrigation mainly applied between 2008 and 2014 and no irrigation after 2014.</footnote>
time and used initial measurements made with steel cylinders.

## 2.3. Soil analysis

All soil samples were analysed for total carbon concentration and ¹³C abundance using an elemental analyser (EURO EA, Eurovector, Italy) coupled to an isotope ratio mass spectrometer (Delta Plus Advantage, Thermo Electron, Germany). The ¹³C signature was expressed as δ¹³C (‰) relative to the international Pee Dee Belemnite standard.

In the “harvest date” part of site 2, the mineral carbon (Cmin) concentration in the soil was high because of the presence of CaCO₃ (up to 277 g CaCO₃ kg⁻¹ in the 30–60 cm layer in 2006). Therefore, carbonates present in these plots were removed prior organic carbon analysis by adding phosphoric acid. In the “water supply” part of site 2, CaCO₃ concentration was measured in all 2006 and 2019 soil samples using a Bernard calcimeter. When the concentration exceeded 10 g CaCO₃ kg⁻¹ (20 % of the samples), carbonates were removed before analysis. Otherwise, the analysis was made without removing carbonates. The organic carbon concentration and its δ¹³C were then obtained from the total carbon by calculation after determination of the inorganic ¹³C composition (δ¹³Cmin = −9.5 ‰). In site 1, the same type of correction was made for some soil samples containing small amounts of CaCO₃.

## 2.4. Crop residues and belowground biomass

Crop residues (> 2 mm) remaining at soil surface were collected just before soil sampling in the micro-plots (3.8 m² for miscanthus and 2.5 m² for switchgrass in site 1; 2.0 m² for both crops in site 2). Crop residues were composed of stem bases, large stem fragments (> 10 mm) and fallen leaves. Small stem fragments and leaf debris present at soil surface were collected (only at site 1) in six areas (27 × 27 cm) within each micro-plot, corresponding to the location of the soil cores. Stem fragments below soil surface, as well as rhizomes and roots were separated manually from the soil cores and weighted. Samples from the six areas were pooled, as well as samples from the six soil cores. Rhizomes and roots were washed and all samples were dried at 65 °C for 96 h, weighed and ground before analysis. All samples were analyzed for carbon concentration using an elemental analyzer (Flash EA 112 series, Thermo Electron).

## 2.5. Calculation of soil organic carbon stocks and mean weighted δ¹³C

SOC stocks and mean weighted δ¹³C (δ¹³Cw) were calculated at equivalent soil mass (ESM) for a given site, in order to take into account differences in soil bulk density between treatments and years (Ellert and Bettany, 1995; Wendt and Hauser, 2013). In a first step, the SOC concentration, δ¹³C and bulk density data were averaged by plot for each layer and year. The large belowground biomass found in some soil cores resulted in a very variable soil mass between soil cores taken in the layers 0–5 and 5–20 cm. The SOC concentration and δ¹³C signature were therefore weighted by the soil mass in the cores for these two layers. Calculations of SOC stocks at equivalent soil mass were then made using a dedicated R script called “SimpleESM” and available at https://github.com/fabienferchaud/SimpleESM (Ferchaud et al., 2023). We used the method referred as “classical” in the script (Ellert and Bettany, 1995; Lee et al., 2009). The soil is discretized into elementary layers of 1 mm thickness. For a given plot and year, a soil mass, a SOC concentration and a δ¹³C are assigned to each elementary layer k, depending on the measured layer to which it belongs. The depth of SOC stock calculation (z) is then determined to obtain a soil mass as close as possible to the reference soil mass: additional 1 mm soil layers are added until the reference soil mass is reached. The cumulative SOC stock in the layer 0-z (SOC(z), in t C ha⁻¹) is calculated according to Eq. (1):

$$SOC(z) = 0.01 \cdot \sum_{k=1}^{z} \rho(k) \cdot C(k) \quad (1)$$

where ρ(k) and C(k) are respectively the bulk density (g cm⁻³) and the SOC concentration (g kg⁻¹) of the elementary layer k. The mean weighted δ¹³C of the SOC in the layer 0-z (δ¹³Cw(z), in ‰), is calculated as follows in Eq. (2):

$$\delta^{13}C_w(z) = \frac{0.01}{SOC(z)} \cdot \sum_{k=1}^{z} \rho(k) \cdot C(k) \cdot \delta^{13}C(k) \quad (2)$$

where δ¹³C(k) is the measured δ¹³C (‰) of the elementary layer k.

The calculations were made for five soil layers in site 1 and four soil layers in site 2, using the reference soil masses calculated for each site according to the mean bulk densities measured in 2006. At Estrées-Mons (site 1), calculations were made for: L1 (715 t soil ha⁻¹ corresponding to ~0–5 cm), L2 (2150 t ha⁻¹, ~5–20 cm), L3 (2040 t ha⁻¹, ~20–34 cm), L4 (930 t ha⁻¹, ~34–40 cm) and L5 (3137 t ha⁻¹, ~40–60 cm). At Montgaillard-Lauragais (site 2), calculations were made for: L1 (730 t soil ha⁻¹ corresponding to ~0–5 cm), L2 (2190 t ha⁻¹, ~5–20 cm), L3 (1600 t ha⁻¹, ~20–30 cm) and L4 (4920 t ha⁻¹, ~30–60 cm). In both sites, the cumulative layer L1–3 (4905 t ha⁻¹ in site 1 and 4520 t ha⁻¹ in site 2) corresponds to the old ploughed layer.

## 2.6. Calculation of “new” and “old” soil organic carbon stocks

At the start of the experiment, the SOC was mainly derived from C3 crops, with a small proportion of C4 crops (corn). Since miscanthus and switchgrass are C4 crops, the proportion of the final SOC stock derived from these crops (the “new” SOC stock) can be calculated since the start of the experiment. The proportion α of “new” SOC in the total SOC was calculated according to Eq. (3) (Andriulo et al., 1999):

$$\alpha = \frac{\delta - \delta_0}{\delta_1 - \delta_0} \quad (3)$$

where δ is the final δ¹³C signature of SOC, δ₀ is the initial δ¹³C signature of SOC and δ₁ is the δ¹³C signature of the new crop. δ₁ was obtained as the average of all δ¹³C analyses in the aboveground and belowground plant organs, for a given crop and site: −12.7 ‰ and −13.0 ‰, for miscanthus and switchgrass respectively at site 1; −13.4 ‰ and −13.9 ‰, for miscanthus and switchgrass respectively at site 2.

