# Long-term steady state 13C labelling to investigate soil carbon turnover in grasslands

K. Klumpp, J. F. Soussana, R. Falcimagne

► To cite this version:

K. Klumpp, J. F. Soussana, R. Falcimagne. Long-term steady state 13C labelling to investigate soil carbon turnover in grasslands. Biogeosciences, 2007, 4 (3), pp.385-394. hal-00330321

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# Long-term steady state ¹³C labelling to investigate soil carbon turnover in grasslands

K. Klumpp, J. F. Soussana, and R. Falcimagne

INRA, UR874 Grassland Ecosystem Research, Clermont-Ferrand, France

Received: 2 February 2007 – Published in Biogeosciences Discuss.: 7 March 2007

Revised: 7 June 2007 – Accepted: 11 June 2007 – Published: 26 June 2007

**Abstract.** We have set up a facility allowing steady state ¹³CO₂ labeling of short stature vegetation (12 m²) for several years. ¹³C labelling is obtained by scrubbing the CO₂ from outdoors air with a self-regenerating molecular sieve and by replacing it with ¹³C depleted (–34.7±0.03‰) fossil-fuel derived CO₂. The facility, which comprises 16 replicate mesocosms, allows to trace the fate of photosynthetic carbon in plant-soil systems in natural light and at outdoors temperature. This method was applied to the study of soil organic carbon turnover in temperate grasslands. We tested the hypothesis that a low disturbance by grazing and cutting of the grassland increases the mean residence time of carbon in coarse (>0.2 mm) soil organic fractions.

Grassland monoliths (0.5×0.5×0.4 m) were sampled from high and low disturbance treatments in a long-term (14 yrs) grazing experiment and were placed during two years in the mesocosms. During daytime, the canopy enclosure in each mesocosm was supplied in an open flow with air at mean CO₂ concentration of 425 µmol mol⁻¹ and δ¹³C of –21.5±0.27‰. Fully labelled mature grass leaves reached a δ¹³C of –40.8 (±0.93) and –42.2‰ (±0.60) in the low and high disturbance treatments, respectively, indicating a mean ¹³C labelling intensity of 12.7‰ compared to unlabelled control grass leaves. After two years, the delta ¹³C value of total soil organic matter above 0.2 mm was reduced in average by 7.8‰ in the labelled monoliths compared to controls. The isotope mass balance technique was used to calculate for the top (0–10 cm) soil the fraction of ¹³C labelled carbon in the soil organic matter above 0.2 mm (i.e. roots, rhizomes and particulate organic matter). A first order exponential decay model fitted to the unlabelled C in this fraction shows an increase in mean residence time from 22 to 31 months at low compared to high disturbance. A slower decay of roots, rhizomes and particulate organic matter above 0.2 mm

is therefore likely to contribute to the observed increased in soil carbon sequestration in grassland monoliths exposed to low disturbance.

## 1 Introduction

About two-thirds of terrestrial C is found belowground and the amount of organic carbon that is stored in the soil (1.5×10¹⁸g C) is globally about twice that of the total C in atmosphere (Schlesinger, 1997). Below-ground C generally has slower turnover rates than above-ground carbon, as most of the organic carbon in soils (humic substances) is produced by the transformation of plant litter into more persistent organic compounds (Jones and Donelly, 2004). Transformation of plant litter is a key process of the carbon and nutrient cycles, which drives C mineralization and C accumulation in the soil organic matter (Tateno and Chapin, 1997). Detrital carbon accumulation accounts for most of an ecosystem's capacity to store organic carbon belowground within a few years and is largely conditioned by the plant turnover rate (Cebrian and Duarte, 1995). Root litter transformation is an important determinant of the carbon cycle in grassland ecosystems, which is affected both by the root litter quality and by the rhizosphere activity (Personeni and Loiseau, 2004, 2005).

Carbon sequestration can be determined directly by measuring changes in C pools (Conant et al., 2001) and, or, by simulation (e.g. Smith et al., 2005). However, to gain further understanding on C turnover in different soil fractions, the use of radiocarbon tracers and of stable isotopes has proven to be essential (e.g. Trumbore, 2000; Verburg et al., 2004). Isotope techniques, often in combination with other methods (e.g. gas exchange) stand out among the few tools available to track C fluxes in terrestrial ecosystems. Carbon isotopes have been used as tracers including radioactive short lived ¹¹C (half time 20.5 min) and long lived ¹⁴C (5760 yr)

<footnote>Correspondence to: K. Klumpp
(kklump@clermont.inra.fr)</footnote>
(Stevenson, 1986), as well as stable ¹³C (Balesdent et al., 1988). The natural atmospheric ¹⁴C activity can be used to date the accumulation of C in fractions with slow turnover time (>50 year). Artificial atmospheric ¹⁴C activity, such as the “bomb ¹⁴C” studies, can date shorter lived (>10 years) C pools from undisturbed soils (e.g. Trumbore, 2000).

Under the Kyoto Protocol (available at http://www.unfccc.de), biospheric sinks and sources of C can be taken into account in attempts to meet “Quantified Emission Limitation or Reduction Commitments” (QELRCs) for the first commitment period (2008–2012). To better understand how land use and management factors affect carbon turnover in the short-term (<5 yrs), the fate of photosynthesates in plant-soil systems should be traced at this time scale.

