# Seasonal Mineralisation of Organic Matter in Alpine Soils and Responses to Global Warming: An In Vitro Approach

Nicolas Bonfanti, Jean-christophe Clement, Annie Millery-Vigues, Tamara

Münkemüller, Yves Perrette, Jérôme Poulenard

► To cite this version:

Nicolas Bonfanti, Jean-christophe Clement, Annie Millery-Vigues, Tamara Münkemüller, Yves Perrette, et al.. Seasonal Mineralisation of Organic Matter in Alpine Soils and Responses to Global Warming: An In Vitro Approach. European Journal of Soil Science, 2025, 76 (1), pp.e70050. 10.1111/ejss.70050. hal-04934905

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# Seasonal mineralization of organic matter in alpine soils and responses to global warming: an *in-vitro* approach

BONFANTI Nicolas<fnref n="1" />,<fnref n="2" />, CLEMENT Jean-Christophe<fnref n="1" />, MILLERY-VIGUES Annie<fnref n="1" />,<fnref n="3" />, MÜNKEMÜLLER Tamara<fnref n="3" />, PERRETTE Yves<fnref n="2" />, POULENARD Jérôme<fnref n="2" />

Contact: nicolas.bonfanti@univ-smb.fr

**Key words**

Carbon Nitrogen & Phosphorus mineralization, Incubations, Mountain soils, Soil Organic Matter

**Abstract**

Mountains are particularly vulnerable to climate change, as they are warming at a rate that exceeds the global average, significantly impacting cold-adapted ecosystems. In these environments, soil organic matter (SOM) stocks are often considerably higher than at lower elevations. These stocks are therefore highly susceptible to global warming and the associated risk of greenhouse gas (CO₂, CH₄, N₂O) emissions driven by temperature-induced increases in SOM mineralization. In order to quantify these emissions and the change of mineralization rates under warming, it is necessary to gain an understanding of the annual mineralization balance. We investigated how warming impacts the duration and intensity of mineralization at different seasons. The main aim of the present study is to quantify alpine SOM mineralization rates and greenhouse gas production under a range of seasonal conditions, including those associated with warming. An *in vitro* approach was employed to expose alpine topsoils (0-10 cm) to the conditions of key seasonal periods: snow-cover, growing season and rainfall/snowmelt. This was achieved by experimentally varying temperature and inflow of precipitation water. Additionally, the soil samples were subjected to a temperature increase of 4°C. The short-term responses of carbon (C), nitrogen (N) and phosphorus (P) mineralization and greenhouse gas production were monitored. The results demonstrated that alpine soil respiration rates exhibited a twofold increase with a 4°C warming, while the relative proportion of labile SOM demonstrated a decline with rising temperatures. Water saturation from simulated rain and snowmelt played a crucial role in organic matter mineralization, and increased the mineralization of carbon (+12 to +53 %), nitrogen (+20 to +80% of net ammonification) and phosphorus (+50% of net phosphate production). This suggests that nutrients present in the snowpack or in the rain were added to the soil.

<footnote n="1">¹ Univ. Savoie Mont Blanc, INRAE, CARRTEL, Thonon-Les-Bains, France</footnote>

<footnote n="2">² Univ. Savoie Mont Blanc, CNRS, EDYTEM, Chambéry, France</footnote>

<footnote n="3">³ Univ. Grenoble Alpes, Univ. Savoie Mont Blanc, CNRS, LECA, Grenoble, France</footnote>
In contrast, soil water saturation decreased net nitrate production by between 10 and 90%. The Results of this study highlight the potential for alpine soil warming to release labile SOM, and demonstrate the influence of snow regime on nutrient and carbon fluxes.

Introduction

Alpine and high-latitude ecosystems are particularly vulnerable to climate warming, exhibiting accelerated warming compared to other ecosystems (Beniston et al. 1997; IPCC 2023). Concurrently, these ecosystems play a pivotal role in climate regulation (Davidson and Janssens 2006). Low temperatures in these ecosystems slow down soil organic matter (SOM) mineralization and increase its mean residence time, resulting in higher SOM stocks than those observed in warmer ecosystems (Davidson and Janssens 2006; RMQS 2017). Furthermore, alpine SOM stocks are distinguished by a high proportion of labile SOM, comprising approximately 40-60% of labile C that is readily mineralizable (Budge et al. 2011). Global warming can exert a twofold influence on SOM dynamics. On the one hand, warming can increase SOM mineralization, thereby further destabilizing labile C stocks (Poulenard et al. 2020; Khedim et al. 2022). These stocks are vulnerable to temperature increase with Q10 values ranging from 2 to more than 170 (Bekku et al. 2003; Hamdi et al. 2013; Schädel et al. 2016). Consequently, warming can increase the production of greenhouse gases, including CO2 and CH4 (Brummell et al. 2014). On the other hand, warming can increase plant growth and biomass production (Choler et al. 2021; Rumpf et al. 2022), and thus increase SOM inputs. These two processes, mineralization and productivity, are not independent of one another, as mineralization affects nutrient availability, especially for nitrogen and phosphorus, and thus plant growth (Nadelhoffer et al. 1991). The impact of climate warming on alpine soils depends on whether the increase in mineralization or productivity is greater. If mineralization is the dominant process, soils act as carbon sources, whereas if productivity is the dominant process, soils act as carbon sinks. This has a negative or positive impact on climate regulation (Kirschbaum 2000). Consequently, modelling the dynamic alterations in soil organic matter (SOM) stocks represents a pivotal challenge for alpine ecology research. To this end, it is imperative to comprehend the seasonal patterns of mineralization rates and their responses to warming. In particular, it is essential to quantify the impact of warming on the annual balance of SOM mineralization rates and the stability of SOM pools.

SOM stability depends on three main factors: the chemical composition of SOM (quality) (Lorenz et al. 2007; Amelung et al. 2008), organo-mineral interactions (Kögel-Knabner et al. 2008), and environmental conditions (climate, topography) which exert control over the abundance, composition, and activity of microbial communities involved in SOM mineralization (Doetterl et al. 2015). In alpine environments, SOM stability is significantly influenced by low temperatures but also by the soil
moisture regime (Kirschbaum 2000; Davidson and Janssens 2006; Miller et al. 2007). Accordingly, a year can be divided in two main periods: a snow-covered season (SCS) and a snow-free, growing season (GS) (Grogan and Chapin 1999), with particularly moist periods marked by precipitation water inflows due to rainfall and snowmelt (Water Inflow Periods: WIPE) (Durand et al. 2009; Morse et al. 2015; Choler 2015) (Figure 1). The SCS is distinguished by a persistent snow cover which insulated the soil from low temperatures, maintaining a temperature of app. 0°C with moderate freeze-thaw alternation (Freppaz et al. 2006). During SCS, mineralization activity is reduced, but still occurs (Sommerfeld et al. 1991, 1993; Saccone et al. 2013; Bernard et al. 2019). Soil conditions during the GS can vary considerably on a daily (day/night), inter-day (sunny/rainy, hot/cold), and inter-annual (hot year/rainy year) basis. WIPE occurs during the transition between the snow cover season (SCS) and the growing season (GS), during SCS with rainfall on the snowpack, or during GS with rainfall and early or late snowfall. It causes periodic soil saturation with water from melting or precipitation. It is a critical time for the dynamics of alpine SOM as it exerts a profound influence on ecosystem processes, including C and N dynamics (Freppaz et al., 2008; Clément et al., 2012), microbial biomass and activity (Lipson et al. 2000; Jusselme et al. 2016), and plant phenology and productivity (Choler 2015; Zhang et al. 2023). Additionally, the anaerobic conditions generated by saturation favour other processes of SOM mineralization, especially by inhibiting aerobic respiration and promoting methanogenesis (Mosier et al. 1993; Wang and Bettany 1995, 1997), as well as denitrification (Barnard et al. 2005; Morse et al. 2015).

The impact of climate warming on mineralization rates is contingent upon the duration of each season and the prevailing environmental conditions. Consequently, the effect of climate warming on mineralization rates differs between seasons. Firstly, the SCS tends to become shorter, whereas the opposite is observed for the GS (Beniston et al. 1997; Serquet et al. 2011; Gobiet et al. 2014; Notarnicola 2020; Rumpf et al. 2022). Secondly, the dynamic of WIPE undergoes changes, with snowmelt episodes occurring earlier and with greater frequency during the year (alternating snow-free soil/snowfall) (Stone et al. 2002). Precipitation events become rarer during the GS (Durand et al. 2009; Rousselot et al. 2012). Some studies have highlighted the impacts of early snowmelt (Legay et al. 2013; Gavazov et al. 2017), and rainfall change on SOM mineralization (Wang et al. 2016; Liu et al. 2024). Thirdly, climate change is increasing the intensity and frequency of extreme events such as heat waves, droughts and floods (Calanca 2007; Wipf et al. 2013). All these consequences of climate change modify most biogeochemical cycles by affecting mineralization activity (Groffman et al. 2001; Koch et al. 2007). This may result in an elevated mineralization of the more labile forms of SOM in alpine environments (Prietzel et al. 2016; Khedim et al. 2023). Vegetation studies have demonstrated that warming results in taxonomic changes in primary producers (Alexander et al. 2018), modifications in
their functional traits (Bernard et al. 2020; Bektaş et al. 2021), and vertical migrations of lowland plants (Mayor et al. 2017; Walker et al. 2021). Consequently, the quantity and chemical composition of OM inputs are modified. The labile SOM that is maintained in alpine soils by low temperatures can also be rapidly mineralized (Khedim et al. 2023). Therefore, warming affects not only the quantitative fluxes of alpine SOM mineralization but also alpine SOM chemical stability.