The change in “new” SOC stock (ΔSOC<sub>New</sub>, in t C ha⁻¹) was calculated as shown in Eq. (4) (Ferchaud et al., 2016):

$$\Delta SOC_{New} = \alpha \cdot SOC \quad (4)$$

where SOC is the final SOC stock. The change in “old” SOC stock (ΔSOC<sub>Old</sub>, in t C ha⁻¹) was calculated by difference (Eq. (5)):

$$\Delta SOC_{Old} = SOC - SOC_{0} - \Delta SOC_{New} \quad (5)$$

where SOC₀ is the initial SOC stock.

## 2.7. Statistical analysis

All the statistical analyses were performed using R version 4.0.3 (R Core Team, 2020). Although the two sites shared the same perennial bioenergy crops, the statistical analyses were made separately for each experimental site because their treatments and spatial arrangements were different.

In site 1, an analysis of variance (ANOVA) was performed for each soil layer to test the effect of three factors and their interactions: “crop-harvest” (Miscanthus Early, Miscanthus Late, Switchgrass Early and Switchgrass Late), nitrogen fertilization (N- and N + ) and year (2006 and 2018). These analyses were made for SOC concentration, cumulative SOC stock, δ¹³C and mean weighted δ¹³C. The same type of ANOVA was achieved for the change in “new” and “old” SOC stock and for the carbon stock in living belowground biomass and dead residues, but without the year factor. A linear mixed-effect model adapted to the split-
block design of the experiment (with blocks, “crop-harvest” × blocks and nitrogen × blocks interactions as random factors) was applied using the *lme* function of the *nlme* package (Pinheiro et al., 2020). When significant effects were observed with the ANOVA, significant differences between modalities (p < 0.05) were then identified using the *emmeans* function (Lenth, 2020).

Site 2 included two separated parts (“harvest date” and “water supply”) and within each part, miscanthus and switchgrass were grown in separated zones. We did not include the crop factor (miscanthus vs switchgrass) into our statistical analyses because of this absence of randomisation. A separate ANOVA was therefore performed within each experimental part and crop zone to test the effect of two factors and their interaction: the crop management (Early vs Late in the “harvest date” part, Rainfed vs Irrigated in the “water supply” part) and the year (2006 and 2019). The analyses were made for each soil layer and for the same variables than in site 1. A linear model was applied using the *lm* function of R.

In both sites, the assumptions of the linear model were checked by visual examination of the residuals against predicted values and using Shapiro-Wilk and Levene's tests. Log transformed data were used when necessary to meet these assumptions.

# 3. Results

## 3.1. Carbon in living belowground biomass and dead residues

The living belowground biomass (rhizomes and roots) of the perennial bioenergy crops represented a large carbon pool in the two sites, twelve (site 1) to thirteen (site 2) years after establishment. It was on average lower in site 1 than in site 2: 8.3 vs 11.9 t C ha⁻¹ in the layer 0–60 cm. Switchgrass had a higher root carbon stock than miscanthus at site 1 (Table 3). Root carbon was also negatively affected by nitrogen fertilization. Rhizome carbon and total belowground carbon did not differ significantly between treatments. At site 2, the only significant effect of the crop management was observed for switchgrass in the “harvest date” part: rhizome carbon and total belowground carbon were higher in the early compared to the late harvest (Table 4). At both sites, the carbon stock of rhizomes represented a much higher proportion of the total belowground carbon for miscanthus than for switchgrass: 70 vs 40 % in site 1 and 76 vs 28 % in site 2.

The dead residues also contained a large carbon stock: on average, 4.4 t C ha⁻¹ in site 1 and 8.5 t C ha⁻¹ in site 2. At site 1, the aboveground residues (stem bases, stem fragments and fallen leaves for miscanthus harvested late) contained more carbon in the late than in the early harvest treatment (4.6 vs 2.1 t C ha⁻¹ on average) but similar amounts between crops and nitrogen rates (Table 3). The belowground residues (mainly stem fragments found in the 0–5 cm soil layer) contained 1.1 t C ha⁻¹ on average without any significant effect of the experimental factors. At site 2, total residue carbon was on average higher for miscanthus (10.7 t C ha⁻¹) than for switchgrass (6.2 t C ha⁻¹). Among the tested crop management practices, only the water supply had a significant effect for switchgrass, with higher amount of residue carbon under irrigation compared to rainfed. However, it should be remembered that the differentiation between treatments stopped in 2014 (harvest date) or 2015 (water supply), i.e. four to five years before the sampling.

## 3.2. Changes in soil organic carbon concentrations

At the onset of the experiment (2006), SOC concentrations at site 1 were very similar between treatments (Fig. 1a). The mean concentration was 11.2 ± 0.1 g kg⁻¹ on average in layers L1 (~0–5 cm) and L2 (~5–20 cm) and 10.0 ± 0.1 g kg⁻¹ on average in layer L3 (~20–34 cm). In 2018, SOC concentrations had significantly and strongly increased in layer L1 for all treatments (+7.4 g kg⁻¹ on average). Conversely, SOC concentrations had slightly decreased in layers L2 and L3, by −0.5 g kg⁻¹ on average (significant decrease for all treatments except

<table><caption>**Table 3**
Carbon stock in living belowground biomass (0–60 cm) and dead crop residues measured in 2018 for site 1. Values in brackets are standard deviations. Different letters indicate significant differences between treatments (p < 0.05).</caption><thead><tr><th rowspan="2"></th><th colspan="2">Miscanthus</th><th colspan="2">Late</th><th colspan="2">Switchgrass</th><th colspan="2">Late</th><th rowspan="2">N+</th></tr><tr><th>Early</th><th>N-</th><th>N-</th><th>N+</th><th>Early</th><th>N+</th><th>N-</th><th>N+</th></tr></thead><tbody><tr><td>Living biomass (t C ha⁻¹)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Rhizomes</td><td>3.0 (0.8)</td><td>a</td><td>5.0 (1.1)</td><td>a</td><td>6.2 (2.7)</td><td>a</td><td>3.4 (2.7)</td><td>a</td><td>4.7 (1.9)</td></tr><tr><td>Roots</td><td>2.8 (0.8)</td><td>ab</td><td>2.1 (0.3)</td><td>a</td><td>2.5 (0.7)</td><td>ab</td><td>5.0 (2.2)</td><td>bc</td><td>7.2 (0.4)</td></tr><tr><td>Total</td><td>5.8 (0.3)</td><td>a</td><td>7.1 (1.0)</td><td>a</td><td>8.7 (2.8)</td><td>a</td><td>8.4 (4.5)</td><td>a</td><td>11.9 (1.9)</td></tr><tr><td>Dead residues (t C ha⁻¹)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Aboveground</td><td>1.9 (0.2)</td><td>a</td><td>2.4 (0.5)</td><td>a</td><td>5.0 (0.8)</td><td>b</td><td>4.7 (0.6)</td><td>b</td><td>4.2 (0.2)</td></tr><tr><td>Belowground</td><td>0.7 (0.5)</td><td>a</td><td>1.1 (0.4)</td><td>a</td><td>1.8 (0.4)</td><td>a</td><td>1.0 (0.8)</td><td>a</td><td>1.3 (0.6)</td></tr><tr><td>Total</td><td>2.6 (0.4)</td><td>a</td><td>3.5 (0.5)</td><td>ab</td><td>6.8 (1.2)</td><td>c</td><td>5.7 (1.1)</td><td>c</td><td>5.5 (0.5)</td></tr></tbody></table>
<caption>**Table 4**</caption>

Carbon stock in living belowground biomass (0–60 cm) and dead crop residues measured in 2019 for site 2. Values in brackets are standard deviations. Asterisks indicate significant differences between treatments (* p < 0.05; ** p < 0.01; *** p < 0.001).

