Rapid response of soil GHG emissions and microbial parameters to the addition of biochar and the freeze-thaw cycle*

. A reduction in snow cover makes soil more exposed to freezing-thawing processes. We tested the rapid response of greenhouse gas (GHG) emissions in fertilized soil to biochar addition under the freeze-thaw cycle. The soil (at a moisture of 55% water holding capacity) was enriched with biochar at a rate of 20 t ha -1 , frozen at -20°C for 12 h and thawed at 20°C. The control was soil without biochar and with biochar incubated at temperature of 20°C. Unfrozen soil was a CO 2 and N 2 O source and a weak sink for CH 4 . The GHG fluxes were not significantly altered by biochar, although biochar-treated soil emitted CH 4 . The frozen soil emitted all the tested GHGs during the thawing period. A pulse of CO 2 and CH 4 emissions (and N 2 O in soil with biochar) occurred in the period of rapid temperature growth, while GHG fluxes reached levels similar to unfrozen soil after temperature stabilization at 20°C. In frozen soil, the addition of biochar significantly increased the CH 4 and N 2 O peak only in the first phase of thawing with a dynamic temperature growth. Biochar changed microbial parameters, therefore we assume that both physical and biological mechanisms could be responsible for GHG emissions in frozen soils.


INTRODUCTION
The soil ecosystem contributes to global warming by emitting and absorbing greenhouse gases (GHGs).Carbon dioxide (CO 2 ) is emitted as a result of microbial decomposition of soil organic matter, root respiration, rhizomicrobial respiration and the priming effect, and from human activity including land use changes, deforestation and forest fires (Kuzyakov, 2006;Li et al., 2015;Rahman, 2013).Methane (CH 4 ) is a gas with a 27-fold higher global warming potential (GWP) than CO 2 (IPCC, 2021).It can be produced in soil under anaerobic conditions (methanogenesis) or can be absorbed through oxidation by methanotrophic bacteria (methanotrophy) (Wnuk et al., 2020).Nitrous oxide (N 2 O) is a gas with 273-fold greater contribution to global warming than CO 2 (IPCC, 2021).Soil N 2 O formation involves biotic and abiotic processes, but is not clearly understood (Butterbach-Bahl et al., 2013).The agricultural sector is a major source of N 2 O from soil fertilization and manure management.
Global climate change alters the frequency and intensity of extreme weather events, causing droughts, floods and freezing-thawing from a reduction of snow cover during winter (Kim et al., 2012;Nabizada et al., 2023).A decrease in snow cover occurs in most regions and has been confirmed in Poland (Tomczyk et al., 2021), although the opposite phenomenon has been observed in some regions (Choi et al., 2010).During the winter, the cold periods vary in length and are interrupted by periods of thaw, therefore soil freeze-thaw cycles are common in the temperate zone (Miranda-Vélez et al., 2023).Such changes in soil temperature are stressful for soil microorganisms and alter the intensity of various biochemical processes (Rosinger et al., 2022).The influence of air temperature on soil dynamics is also determined by land use and management.In 2016 in the European Union (EU), 23% of arable land was left as bare soil without any vegetation cover during winter (Eurostat, 2022).Bare soils are more exposed to erosion, leaching of nutrients and freezing than land covered with vegetation (Bo et al., 2021;Eurostat, 2022).Larger temperature fluctuations during freezing and thawing have been seen for the surface layer (up to 10 cm deep) (Bo et al., 2021), which simultaneously contributes significantly to gas exchange with the atmosphere.In addition to the frozen state of the soil itself, dynamic changes in soil temperature, hydrology and release of nutrients during freeze-thaw events particularly affect GHGs.Studies on agricultural soils have shown stimulation of respiration and N 2 O emission under freezing-thawing because of the release of decomposable organic C and N which increases denitrification (DeLuca et al., 1992;Mørkved et al., 2006).Moreover, thawing causes the gases accumulated in the soil profile during winter to be rapidly released in spring (Maljanen et al., 2007).