While a substantial body of knowledge exists regarding the primary drivers of SOM mineralization, the season-specific effects of climate warming in alpine soils remain largely unexplored. Our study aims to: 1) estimate flux and process-related to alpine SOM mineralization under distinctive seasonal conditions (SCS, GS and WIPE); 2) assess the impact of warming on alpine SOM mineralization rates throughout each season; 3) compare warming consequences with the mineralization rates of a warmer ecosystem (subalpine soil), as a space-for-time comparison. An *in vitro* approach was employed to simulate alpine seasonal conditions of the SCS (0°C ± 1.5°C) and the GS (8°C ± 1.5°C) with or without WIPE (8°C and addition of precipitation water). Furthermore, alpine soils were incubated under conditions simulating a warming of 4°C, which is anticipated in the Alps by 2070. This resulted in a warmed GS (12°C ± 1.5°C) with or without WIPE (12°C and melting water inflow). Furthermore, we compared these dynamics with those of subalpine soils situated 550 m below the alpine soils that had an average temperature equivalent to the simulated warming conditions of alpine soils (SCS: 0°C; GS: 12°C with and without WIPE). The short-term responses of carbon, nitrogen, and phosphorus mineralization rates were measured for each alpine and subalpine soil incubation, and the quality of the soil organic matter was characterized before and after incubation. We hypothesize that:

H1) WIPE boosts mineralization rates, as well as methanogenesis and denitrification. Therefore, climate-related changes in the frequency and intensity of these episodes can increase the annual balance of mineralization rates and GHG production;

H2) Warming increases mineralization rates during GS and WIPE and destabilizes the labile pools of organic matter. This results in the loss of organic matter in gaseous and aqueous forms.

H3) As a consequence of H2, mineralization rates for alpine soils that have been warmed are higher than those of subalpine soils with an equivalent average temperature.
Study sites and sampling

Alpine and subalpine research sites were located in the French Alps near the Galibier pass (Figure 1, see Bektaş et al. (2021) for more information on sites). The elevation of the sites was 2470 m a.s.l. and 1920 m a.s.l., for alpine and subalpine respectively. Both study areas (≈ 250 m²) exhibited the same geological and topographical characteristics and similar land uses. They were exposed to the southeast on a sandstone flysch substratum (“flysch des aiguilles d’Arves”, base-rich). During the summer period, extensive pastoral practices were conducted, involving the grazing of sheep and cattle. The plant communities at the subalpine site were dominated by *Patzkea paniculata* (EUNIS code: E4.331) and by *Carex sempervirens* & *Festuca nigricens* (EUNIS code: E4.4) at the alpine site. Soils in both sites were Dystric Cambisols (Table S1), non-carbonated (WRB 2022).

In February 2022, the snowpack was excavated to collect homogeneous topsoil samples, corresponding to the A horizon (litter excluded), from six randomly selected soil pits with a depth of 0 to 10 cm at each site (1 kg per soil pit) (Figure 1, and see Table S1 for soil properties). During sampling, soil temperature and moisture were measured in the 0-10 cm horizon with a temperature probe (Hanna HI151) and a moisture probe (Campbell Scientific Hydrosense II). The samples were transported to the laboratory in a cool box and stored at a temperature and humidity close to field conditions until the commencement of the incubation period (0.0°C ± 0.3, 42.0% ± 6.4 for alpine soil and 0.1°C ± 0.15, 33.5% ± 5.2 for subalpine soil). Additionally, three soil replicates of 100 cm³ were collected with a cylinder to assess bulk density at each site.

The initial soil properties (prior to incubation) were analyzed at each site using the six pit soil samples (see Table S1 and the following section for soil analysis methods). For this, soils were sieved to 5 mm and hand-sorted in the laboratory to remove mineral and organic fragments (plants, roots, rocks, etc.) (Robertson et al. 1999; Miller et al. 2007). Prior to incubation, the six samples from one site were combined to form a composite sample (Figure 2). The composite samples were then divided for the incubations.

In order to simulate rainfall/snowmelt water saturation, twenty litres of liquid water equivalent of snow were collected from the entire height of the snowpack near the subalpine site at two different locations. Snow was melted at room temperature (approximately 18-20°C) for 10 hours, and then stored at -20°C until use (1-2 months). Snow chemical composition was analysed (inorganic N, DOC, see below for methods).

Experimental design
The composite soils were incubated in the dark in thermostatic chambers (Electrolux AR6412T) for a period of two months under 11 different incubation treatments, with seven treatments for alpine and four treatments for subalpine soils (Figure 2). Each treatment was replicated six times, resulting in a total of 66 samples. Each sample comprised 200 g of composite soil placed in an independent incubation unit. Alpine soils were incubated under conditions representative (i.e., average temperature) of the SCS (simulated conditions: 0°C ± 1.5°C), alpine GS (8°C ± 1.5°C) without and with WIPE (adding precipitation water), and the GS under 4°C of warming (12°C ± 1.5°C) without and with WIPE. According to the temperature probes at the sampling sites, the average temperature of GS at the alpine site was 8°C and at the subalpine site 12°C. To estimate mineralization potential at high temperatures, alpine soils were additionally incubated in an oven (Memmert loading model 100-800) at 30°C with and without WIPE. In the same way, subalpine soils were incubated under conditions representative of the SCS (0°C ± 1.5°C) and the subalpine GS (12°C ± 1.5°C) without and with WIPE. To ascertain the short-term responses of SOM mineralization and respiration rates, we performed for each of the 66 samples two measurements of gas fluxes per week and one leaching per month (leaching at the end of the first month named "L2", second month: "L3") (Figure S2, see *Leachate analysis* below). To determine the responses of soil properties to the incubation treatments, we analysed soil properties again at the end of the experiment.

Soils were incubated in hand-made incubation units using inert materials (Figure S1), which enabled the measurement of gas production, leaching of the soils, and saturation with precipitation water (Figure S1) (Nadelhoffer 1990). To confirm that the release of carbon and nitrogen was limited and that the incubation units were airtight, release tests (leachate and gas) were carried out on blank units without soil (see Figure S1 for details of test protocols and §Comments on method in Supp. materials). Throughout the experiment, for each treatment, blank units were subjected to the same treatments and analyzed.

To start the experiment, 200 g of fresh, sieved composite soils were placed into the incubation units (total volume of 600 cm³). An initial leaching with distilled water (to avoid adding nutrients) was done to ensure that all replicates had similar moisture conditions. During this leaching (L1), the incubation units were filled with water and weighed. Subsequently, following the leaching process, the samples were weighed once more, and the difference in mass was converted to headspace volume, which was then used for gas flux calculations. To follow nitrogen and phosphorus net release (mineralization and sorption/desorption for P) throughout the experiment, two additional leaching events with distilled water were carried out (L2, L3) after one and two months of incubation respectively. These leaching operations aimed to export inorganic forms of nitrogen and phosphorus for subsequent measurement, and also to quantify dissolved organic carbon. Leaching were made with
250mL of distilled water (pH 5.6) per incubation unit. The water was brought to incubation temperature to avoid thermal shift. Distilled water was used so that no extra nutrients were added to the soil. Water was introduced into the incubation unit over the soil through the upper pore (Figure S1). A five-minute waiting period was observed to ensure complete saturation and allow for soil-water equilibrium. The water was then removed through the lower pore using a vacuum pump (0.06 MPa) for further analysis (see § Leachate analysis).

Prior to flux measurements, the incubation units were subjected to the incubation temperature for a six-day acclimation period, ensuring that the entire soil sample was maintained at the desired temperature. Throughout the course of the experiment, the incubation units were relocated on a regular basis within the thermostatic chambers to ensure that no positional effects were present. Additionally, soil moisture was maintained at 0.06 MPa (with the use of a vacuum pump) for all treatments, with the objective of maintaining them at 60-70% WHC. To ascertain the stability of soil moisture, the incubation units were weighed on a weekly basis, and distilled water was added when necessary. The observed variations were minimal, with a maximum loss of approximately 8% at 30°C, as reported in other studies (Nadelhoffer 1990). WIPE events were simulated on four occasions throughout the course of the experiment (W1, W2, W3, and W4) through the saturation of the soil with 150 mL of precipitation water at temperatures of approximately 1-2°C. This period of four days is consistent with observations made in the field, where melting occurs through a succession of episodic saturated and unsaturated soil conditions. Alternatively, this process can be used to simulate rainfall events. Precipitation water was supplied slowly from above until the soil was saturated.

## Laboratory analyses

### Soil analysis

Analyses were first performed on the sample of the soil pits and on the composite samples to characterize them before incubation. Some were repeated at the end of the incubation period: C organic, N<sub>tot</sub>, NO<sub>3</sub><sup>-</sup>, NH<sub>4</sub><sup>+</sup> content, P available, Diffuse Reflectance Infrared Fourier Transform spectroscopy (DRIFT).