The development of the ¹³C isotope mass balance technique has allowed to calculate the amount of “new” carbon in soils after shifting cultivation from C₃ to C₄ plant species (or conversely from C₄ to C₃) (Balesdent et al., 1988; Conen et al., 2006; Derner et al., 2006). However, this method cannot be applied to temperate and high latitude/altitude ecosystems which lack C₄ species.

Recently, novel approaches were developed by using CO₂ enrichment experiments (FACE) to obtain a ¹³C depleted signature in plant-soil systems (e.g. Loiseau and Soussana, 1999; Personeni et al., 2004; Trueman and Gonzales-Meler, 2005; Dijkstra et al., 2004). However, this method cannot be used to investigate C cycling under ambient CO₂.

¹³C pulse-labeling experiments in the field allow detailed studies of the temporal relationships between carbon fixation and its delivery to a defined sink (e.g. exudates, soil biota) (Ostle et al., 2000). Nevertheless, ¹³C pulse labeling studies cannot be used to understand multi-year processes as the pulse is diluted over time.

To investigate the fate of carbon in plant-soil systems, we have further developed a steady-state ¹³CO₂ labeling technique, which was first used with seedlings grown hydroponically or on sand in phytotrons (Deléens et al., 1983; Schnyder et al., 1992, 2003).

This method has been applied to the mesocosm experiment previously described by Klumpp et al. (2007), showing reduced soil carbon sequestration at high compared to low disturbance by grazing and cutting of the grassland increases the mean residence time of carbon in coarse (>0.2 mm) soil organic fractions.

The aim of the present study was: i) to change during several months the ¹³C isotope signature of photosynthesates from plant-soil systems grown in natural light and at outdoors temperature using a steady state ¹³CO₂ labelling facility; ii) to apply this method to study the role of disturbance for soil carbon turnover in grassland ecosystems.

## 2 Materials and methods

### 2.1 Set-up of controlled system for long-term ¹³CO₂/¹²CO₂ labelling

The facility is shown schematically in Fig. 1 and comprises four components: labeling system, air dilution, 16 mesocosms and a gas exchange system (previously described by Klumpp et al., 2007). More precisely, ambient air is compressed, CO₂ and H₂O of ambient air is scrubbed by passing through a molecular sieve and then replaced by fossil-fuel derived CO₂ being depleted in ¹³C by –21‰ compared to outdoor air (δ¹³C –34.7±0.03‰). Thereafter, the ¹³C depleted air is humidified, mixed with outdoor air, temperature regulated and distributed to canopy enclosures. The main parts of the labeling system are: a screw compressor, a self-regenerating adsorption dryer (Schnyder et al., 1992) generating CO₂ free dry air at a rate of up to 5000 standard liters per minute (SLPM) and a residual CO₂ below 1 µmol CO₂ mol⁻¹, an air tank, gas cylinders containing fossil fuel derived CO₂ and a humidifier (1 m³; 1 m² cross corrugated cellulose pads). The flow rates of the labeling system are controlled by pressure regulators and the CO₂ injection rate by a mass flow meter (Tylan, 0–3 SLPM). During daytime, to regenerate the molecular sieve of the adsorption dryer, approximately 30% of the CO₂ free dry air is diverted to the adsorber chamber, where drying and regeneration alternated in 6 min cycles. At night time (one hour after sunset until one hour before sunrise), the supply of CO₂ free dry air was stopped which allowed for a full regeneration of the adsorption dryer.

The labeling system was coupled to an air dilution system. This allowed to vary the degree of ¹³C labeling by mixing ¹³C depleted air with outdoor air in variable proportions and to apply the labeling system to continental climate with hot summers (e.g. 38°C during summer 2003). For air dilution, ambient air was sampled at a height of 3 m by a centrifuge fan at flow rates up to 400 L min⁻¹. Then ambient air passed through a cooler (378 kWh) being automatically regulated by the temperature difference between enclosure outlets and ambient air. Finally, ambient air and ¹³C-depleted air were mixed in a 1 m³ metal box (Fig. 1). At the outlet of the mixing box, thermally insulated PVC pipes delivered the air to the 16 canopy enclosures.

#### 2.1.1 Gas exchange measurement system

The gas exchange system was previously described and discussed by Klumpp et al. (2007). Briefly, each open-flow canopy enclosure (Fig. 1) had an average flow rate between 500 and 650 L min⁻¹ during day, which gave with the internal volume of 742 L per enclosure an air exchange rate of 40–50 times per hour and a mean air residence time of 1 min 30 s. During night (one hour after sunset until one hour before sunrise), the flow rate was automatically reduced to
<figure><img src="image_3.png" /></figure>