<table><thead><tr><th rowspan="3"></th><th colspan="6">Harvest date</th><th colspan="6">Water supply</th></tr><tr><th colspan="2">Miscanthus</th><th colspan="2">Switchgrass</th><th colspan="2">Miscanthus</th><th colspan="2">Switchgrass</th><th colspan="2">Miscanthus</th><th colspan="2">Switchgrass</th></tr><tr><th>Early</th><th>Late</th><th>Early</th><th>Late</th><th>Rainfed</th><th>Irrigated</th><th>Rainfed</th><th>Irrigated</th><th>Rainfed</th><th>Irrigated</th><th>Rainfed</th><th>Irrigated</th></tr></thead><tbody><tr><td colspan="14">Living biomass (t C ha<sup>-1</sup>)</td></tr><tr><td>Rhizomes</td><td>10.5</td><td>(1.8)</td><td>8.3</td><td>(5.1)</td><td>4.0</td><td>(0.3)</td><td>1.9</td><td>(0.5)</td><td>9.9</td><td>(4.4)</td><td>11.1</td><td>(2.4)</td><td>1.9</td><td>(1.3)</td><td>4.1</td><td>(1.2)</td></tr><tr><td>Roots</td><td>4.0</td><td>(0.6)</td><td>3.4</td><td>(0.9)</td><td>9.6</td><td>(0.4)</td><td>7.2</td><td>(1.9)</td><td>2.5</td><td>(0.8)</td><td>3.1</td><td>(0.5)</td><td>5.3</td><td>(1.9)</td><td>8.6</td><td>(2.7)</td></tr><tr><td>Total</td><td>14.5</td><td>(1.4)</td><td>11.6</td><td>(5.7)</td><td>13.7</td><td>(0.6)</td><td>9.1</td><td>(2.4)</td><td>12.4</td><td>(4.6)</td><td>14.2</td><td>(2.0)</td><td>7.2</td><td>(3.1)</td><td>12.8</td><td>(2.2)</td></tr><tr><td colspan="14">Dead residues (t C ha<sup>-1</sup>)</td></tr><tr><td>Total</td><td>11.3</td><td>(4.9)</td><td>11.2</td><td>(3.3)</td><td>5.4</td><td>(0.9)</td><td>6.0</td><td>(0.1)</td><td>9.7</td><td>(1.7)</td><td>10.8</td><td>(1.0)</td><td>4.9</td><td>(0.8)</td><td>8.4</td><td>(1.0) **</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Profile of a) soil organic carbon (SOC) concentration and b) δ¹³C of soil organic carbon, at equivalent soil mass in 2006 and 2018 for site 1. The 2006 data are averaged between N- and N+. Asterisks indicate significant differences between years for a given soil layer (* p < 0.05; ** p < 0.01; *** p < 0.001).</figcaption></figure>

Switchgrass Early). SOC concentrations remained constant in layer L4 (~34–40 cm) between 2006 and 2018. It was not possible to test the evolution in layer L5 (~40–60 cm) because this layer was not sampled in 2006. In 2018, only SOC concentration in layer L1 was significantly affected by the treatments: it was on average higher in late than early harvest (20.1 vs 17.1 g kg⁻¹) and in N + than N- (19.4 vs 17.8 g kg⁻¹).

At site 2, the initial SOC concentrations in 2006 were lower in the “harvest date” part of the experiment than in the “water supply” part: 7.8 ± 0.2 vs 8.9 ± 0.2 g kg⁻¹ on average in layers L1–3 (~0–30 cm) and 4.1 ± 0.2 vs 6.0 ± 0.4 g kg⁻¹ on average in layer L4 (~30–60 cm). However, SOC concentrations within each part did not differ significantly between miscanthus and switchgrass zones. Between 2006 and 2019, the SOC concentration strongly increased in layer L1 (+13.3 g kg⁻¹ on average), with a significant difference between years for all treatments (Fig. 2a). The SOC concentration also significantly increased in layer L2 (except for Miscanthus Early), by 1.9 g kg⁻¹ on average. In layer L3, the SOC concentration either slightly decreased (miscanthus, −1.0 g kg⁻¹ on average) or did not change (switchgrass) between 2006 and 2019. It remained stable in layer L4. In 2019, the SOC concentration in layer L1 was significantly affected by harvest for miscanthus: it was lower after early harvest (18.9 g kg⁻¹) than after late harvest (22.6 g kg⁻¹). There was no other significant effect of the crop management.

### 3.3. Changes in the isotopic composition of soil organic carbon

At site 1, the initial δ¹³C values were very homogeneous between treatments (−26.29 ± 0.02 ‰ on average in layers L1–2) and between soil layers (Fig. 1b). The δ¹³C values in the soil profile showed a general increase between 2006 and 2018, demonstrating carbon inputs from C4 plants into the soil organic matter. The increase was significant in all soil layers and all treatments and was particularly high in layer L1: it was on average +6.3, +1.7, +0.8 and +0.5 ‰ in layers L1, L2, L3 and L4 respectively. In 2018, the δ¹³C of layer L1 was significantly higher in the late than in the early harvest treatments (−18.8 vs −21.2 ‰).