Basic analyses were carried out on the 14 sieved soil samples (6 alpine pits, 6 subalpine pits, alpine and subalpine composites). Soil pH (pH<sub>H2O</sub>, pH<sub>KCl</sub>), CEC and granulometry were measured in accordance with the international standards ISO 10390-2005, NF X 31-130 (1993) and AFNOR X31 107 respectively. The SOM content was determined through loss on ignition (4H at 550°C). The soil organic carbon and nitrogen contents were determined on dry and ground soil using a CHN elemental analyzer (Flash EA 1112, Thermo Electron Corporation, ThermoFisher). Soil inorganic nitrogen (NO<sub>3</sub><sup>-</sup> and NH<sub>4</sub><sup>+</sup>)
contents were extracted with a K₂SO₄ solution (0.5 M) and subsequently determined using a sequential analyzer (Gallery +, ThermoFisher). Bioavailable phosphorus was determined through the Olsen method, which involved extraction with 0.5M NaHCO₃ (Olsen 1954; Fardeau et al. 1988; Le Noë et al. 2016).

Following an overnight drying period at 40°C to standardize the water content of the samples, each soil was finely ground and analyzed by DRIFT spectroscopy to assess its organic carbon functional groups composition. This analysis was conducted using a Nicolet iS10 spectrometer (Thermo Fischer Scientific). The spectra obtained extends from 400 cm⁻¹ to 4000 cm⁻¹ with a step of 0.4 cm⁻¹(Farmer 1974; Kaiser et al. 1997; Celi et al. 1997). They have been corrected for atmospheric interferences (H₂O and CO₂), offset by using a reference region (4000-3950 cm⁻¹), and setting the minimum absorbance value to zero. Then, the integration of the spectrum over wavelength ranges allowed the calculation of functional group index: aliphatic group (CH₂, CH₃) between 3000 and 2750 cm⁻¹; carbonyl and carboxyl group (C=O) between 1750 and 1670 cm⁻¹; aromatic group (C=C) between 1618 and 1576 cm⁻¹ (Figure S5). These regions were selected because they are representative of carbon functional groups (Truche 2011; Saenger 2013; Saenger et al. 2015; Puissant et al. 2017), and are weakly impacted by soil mineralogical composition (Soucémarianadin et al. 2019). Relative ratios were calculated (equation 1; Khedim et al., 2021) on samples before and after incubation. The aliphatic index characterises organic matter rich in aliphatic groups which are known to be labile and easily mineralized. The aromatic and carbonyl indexes characterise organic matter that is more recalcitrant to mineralization.

Equation (1): DRIFT index calculation:

$$Relative index_i = \frac{integration area index_i}{\sum integration area of index_{CH,CO,CC}}$$

With: i: aliphatic (CH), carbonyl-carboxyl (C=O) or aromatic (C=C)

## Gas analysis

Greenhouse gas production (CO₂, CH₄, N₂O) was measured twice a week during incubation on each incubation unit. For this, the change in the air composition was measured by difference between the closing time of the incubation unit (tᵢ) and the final measurement time (tᵣ). This allowed for the quantification of the accumulated gases produced within the unit during incubation period (tᵣ-tᵢ). Gas sample was taken directly from the headspace volume of the incubation units. Outside the closing time, the incubation units were opened to ensure equilibrium with the atmosphere.

Gas composition was determined using an Agilent 490 Micro-Gas Chromatograph (µGC, SRA Instruments, France) with an unheated injector and a thermal conductivity detector (µTDC; column used: Molsieve MS 5 Å for CH₄ and a Poraplot Q for N₂O and CO₂ at 110°C and 28 Psi). The carrier gas
was helium (99.9999 % purity, flux of 2.7 mL.min⁻¹). The analysis time was 46 s and the backflush time was 3.9 s. The µGC was calibrated with certified CH₄, N₂O and CO₂ standards of 5000 ppm and a dilutor using helium (Zéphir, Alytech, France). To quantify gas production, the incubation unit was vented with pressurized air, and the initial gas content (Cᵢ, ppm) was subsequently analyzed. Subsequently, the incubation unit was sealed. Gases produced by the soils accumulated in the headspace during the incubation period. Incubation times were adapted to achieve CO₂ concentrations in the upper half of µGC range (3000-5000 ppm), i.e., 1h, 2h, 4h and 10h at 30°C, 12°C, 8°C and 0°C respectively. Beyond these incubation times, CO₂ concentrations were out of range (>5000 ppm). The gas content was measured at the end of the incubation period (Cᵣ, ppm). The gas content value was obtained by averaging the results of three consecutive gas sample analyses, thereby validating the reproducibility of the measurement. It should be noted that the variations between analyses on the same replicate were generally less than 5%. Given that the total volume of gas sampled for concentration measurements was very small (less than 5% of the total headspace volume), the slight under pressure generated in the incubation unit did not affect the measurement. The gas flows were calculated using equations (2) and integrated both CO₂ increase in headspace volume of incubation units (Robertson et al. 1999) and dissolved CO₂ in soil water (H₂CO₃, HCO₃⁻, CO₃²⁻) (Millero et al. 2007; Sander 2015).

The calculation of the accumulated gases (µg C or N/g OM) corresponded to an extrapolation of the closest and the most consistent (e.g., post-melting) measured daily rate (µg C or N/g OM/day) and summed for each day.

Equations (2): Equations used for calculated C respiration flux (F<sub>C mineralization</sub>), including both CO₂ increase in the headspace volume and DIC in soil water. This flux was obtained by difference between carbon content before (C<sub>tot initial</sub>) and after (C<sub>tot final</sub>) incubation period divided by the duration of incubation period (d) and normalized by dry soil mass (W).

$$F_{C \text{ mineralization}} = \frac{C_{\text{tot final}} - C_{\text{tot initial}}}{d * W}$$

with:

$$C_{\text{tot final}} = C_{\text{CO2 final}} + DIC_{\text{final}}$$

$$C_{\text{tot final}} = C_{\text{CO2 final}} + [H_2CO_3_{\text{final}} + HCO_3^-_{\text{final}} + CO_3^{2-}_{\text{final}}]$$

$$C_{\text{tot final}} = \left[ \frac{C_f * M * P * V}{R * T} \right] + \left[ K_H * (C_f * 10^{-6} * P) \left( 1 + \frac{Ka1}{10^{-pH}} + \frac{Ka1 * Ka2}{(10^{-pH})^2} \right) * M * V_w * 10^6 \right]$$

$$C_{\text{tot initial}} = C_{\text{CO2 initial}} + DIC_{\text{initial}}$$

$$C_{\text{tot initial}} = C_{\text{CO2 initial}} + [H_2CO_3_{\text{initial}} + HCO_3^-_{\text{initial}} + CO_3^{2-}_{\text{initial}}]$$

$$C_{\text{tot initial}} = \left[ \frac{C_i * M * P * V_{\text{air}}}{R * T} \right] + \left[ K_H * (C_i * 10^{-6} * P) \left( 1 + \frac{Ka1}{10^{-pH}} + \frac{Ka1 * Ka2}{(10^{-pH})^2} \right) * M * V_w * 10^6 \right]$$

Where:
- C<sub>tot final</sub>/C<sub>tot initial</sub>: inorganic carbon in incubation unit (CO<sub>2</sub>+DIC) at the end/beginning of incubation period (µg C);

- C<sub>f</sub>/C<sub>i</sub>: Final/initial CO<sub>2</sub> content in the headspace, measured with µGC (ppm);

- M: molecular weight of C-CO<sub>2</sub> (12 µg. µmol<sup>-1</sup>);

- P: barometric pressure (1 atm);

- V<sub>air</sub>: Headspace volume (L);

- R: universal gas constant (0.0820575 L.atm.<sup>-1</sup>.K.mol);

- T: incubation temperature (°K);

- K<sub>H</sub>: Henry's Law constant for CO<sub>2</sub> (0.073 mol/L·atm at 0°C, 0.085 mol/L·atm at 8°C, 0.091 mol/L·atm at 12°C, 0.033 mol/L·atm at 30°C accordingly to Sander (2015);

- Ka1: equilibrium constant of H<sub>2</sub>CO<sub>3</sub><sup>-</sup>↔HCO<sub>3</sub><sup>-</sup>+H<sup>+</sup> (4.45×10<sup>-7</sup>);

- Ka2: equilibrium constant of HCO<sub>3</sub><sup>-</sup>↔CO<sub>3</sub><sup>2-</sup>+H<sup>+</sup> (4.69×10<sup>-11</sup>);

- pH: pH of the water (~5.6);

- V<sub>w</sub>: water volume (L);

- W: dry soil mass equivalent (g);

- d: duration of accumulation period (day);

- F<sub>C mineralization</sub>: flux of C mineralisation (µgC-CO<sub>2</sub>/day/g<sub>soil</sub>)

The temperature sensitivity of SOM mineralization was estimated using Q<sub>10</sub> (equations 3, Miller et al. 2007), as described by Bekku et al. (2003). The mean respiration rates of each replicate were averaged with all combinations of values between two temperatures (n = 36) to calculate the Q10 value (Heitkamp et al. 2012). This method has been shown to be biased towards substrate supply, however it allows to compare our values with other studies on similar soils under similar experimental conditions.