<table><caption>**Fig. 1.** Long term steady state ¹³C labelling facility to investigate carbon turnover in plant-soil system.</caption><thead><tr><th></th><th></th><th>Abbreviation</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">Labelling system</td><td>SC</td><td>Screw compressor</td><td>Air Worthington-Creyssenac, 50AX6, Lyon, France</td></tr><tr><td>AD</td><td>Adsorption dryer</td><td>KEN300, Zander Essen, Germany, molecular sieve with zeolith, activated aluminum oxide</td></tr><tr><td>AR</td><td>Air reservoir (1 m³)</td><td>Zander, Essen, Germany</td></tr><tr><td>F1</td><td>Oil and water condensate drain</td><td>Zander, Essen, Germany</td></tr><tr><td>F2</td><td>Oil-, water- and particle filter</td><td></td></tr><tr><td>PR</td><td>Pressure regulator</td><td>Tylan General, Eching Germany</td></tr><tr><td>MFC</td><td>Mass flow controller 0-5 l min<sup>-1</sup></td><td>Messer, Lyon, France</td></tr><tr><td>CO<sub>2</sub></td><td>Cylinder containing CO<sub>2</sub> of fossil origin</td><td></td></tr><tr><td>MVB</td><td>Multi valve block</td><td></td></tr><tr><td rowspan="4">Air dilution system</td><td>HF</td><td>Humidifier</td><td>CMF, Varades, France</td></tr><tr><td>MB</td><td>Mixing vessel (1 m³)</td><td></td></tr><tr><td>C</td><td>Temperature regulated cooler</td><td>AuverFroid, Clermont-Ferrand, France</td></tr><tr><td>V</td><td>Air flow controllable centrifuge fan</td><td></td></tr><tr><td rowspan="10">Enclosure</td><td>I<sub>A</sub></td><td>Inlet aboveground</td><td></td></tr><tr><td>O<sub>A</sub></td><td>Outlet aboveground</td><td></td></tr><tr><td>T<sub>A</sub></td><td>Temperature sensor aboveground</td><td></td></tr><tr><td>O<sub>s</sub></td><td>Outlet belowground</td><td></td></tr><tr><td>P<sub>s</sub></td><td>Pump belowground</td><td></td></tr><tr><td>WT</td><td>Water trap</td><td></td></tr><tr><td>C<sub>D</sub></td><td>Canister for drainage water</td><td></td></tr><tr><td>IRGA</td><td>Infrared gas analyzers</td><td>LI6262, LICOR, Nebraska, USA; Maihak, Unor 100, Germany</td></tr><tr><td></td><td></td><td></td></tr><tr><td>PC</td><td>Computer</td><td></td></tr></tbody></table>

150–250 L min⁻¹ to regenerate the molecular sieves of adsorption dryer. The air flow in each enclosure was monitored continuously by an Averaging Pitot tube connected to a differential pressure gauge (Annubar Rosemount, Dietrich Standard Inc., North, USA).

From start of growing season, in April 2003, the CO₂ concentration in enclosures was held from sunrise to sunset, at 425±39 µmol mol⁻¹ (mean ± s.d. of 30 min measurements, data not shown) by injection of ¹³C depleted CO₂ (−34.7±0.03‰). The mean CO₂ concentration inside the enclosures was close to outdoors CO₂ concentration (mean difference of 13.2±9.5 µmol mol⁻¹). During night time, the enclosures were provided with outdoors air.

Following Casella and Soussana (1997) and Schapendonk et al. (1997), a fraction of the air flowing through the canopy enclosure of a mesocosms was pumped (KNF, Neuberger,

Germany) at a constant flow rate (3.5±0.2 L min⁻¹) through the soil column of each monolith. A small pressure head was maintained by the open-flow system. This pressure head and the depression caused by the pump eliminated back-diffusion of CO₂ from the soil (e.g. Casella and Soussana, 1997). The flow rate though the soil column was adjusted according to preliminary trials, showing, that a flow rate of 3.5 L min⁻¹ suppresses diffusion of soil CO₂ in the above ground compartment without changing significantly soil respiration rates. The CO₂ concentration between outlet and inlet of each enclosure was measured in differential mode every 20 min for a period of 1 min with an IRGA (LI6262, LICOR Nebraska, USA). Both the ambient air and the inlet air CO₂ concentrations were measured in absolute mode with a second IRGA (Maihak, UNOR100, Germany). The two IRGAs were calibrated every two weeks with a 480 µL CO₂
$L^{-1}$ standard (Messer-Griesheim, Germany). Soil respiation (Klumpp et al., 2007), soil temperature and air humidity of each enclosure as well as external PAR, temperature and humidity were monitored every 30 and 40 min, respectively.

## 2.1.2 Mesocosms

A mesocosm consists of an aboveground canopy enclosure of $0.74 \text{ m}^3$ (L $1.8 \times W \ 0.55 \times H \ 0.75 \text{ m}$) and a belowground compartment of $0.30 \text{ m}^3$ (L $1.5 \times W \ 0.5 \times H \ 0.4 \text{ m}$). The canopy enclosure consists of a thin metal frame covered with a transparent polyethylene film ($60 \ \mu\text{m}$, transmitting 90% of the incident PAR). One side in polycarbonate was equipped with two plastic tubes of 20 cm length and 8 cm inner diameter, used as air inlet and outlet (Fig. 1). Air mixing inside enclosures was provided by a fan. A temperature sensor and a gas sample tube connected to an IRGA through a multi valve block allowed to monitor air temperature and $CO_2$ concentration at the outlet.

The belowground compartment consisted of three metal containers, each with a volume of $0.14 \text{ m}^3$ (L $0.6 \times W \ 0.6 \times H \ 0.4 \text{ m}$) and a hole at the bottom for drainage water (Fig. 1). Each of these containers contained a grassland monolith (L $0.5 \times W \ 0.5 \times H \ 0.4 \text{ m}$) framed in a 5 side stainless steel box with drain holes at the bottom. Gaps between monolith and box were filled with a thermally insulating material (vermiculite) and the surface was airtight sealed. The drainage holes of the three boxes were connected to a 20 L canister (Fig. 1). A pump connected to the canister pumped air continuously through the soil column (see gas exchange measurements).