At site 2, the initial δ¹³C values were also homogeneous between treatments and soil layers (Fig. 2b): on average −25.32 ± 0.20 ‰ in layers L1 to L3 and −25.1 ± 0.2 ‰ in layer L4. The δ¹³C values increased between 2006 and 2019 in all layers. The average increase was 7.2, 3.5, 1.4 and 0.6 ‰ in layers L1, L2, L3 and L4 respectively. The increase was significant in all treatments in layers L1, L2 and L3, and for four treatments in layer L4. In 2019, the δ¹³C in layer L1 was significantly higher after late harvest than after early harvest for miscanthus (−17.3 vs −19.1 ‰), similarly to the SOC concentration. A significant effect of the water supply was also observed in layer L3, with slightly higher δ¹³C in irrigated compared to rainfed for both crop species (−23.4 ‰ vs −24.2 ‰ on average).
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Profile of a) soil organic carbon (SOC) concentration and b) δ¹³C of soil organic carbon, at equivalent soil mass in 2006 and 2019 for site 2. Asterisks indicate significant differences between years for a given soil layer (\* p < 0.05; \*\* p < 0.01; \*\*\* p < 0.001).</figcaption></figure>

### 3.4. *Changes in soil organic carbon stocks*

At site 1, the initial SOC stock (2006) in the old ploughed layer (layer L1–3, 4905 t ha⁻¹, ~0–34 cm) was 52.4 t C ha⁻¹ (Table S3) with a moderate spatial variability: the SOC stock ranged from 49.1 to 55.2 t C ha⁻¹ among the 24 plots (standard deviation = 1.7 t C ha⁻¹). At site 2, the initial SOC stock (2006) in the old ploughed layer (layer L1–3, 4520 t ha⁻¹, ~0–30 cm) was lower in the “harvest date” part of the experiment than in the “water supply” part (34.2 ± 1.6 vs 40.4 ± 2.3 t C ha⁻¹) but did not differ between miscanthus and switchgrass zones within each part (Table S4). The same trend was observed in the whole soil profile (layer L1–4, 9440 t ha⁻¹, ~0–60 cm).

SOC stocks in the old ploughed layer L1–3 increased significantly under perennial bioenergy crops in the two sites and under all treatments (Table 5). The mean rate of SOC storage in this layer was more than three times lower in the northern site (site 1) than in the southern site (site 2): 0.26 vs 0.96 t C ha⁻¹ yr⁻¹. It was fairly similar between miscanthus and switchgrass: 0.22 vs 0.30 t C ha⁻¹ yr⁻¹ for site 1 and 1.03 vs 0.89 t C ha⁻¹ yr⁻¹ for site 2. In both sites, the analysis of variance did not show any significant interaction with the experimental factors

(crop, harvest, nitrogen fertilization or irrigation). Final SOC stocks did not differ significantly between treatments in layers L1–3, L1–4 and L1–5, both in site 1 (Table S5) and in site 2 (Table S6). Despite this lack of significant effect, there was a tendency across both sites and both crops towards a greater SOC storage under late than early harvest: the extra storage due to late harvest was on average +0.07 t C ha⁻¹ yr⁻¹ in L1–3 in site 1 and +0.21 t C ha⁻¹ yr⁻¹ in site 2. Furthermore, the difference was higher for miscanthus than for switchgrass: early harvest reduced SOC storage on average by 33 % for miscanthus (32 % in site 1 and 34 % in site 2) and by 12 % for switchgrass (15 % in site 1 and 10 % in site 2). At site 1, there were only small differences between N- and N + treatments. At site 2, irrigation increased SOC storage, particularly for switchgrass (+0.09 t C ha⁻¹ yr⁻¹ for miscanthus and +0.47 t C ha⁻¹ yr⁻¹ for switchgrass). In both sites, the SOC stock change in layer L1–4 was very similar to the SOC stock change in layer L1–3 because the SOC stock in layer L4 remained constant during the experiments.

### 3.5. *Changes in the isotopic composition of soil organic carbon stocks*

During the experimental period (12 or 13 years), the mean weighted
<caption>**Table 5**</caption>

Rate of change of cumulative soil organic carbon stock (ΔSOC) and mean weighted δ¹³C (Δδ¹³Cw) between 2006 and 2018 (site 1) or 2006 and 2019 (site 2), at equivalent soil mass. Values in brackets are standard deviations.