Equations (3): Q<sub>10</sub> calculation:

$$B = \frac{\ln \frac{R_2}{R_1}}{(T_2 - T_1)} ; \quad Q_{10} = e^{(10*B)}$$

With: R<sub>1</sub> the mineralization rate at temperature T<sub>1</sub> and R<sub>2</sub> the mineralization rate at temperature T<sub>2</sub>

## Leachate analysis

All water samples (WIPE water and soil leachate water: L1, L2, L3) were analysed for dissolved elements. After filtration at 0.7 µm (GF/F glass fibre filter) (Miller et al. 2007), non-purgeable organic carbon (NPOC) and total dissolved nitrogen (TDN) were analysed on one subsample using a TOC-meter analyser (Shimadzu TOC-V analyser). The samples were first acidified with 1 mL of 10 % sulphuric acid per 120 ml of water to obtain a pH below 2 for storage at -20°C for a maximum of two weeks. This procedure also removed inorganic carbon (carbonates). The dissolved organic carbon (DOC) then underwent catalytic combustion at 800°C in the presence of air. The CO<sub>2</sub> formed was measured in the infrared. Total dissolved nitrogen (TDN) content was measured using a chemiluminescence detector coupled to the TOC analyser. Inorganic nitrogen (NH<sub>4</sub><sup>+</sup> and NO<sub>3</sub><sup>-</sup>) and dissolved phosphorus (PO<sub>4</sub><sup>3-</sup>) were analysed on a second subsample by colorimetric method (Gallery+, ThermoFischer). We calculated total nitrate extracted as the sum of nitrate leached during the experiment (L2, L3, F1, F2, F3, F4) plus nitrate extracted from the soil after the incubation (corresponded to the balance between nitrification
minus denitrification and DNRA during the incubation period, i.e., the net nitrate production). The same methodology was employed to calculate the total ammonium and total phosphate extracted.

The chemical composition of the OM present in the leachates was assessed by three-dimensional ultraviolet (UV) spectrofluorescence measurements with a spectrofluorometer (Varian Cary-Eclipse). The samples were placed in a high precision quartz suprasil cell (Hellma Analytics) and were excited from 230 to 350 nm with a 10 nm step. Fluorescence measurements were conducted for each excitation wavelength between 250 and 600 nm. The absorbance of the samples was performed at 280 nm (Kalbitz et al. 2003) and 254 nm (Weishaar et al. 2003) to estimate the aromaticity (SUVA) and to ensure that the internal filter was insignificant (dilution was performed if the absorbance was higher than 0.1). The emission spectra were simulated by the linear combination of log-normal functions (Siano and Metzler 1969). The simulations were performed by a constrained least squares simulation (for center location and width) of a set of user-defined bands (number and positions) using a matlab optimization toolkit (Puissant et al. 2017). This method enables the detection of protein-like fluorophores whose emissions are masked by Rayleigh or Raman scattering. Excitation/emission couples were assigned to tyrosine-like (230 nm/310 nm), tryptophane-like (270 nm/350 nm), fulvic-like (340 nm/420 nm) and humic-like (340 nm/480 nm) compounds (see Figure S5) (Puissant et al. 2017). Sum of tyrosine-like and tryptophane-like represented protein-like (PL) molecules and sum of fulvic-like and humic-like represented Humic-like (HL). An organic matter fluorescence index (OM<sub>fluorescence index</sub>) and a SUVA index (SUVA<sub>254</sub>) of leachates were calculated according to equations 4.

Equations (4): Spectrophotometric analysis of OM in leachate:

$$OM_{fluorescence\ index} = \frac{HL + PL}{HL - PL}; SUVA_{254} = \frac{absorbance_{254nm}}{NPOC}$$

With: HL and PL humic-like and protein-like index; NPOC = non-purgeable organic carbon

Statistical analyses

For each incubation treatment, we measured the following variables: gaseous losses (CO₂, N₂O, CH₄ production), leaching losses (NPOC, TDN, DIN, DON), N and P mineralization (net nitrate, ammonium and phosphates production and P availability), and the change in SOM stability (DRIFT, SUVA, UV fluorescence). To test our three hypotheses: H1) we compared the effect of WIPE on these variables for alpine soils (within the same incubation temperature, i.e., alpine GS at 8°C with alpine at 8°C + WIPE; alpine GS at 12°C with alpine at 12°C + WIPE; alpine GS at 30°C with alpine at 30°C + WIPE); H2) we compared these variables for the alpine soils between 8°C and 12°C and 30°C (within the same WIPE treatment but also the interaction effect between warming and WIPE, i.e., alpine GS at 8°C with alpine GS at 12°C; alpine GS at 8°C with alpine GS at 30°C; alpine at 8°C + WIPE with alpine at 12°C +
WIPE; alpine at 8°C + WIPE with alpine at 30°C + WIPE); and H3) we compared alpine and subalpine sites for the same temperature (0°C, 12°C and 30°C) and WIPE treatment (i.e., alpine GS at 12°C with subalpine GS at 12°C; alpine at 12°C + WIPE with subalpine at 12°C + WIPE; alpine 30°C with subalpine 30°C). We expected that WIPE (H1) and warming (H2) would enhance the production of gases, increase leaching losses, and accelerate the mineralization of nitrogen and phosphorus. Additionally, it was hypothesized that mineralization rates would be higher in alpine soils than in subalpine soils when subjected to the same temperature (12 and 30°C) (H3).

Given the considerable discrepancy in OM content between subalpine and alpine topsoils, we standardized each response per grams of organic carbon (gSOC⁻¹). A one-way ANOVA was conducted to compare the effect of the 11 treatments (site + temperature + WIPE) on the total amount of leached elements (NPOC, TDN, DIN, DON), the total amount of extracted elements (NO₃⁺, NH₄⁻, PO₄³⁻, POlsen), and the OM stability indices at the end of incubation, and post hoc Tukey's Studentized Range (HSD) test was used to examine a posteriori pairwise difference among treatment. With the post-hoc test, we compared Alpine GS with alpine GS + WIPE, alpine GS warmed with alpine warmed + WIPE and alpine at 30°C with alpine at 30°C + WIPE (H1); GS alpine with GS alpine warmed with 30°C alpine and alpine GS + WIPE with alpine GS warmed + WIPE with 30°C alpine (H2); alpine SCS with subalpine SCS, alpine GS warmed with subalpine GS, alpine GS warmed + WIPE with subalpine GS + WIPE and 30°C alpine with 30°C subalpine (H3) (Table 3). Respiration measurements were made several times during incubation, therefore measurement day was considered as random effect, and we used mixed effects models for this variable. Variables were square root or log-transformed as necessary to meet assumptions of normality and homoscedasticity of residuals in the regression models. We removed outliers for each measurement series (i.e., each repetition per treatment) using Grubbs tests (significance level: p<0.01).

All data management and analysis were performed with Excel and R 4.0.2 (R Core Team 2023) software using the R packages "tidyverse" (Wickham et al. 2019), "lme4" (Bates et al. 2015), "ChemoSpec" (Hanson 2016), "lmerTest" (Kuznetsova et al. 2020) and "outliers" (Komsta 2022).
## Results

To enhance readability, results are organized by variables: gas production, leaching losses, N and P mineralization, and changes in SOM stability.

### GHG production (CO₂, CH₄, N₂O)

The total cumulative C respiration during the incubation was the same for GS and GS + WIPE (Water Inflow Periods) but there were clear differences in temporal dynamics (Figure 3A). C respiration of non-saturated soils was relatively constant over time. In contrast, in the WIPE treatment, soils showed no C respiration during the water saturation period (Figure 3B). However, following the saturation period, these soils exhibited a notable increase in respiration rates compared to the non-saturation treatment (p < 0.01). No methane or nitrous oxide were detected in the WIPE treatment, including when the soil was saturated with water (data not shown). However, redox traits (stains) were observed in soils of these treatments and were more pronounced at higher temperatures.

Alpine warmed GS exhibited a twofold increase in total C respiration with and without WIPE (Figure 3A, Table 3.H2). Accordingly, the Q₁₀ value exhibited a markedly elevated ratio across these temperature ranges (Table 1). Similarly, C respiration at 30°C was 4-5 times more important than in the 8°C treatment.

By comparing C respiration between alpine and subalpine soils at the same temperature, we first noted that alpine GS respiration was higher than subalpine GS respiration (Figure 3 and Table 3.H3). During SCS, alpine respiration was higher than subalpine respiration and we measured average N₂O fluxes of 0.56 (± 0.52) µgN-N₂O/gOM/day for alpine soil. Similarly, at 30°C, alpine respiration was higher than subalpine respiration, where N₂O fluxes were app. 6.41 (± 2.07) µgN-N₂O/gOM/day at 30°C.

### Leaching losses (dissolved C, N)

First, we noted that raw snowmelt water contained NPOC with high OM fluorescence index, (i.e., labile, easily usable for microbiological activity) and dissolved nitrogen (2.13 mg C-NPOC/L; 2 µg N-NO₃⁻/L; 16 µg N-NH₄⁺/L; 0.16 µg PO₄³⁻/L),

At the same temperature, soils with WIPE released more NPOC, TDN and DON than soils without WIPE both during SCS and GS. However, WIPE had no effect on leached inorganic nitrogen except at 30°C (Figure 4; Table 3.H1). Warming of the alpine soil from 8°C to 12°C increased leached TDN, mainly through DIN loss (Table 3.H2). Comparison between alpine and subalpine soils showed that, prior incubation, alpine soil leaching had lower NPOC concentration than subalpine soil but
similar TDN concentration (Figure S2). For both soils, dissolved organic nitrogen (DON) and NPOC lost through leaching was higher during SCS than during GS (Table 3.H3).