## 2.2 Experiment with grassland monoliths and management

The grassland (soil, plant community structure) type and procedures to select and extract grassland monoliths were described by Klumpp et al. (2007). Briefly, in June 2002, 56 monoliths (L $0.5 \times W \ 0.5 \times H \ 0.4 \text{ m}$) were extracted from two semi-natural grassland plots, being subjected to two contrasted sheep grazing treatments during the last 14 years (Louault et al., 2005). Plot 1 was cut once and sheep grazed 4 times $yr^{-1}$ and, hence, represented a high disturbance regime (H). Plot 2 was sheep grazed only once a year (low disturbance regime, L). 24 monoliths of each treatment were placed in the 16 mesocosms (3 monoliths per enclosure). The enclosures were placed in natural light and at outdoors temperature and air humidity was adjusted to field conditions. 4 monoliths of each field treatment were kept outdoors as unlabelled controls. Depending on season, monoliths were watered once to three times a week to target a soil volumetric water content of 33% corresponding to a soil water potential of ca. -30 kPa. All enclosures received the same irrigation volumes.

On five occasions per year, a high current disturbance was applied (24 monoliths in enclosures and 4 outdoor controls)

by cutting simultaneously at 5 cm stubble height and applying artificial urine ($5 \text{ g N m}^{-2}$) consisting of 80% urea, 10% hippuric acid, 5% allantoin N and 5% creatine N (Doak, 1952) in order to simulate N returns at grazing (see Klumpp et al., 2007). The harvested phytomass was separated into live and dead plant parts, oven dried and analysed for $\delta^{13}\text{C}$. For the low current disturbance monoliths were neither cut nor fertilised. At the start of the experiment, half of the enclosures of the low and high field disturbance were switched to the opposite disturbance treatment (Klumpp et al., 2007).

## 2.3 Monitoring of ¹³C labeling

### 2.3.1 Supplied CO₂

To determine $\delta^{13}\text{C}$ of $CO_2$ in canopy enclosures, air was sampled every 14 days at enclosure outlets during daytime. Air samples were collected in 10 ml air-tight vials (BD Vacutainer, UK) through a y-branch-connection (equipped with 2 needles) which was inserted in the continuous air flow going to the IRGA. Vials were flushed for 2 min and analyzed monthly for $\delta^{13}\text{C}$ (see ¹³C isotope analyses).

### 2.3.2 Plant material

To monitor the ¹³C-signature of the aboveground vegetation, 20 last mature green grass laminae (tip to ligula) were harvested monthly in each enclosure and in outdoor controls by clipping. For each enclosure (and outdoor control) harvested leaves were oven dried and analyzed for their $\delta^{13}\text{C}$ signature.

## 2.4 Soil organic matter fractions

Soil harvest and analyses were described by Klumpp et al. (2007). Briefly, soils were sampled once before start of ¹³C labeling and then 5 times during the experiment (in June and September 2003, April and September 2004 and April 2005). At each soil harvest a vertical soil slice ($40 \times 6 \times 10 \text{ cm}$) was split into horizontal layers (0–10, 10–20 and 20–30 cm depth) in each mesocosm. The soil layers were air dried and the free organic matter fractions (OM) were separated with water by passing through a series of three brass sieves with successive mesh sizes (1.0, 0.2 and 0.05 mm) (wet sieving). The remaining material in each sieve was separated into the organic and mineral fraction by density flotation in water (Loiseau and Soussana, 1999; Six et al., 2001). Organic fractions were oven dried and analyzed for $\delta^{13}\text{C}$.

The $\delta^{13}\text{C}$ of the total soil organic matter (SOM) above 0.2 mm, containing roots, rhizomes, coarse (>1 mm) and fine (1 mm> × >0.2 mm) particulate organic matter (POM) and aggregated organic matter (AOM, 0.2 mm> × >0.05 mm) was calculated by weighing the $\delta^{13}\text{C}$ signature of each organic fraction by its dry weight.
## 2.5 ¹³C-determination and calculation

### 2.5.1 ¹³C-isotope analyses

Soil and vegetation samples were oven dried for 48 h at 60°C, grounded to homogenous powder and analyzed for C-content and δ¹³C by elemental analyzer (EA1110, Carlo Erba, Milano, Italy) coupled (Conflo III) with a mass spectrometer (Delta plus; FinniganMAT, Bremen, Germany). The δ¹³C in CO₂ from outdoors and labelling atmosphere were measured on a gas chromatography isotope ratio mass spectrometer (Gas system, Fisons, Loughborough, UK).

### 2.5.2 ¹³C methods and terminology

Isotope ratios are reported as δ¹³C values relative to V-PDB standard (‰) and expressed as:

$$ \delta^{13}C = \left[ \frac{(R_{\text{sample}} - R_{\text{standard}})}{R_{\text{standard}}} \right] \times 10^3 \quad (1) $$

where R is ¹³C/ ¹²C ratio of the standard and sample. The discrimination (Δ) between product (δ¹³C sample) and source was:

$$ \Delta = \left[ \frac{(\delta^{13}C_{\text{source}} - \delta^{13}C_{\text{sample}})}{(1000 + \delta^{13}C_{\text{sample}})} \right] \times 10^3 \quad (2) $$

Gas samples and solid samples (soil and plant material) were measured against working laboratory standard gases, previously calibrated against IAEA secondary standards. The fraction of “new” C derived from ¹³C-labelling (fCnew) in soil organic fractions was calculated by a mass balance equation:

$$ fC_{\text{new}} = \frac{(\delta^{13}C_{\text{sample}} - \delta^{13}C_{\text{control}})}{(\delta^{13}C_{\text{input}} - \delta^{13}C_{\text{control}})} \quad (3) $$

Where δ¹³C sample is the δ¹³C of the sample, δ¹³C control is the δ¹³C value before start of labelling and δ¹³C input is the δ¹³C value of a fully labelled plant derived material entering the soil (see Results).