The significant contribution of agriculture to global warming means that it is important to identify alternatives to traditional fertilization that reduce emissions and increase C sequestration (Lehmann et al., 2011;Radawiec et al., 2023).Among various potential solutions, an ongoing focus of research is the application of biochar, which can improve soil conditions and crop yields (Ding et al., 2016).Enrichment of soils with biochar changes the physical and chemical properties of soil, e.g., it increases soil C content, pH, water and nutrient storage, improves soil aeration and thus affects soil microbiota (as summarized, e.g., by Ding et al. (2016) and Lehmann et al. (2011)).Beside practical benefits, it is important to recognize the environmental consequences of biochar application, which are not explained by freeze-thaw cycles (Li et al., 2022).Biochar may reduce GHG emissions in agricultural soil, although the mitigation potential is determined by different factors such as soil type, biochar properties, ageing and pyrolysis temperature (Feng et al., 2022;Méndez et al., 2013;Sha et al., 2019).As GHG pulse emissions occur during soil thawing (Kim et al., 2012), it is useful to recognize how biochar in soil may modify GHG fluxes during freeze-thaw cycles.The few studies in this field suggest that the GHG pulse can contribute significantly to their annual balance.Usage of a higher dose of biochar can increase CO 2 emissions more than thawing, but biochar increases the capacity of soil to absorb CH 4 by improving aeration (Liu et al., 2017).The presence of biochar in cultivated soil during the freezing-thawing cycle has been shown to reduce N 2 O emissions through decreased soil nitrification (Liu et al., 2016a), but this effect has not been confirmed in all studies (Zhou et al., 2017b).
The aim of the present study was to assess the rapid response of GHG (CO 2 , CH 4 , N 2 O) emissions and microbial parameters of arable soil with and without biochar and under the controlled simulation of the freeze-thaw event.

MATERIALS AND METHODS
A laboratory experiment was conducted on soil samples collected in the Lublin Upland, Poland (coordinates 51°14'69''N, 22°89'39''E).In recent years before sampling, data from the weather station located next to the Institute of Agrophysics, PAS in Lublin (51°13'11''N, 22°37'32''E, 207 m above sea level) confirmed that the average annual temperature in this region was between 8.5 and 10°C and the annual rainfall was between ~500 and 700 mm.During the winter period preceding soil sampling, the average temperature ranged from -1.08°C in December 2022 to 0.82°C in February 2023.Soil samples were collected in April 2023 from a fertilized field under winter wheat.The soil was classified as a fertile arable land, the management of which included a multi-year fertilization regime with annually cultivated winter wheat or oilseed rape.Five representative soil samples (surface layer, i.e., 0-20 cm depth) were collected at 1 m intervals, thoroughly homogenized to provide a representative sample and sieved to < 2 mm.The soil was classified as a Luvisol soil, with a pH of 7.61, had silt loam texture, 3.44% of clay, 54.4% of silt, 42.2% of sand, and 22.08 g kg -1 of organic C concentrations.
The physico-chemical soil parameters were determined under controlled laboratory conditions (n = 3).Particle size distribution (PSD: clay (diameter < 2 µm), silt (diameter 50-2 µm) and sand (diameter 2000-50 µm)) was determined using a Mastersizer 2000 laser diffractometer with a Hydro G dispersion unit (Malvern Ltd., Malvern, UK) (Polakowski et al., 2023).Soil organic carbon was determined with a TOC-VCPH analyser (Shimadzu, Kyoto, Japan).Soil pH was measured potentiometrically at room temperature in a soil and water slurry, with soil:water ratio of 1:2.5 w/w.Nitrate (NO 3 -) concentrations were determined potentiometrically in the soil solution using an ion-selective electrode and reagents for nitrate contents (HQ40D Portable Multi Meter analyser).Water holding capacity (WHC) of the soil and of the soil with biochar was measured based on the difference in weight of soil samples wetted in water and then dried (Priha and Smolander, 1999).
The biochar was produced in 2018 at 650°C from fir sawdust, with a pH of 7.14, concentrations of 78.46% C and 0.19% of N, and a density of 220 kg m -3 , as described previously by Walkiewicz et al. (2020).The structure and surface morphology of the biochar were detected by scanning electron microscopy (SEM).The biochar has been stored in the dark at room temperature.