<table><thead><tr><th rowspan="2">Site 1</th><th rowspan="2">Soil layer</th><th colspan="2">Miscanthus Early</th><th colspan="2">Miscanthus Late</th><th colspan="2">Switchgrass Early</th><th colspan="2">Switchgrass Late</th></tr><tr><th>N-</th><th>N+</th><th>N-</th><th>N+</th><th>N-</th><th>N+</th><th>N-</th><th>N+</th></tr></thead><tbody><tr><td rowspan="2">ΔSOC<br>(t C ha<sup>-1</sup><br>yr<sup>-1</sup>)</td><td>L1</td><td>0.33<br>(0.07)</td><td>0.34<br>(0.10)</td><td>0.51<br>(0.11)</td><td>0.57<br>(0.19)</td><td>0.29<br>(0.07)</td><td>0.43<br>(0.10)</td><td>0.42<br>(0.10)</td><td>0.58<br>(0.09)</td></tr><tr><td>L1-2</td><td>0.26<br>(0.22)</td><td>0.24<br>(0.07)</td><td>0.43<br>(0.22)</td><td>0.38<br>(0.21)</td><td>0.28<br>(0.20)</td><td>0.38<br>(0.20)</td><td>0.32<br>(0.06)</td><td>0.47<br>(0.15)</td></tr><tr><td rowspan="5">Δδ<sup>13</sup>Cw<br>(‰ yr<sup>-1</sup>)</td><td>L1-3</td><td><strong>0.18</strong><br>(0.28)</td><td><strong>0.18</strong><br>(0.13)</td><td><strong>0.32</strong><br>(0.28)</td><td><strong>0.21</strong><br>(0.22)</td><td><strong>0.24</strong><br>(0.20)</td><td><strong>0.32</strong><br>(0.19)</td><td><strong>0.26</strong><br>(0.07)</td><td><strong>0.39</strong><br>(0.23)</td></tr><tr><td>L1-4</td><td>0.18<br>(0.30)</td><td>0.22<br>(0.19)</td><td>0.34<br>(0.37)</td><td>0.22<br>(0.21)</td><td>0.26<br>(0.19)</td><td>0.37<br>(0.23)</td><td>0.26<br>(0.07)</td><td>0.40<br>(0.30)</td></tr><tr><td>L1</td><td>0.47<br>(0.05)</td><td>0.41<br>(0.11)</td><td>0.63<br>(0.03)</td><td>0.65<br>(0.07)</td><td>0.40<br>(0.03)</td><td>0.41<br>(0.11)</td><td>0.60<br>(0.06)</td><td>0.63<br>(0.05)</td></tr><tr><td>L1-2</td><td>0.26<br>(0.03)</td><td>0.21<br>(0.05)</td><td>0.33<br>(0.04)</td><td>0.32<br>(0.05)</td><td>0.22<br>(0.02)</td><td>0.24<br>(0.08)</td><td>0.34<br>(0.04)</td><td>0.34<br>(0.06)</td></tr><tr><td>L1-3</td><td><strong>0.20</strong><br>(0.02)</td><td><strong>0.14</strong><br>(0.03)</td><td><strong>0.24</strong><br>(0.03)</td><td><strong>0.22</strong><br>(0.04)</td><td><strong>0.17</strong><br>(0.01)</td><td><strong>0.19</strong><br>(0.06)</td><td><strong>0.26</strong><br>(0.04)</td><td><strong>0.24</strong><br>(0.05)</td></tr><tr><td>L1-4</td><td>0.19<br>(0.01)</td><td>0.13<br>(0.03)</td><td>0.22<br>(0.03)</td><td>0.20<br>(0.04)</td><td>0.16<br>(0.01)</td><td>0.17<br>(0.05)</td><td>0.24<br>(0.04)</td><td>0.22<br>(0.04)</td></tr><tr><th rowspan="2">Site 2</th><th rowspan="2">Soil layer</th><th colspan="2">Miscanthus</th><th colspan="2">Switchgrass</th><th colspan="2">Miscanthus</th><th colspan="2">Switchgrass</th></tr><tr><th>Early</th><th>Late</th><th>Early</th><th>Late</th><th>Rainfed</th><th>Irrigated</th><th>Rainfed</th><th>Irrigated</th></tr><tr><td rowspan="2">ΔSOC<br>(t C ha<sup>-1</sup><br>yr<sup>-1</sup>)</td><td>L1</td><td>0.62<br>(0.05)</td><td>0.83<br>(0.07)</td><td>0.54<br>(0.17)</td><td>0.57<br>(0.13)</td><td>0.91<br>(0.36)</td><td>0.90<br>(0.22)</td><td>0.66<br>(0.13)</td><td>0.82<br>(0.15)</td></tr><tr><td>L1-2</td><td>0.73<br>(0.16)</td><td>1.13<br>(0.03)</td><td>0.78<br>(0.20)</td><td>0.84<br>(0.20)</td><td>1.33<br>(0.13)</td><td>1.40<br>(0.19)</td><td>0.86<br>(0.02)</td><td>1.30<br>(0.21)</td></tr><tr><td rowspan="5">Δδ<sup>13</sup>Cw<br>(‰ yr<sup>-1</sup>)</td><td>L1-3</td><td><strong>0.65</strong><br>(0.13)</td><td><strong>0.98</strong><br>(0.02)</td><td><strong>0.72</strong><br>(0.02)</td><td><strong>0.79</strong><br>(0.05)</td><td><strong>1.19</strong><br>(0.08)</td><td><strong>1.28</strong><br>(0.02)</td><td><strong>0.79</strong><br>(0.07)</td><td><strong>1.25</strong><br>(0.03)</td></tr><tr><td>L1-4</td><td>0.77<br>(0.06)</td><td>1.06<br>(0.09)</td><td>0.82<br>(0.18)</td><td>0.74<br>(0.09)</td><td>1.34<br>(0.08)</td><td>1.34<br>(0.06)</td><td>0.75<br>(0.16)</td><td>1.09<br>(0.15)</td></tr><tr><td>L1</td><td>0.45<br>(0.06)</td><td>0.59<br>(0.05)</td><td>0.42<br>(0.12)</td><td>0.52<br>(0.08)</td><td>0.64<br>(0.06)</td><td>0.69<br>(0.04)</td><td>0.51<br>(0.11)</td><td>0.61<br>(0.03)</td></tr><tr><td>L1-2</td><td>0.30<br>(0.04)</td><td>0.42<br>(0.06)</td><td>0.28<br>(0.09)</td><td>0.36<br>(0.08)</td><td>0.44<br>(0.06)</td><td>0.50<br>(0.05)</td><td>0.35<br>(0.07)</td><td>0.44<br>(0.05)</td></tr><tr><td>L1-3</td><td><strong>0.25</strong><br>(0.03)</td><td><strong>0.34</strong><br>(0.04)</td><td><strong>0.24</strong><br>(0.09)</td><td><strong>0.30</strong><br>(0.08)</td><td><strong>0.37</strong><br>(0.05)</td><td><strong>0.42</strong><br>(0.05)</td><td><strong>0.29</strong><br>(0.06)</td><td><strong>0.37</strong><br>(0.05)</td></tr><tr><td>L1-4</td><td>0.19<br>(0.03)</td><td>0.25<br>(0.03)</td><td>0.18<br>(0.06)</td><td>0.22<br>(0.06)</td><td>0.25<br>(0.03)</td><td>0.30<br>(0.04)</td><td>0.21<br>(0.04)</td><td>0.28<br>(0.04)</td></tr></tbody></table>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Change in a) “new” and b) “old” soil organic carbon (SOC) stock between 2006 and 2018 (site 1) or 2006 and 2019 (site 2), at equivalent soil mass for the old ploughed layer (L1-3: 4905 t ha⁻¹ in site 1, 4520 t ha⁻¹ in site 2). L1, L2 and L3 are soil layers at equivalent soil mass (respectively ~0-5 cm, ~5-20 cm and ~20-34 cm for site 1 and respectively ~0-5 cm, ~5-20 cm and ~20-30 cm for site 2). Error bars are standard deviations for the layer L1-3.</figcaption></figure>

δ¹³C values in the old ploughed layer L1-3 increased significantly in all treatments at both sites (Table 5). The mean weighted δ¹³C increase in layer L1-3 varied with site, harvest and irrigation treatments. It was similar between miscanthus and switchgrass at both sites. The increase was higher in site 2 (+0.32 ± 0.06 ‰ yr⁻¹) than in site 1 (+0.21 ± 0.04 ‰ yr⁻¹). It was also higher in late than in early harvest (0.28 vs 0.21 ‰ yr⁻¹ on average), although the difference was significant only for switchgrass in site 1 (Table S4) and for miscanthus in site 2 (Table S5). At site 2, irrigation seemed to stimulate the mean weighted δ¹³C increase compared to the rainfed management (0.40 vs 0.21 ‰ yr⁻¹ on average), but the difference was not significant.

### 3.6. Changes in “new” soil organic carbon stocks

New SOC originated from the C4 perennial bioenergy crops

established in 2006 increased in the old ploughed layer (layer L1-3) on average by 0.86 t C ha⁻¹ yr⁻¹ in site 1 and 1.41 t C ha⁻¹ yr⁻¹ in site 2 (Fig. 3a). The new SOC storage rate in layer L1-3 was close for miscanthus and switchgrass: 0.81 vs 0.91 t C ha⁻¹ yr⁻¹ for site 1 and 1.51 vs 1.30 t C ha⁻¹ yr⁻¹ for site 2, respectively. The analysis of variance showed a significant effect of the harvest date at site 1. The increase in new SOC stock was higher for late than early harvest (1.01 vs 0.71 t C ha⁻¹ yr⁻¹), the difference being significant only for switchgrass. No effect of nitrogen fertilization could be observed. At site 2, the storage rate of new SOC was also higher for late than early harvest (1.31 vs 0.91 t C ha⁻¹ yr⁻¹ in layer L1-3) and the difference was significant for miscanthus (1.41 ± 0.27 vs 0.96 ± 0.10 t C ha⁻¹ yr⁻¹). There was also a tendency towards a higher new SOC storage in irrigated versus rainfed treatment, with a larger difference for switchgrass: 1.19 ± 0.31 t C ha⁻¹ yr⁻¹ in the rainfed and 1.86 ± 0.46 t C ha⁻¹ yr⁻¹ in the irrigated
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Relationship between the rate of change of “new” and “old” soil organic carbon (SOC) stocks in the old ploughed layer L1–3 (4905 t ha⁻¹ in site 1, 4520 t ha⁻¹ in site 2) for a) site 1 and b) site 2 (Mis = Miscanthus; Swi = Switchgrass; E = Early; L = Late). Error bars are standard deviations. Asterisks indicate the level of significance (**) p < 0.01; (***) p < 0.001).</figcaption></figure>