### 2.5.3 Follow up of ¹³C labeling

Additional to the monthly air sampling, we estimated (in 30 min time steps) the ¹³C signature of CO₂ provided to enclosures inlet (δ¹³C estimated) with a mass balance equation:

$$ \delta^{13}C_{\text{estimated}} = \frac{q\text{CO}_{2\text{air}} \cdot \delta^{13}\text{C}_{\text{air}} + q\text{CO}_{2\text{inject}} \cdot \delta^{13}\text{C}_{\text{inject}} + q\text{CO}_{2\text{decarb}} \cdot \delta^{13}\text{C}_{\text{decarb}}}{q\text{CO}_{2\text{air}} + q\text{CO}_{2\text{inject}} + q\text{CO}_{2\text{decarb}}} \quad (4) $$

where δ¹³C air, δ¹³C inject and δ¹³C decarb are the measured δ¹³C values of CO₂ from outdoors air, from CO₂ cylinders and from decarbonated air, respectively. qCO₂air, qCO₂inject and qCO₂decarb are the flow rates of CO₂ supplied by outdoors air, by CO₂ injection from cylinders and by decarbonated air, respectively. qCO₂inject was adjusted to 1.55 standard L min⁻¹ by a mass flow-meter. qCO₂air reached 2.01±0.14 standard L min⁻¹ and qCO₂decarb was equal to zero (except for a few days above 35°C in summer 2003).

## 2.6 Data analysis

Differences in ¹³C values for leaves and for gas samples were analyzed by repeated measure ANOVA. The amounts of “new” and of “old” C were fitted using non linear regression models with log transformed data. Since in these regression models there were no significant differences among the two current disturbance treatments (p>0.95), the data from the two current disturbance treatments were pooled. Hence, we compare the two field disturbance treatments (H and L), each with 8 replicate enclosures.

# 3 Results and discussion

## 3.1 System accuracy and δ¹³C signature

### 3.1.1 ¹³C labelling

After scrubbing CO₂ and H₂O from ambient air, the residual CO₂ concentration in CO₂-free air was below 1 ppm, except for some hours of high outdoor air temperature (>35°C) during summer 2003 heat wave where residual CO₂ concentration could reach 10 ppm (data not shown).

Inlet and outlet δ¹³C values were compared for enclosures with vegetation during the experiment (i.e. monthly air sampling at noon) and for enclosures without vegetation. In the latter case, two enclosures were placed on an even gas-tight surface under the same conditions (air flow, natural light and temperature). The comparison resulted in a mean absolute δ¹³C-difference between enclosure inlet and outlet of -0.35±0.39‰ (P>0.5, repeated measure ANOVA) and -0.19±0.47‰ (P>0.5) for enclosures with and without vegetation, respectively (data not shown). These non significant δ¹³C differences between enclosure inlet and outlet indicate that δ¹³C fractionation processes caused by photosynthesis were not measurable due to high air flows.

The CO₂ concentration delivered by the labelling system reached, on average, 425±5 µmol mol⁻¹ (daily mean, ±s.e.). However, due to mixing with outdoors air, the CO₂ concentration at enclosure inlet followed a seasonal cycle, characterized by a higher CO₂ concentration is higher in winter than in summer (e.g. Pataki et al., 2004) (Fig. 2a).

¹³C fractionation processes during photosynthesis modify instantly the δ¹³C of CO₂ (O'Leary 1981), thereby the CO₂ in the atmosphere surrounding the plants becomes ¹³C enriched relative to the CO₂ at the inlet. In a well-mixed gas exchange enclosure, the ¹³C signature of CO₂ sensed by plants during photosynthesis corresponds to the δ¹³C measured at the enclosure outlet (Evans et al., 1986; Schnyder et al., 2003). In our experiment, the δ¹³C measured at enclosure outlet indicated ¹³C depletion in winter and enrichment in summer time (Fig. 2b). This was explained by CO₂ mixing with outdoors air, which contributed on average to 57% of the CO₂ flux supplied to the enclosures. The CO₂ from outdoors air (mean δ¹³C -11.4) had a depleted ¹³C signature from
<figure><img src="image_4.png" /></figure>

**Fig. 2.** (A) Measured CO₂ concentration at the canopy enclosure inlet, (B) δ¹³C at canopy enclosure outlet, (C) estimated δ¹³C of CO₂ supplied to canopy enclosure inlet and (D) δ¹³C of labelled C₃ grass leaves during experimental period (April 2003 to March 2005) and of unlabelled C₃ control grasses grown outdoors. Data are means (±SE) of 16, 14 and 4 replicates for labelled C₃ and unlabelled C₃ controls, respectively. Dotted lines represent the mean δ¹³C values of supplied CO₂ (−21.5‰) (A, B) and of C₃ leaves of labelled (−41.4‰) and unlabelled control (−28.7‰) plants (D).

fossil fuel combustion and plant/soil respiration in winter time and an enriched ¹³C signature from plant photosynthesis in summer and spring time (Pataki et al., 2004). The plant supplied CO₂ had on average a δ¹³C offer of −21.5±0.27‰ during the two growth periods.