Fresh soil samples of 450 g dry weight were placed in 5.2 dm -3 airtight dark vessels (n = 3).Biochar was added to half of the soil samples at an amount corresponding to a rate of 20 t ha -1 .The soil was enriched with biochar two weeks before the main experiment and preincubated at 20°C in darkness to adapt the microorganisms.Next, the samples were moistened with distilled water to a level corresponding to 55% WHC.After a three-day preincubation period, half of the samples were incubated at temperature of 20°C and the other half placed under frozen conditions (-20°C in a freezer) for 12 h and thawed at 20°C.A similar temperature gradient was used by Müller et al. (2002).After sealing the vessels, measurements of changes in gas concentrations (CO 2 , CH 4 and N 2 O) in the headspace were taken for 10 h at 20°C with a frequency of half or one hour.In frozen samples, GHG measurements started once the vessels were removed from the freezer.Changes in headspace temperature inside the vessels were monitored at each GHG measurement.At the end of the incubation period, soil samples were taken from each variant to measure pH, concentrations of C and NO 3 -, and microbial parameters (basal respiration (BR) and soil microbial biomass (C mic )).A visual overview of the experimental design is shown in Fig. 1.
GHG concentrations (CO 2 , CH 4 , and N 2 O) in the headspace were measured using a portable Gasmet DX-4040 Fourier-Transform Infrared Gas Analyzer (FTIR-GA) (Gasmet Technologie Oy, Helsinki, Finland).Prior to the measurements, the analyser was zeroed with helium.Microbial parameters included BR, C mic and metabolic quotient qCO 2 .Soils in a moisture content corresponding to 55% WHC were weighed (3 g dry mass, n = 3) into 60 cm 3 glass vessels.Soil BR was determined based on CO 2 emission after 2 h incubation at 25 °C.C mic was measured using the substrate-induced respiration (SIR) method recommending enrichment of the soil with a glucose solution (10 mg per gram of soil) and CO 2 emission analysis after 2 h incubation at 25°C (Anderson and Domsch, 1978).The CO 2 concentrations were measured chromatographically using a Shimadzu GC-14A equipped with a thermal conductivity detector (TCD) and with the use of a 2 m column (3.2 mm diameter) packed with Porapak Q (Shimadzu Corp., Kyoto, Japan).
GHG emission rates were calculated from the slope of the change in concentration with time, according to the commonly used equations taking into account air temperature, area and volume of vessel (Flessa et al., 1998;Cowan et al., 2014).The rates were calculated for each variant separately and were expressed in mg CO In the variant with soil freezing, the GHG emission rates were calculated by dividing the incubation time into two stages depending on the headspace temperature in the vessel: the initial stage of thawing (with a dynamic temperature growth) and the final stage of thawing (after temperature stabilization).Microbial biomass content was expressed in CO 2 g -1 h -1 and calculated according to Šimek and Kalčík (1998).The qCO 2 was calculated based on the BR:C mic ratio, and expressed as µg The results were statistically analysed with Statistica 13 software (StatSoft Inc., Tulsa, OK, USA).The analysis of variance (ANOVA) test was used to evaluate the significance (at the 5% level) of the differences in soil parameters between variants and in GHG emission rates, separately for each gas.Multifactorial analysis of variance (MANOVA) was used to test which of the factors (biochar, freezing-thawing or their interaction) significantly affected the GHG fluxes.Correlation analyses were conducted (at the 5% level) to recognize significant relationships between different parameters.

RESULTS
Both biochar enrichment and the freeze-thaw cycle changed soil parameters (Table 1).The addition of biochar significantly increased the water storage capacity of soil and doubled the organic C concentration.Soil pH was significantly higher in soil with biochar, which was not altered by freezing.The concentrations of NO 3 -were the lowest in unfrozen soil without biochar, significantly higher in unfrozen soil with biochar and non-enriched frozen soil, and the highest in frozen soil with biochar.Among microbiological parameters, the combination of biochar and freezing-thawing significantly increased BR; the lowest BR was in unfrozen soil with biochar and the highest in thawing soil.C mic increased significantly after the addition of biochar, both in unfrozen and frozen soil.qCO 2 was significantly decreased by biochar and insignificantly increased by the freezing-thawing cycle.
The headspace air temperature in the vessel was maintained at 20°C during GHG measurements in the unfrozen soil which was a source of CO 2 and N 2 O, a weak sink for atmospheric CH 4 .The GHG fluxes were not significantly altered by biochar, although soil without biochar consumed CH 4 , while with biochar emitted CH 4 (Fig. 2).
In the frozen soil variant, the vessel placed at room temperature showed an initial dynamic from 11°C to 20°C which lasted about 2 h.Thereafter, the air temperature in the vessel remained constant at 20°C during soil thawing.