treatment. In both sites, most of the new SOC storage occurred in layers L1 and L2 (90 % on average in both sites) and more than half in layer L1 (61 % in site 1 and 54 % in site 2).

### 3.7. Changes in “old” soil organic carbon stocks

As expected, the old SOC stock (i.e. the SOC already present at the onset of the experiments) decreased over time in all treatments due to SOC mineralization (Fig. 3b). The decrease in the old ploughed layer L1–3 was on average slightly higher at site 1 (−0.60 t C ha⁻¹ yr⁻¹) than at site 2 (−0.45 t C ha⁻¹ yr⁻¹). The decay rate of the old SOC was similar for miscanthus and switchgrass: −0.59 vs −0.61 t C ha⁻¹ yr⁻¹ at site 1 and −0.49 vs −0.41 t C ha⁻¹ yr⁻¹ at site 2, respectively. At site 1, there was no significant difference between treatments in L1–3. However, the old SOC decrease rate in layer L1 was significantly lower in early than in late harvest (−0.03 vs −0.15 t C ha⁻¹ yr⁻¹ on average) and the same trend was observed for layer L1–3 (−0.48 ± 0.09 vs −0.71 ± 0.08 t C ha⁻¹ yr⁻¹). No significant effect of the crop management appeared at site 2, although the decrease in layer L1–3 was almost two times lower in early than in late harvest (−0.24 vs −0.42 t C ha⁻¹ yr⁻¹ on average). The calculated old SOC stock even slightly increased in layer L1 in the early harvest treatments (+0.08 t C ha⁻¹ yr⁻¹ on average). This paradoxical result could be due to the C inputs of C3 weeds which were much more abundant in early harvest. There was also a tendency towards a higher old SOC decrease in irrigated compared to rainfed treatments (−0.70 vs −0.47 t C ha⁻¹ yr⁻¹ in layer L1–3). Contrary to the new SOC storage, most of the old SOC decrease calculated in layer L1–3 occurred in layers L2 and L3 for both sites, consistently with the soil mass of these layers.

### 3.8. Relationship between “new” and “old” soil organic carbon change rates

The rates of change of the old SOC stock can be compared to the rates of change of the new SOC stock in the old ploughed layer L1–3. We found at each site a strong and significant linear relationship between these variables (Fig. 4). This relationship suggests that the decay rate of the old SOC was stimulated by the rate of new SOC storage. The slope of the relationship was higher at site 1 than at site 2, indicating a greater old SOC depletion for an equivalent new SOC storage. The same kind of relationship was also observed in each individual soil layer, although it was not always significant (Table S7).

## 4. Discussion

### 4.1. Soil organic carbon stock changes under perennial bioenergy crops

The SOC stock in the old ploughed layer significantly increased in both sites after 12–13 years under perennial bioenergy crops, confirming our first initial hypothesis. Although SOC stock changes after conversion of arable land to miscanthus or switchgrass are generally positive (Knight et al., 2024; Qin et al., 2016), individual studies often failed to detect significant increases (e.g. Chimento et al., 2016; Dou et al., 2013; Robertson et al., 2017). Indeed, SOC stock change rates reported in the literature show a large variability for both species (Qin et al., 2016). In our study, we found similar stock change rates for miscanthus and switchgrass at each site. This result is consistent with the few studies available in the literature comparing SOC stocks under miscanthus and switchgrass at the same site (Chimento et al., 2016; Dheri et al., 2022; Gauder et al., 2016; Martani et al., 2021). Indeed, these studies could not find a significant effect of the crop species. Comparisons between miscanthus and switchgrass based on published data (e.g. Qin et al., 2016) should therefore be taken with caution, because the two crops were not present simultaneously in many sites.

In our study, we found that the SOC change rates strongly differed between the two sites, in line with our second hypothesis. The mean storage rate was almost four times higher in site 2 than in site 1 (0.96 vs 0.26 t C ha⁻¹ yr⁻¹). The difference was even higher when the SOC change rate was expressed relative to the initial SOC stock: +5 ‰ yr⁻¹ in site 1 vs +25 ‰ yr⁻¹ in site 2. Our results in the northern site (site 1) fall in the lower range of the data reported by Qin et al. (2016), while those obtained in the southern site (site 2) are closer to the median of these data. In the case of miscanthus harvested late, our results are slightly lower than the default SOC storage rate proposed by Poeplau and Don (2014) (0.40 ± 0.20 t C ha⁻¹ yr⁻¹ in 0–30 cm) for site 1 (0.26 t C ha⁻¹ yr⁻¹ over ~0–34 cm), but higher for site 2 (1.09 t C ha⁻¹ yr⁻¹ over ~0–30 cm). This strong site effect, which could be due to differences in soil and climate conditions and in crop management practices (cutting height at harvest) is discussed further in Sections 4.3 and 4.4.

Previous results obtained at site 1 could not detect a significant change in SOC stocks under perennial bioenergy crops after 5 years in the old ploughed layer or deeper (Ferchaud et al., 2016). Assuming a constant storage rate, the change in SOC stock after 5 years (~1.3 t C ha⁻¹) could have been too small to be detected. Furthermore, the storage rate might have been smaller during the first years, due to lower carbon
inputs to the soil than after full crop establishment. Published data showed that the effects on the SOC stock were often greater in longer duration trials (>10 years vs 0–5 years) with miscanthus and switchgrass (Qin et al., 2016). Significant SOC storage has generally been reported for plantations older than 8 years (Cattaneo et al., 2014; Dufossé et al., 2014; Felten and Emmerling, 2012; Follett et al., 2012; Hansen et al., 2004). When calculating SOC change rates with the paired plot approach, short-term studies (0–5 years) often show a greater variability than long-term studies. For example, based on published data, Zang et al. (2018) reported unrealistic SOC change rates ranging from –4 to +7 t C ha⁻¹ yr⁻¹ during the first 5 years after planting miscanthus, while the range was much narrower after 10 years. This could be due to a higher relative impact of confusing factors, particularly spatial variability, in short duration studies.