The calculated labelling intensity, δ¹³C<sub>estimated</sub> (Fig. 2c), and the measured δ¹³C at the enclosure outlet did not differ significantly during the time course of the experiment

(P>0.1; repeated measure ANOVA, data not shown, absolute δ¹³C difference of −0.47±0.5‰). Therefore, even during periods without direct δ¹³C analyses, the labelling intensity can be estimated by calculating δ¹³C<sub>estimated</sub>. The seasonal pattern of δ¹³C<sub>estimated</sub>, which was close to the monthly measured δ¹³C at enclosure outlet (Fig. 2b) and was most susceptible to changes in δ¹³C of outdoor air (δ¹³C<sub>air</sub>). In our experiment the δ¹³C of outdoors air was in average −11.4‰ due to urban activity and could not be controlled by the experimental set up.

In our experiment, a small pressure head was maintained in the aboveground compartment by the open-flow system and air was sampled from the soil, which eliminated back-diffusion of CO₂ from the soil surface. This may have disturbed the O₂ and CO₂ concentration profile in the soil. Nevertheless, the CO₂ concentration at the outlet of the below-ground compartment varied between 1000 and 2000 ppm and was thus far above the concentration inside the shoot enclosure. We have used a low air flow rate (3.5 L min⁻¹) and a well structured sandy loam (50% sand) soil with a large macroporosity. Therefore, only 4–6% of the air filled pore space was sampled each minute, which reduced the disturbance of the soil gas concentration profiles.

### 3.1.2 Plant material

The C₃ grass leaves harvested each month had mean δ¹³C values of −41.4±0.67 and −28.7±0.39‰ for ¹³C-labelled and control monoliths, respectively (Fig. 2d, means of April to November 2004). The C₃ grass leaves grown in labelled mesocosms were therefore more depleted in ¹³C (P<0.001; repeated measure ANOVA) than unlabelled controls grown outdoors. The lower δ¹³C values of C₃ leaves compared to air surrounding the leaves indicated the selectivity for ¹²C (i.e. discrimination Δ, O'Leary, 1981) of the carbon fixing enzyme Rubisco. The mean discrimination (Δ¹³C) over the experimental period reached 17.0±0.6 (April 2003 to April 2005). This was irrespective of the disturbance level by cutting in the mesocosms (P>0.1, repeated measure ANOVA, data not shown). Labelled and control leaves showed a seasonal pattern (Fig. 2d), being isotopically enriched in summer (−40.4±0.3‰ in the second year) and depleted in winter (−44.0±0.6‰), indicating changes in plant metabolism induced by abiotic factors such as water stress, high temperatures and high radiation intensities which decrease stomatal conductance (Farquhar et al., 1989; Brugnoli and Farquhar, 2000). Interestingly, seasonal changes in δ¹³C were similar inside and outside (controls) of the labelled mesocosms (Fig. 2d). Hence, during the experiment, the mean change in δ¹³C signature of C₃ grass leaves caused by labelling was equal to −13.6±0.7‰. This shows that the labelling facility generated a fairly constant change in delta ¹³C signature of leaves, despite seasonal variations, occurring in both, outdoors and inside the mesocosms.
<figure><img src="image_5.png" /></figure>

**Fig. 3.** Changes of the δ¹³C of total soil organic matter above 0.2 mm (SOM>0.2 mm, including roots, rhizomes and particulate organic matter) in the top soil (0–10 cm) during the labelling experiment. Monoliths acclimated to low (solid symbols) and to high (open symbols) field disturbance. Data are means ±SE of 8 replicate mesocosms.

The mean δ¹³C of fully labelled plant shoots reached –41.4‰. Several studies reported a δ¹³C difference of 1–2‰ between roots and shoots in grass plants grown under controlled and field conditions (Klumpp et al., 2005; Hobbie and Werner, 2004; Schweizer et al., 1999), showing that roots are isotopically enriched compared to shoots. Accordingly, we set the δ¹³Cinput of “new” C entering the belowground compartment at a δ¹³C of –40.4‰, which corresponds to a ¹³C enrichment of 1‰ of roots compared to shoots.

## 3.2 Effects of grassland disturbance level on soil carbon turnover

Klumpp et al. (2007) have shown that belowground carbon storage was higher for monoliths previously acclimated for 14 yrs to low than to high disturbance by grazing and cutting. Notably, values for belowground carbon storage calculated from the balance of gas exchanges were consistent with the increment in soil organic carbon content directly measured during the experiment.

Changes in δ¹³C values of SOM>0.2 mm (i.e. roots, rhizomes and particulate organic matter) in the top soil layer (0–10 cm) during the labelling experiment are shown in Fig. 3. Interestingly, initial δ¹³C values of SOM>0.2 mm were significantly different (P<0.001) between monoliths adapted to high (–28.7±0.10‰) and to low (–27.4±0.14‰) disturbance, illustrating the effects of grassland management by grazing and cutting on plant community structure and carbon cycling (Hooper and Vitousek, 1998). Plant community structure was suggested to affect ¹³C signature of root litter in grasslands (Dijkstra et al., 2004). Personeni and

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

**Fig. 4.** Changes in the contents (mg C g⁻¹ dry soil) of “old” (A) and “new” C (B) in the SOM above 0.2 mm of the top soil (0–10 cm) layer after start of ¹³C labelling, for monoliths acclimated for 14 yrs to low (full symbols) and high (open symbols) disturbance in the field. The fate of “old” C and the input of “new” C were fitted to first order exponential decay and rise to maximum models, respectively: Cold=a·e⁻⁰.⁰¹t; Cnew=a'·(1-e⁻⁰.⁰¹t) (abbreviations and coefficients of regression lines see Table 1).