### Soil nutrient dynamics (Net ammonium, nitrate and phosphate productions, P availability)

WIPE strongly increased total extracted ammonium at all temperatures (Figure 4A; Table 3.H1). In contrast, total extracted nitrate was negatively impacted by WIPE. For soils subjected to WIPE net nitrate production was negatively influenced by temperature. Total extracted phosphate in soils was higher when WIPE occurred.

Alpine soil warming had no effect on extracted ammonium between 8°C and 12°C but it increased significantly at high temperature (30°C). Warming positively influenced extracted nitrate (doubling between 8°C and 12°C) but decreased soil available P contents (Table 3.H2).

Total extracted ammonium and phosphate were lower for alpine GS warmed than subalpine GS. However, available P and total nitrate extraction were higher for alpine GS warmed than subalpine GS (Table 3.H3).

### OM characterisation in soil (DRIFT) and leachate (SUVA, UV fluorescence)

Relative aliphatic index on post-incubation soils decreased with WIPE. Conversely, CC and CO indices increased with WIPE (Figures 6). WIPE had no effect on the chemical structure of OM found in the leachates (Figures 6). The OM fluorescence index in water that saturated the soil decreased with temperature and decreased over the course of the incubation (F1, F2, F4; Table 3.H1).

The aliphatic index was found to decrease as a result of warming, while the C=O and C=C indexes exhibited an increase. A decrease of the OM fluorescence index in leachates with temperature was observed: i.e., there was a decrease of the proportion of protein-like compared to the proportion of humic-like molecules (Figures 6.D & Table 3.H2). Similarly, we observed an increase in SUVA_254 proportional to temperature (Figures 6.C & Table 3.H2). The results were similar at the 2^nd^ (L2; Figure S3) and 3^rd^ leaching (Figure 6).

Alpine soil IR analysis before incubation showed higher CH and C=O indices and lower C=C index than for subalpine soils (Figure 6). The values at 0°C were not in line with the ones at other temperatures. A lower CH index and higher C=C and C=O indices were observed at 0°C than at 8 and 12°C. Although the CH index was higher for alpine soils than for subalpine before incubation, it became similar for warmed alpine GS and subalpine GS at the end of incubation (Table 3.H3).
WIPE effects on mineralization processes

First, we observed a boost of C-mineralization (respiration) and ammonification following saturation with precipitation water (Figure 3B). This could be an experimental consequence caused by the degradation of the soil structure and of the physical mineral protection of SOM or clay opening due to water (Virto et al. 2010; Moni et al. 2010). However, all treatments were disturbed by water flow during leaching, and increase in mineralization was not observed. Furthermore, previous studies have demonstrated the impact of precipitation water inflows (snowmelt/rainfall) on mineralization (Legay et al. 2013; Gavazov et al. 2017). This period probably led to an increase in OM mineralization due to nutrient supply (Kuzyakov et al. 2000; Kuzyakov 2010). Indeed, the snowpack acted as an atmospheric filter, accumulating nutrients that reached the soil during snowmelt (Harder et al. 2000; Clément et al. 2012), while rainfall events captured volatile nutrients from the atmosphere (Rose et al. 2015; Bourgeois et al. 2018, 2019). NPOC and nutrients contained in raw snowmelt water were therefore supplied to the soil during WIPE and could generate an increase in SOM mineralization (priming), and particularly labile SOM mineralization. Interestingly, the over-mineralization of C after water saturation compensated for the lack of respiration during saturation due to anoxic conditions. Contrastingly, WIPE had limited effect on phosphorus dynamics, despite the potential for significant phosphorus losses due to leaching as phosphate (Su et al. 2011). Phosphorus dynamics are influenced not only by mineralization but also by sorption and desorption processes, which are affected by pH, temperature, iron and aluminium oxides (CBD-extractable iron ≈ 2 g/kg, CBD-extractable aluminium ≈ 0.4 g/kg for both sites), and by the ion balance between charged soil particles and the soil solution (Wu et al. 2013; Asomaning 2020). For example, Hazlett et al. (1992) showed *in situ* that snowmelt could act as ionic pulse, leading to increase in $NO_3^-$ content and desorption of other ions like $SO_4^{2-}$ or $PO_4^{3-}$.

It was anticipated that WIPE would create anaerobic soil conditions; however, we did not measure any discernible emissions of $CH_4$ and $N_2O$ (data not shown). The detection limit of 20 ppm for methane with our Agilent 490 µGC was probably too high to detect current methane (Table 2). It is possible that WIPE-related anaerobic soil conditions may have either inhibited nitrification and promoted denitrification and/or dissimilatory nitrate reduction to ammonium (DNRA), as suggested by the lower net quantity of nitrate produced for soils with WIPE. These results align with those of other studies indicating that WIPE (snowmelt or rainfall) enhanced denitrification, and constituted a pivotal period for annual nitrogen losses from the ecosystem (Morse et al. 2015). The higher production of ammonium in soils subjected to WIPE may be attributed to DNRA. Although fluctuating aerobic-anaerobic conditions were expected to favour $N_2O$ production (Wrage-Mönnig et al. 2018), we did not
detect any N₂O during the WIPE unless N₂O emissions were below the detection limit (20 ppm) (Table 2). Nevertheless, the observed redox traits suggest that the redox conditions may have been conducive to methanogenesis and denitrification. To confirm these hypotheses, further analysis of the enzymatic activity of methanogenesis and denitrification (DEA) is necessary.

Our findings indicated that WIPE played a pivotal role in nutrient loss through leaching (NH₄⁺ and PO₄³⁻) and potentially through denitrification (NO₃⁻), a result that is consistent with the findings of other studies (Campbell et al. 2002; Piatek et al. 2005). Additionally, the supply of nutrients and labile organic matter (OM) resulted in enhanced mineralization (both C and N).

## Sensitivity of mineralization rates to warming

### Warming over the growing season

Prior research has indicated a pronounced increase in C respiration in response to elevated temperatures above 10°C in cold environments (Nadelhoffer et al. 1991; Reichstein et al. 2000; Bekku et al. 2003; Moni et al. 2010). Here, our results suggested a strong sensitivity of alpine SOM mineralization to a 4°C increase, with a doubling of the respiration rate, and Q₁₀ values (9.42) between 8 and 12°C (Tables 1 & 2). The low Q₁₀ values between 8/12°C and 30°C suggested that the optimum temperature for respiration was exceeded at 30°C. SOM characteristics were affected by the majority of treatments, indicating that the majority of mineralization occurred in labile SOM throughout the course of the experiment. The IR indexes substantiated the hypothesis that alpine soils contain greater stocks of labile organic matter (Leifeld et al. 2005, 2009; Budge et al. 2011; RMQS 2017; Ginet-calloud 2020) compared to most other soil types. This is due to the fact that lower temperatures further limit mineralization (Leifeld et al. 2005, 2009; Budge et al. 2011; RMQS 2017; Ginet-calloud 2020). By slightly increasing the temperature, this limitation became less important (high Q₁₀ value), and labile OM could be quickly mineralised (Khedim et al. 2023). Another mechanism to explain the carbon loss associated with global warming could be changes in the enzymatic activity of soil microorganisms. For example, Chen et al. (2020) had shown that reductions in soil C stocks with warming were associated with increased ratios of ligninase to cellulase activity. Analysis of extracellular enzyme activity would be useful to check these results, although such changes are unlikely given the short duration of the experiment.

Warming from 8 to 12°C tended to increase the proportion of nitrate compared to the proportion of ammonium in soils, suggesting that nitrification was promoted. The response to warming of leached phosphates was not significant, aligning with findings from prior studies indicating no increase in phosphatase activities with warming (Zhou et al. 2021) (Table 2). Our findings suggest that
climate warming exerts limited influence on soil phosphorus concentrations, yet it may potentially exacerbate the scarcity of this nutrient in alpine ecosystems (Luo et al. 2022).

## Warming over the WIPE

Climate change modelling scenarios in the Alps have predicted an earlier melting of the snowpack, a sharper increase in temperature at the end of winter (warm spell), and an increase in the number of snowmelt and rain-on-snow events during the year (Gobiet et al. 2014). Consequently, higher nutrient losses could be expected during these key moments for alpine ecosystems. Yet, it was shown that losses were limited by the development of the microbial biomass (which acted as a nutrient buffer through immobilisation) at the time of snowmelt (Brooks et al. 1998). However, this microbial nutrient immobilisation was suspected to become less effective with earlier snowpack removal due to warming, and inorganic N losses could therefore increase (Brooks et al. 1998; Jusselme et al. 2016). Finally, the results of our study indicated that if the temperature increases during snowmelt, N mineralization (ammonification) and denitrification could also increase potentially resulting in additional net loss of inorganic N for the alpine ecosystem.

## Increase in extreme events

Soil incubation at 30°C illustrated what could happen during heat wave and showed that mineralization rates were highly and positively impacted. Such extreme climatic events, which were becoming longer as the climate warmed up, could have significant effects on SOM mineralization rates and leached elements from the soil related to strong increases in carbon respiration and nitrogen mineralization (net ammonification) and net nitrification (including N₂O production) (Stanford et al. 1975; Leirós et al. 1999; Guntiñas et al. 2012).

Finally, our results suggested that warming in alpine soil led to a significant mineralisation boost (both C and N), especially on labile SOM (i.e., which required less activation energy like aliphatic chain).