In our study, most of the SOC storage occurred in the upper soil layer (~0–5 cm) at both sites while SOC concentrations sometimes decreased below 20 cm. We did not detect any significant change in SOC concentrations below the old ploughing depth (~34 cm in site 1 and ~30 cm in site 2). This is consistent with the published data collected by Qin et al. (2016) for miscanthus suggesting very low SOC sequestration rates in the 30–100 cm soil layer. The strong SOC stratification with depth that we observed under perennial bioenergy crops is consistent with the absence of soil perturbation. Such a stratification is observed in no-till cropping systems (e.g. Dimassi et al., 2014) in which all aboveground carbon inputs are concentrated on the soil surface. However, below-ground carbon inputs (due to rhizome and root turnover and rhizodeposition) were also significant under perennial bioenergy crops as shown by the significant increases in δ¹³C values found below 20 cm and consistently with the amounts of carbon measured in the living below-ground biomass.

## 4.2. Effects of cropping practices

We could not detect any significant effect of the crop management practices on the SOC stock changes in the old ploughed layer. However, early harvest systematically reduced SOC storage compared to late harvest, on average by 33 % for miscanthus and 12 % for switchgrass. Carbon inputs from the perennial bioenergy crops, at least for miscanthus, were higher in late than in early harvest, resulting in an extra accumulation of new SOC in the late harvest treatment. In the case of miscanthus, leaf fall during winter was probably the main source of additional carbon inputs to the soil in late harvest compared to early harvest: it was estimated at 1.4 t C ha⁻¹ yr⁻¹ at site 1 (Amougou et al., 2012). At this site, the amount of mulch derived from fallen leaves (included in the aboveground dead residues) was on average 2.7 t C ha⁻¹ in 2018 for miscanthus harvested late, in accordance with previous measurements (Amougou et al., 2012; Ferchaud et al., 2016). To our knowledge, there was no other published study dealing with the effect of harvest date on SOC storage under miscanthus. For switchgrass, Follett et al. (2012) compared SOC change rate in early and late harvest after 9 years and did not detect a significant difference between harvest dates. Two other authors compared a single and a double annual harvest of switchgrass (Garten and Wullschleger, 1999; Valdez et al., 2017). They did not find an effect of the harvest type on SOC stocks after 5 and 4 years, respectively. The effect of harvest date on SOC dynamics is probably smaller for switchgrass than for miscanthus because switchgrass leaves do not fall during winter, contrary to miscanthus.

In our study, irrigation was tested in site 2 during seven years and appeared to increase SOC storage, particularly for switchgrass. The net SOC increase under switchgrass was 0.47 t C ha⁻¹ yr⁻¹ and was composed of +0.68 t C ha⁻¹ yr⁻¹ of new SOC and -0.21 t C ha⁻¹ yr⁻¹ of old SOC. Our results are consistent with those of Chatterjee et al. (2018) who studied the effect of irrigation on SOC storage under switchgrass in a five-year diachronic study in Oregon. They found that irrigation (140 mm yr⁻¹) increased total SOC by 0.40 t C ha⁻¹ yr⁻¹ over 0–30 cm compared to rainfed conditions, with a net accumulation of new SOC

increased by 0.67 t C ha⁻¹ yr⁻¹ and a stimulation of the decay rate of old SOC of 0.27 t C ha⁻¹ yr⁻¹.

Nitrogen fertilization, which was tested in site 1, did not exert a significant effect on crop residue carbon, belowground biomass carbon and SOC stock change rates (total, new, old) in the old ploughed layer, although it increased crop production (Cadoux et al., 2014). Cattaneo et al. (2014) and Gauder et al. (2016) also reported a non-significant effect of nitrogen fertilization on SOC stock or SOC change rate under miscanthus after respectively 9 and 11 years. Contrasted results were reported for switchgrass: either no effect of nitrogen fertilization on SOC stocks (Gauder et al., 2016; Lai et al., 2018), positive (Follett et al., 2012; Jung and Lal, 2011) or even negative (Valdez et al., 2017) effects. Biological nitrogen fixation by diazotrophic bacteria associated to perennial bioenergy crops could play a role in their nitrogen budget, particularly in the absence of nitrogen fertilization (Wewalwela et al., 2020), and should be further studied. Since mineral nitrogen fertilizers emit greenhouse gases during their production and stimulate nitrous oxide emissions in the field (Ferchaud et al., 2020), mineral nitrogen fertilization of perennial bioenergy crops should be kept to a minimum.

## 4.3. Changes in “new” soil organic carbon stocks

In our study, new SOC accumulation rates did not differ between species (miscanthus and switchgrass) but differed between sites. They were higher for site 2 (1.41 ± 0.42 t C ha⁻¹ yr⁻¹ in layer L1–3) than for site 1 (0.86 ± 0.18 t C ha⁻¹ yr⁻¹). These values are consistent with the results of previous studies also using natural ¹³C tracing. Zang et al. (2018) analysed 12 published studies (41 observations) and calculated a mean C4 soil carbon sequestration rate of 1.0 ± 0.6 t C ha⁻¹ yr⁻¹ in the topsoil (0–20 or 0–30 cm) for miscanthus established on cropland. This is slightly higher than the mean value previously obtained for cropland transition to miscanthus (19 paired sites) by Poeplau and Don (2014): 0.85 ± 0.68 t C ha⁻¹ yr⁻¹ in 0–30 cm. There are fewer available data for switchgrass in the literature but the published values are in the same range: from 0.5 to 1.3 t C ha⁻¹ yr⁻¹ in 0–30 cm (Chatterjee et al., 2018; Follett et al., 2012).

The greater storage rate of new SOC in site 2 results very likely from higher carbon inputs derived from the C4 perennial bioenergy crops, as suggested by the higher amount of carbon found in crop residues and in belowground biomass in site 2 compared to site 1. Indeed, we found a correlation (R² = 0.57, p < 0.001) between the new SOC storage rate in layer L1 (~0–5 cm) and the total amount of crop residues present in the soil at the end of the experiment (Figure S3). The cutting height at harvest was estimated to be twice higher in site 2 than in site 1, leading to higher amount of aboveground carbon inputs. The higher storage rate in site 2 could also be linked to temperature, in accordance with the positive correlation between C4 soil carbon accumulation rate and mean annual temperature found by Poeplau and Don (2014) for miscanthus. During our study, the mean annual temperature was 10.7 °C in site 1 and 13.7 °C in site 2. Root and rhizome turnover could be accelerated by higher temperatures, increasing belowground carbon inputs. In an Italian site with similar climatic conditions to our southern site, Cattaneo et al. (2014) found a C4 soil carbon accumulation rate of 1.7 t C ha⁻¹ yr⁻¹ in 0–30 cm after 9 years of miscanthus, close to our values for miscanthus in site 2 (1.41 ± 0.27 t C ha⁻¹ yr⁻¹ for Miscanthus Late and 1.70 ± 0.38 t C ha⁻¹ yr⁻¹ for Miscanthus Rainfed). The sites compiled by Poeplau and Don (2014), with a mean C4 soil carbon accumulation rate of 0.85 ± 0.68 t C ha⁻¹ yr⁻¹, had climate conditions closer to our northern site for which the new SOC accumulation rate was 0.94 t C ha⁻¹ yr⁻¹ for Miscanthus Late.