Loiseau (2004) reported more depleted δ¹³C values for particulate organic matter fractions (POM) of *Lolium perenne* compared to *Dactylis glomerata*. Possible reasons for these differences in isotopic signatures in grasslands exposed to contrasted disturbance levels are: i) contrasted decomposition rates of distinct biochemical components of plant litter (Melillo et al., 1989; Agren et al., 1996) and ii) the contribution and δ¹³C values of different particle sizes to soil OM (Balesdent et al., 1988; Personeni and Loiseau, 2004). Findings from the same field site, showing a higher cellulose content in the plant material at low compared to high disturbance (Picon-Cochard et al., 2004) confirm the role of differences in biochemical components such as cellulose, which is enriched in ¹³C (Schweizer et al., 1999; Gleixner et al., 1993). Moreover, monoliths adapted to high disturbance in the field had a higher fraction of particulate OM (0.2×> x > 0.05 mm) (P<0.01, data not shown) with a more depleted δ¹³C value than monoliths adapted to low disturbance (data not shown).

During the 24 months of labelling, the gradual δ¹³C depletion of SOM (>0.2 mm) in the upper most 10 cm (Fig. 3) indicated that the soil litter continuum was steadily filled with “new” ¹³C-depleted carbon (P<0.001). Similar trends were found for the 10–20 cm and 20–30 cm soil depths, where
<table><caption>**Table 1.** First order exponential models of “old” C decay and “new” C accumulation in pasture monoliths adapted to low (L) and high (H) disturbance and continuously labelled with ¹³C. The fate of “old” C (C<sub>old</sub>) and the accumulation of “new” C (C<sub>new</sub>) into SOM (>0.2 mm) in 0–10 cm soil layer were fitted to first order exponential decay and rise to maximum models, respectively: C<sub>old</sub>=a.e<sup>-k.t</sup>; C<sub>new</sub>=a'(1-k'.t), where t is time in months; a is the initial amount of old C at t=0 (start of the labelling experiment); a' is the potential accumulation of “new” C (mg C g<sup>-1</sup> soil) in the compartment. k is the first order decay constant of “old” organic C. k' is the rate constant of accumulation of “new” C. Mean residence time of “old” C (MRT<sub>old</sub>, months) was calculated as: MRT<sub>old</sub>=1/k; the time for half-potential accumulation of “new” C (MT<sub>1/2–new</sub>) was calculated as: MT<sub>1/2–new</sub>=ln(1/2)/k'. Results are means of 8 replicates per treatment.</caption><thead><tr><th>C pool</th><th>Disturbance level</th><th>Coefficient</th><th>(mean ± s.e.)</th><th>P</th><th>Model R<sup>2</sup></th></tr></thead><tbody><tr><td rowspan="6">“Old” C</td><td rowspan="3">High</td><td>a (mg C g<sup>-1</sup> soil)</td><td>3.94±0.43</td><td>&lt;0.001</td><td>0.79*</td></tr><tr><td>k (month<sup>-1</sup>)</td><td>0.045±0.014</td><td>&lt;0.05</td><td></td></tr><tr><td>MRT<sub>old</sub> (month)</td><td>22.0</td><td></td><td></td></tr><tr><td rowspan="3">Low</td><td>a (mg C g<sup>-1</sup> soil)</td><td>7.35±0.38</td><td>&lt;0.001</td><td>0.91**</td></tr><tr><td>k (month<sup>-1</sup>)</td><td>0.032±0.005</td><td>&lt;0.01</td><td></td></tr><tr><td>MRT<sub>old</sub> (month)</td><td>31.2</td><td></td><td></td></tr><tr><td rowspan="6">“New” C</td><td rowspan="3">High</td><td>a' (mg C g<sup>-1</sup> soil)</td><td>2.27±0.05</td><td>&lt;0.001</td><td>0.99***</td></tr><tr><td>k' (month<sup>-1</sup>)</td><td>0.199±0.018</td><td>&lt;0.001</td><td></td></tr><tr><td>MT<sub>1/2–new</sub> (month)</td><td>3.48</td><td></td><td></td></tr><tr><td rowspan="3">Low</td><td>a' (mg C g<sup>-1</sup> soil)</td><td>3.047±0.298</td><td>&lt;0.001</td><td>0.92**</td></tr><tr><td>k' (month<sup>-1</sup>)</td><td>0.196±0.072</td><td>&lt;0.05</td><td></td></tr><tr><td>MT<sub>1/2–new</sub> (month)</td><td>3.54</td><td></td><td></td></tr></tbody></table>

the δ¹³C also declined towards more ¹³C depleted values (P<0.001, data not shown). SOM above 0.2 mm (P<0.001) had less negative δ¹³C values in monoliths adapted to low compared to high disturbance, suggesting a higher fraction of “new” (¹³C depleted) carbon in the high compared to low disturbance treatment (Fig. 3).