## *Changes in alpine seasonal pattern of mineralization rates: towards subalpine dynamics?*

Comparison between alpine and subalpine sites should be done carefully since they exhibited different soil properties (Table S1) and vegetations. We measured higher respiration rate for alpine GS warmed at 12°C than for subalpine GS. With warming, an increase in the productivity of the alpine ecosystem was anticipated to approach the average productivity of the subalpine ecosystem. However, we suggested that the rate of alpine SOM mineralization could be higher than in the subalpine ecosystem with warming, and this could potentially lead to a SOM decrease in alpine soils (Alatalo et al. 2017). Comparing the alpine soil DRIFT indexes (also supported by OM fluorescence index and SUVA on leachates), we found that the change between pre- and post-incubation was more
important for the alpine than for the subalpine soils, especially at 12°C (Figure 6). This indicated that the proportion of labile SOM (CH index) decreased more significantly in the alpine soils than in the subalpine ones.

Our findings corroborate those of previous studies indicating the presence of labile elements in alpine SOM. NPOC (used as an indicator of OM stability, Marschner and Kalbitz 2003; Guigue et al. 2014), SUVA and UV fluorescence all showed that soil organic matter at 0°C remained labile, though IR indices were not in agreement with this observation. We suggested that leaching events removed a considerable portion of labile OM from the soil, reducing the soil CH index at the end of incubation. In view of the high NPOC and DON content, we hypothesised that dead microbial biomass due to low temperatures (cell lysis) participated to this loss of labile OM (Gavazov et al. 2017). The higher respiration rates in alpine than in subalpine soils confirmed that alpine microbial communities were more adapted to low temperatures than subalpine communities, probably because the taxonomic composition of microorganisms influenced mineralization rates (Baptist et al. 2008; Ding et al. 2016).

Likewise, nitrogen dynamics at temperatures close to 0°C were known to be strongly dependent on soil temperature variations (between -2°C and 2°C) and soil moisture (Chapuis-Lardy et al. 2007; Miller et al. 2007). For example, in tundra soils, wet soils showed nitrogen mineralization only at temperatures above 0°C (Giblin et al. 1991; Clein and Schimel 1995), while dry soils showed nitrogen mineralization only at negative temperatures (Brooks et al. 1996, 1997; Schimel et al. 2004). The thermal amplitude of the thermostatic chamber could lead to a shift in nitrogen dynamics, which may explain the observed variations in N₂O measurements since freezing may stimulate N₂O fluxes (Nielsen et al. 2001). It has been shown that, in cold ecosystems, N₂O dynamics can vary from production, proportional to CO₂ respiration (Brummell et al. 2014), to consumption (Chapuis-Lardy et al. 2007) depending on light conditions (Stewart et al. 2012). It can be assumed that this production was due to the inhibition of nitrous oxide reductase enzyme activity (Nos) during denitrification (Barnard et al. 2005; Griffith 2016), with denitrification at 0°C being favoured due to frozen soil anaerobic conditions. Finally, N₂O measured in our study could also result from the predominance of denitrification by nitrifiers in cold environments (Ma et al. 2007; Siciliano et al. 2009).

## Extrapolation to annual and catchment scales

By extrapolating our results to a yearly scale, we suggested a significant shift due to warming in annual mineralization rates (heterotrophic respiration, ammonification). A change in the nature of the SOM stocks was also expected with a relative increase in the C=C index and a relative decrease in the CH index (Khedim et al. 2023). Furthermore, labile organic matter (OM) stocks may undergo mineralization with subsequent release as greenhouse gases (GHGs). It can be reasonably deduced
that climate warming, both during SCS (Nielsen et al. 2001) and GS (Alatalo et al. 2017), could
potentially result in elevated nitrogen and carbon losses for the ecosystem. Yet, the soil behaviour as
a source or sink of C and N remained to be determined because the increase in productivity and
subsequent SOM inputs remained to be integrated in the balance.

Conclusion

To conclude, we found that mineralisation rates (C, N, P) in alpine soils were structured seasonally and significant throughout the year. Snowmelt (i.e., WIPE) was confirmed as a key period for alpine soil C, N, P cycles, as it initiated a mineralisation boost significantly increasing nutrient availability for plants during the following GS. We suggest that this mineralisation boost was related to the nutrients contained in the snowpack (atmospheric deposition), which was released during snowmelt into the soil. We also showed that warming might destabilize alpine SOM stocks by modifying their mineralization rates and seasonal distribution, as suggested by the significant increase of mineralization rates with a 4°C warming, and the joined decrease in labile SOM. Further in-depth studies on related alpine soil microbial activities remain needed to better model the annual mineralization budget, and to anticipate the soil behaviour as a source or sink of greenhouse gases. These analyses must concentrate on the alterations in the duration and frequency of snow melts and extreme events, as well as temperature and precipitation patterns, which are anticipated to undergo transformation with the advent of warming.

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## Acknowledgments, Statements & Declarations

This study was funded by the French Agence Nationale de la Recherche (ANR) through the TransAlp project (ANR-20-CE02-0021), and was conducted with the help of the Lautaret Garden- UMS 3370 (Univ. Grenoble Alpes, CNRS, SAJF, 38000 Grenoble, France), a member of the Zone Atelier Alpes
and eLTER network and of AnaEE-France (ANR-11-INBS-0001AnaEE-Services, Investissements d'Avenir frame). This research was conducted within the Long-Term Socio-Ecological Research (LTSER) platform Lautaret-Oisans, a site of the European Research Infrastructure eLTER. This work was carried out by Nicolas BONFANTI, Jean-Christophe CLEMENT, Annie MILLERY-VIGUES, Tamara MÜNKEMÜLLER, Yves PERRETTE and Jérôme POULENARD. The authors declare that no other funds, grants, or other support were received during the preparation of this manuscript. The authors have no relevant financial or non-financial conflict-of-interests to disclose. All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Nicolas BONFANTI, Jean-Christophe CLEMENT, Annie MILLERY-VIGUES, Tamara MÜNKEMÜLLER, Yves PERRETTE and Jérôme POULENARD. The first draft of the manuscript was written by Nicolas BONFANTI and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. The datasets generated during and analysed during the current study are not publicly available but are available from the corresponding author on reasonable request.
<figure><img src="image_2.png" /><figcaption>Figure 1: Climatic and phenological conditions of sampling locations (soil temperature and moisture were at 5cm depth during one year) and map of sampling sites. 'Study area' delimited a homogeneous property zone (vegetation, soil, topography, etc.). APx were the 6 soil pits where we sampled the soil in alpine site and SPx in subalpine site.</figcaption></figure>
<figure><img src="image_3.png" /></figure>

Figure 2: Experimental design from sampling to post incubation analysis; each modality was replicated with 6 samples. APx were the 6 soil pits where we sampled the soil in alpine site and SPx in subalpine site, pooled together to form a composite sample. WIPE was water inflow period, i.e., when soil was saturated with snowmelt water. We conducted 4 WIPE throughout the experiment (W1, W2, W3, W4). L1, L2, L3 were the leaching with distilled water to follow nitrogen and phosphorus dynamics.
<figure><img src="image_4.png" /><figcaption>Figure 3: Respiration rates - A: Cumulative C respiration (mean ±sd); B: C respiration throughout the incubation. ANOVA results are shown in Table 3. SCS=snow cover season; GS=growing season; WIPE=water inflow periods</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 4: Mean ± sd of carbon and nitrogen in soil leachates (L2+L3) for each treatment (log y scale): A: non-purgeable organic carbon; B: total dissolved nitrogen; C: dissolved inorganic nitrogen; D: dissolved organic nitrogen</figcaption></figure>

ANOVA results are shown in Table 3. SCS=snow cover season; GS=growing season; WIPE=water inflow periods
<figure><img src="image_6.png" /><figcaption>Figure 5: Total extraction (from leachate and soil, mean ± sd) for each treatment of ammonium (A), nitrate (B) and phosphate extracted (C); P available before and after incubation (D); note the log y scale for A, B & C</figcaption></figure>

ANOVA results are shown in Table 3. SCS=snow cover season; GS=growing season; WIPE=water inflow periods
<figure><img src="image_7.png" /><figcaption>Figure 6: Change in OM chemistry for all treatments: - in soil: CH index (A); C=C index (B); - in leachate (post incubation leaching: L3): SUVA_254 (C); OM fluorescence index (note the log y scale) (D)</figcaption></figure>

ANOVA results are shown in Table 3. SCS=snow cover season; GS=growing season; WIPE=water inflow periods
Tables

<table><caption>Table 1: Q₁₀ values for alpine and subalpine calculated between C respiration for two temperatures (mean ± sd)</caption><thead><tr><th rowspan="2">Temperature</th><th colspan="3">Alpine</th><th colspan="2">Subalpine</th></tr><tr><th>8°C</th><th>12°C</th><th>30°C</th><th>12°C</th><th>30°C</th></tr></thead><tbody><tr><td>0°C</td><td>4,82 (0,99)</td><td>6,03 (0,82)</td><td>2,20 (0,09)</td><td>6,73 (1,40)</td><td>2,67 (0,19)</td></tr><tr><td>8°C</td><td></td><td>9,42 (3,32)</td><td>1,65 (0,1)</td><td></td><td></td></tr><tr><td>12°C</td><td></td><td></td><td>1,12 (0,07)</td><td></td><td>1,44 (0,10)</td></tr></tbody></table>