Regarding the distribution of new SOC in the soil profile, the fact that most of the new SOC accumulation occurred in the ~0–20 cm soil layer in both sites is consistent with other studies (Cattaneo et al., 2014; Felten and Emmerling, 2012; Poeplau et al., 2019; Poeplau and Don, 2014; Richter et al., 2015; Schneckenberger and Kuzyakov, 2007) showing that new SOC was concentrated above 30 cm. However, a few other
studies found large proportions of total new SOC in deeper soil layers (Follett et al., 2012; Gregory et al., 2018).

## 4.4. Changes in “old” soil organic carbon stocks

The decay rate of the old SOC was on average higher at site 1 than at site 2 (−0.60 vs −0.45 t C ha⁻¹ yr⁻¹) and similar between miscanthus and switchgrass. Reported values in the literature (obtained with the same methodology) are extremely variable: Poeplau and Don (2014) calculated from 19 paired sites a mean positive rate of 0.83 ± 3.24 t C ha⁻¹ yr⁻¹ in 0–30 cm for miscanthus. In the absence of C3 carbon inputs such as exogenous organic matter inputs or C3 weeds, this value should be negative because of SOC mineralization. Using RothC to simulate the old SOC mineralization, Poeplau and Don (2014) obtained for the same sites a mean change of −0.60 ± 0.43 t C ha⁻¹ yr⁻¹, consistent with our results. This illustrates the limits of the paired plot approach to quantify precisely SOC change rates under bioenergy crops.

The initial SOC stock was lower in site 2 than in site 1 and therefore the old SOC decrease relative to the initial SOC stock was similar between the two sites (~1.2 % yr⁻¹). The higher mean temperature in site 2 should have stimulated SOC mineralization but the higher clay content (27 % in site 2 vs 18 % in site 1) and the higher water deficit (−257 vs −64 mm yr⁻¹) could have limited SOC mineralization in site 2 (Clivot et al., 2017) and compensated the temperature effect.

Another striking point in our study was the linear relationship observed between the new and old SOC change rates. This relationship could be partly explained by some indirect effects of the cropping practices. In site 2, it is likely that irrigation increased soil moisture during summer and therefore favoured SOC mineralization. In both sites, the topsoil (~0–5 cm) moisture under miscanthus was probably higher in late than early harvest because the leaf mulch limited soil evaporation (Peyrard et al., 2017), which could also have favoured SOC mineralization. Weeds could also have been an additional source of (C3) carbon inputs in early harvest (they were much more abundant in early than in late harvest), leading to a lower apparent old SOC decrease, particularly in the upper soil layer. However, this relationship was observed at both sites in all soil layers and across all treatments. It therefore strongly suggests that the intensity of the carbon inputs favours SOC accumulation but also stimulates SOC mineralization, due to the priming effect. This mechanism (i.e. the stimulation of the soil organic matter mineralization with the addition of fresh organic matter) has been shown to be a systematic process in all soils (Perveen et al., 2019) and to increase with the carbon input rate (e.g. Liu et al., 2020). Mary et al. (2020) found that the relationship between SOC mineralization and carbon inputs can be considered to be about linear for plant carbon inputs varying between 0 and 5 g C kg⁻¹ soil.

## 5. Conclusion

This study demonstrated the interest of using long-term experiments with a thorough initial characterization, rather than a paired plot approach, and using the δ¹³C tracing technique which allowed to distinguish changes in C4 and C3 soil carbon stocks. It showed that perennial bioenergy crops established on cropland increased SOC stocks significantly after 12 or 13 years, the storage occurring almost exclusively in the topsoil (~0–20 cm). We found similar net SOC storage rates for miscanthus and switchgrass but a large effect of the site, with on average an almost four times greater SOC change rate in the southern than in the northern site. This site effect was mainly explained by higher carbon inputs in site 2, as shown by the greater accumulation of dead residues and living biomass in soil and by the higher storage rate of new SOC, and to a lesser extent by a lower decay rate of the old SOC stock. Two crop management practices had a smaller impact on the net SOC change rate: late vs early harvest and irrigated vs rainfed conditions resulted in a greater storage of new SOC, partly compensated by a larger decrease of old SOC. Mineral nitrogen fertilization did not affect any of

the SOC storage rates. These C3-C4 soil carbon data are valuable for model calibration and/or evaluation (e.g. Clivot et al., 2019) in order to predict SOC evolution under bioenergy crops on the longer term and/or in other soil and climate conditions. Further studies, such as Autret et al. (2019), are required to evaluate the complete greenhouse gas balance of perennial bioenergy crop production, integrating SOC storage, nitrous oxide emissions and indirect greenhouse gas emissions. Finally, the effect of reverting perennial bioenergy crops back to arable crops on SOC evolution has to be studied and integrated into the greenhouse gas balance of bioenergy crops.

## Funding sources

This work was supported by ADEME - French Agency for Ecological Transition - in the framework of the GRAINE program (CE-CARB project, grant number 17-03-C0005, 2017).

## CRediT authorship contribution statement

**Mary Bruno:** Writing - review & editing, Visualization, Formal analysis.
**Ferchaud Fabien:** Writing - original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.
**Marsac Sylvain:** Writing - review & editing, Investigation.

## Declaration of Competing Interest

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

## Acknowledgements

We want to thank the staff involved in the maintenance of the long term experiments and particularly Emilie Mignot (INRAE GCIE unit, site 1) and Florianne Couloumies (Arvalis, site 2). We are grateful to Nicolas Collanges, Caroline Dominiarczyk, Simon Drochon, Eric Gréhan, Frédéric Mahu, Emilie Mignot, Anita Teixeira, Eric Venet and Laurent Vieville, who participated to the sampling campaigns and/or to the sample preparation. We also thank Olivier Delfosse (INRAE, FARE unit) for the soil analyses.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.agee.2025.109637.

## Data availability

Raw data are available at doi.org/10.57745/SEVWMQ for both sites (SOC concentration and δ¹³C value, soil bulk density, reference soil mass).

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