The fate of “old” C (unlabelled) and the input of “new” C (¹³C labelled) (mg C g⁻<fnref n="1" /> soil) into SOM (>0.2 mm) were fitted to first order exponential decay (Loiseau and Soussana, 1999; Personeni and Loiseau, 2005) and rise to maximum models, respectively (Fig. 4):

$$C_{\text{old}} = a e^{-k.t} \qquad (5)$$

$$C_{\text{new}} = a'(1 - e^{-k'.t}) \qquad (6)$$

where t is time in months; a is the initial amount of old C at t=0 (start of the labelling experiment); a' is the potential accumulation of “new” C (mg C g⁻<fnref n="1" /> soil) in the compartment. k is the first order decay constant of “old” organic C that leaves the OM compartment above 0.2 mm. k' is the rate constant of accumulation of “new” C in the soil in SOM above 0.2 mm. The mean residence time of “old” C (MRT<sub>old</sub>, months) was calculated as:

$$MRT_{\text{old}} = 1/k \qquad (7)$$

The time for half-potential accumulation of “new” C in the SOM above 0.2 mm (MT<sub>1/2–new</sub>) was calculated as:

$$MT_{1/2–new} = -\ln(1/2)/k' \qquad (8)$$

k values of 0.032 and 0.045 were found, giving mean residence time of “old” C in SOM>0.2 mm of 31.2 and 22.0 months (Fig. 4; Table 1) for monoliths acclimated to low and high disturbance, respectively. The different k values indicated a slower decay at low compared to high disturbance level.

With *Lolium perenne*, mean residence times of 55 and 11 months (for litter fractions >0.2 mm) were reported for a monoculture exposed for 10 yrs to elevated CO₂ (van Kessel et al., 2006) and in a two year decomposition experiment using labelled root litter (Personeni and Loiseau, 2004), respectively. In the latter experiment, which compared grass species in monocultures, 95% of the variability in litter decomposition rates was explained by a root morphology trait, the mean specific root length.

In our experiment, the plant species richness reached 41 species and the abundance of *Lolium perenne* in the plant community was always below 10% (Klumpp et al., 2007). The low disturbance treatment was dominated by plant species with coarse and dense roots and rhizomes (Klumpp and Soussana, 2007¹), which are likely to last longer and decompose slower than the fine roots observed at high disturbance level. However, differences in microbial communities and in mycorrhizae between disturbance treatments could also play a significant role in the decay rate of carbon in the detrital pathway (Rangel-Castro et al., 2005).

<footnote n="1">¹Klumpp, K. and Soussana, J. F.: Plant functional traits and species richness control carbon sequestration in grassland mesoscosms, Ecol. Lett., in revision, 2007.</footnote>
First order exponential models indicated that the poten-
tial accumulation of “new” C was higher at low compared to
high disturbance (a', 3.0 and 2.3 mg C g⁻¹ soil, at L and H;
P<0.01) and was for both treatments substantially lower than
the initial amount of “old” C at the start of the experiment (a,
7.4 and 4.0 mg C g⁻¹ soil at L and H) (Table 1). Thus, soil
organic C pools were not fully filled at the end of the experi-
ment, suggesting that a large part of the “new” C which was
deposited into the soil compartment was decomposed before
the end of the two years of the experiment. This would ex-
plain why the potential accumulation of “new” C (a') was
found to be lower than the initial amount of “old” C (a; Ta-
ble 1). For the same reason, half the maximum accumula-
tion of “new” C was reached after 3.5 month on average, as
some “new” C started to vanish from the compartment above
0.2 mm after a few months only.

During ¹³C labelling, “new” C entered first shoots then roots and rhizomes, before being released in the soil through rhizodeposition (i.e. root exudates) and decomposed litter. However, at the low disturbance level shoot litter decomposition also contributed to the supply of “new” C to the soil, which could explain the higher potential accumulation of “new” C (a') in the L compared to H treatment. Another factor contributing to the disappearance of both “old” and “new” carbon might be the activity of the soil fauna (macro-decomposers, e.g. earthworms) (Ostle et al., 2007; Hooper et al., 2000; Seeber et al., 2006), which could transport part of the litter to deeper soil layers and break down coarse litter into finer fractions. Indeed, there was a high presence of earthworms in the high disturbance treatment (data not shown).

A detailed understanding of the C cycling belowground
can be gained by investigating with this method carbon
turnover in different organic matter size fractions and at dif-
ferent depths. Moreover, the ¹³C signature of the respired
carbon can be studied giving access to the rate of decompo-
sition of the “old” versus “new” carbon pools.

4 Conclusion

These results show the potential of ¹³C steady state labelling
for investigating the fate of carbon in the plant-soil contin-
uum. A large array of applications at different time scales
can be envisaged, ranging from short-term labelling of plant
and soil carbon pools to long-term labelling of more stable
soil organic matter fractions. By combining isotope anal-
yses of soil OM compartments and of soil CO₂ efflux, a
detailed understanding of the fate of carbon can be gained
for herbaceous ecosystems. We have shown here that this
method allows calculating the mean residence time of carbon
in soil organic matter fractions. Moreover, we have shown
that changes in grassland disturbance regime alter the rate
of detrital C mineralization and accumulation in the soil or-
ganic matter. Since detrital carbon accumulation accounts

for most of an ecosystem's capacity to store organic carbon
belowground within a few years, this quantification is impor-
tant for understanding the role of grassland management on
belowground C sequestration.

*Acknowledgements.* We thank H. Schnyder, F. Teyssonneyre and P. Pichon for their scientific and technical contributions during planning and set up of the labelling facility. We also thank and R. Schäufele for helpful comments on an earlier version of this manuscript. This work was supported by a Marie Curie Individual Fellowship (EVK2-CT2002-50026) for K. Klumpp, the European GREENGRASS project (EVK2-CT2001-00105) and MEDD (GESSOL).

Edited by: J. Kesselmeier

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