<table><caption>Table 2: Comparison of our results with other in situ or in vitro studies</caption><thead><tr><th>Variable</th><th>Results in this study</th><th>Results in others studies + references</th></tr></thead><tbody><tr><td>Methane fluxes during WIPE</td><td>0 Detection threshold of GC: 900, 920, 3400 µg C-CH<sub>4</sub>.m<sup>-2</sup>.h<sup>-1</sup> at 8, 12 and 30°C respectively (according to the incubation time and the detection of 20 ppm of GC)</td><td>140 and 440 µg C-CH<sub>4</sub>.m<sup>-2</sup>.h<sup>-1</sup> (Mosier et al. 1993; Wang and Bettany 1995, 1997; Wang et al. 2021)</td></tr><tr><td>N<sub>2</sub>O fluxes during WIPE</td><td>0 Lower net nitrate production with WIPE than without WIPE</td><td>0.5 kg N-N<sub>2</sub>O.ha<sup>-1</sup>.y<sup>-1</sup> (Morse et al., 2015)</td></tr><tr><td>Inorganic nitrogen during WIPE</td><td>Higher net ammonium production with WIPE than without WIPE</td><td>Higher denitrification potential for soils with higher moisture content (Miller et al. 2007)</td></tr><tr><td>Soil respiration change with warming</td><td>Alpine soil: 46 and 105 µg C-CO<sub>2</sub>.g<sub>soil</sub><sup>-1</sup>.day<sup>-1</sup> 8°C and 12°C respectively</td><td>Anaerobic conditions may cause DNRA (Campbell et al. 2007)<br>Tundra soil: 120 and 336 µg C-CO<sub>2</sub>.g<sub>soil</sub><sup>-1</sup>.day<sup>-1</sup> at 8°C and 16°C (Bekku et al. 2003)<br>Subalpine grasslands soil: 10 and 120 µg C-CO<sub>2</sub>.g<sub>soil</sub><sup>-1</sup>.day<sup>-1</sup> at 5°C and 15°C (Reichstein et al. 2000)</td></tr><tr><td>Q<sub>10</sub> value</td><td>9.42 between 8 and 12°C<br>1.12 between 12 and 30°C</td><td>Mean Q<sub>10</sub> value for cold ecosystems ≈ 4 (Barrett et al. 2006; Elberling et al. 2006; Hamdi et al. 2013)</td></tr><tr><td>Cumulative mineralised C</td><td>3 and 5 mg C-CO<sub>2</sub>.g<sub>OM</sub><sup>-1</sup> at 0°C over 8 weeks of incubation for subalpine and alpine soils respectively</td><td>2.5 mg C-CO<sub>2</sub>.g<sub>OM</sub><sup>-1</sup> after 16 weeks of incubation (Miller et al. 2007) in vitro<br>Fluxes between 0.957 to 6.8 g C-CO<sub>2</sub>.m<sup>-2</sup>.day<sup>-1</sup> (Sommerfeld et al. 1993) in situ</td></tr><tr><td>OM stability indexes with warming</td><td>Decrease in IR and UV fluorescence indices</td><td>Decrease in IR and RockEval indices (Khedim et al., 2023)</td></tr><tr><td>N<sub>2</sub>O production at 0°C</td><td>5.9 mg N-N<sub>2</sub>O.m<sup>-2</sup>.day<sup>-1</sup></td><td>2.01 mg N-N<sub>2</sub>O.m<sup>-2</sup>.day<sup>-1</sup> (Miller et al. 2007) in vitro<br>0.065 - 0.405 mg N-N<sub>2</sub>O.m<sup>-2</sup>.day<sup>-1</sup> (Roswall et al. 1975; Sommerfeld et al. 1993) in situ</td></tr><tr><td>P available</td><td>Decrease in bioavailable phosphorus with temperature increase</td><td>Decrease in bioavailable phosphorus with temperature increase (Nadelhoffer et al. 1991; Li et al. 2020)</td></tr></tbody></table>
<table><caption>Table 3: Mean values (n=6; ± sd) of variables and pairwise Tukey post-hoc HSD test for each hypothesis and two-ways ANOVA results for H2 (Temp*WIPE); p < 0.001 ‘***’; <0.01 ‘**’; <0.05 ‘*’; <0.1 ‘.’; = p<0.05; = p > 0.05; SCS=snow cover season; GS=growing season; WIPE=water inflow periods</caption><thead><tr><th rowspan="2"></th><th rowspan="2">Gas analysis</th><th colspan="4">Total leached</th><th colspan="3">Total extracted (leached + post-incubation extraction)</th><th colspan="2">Soil post-incubation</th><th colspan="2">Leachate post-incubation</th></tr><tr><th>Cumul<br>C<sub>respiration</sub><br>mgc/g<sub>soc</sub></th><th>NPOC<br>µgC/g<sub>soc</sub></th><th>TDN<br>µgN/g<sub>soc</sub></th><th>DIN<br>µgN/g<sub>soc</sub></th><th>DON<br>µgN/g<sub>soc</sub></th><th>NH<sub>4</sub><sup>+</sup><br>µgN-NH<sub>4</sub><sup>+</sup>/g<sub>soc</sub></th><th>NO<sub>3</sub><sup>-</sup><br>µgN-NO<sub>3</sub><sup>-</sup>/g<sub>soc</sub></th><th>PO<sub>4</sub><sup>3-</sup><br>µgP-PO<sub>4</sub><sup>3-</sup>/g<sub>soc</sub></th><th>P<sub>available</sub><br>µgP/g<sub>soc</sub></th><th>CH<sub>index</sub><br>RATIO</th><th>SUVA<sub>254</sub><br>RATIO</th><th>OM<sub>fluo_index</sub><br>RATIO</th></tr></thead><tbody><tr><td colspan="15" style="text-align: center; font-weight: bold;">H1 – Consequences of the snowmelt</td></tr><tr><td>Alpine GS</td><td>56.4 (±5.8)</td><td>81.0 (± 9.9)</td><td>16.5 (± 5.3)</td><td>8.5 (± 2.1)</td><td>6.3 (± 1.0)</td><td>3.26 (± 0.51)</td><td>9.25 (± 0.62)</td><td>3.85 (± 1.00)</td><td>167.0 (± 35.9)</td><td>0.381 (± 0.008)</td><td>0.091 (± 0.024)</td><td>1.318 (± 0.086)</td></tr><tr><td>Alpine WIPE</td><td>61.4 (±5.5)</td><td>1487.2 (± 1016.1)</td><td>40.0 (± 7.9)</td><td>16.9 (± 11.9)</td><td>23.7 (± 4.2)</td><td>4.24 (± 1.14)</td><td>14.52 (± 11.88)</td><td>8.95 (± 1.21)</td><td>151.2 (± 51.6)</td><td>0.363 (± 0.017)</td><td>0.125 (± 0.026)</td><td>1.264 (± 0.118)</td></tr><tr><td></td><td>NS</td><td></td><td></td><td>NS</td><td></td><td>NS</td><td></td><td></td><td>NS</td><td>NS</td><td></td><td>NS</td><td>NS</td></tr><tr><td>Alpine GS warmed</td><td>142.0 (±15.7)</td><td>75.1 (± 11.6)</td><td>28.0 (± 16.9)</td><td>20.0 (± 13.2)</td><td>8.0 (± 3.7)</td><td>2.99 (± 0.36)</td><td>22.45 (± 15.45)</td><td>4.26 (± 2.01)</td><td>126.1 (± 9.2)</td><td>0.371(± 0.014)</td><td>0.071 (± 0.003)</td><td>1.242 (± 0.019)</td></tr><tr><td>Alpine WIPE warmed</td><td>111.7 (±14.0)</td><td>1261.5 (± 284.5)</td><td>40.3 (± 4.3)</td><td>9.0 (± 2.2)</td><td>27.0 (± 8.5)</td><td>8.00 (± 1.26)</td><td>5.11 (± 1.25)</td><td>8.14 (± 1.44)</td><td>122.8 (± 7.8)</td><td>0.358 (± 0.013)</td><td>0.093 (± 0.014)</td><td>1.331 (± 0.120)</td></tr><tr><td></td><td>*</td><td></td><td></td><td></td><td></td><td>NS</td><td></td><td></td><td>NS</td><td>NS</td><td></td><td>NS</td><td>NS</td></tr><tr><td>Alpine 30°C</td><td>178.0 (±5.2)</td><td>178.0 (± 53.1)</td><td>65.8 (± 7.9)</td><td>47.4 (± 8.9)</td><td>18.4 (± 2.3)</td><td>13.49 (± 5.37)</td><td>40.59 (± 10.72)</td><td>5.77 (± 1.88)</td><td>163.8 (± 11.9)</td><td>0.360 (± 0.007)</td><td>0.406 (± 0.217)</td><td>1.146 (± 0.114)</td></tr><tr><td>Alpine WIPE 30°C</td><td>162.8 (±12.6)</td><td>3339.3 (± 665.1)</td><td>319.2 (± 57.4)</td><td>165.9 (± 17.1)</td><td>166.9 (± 49.4)</td><td>172.15 (± 17.86)</td><td>3.96 (± 2.61)</td><td>13.67 (± 1.52)</td><td>190.5 (± 30.6)</td><td>0.336 (± 0.008)</td><td>0.498 (± 0.166)</td><td>1.206 (± 0.206)</td></tr><tr><td></td><td>NS</td><td></td><td></td><td>*</td><td></td><td>***</td><td></td><td></td><td>NS</td><td></td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td colspan="15" style="text-align: center; font-weight: bold;">H2 – Warming destabilising SOM stocks</td></tr><tr><td>Alpine GS</td><td>56.4 (±5.8) a</td><td>81.0 (± 9.9) a</td><td>16.5 (± 5.3) a</td><td>8.5 (± 2.1) a</td><td>6.3 (± 1.0) a</td><td>3.26 (± 0.51) a</td><td>9.25 (± 0.62) a</td><td>3.85 (± 1.00) a</td><td>167.0 (± 35.9) a</td><td>0.381(± 0.008) a</td><td>0.090 (± 0.024) a</td><td>1.318 (± 0.086)</td></tr><tr><td>Alpine GS warmed</td><td>142.0 (±15.7) b</td><td>75.1 (± 11.6) a</td><td>28.0 (± 16.9) a</td><td>20.0 (± 13.2) ab</td><td>8.0 (± 3.7) a</td><td>2.99 (± 0.36) a</td><td>22.45 (± 15.45) ab</td><td>4.26 (± 2.01) a</td><td>126.1 (± 9.2) a</td><td>0.371(± 0.014) a</td><td>0.071 (± 0.003) a</td><td>1.242 (± 0.019)</td></tr><tr><td>Alpine 30°C</td><td>178.0 (±5.2) c</td><td>178.0 (± 53.1) b</td><td>65.8 (± 7.9) b</td><td>47.4 (± 8.9) b</td><td>17.0 (± 2.1) b</td><td>13.49 (± 5.37) b</td><td>40.59 (± 10.72) b</td><td>5.77 (± 1.88) a</td><td>163.8 (± 11.9) a</td><td>0.360(± 0.007) a</td><td>0.406 (± 0.217) b</td><td>1.146 (± 0.114)</td></tr><tr><td></td><td>***</td><td></td><td></td><td>*</td><td></td><td>NS</td><td></td><td></td><td>NS</td><td>NS</td><td>***</td><td>NS</td><td>NS</td></tr><tr><td>Alpine WIPE</td><td>61.4 (±5.5) a</td><td>1487.2 (± 1016.1) a</td><td>40.0 (± 7.9) a</td><td>16.9 (± 11.9) a</td><td>23.7 (± 4.2) a</td><td>3.04 (± 0.65) a</td><td>14.52 (± 11.88) a</td><td>8.95 (± 1.21) a</td><td>151.2 (± 51.6) ab</td><td>0.363(± 0.017) a</td><td>0.125 (± 0.026) a</td><td>1.264 (± 0.118)</td></tr><tr><td>Alpine WIPE warmed</td><td>111.7 (±14.0) b</td><td>1261.5 (± 284.5) a</td><td>40.3 (± 4.3) a</td><td>9.0 (± 2.2) a</td><td>24.9 (± 7.9) a</td><td>8.00 (± 1.26) a</td><td>5.11 (± 1.25) ab</td><td>8.14 (± 1.44) a</td><td>122.8 (± 7.8) a</td><td>0.358(± 0.013) ab</td><td>0.093 (± 0.014) a</td><td>1.331 (± 0.120)</td></tr><tr><td>Alpine WIPE 30°C</td><td>162.8 (±12.6) c</td><td>3339.3 (± 665.1) b</td><td>319.2 (± 57.4) b</td><td>165.9 (± 17.1) b</td><td>166.9 (± 49.4) b</td><td>172.15 (± 17.86) b</td><td>3.96 (± 2.61) b</td><td>13.67 (± 1.52) b</td><td>190.5 (± 30.6) b</td><td>0.336(± 0.008) b</td><td>0.498 (± 0.166) b</td><td>1.206 (± 0.206)</td></tr><tr><td></td><td>***</td><td></td><td></td><td></td><td></td><td>***</td><td></td><td></td><td>*</td><td>*</td><td>***</td><td>NS</td><td>NS</td></tr><tr><td>Temp / WIPE</td><td>*** / *</td><td>*** / ***</td><td>*** / ***</td><td>*** / **</td><td>*** / ***</td><td>*** / ***</td><td>NS / ***</td><td>*** / ***</td><td>** / NS</td><td>*** / ***</td><td>*** / ***</td><td>*** / ***</td><td>NS</td></tr><tr><td>Temp*WIPE</td><td>**</td><td>NS</td><td>***</td><td>***</td><td>***</td><td>***</td><td>***</td><td>*</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td colspan="15" style="text-align: center; font-weight: bold;">H3 – Comparison Alpine warmed / subalpine</td></tr><tr><td>Alpine SCS</td><td>16.6 (± 1.1)</td><td>162.2 (± 19.0)</td><td>38.0 (± 12.9)</td><td>11.8 (± 7.3)</td><td>22.6 (± 2.7)</td><td>3.23 (± 0.41)</td><td>12.49 (± 8.27)</td><td>6.31 (± 1.62)</td><td>148.3 (± 21.9)</td><td>0.366(± 0.016)</td><td>0.063 (± 0.026)</td><td>1.780 (± 0.509)</td></tr><tr><td>Subalpine SCS</td><td>9.6 (± 2.2)</td><td>253.3 (± 54.1)</td><td>31.7 (± 8.8)</td><td>1.6 (± 0.9)</td><td>30.1 (± 8.2)</td><td>4.99 (± 1.62)</td><td>0.82 (± 0.73)</td><td>9.24 (± 2.15)</td><td>86.0 (± 7.0)</td><td>0.354(± 0.014)</td><td>0.070 (± 0.031)</td><td>1.833 (± 0.464)</td></tr><tr><td></td><td>***</td><td>NS</td><td>NS</td><td>***</td><td>NS</td><td>NS</td><td>***</td><td>NS</td><td>***</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>Alpine GS warmed</td><td>142.0 (±15.7)</td><td>75.1 (± 11.6)</td><td>28.0 (± 16.9)</td><td>20.0 (± 13.2)</td><td>8.0 (± 3.7)</td><td>2.99 (± 0.36)</td><td>22.45 (± 15.45)</td><td>4.26 (± 2.01)</td><td>126.1 (± 9.2)</td><td>0.371 (± 0.014)</td><td>0.071 (± 0.003)</td><td>1.242 (± 0.019)</td></tr><tr><td>Subalpine GS</td><td>94.1 (±10.3)</td><td>117.4 (± 17.0)</td><td>19.5 (± 1.4)</td><td>12.5 (± 1.2)</td><td>7.1 (± 0.7)</td><td>5.40 (± 0.46)</td><td>12.81 (± 1.03)</td><td>6.95 (± 0.69)</td><td>67.9 (± 17.2)</td><td>0.374 (± 0.009)</td><td>0.152 (± 0.070)</td><td>1.161 (± 0.035)</td></tr><tr><td></td><td>***</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>***</td><td>***</td><td>NS</td><td>NS</td><td>**</td><td>NS</td></tr><tr><td>Alpine WIPE warmed</td><td>111.7 (±14.0)</td><td>1261.5 (± 284.5)</td><td>40.3 (± 4.3)</td><td>9.0 (± 2.2)</td><td>24.9 (± 7.9)</td><td>8.00 (± 1.26)</td><td>5.11 (± 1.25)</td><td>8.14 (± 1.44)</td><td>122.8 (± 7.8)</td><td>0.358 (± 0.013)</td><td>0.093 (± 0.014)</td><td>1.331 (± 0.120)</td></tr><tr><td>Subalpine WIPE</td><td>110.2 (±15.2)</td><td>1443.8 (± 543.9)</td><td>26.7 (± 2.6)</td><td>25.5 (± 34.4)</td><td>22.4 (± 2.8)</td><td>25.59 (± 34.19)</td><td>1.05 (± 0.05)</td><td>14.92 (± 3.93)</td><td>61.9 (± 18.3)</td><td>0.372 (± 0.009)</td><td>0.119 (± 0.026)</td><td>1.181 (± 0.060)</td></tr><tr><td></td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td><td>***</td><td>***</td><td>***</td><td>NS</td><td>NS</td><td>NS</td><td>NS</td></tr><tr><td>Alpine 30°C</td><td>178.0 (±5.2)</td><td>178.0 (± 53.1)</td><td>65.8 (± 7.9)</td><td>47.4 (± 8.9)</td><td>18.4 (± 2.3)</td><td>13.49 (± 5.37)</td><td>40.59 (± 10.72)</td><td>5.77 (± 1.88)</td><td>163.8 (± 11.9)</td><td>0.360 (± 0.007)</td><td>0.406 (± 0.217)</td><td>1.146 (± 0.114)</td></tr><tr><td>Subalpine 30°C</td><td>182.3 (±9.6)</td><td>187.2 (± 44.8)</td><td>123.9 (± 47.4)</td><td>101.6 (± 37.7)</td><td>22.3 (± 10.1)</td><td>48.08 (± 2.70)</td><td>102.86 (± 35.58)</td><td>8.14 (± 0.41)</td><td>82.6 (± 19.0)</td><td>0.351 (± 0.007)</td><td>0.200 (± 0.028)</td><td>1.346 (± 0.311)</td></tr><tr><td></td><td>NS</td><td>NS</td><td>*</td><td>NS</td><td>NS</td><td>***</td><td>NS</td><td>NS</td><td>***</td><td>NS</td><td>*</td><td>NS</td><td>NS</td></tr></tbody></table>