Regardless the biochar presence, the CO 2 emission rates were significantly highest in the first period of soil thawing, with a dynamic increase of ambient temperature.In this time, we observed a significant and positive correla-tion between CO 2 level and ambient temperature (Fig. 3).However, after temperature stabilization, the CO 2 emission in soils without and with biochar reached a level close to unfrozen soil (Fig. 2).Biochar enrichment resulted in a shift from CH 4 uptake into soil CH 4 emission, although the differences in the rates were not statistically significant.Thawing soil initially showed the highest CH 4 emission peak and soil with biochar emitting 3.5 times more CH 4 than the control.During the dynamic growth of temperature, a positive relationship (p < 0.05) was detected between temperature and CH 4 concentration, regardless of biochar presence (Fig. 3).After stabilization of the temperature in the headspace, the soil reabsorbed atmospheric CH 4 , regardless of the presence of biochar.The unfrozen soil emitted N 2 O at similar levels without and with biochar.During the initial thawing stage, a much higher N 2 O emission pulse occurred in the variant with biochar and we observed a significant and positive correlation between air temperature in the vessel and N 2 O concentration, similar to CO 2 and CH 4 (Fig. 3).During stabilized temperature in the headspace, the amount of N 2 O emitted was similar to the unfrozen control and slight N 2 O uptake occurred after the addition of biochar.
MANOVA confirmed that all the tested factors and their interactions significantly (p < 0.0001) affected GHG emission rates (considering the rate in unfrozen soil and in the initial thawing stage) in the following order: freezing-thawing (F = 397.3)> biochar × freezing-thawing (F = 200.8)> biochar (F = 192.5).

DISCUSSION
Given the effects of ongoing climate change, it is important to recognize the soil processes occurring during the non-growing season.In recent years, there have been around 95 freezing days (with a daily minimum temperature below 0°C) per year in Poland, which shows a decreasing trend (IOŚ-BIP, 2020).Although the lowest temperatures occur in the winter season (December-February), a longterm analysis for Lublin showed that between 1966 and  Biochar changes soil parameters creating different conditions for soil microorganisms (Lehmann et al., 2011).The increased water storage capacity (as a result of porous structure of biochar and water storage in small pores) and pH (since biochars are often alkaline) of unfrozen soil (not altered by freezing-thawing cycle) reported previously (Karhu et al., 2011) was confirmed in our study.The addition of biochar, carbon-rich material, doubled the organic C content, as in other studies (Paetsch et al., 2018).Combination of the addition of biochar and soil thawing significantly in our study increased extractable NO 3 -concentrations.This N form is a product of nitrification but it may have also originated from other sources.The promotion of nitrification or the slowing down of denitrification caused by the presence of biochar has been reported in other studies (Wang et al., 2011;Yang et al., 2020).Biochar increased soil aeration and pH approaching the optimum level for autotrophic nitrifiers from 7.5 to 8 (Szarlip et al., 2010), which creates the proper conditions for nitrification.The moisture content of 55% WHC in our experiment is suitable for this process (Case et al., 2012).Therefore, based on higher NO 3 -concentrations and altered soil conditions after the addition of biochar, we may suppose that NO 3 -originated partly from nitrification during thawing.However, other studies showed that biochar decreased extractable NO 3 -concentrations because of chemical adsorption on the biochar surface (Kameyama et al., 2012), immobilization within microbial biomass (Andersen and Petersen, 2009) or decreased soil nitrification (Liu et al., 2016a), which was not confirmed by our results.In the freezing-thawing cycle, studies on fertilized soils have shown that different processes can be involved in N transformations depending on the freeze-thaw stage, regulated by soil temperature (Müller et al., 2002).The decrease in soil aeration and the increase in moisture content in frozen-thawed soil may have created conditions for the denitrification process in the frozen soil variant (Li et al., 2023;Mørkved et al., 2006).Higher NO 3 concentrations promoted by thawing-freezing have also been revealed in other soils, suggesting N mineralization as its source (Li et al., 2023).Moreover, the meta-analysis revealed that the freeze-thaw effect significantly promotes ammonification (Song et al., 2017), which also may have occurred in our tests as ammonia emissions were detected (data not shown).
Among soil microbial parameters, the addition of biochar decreased basal respiration and increased soil microbial biomass C. Lower BR has been promoted by thawing as the effect of increased decomposition of thawed C (Natali et al., 2015), although most of the studies show higher BR in soil with biochar (Steiner et al., 2008).C mic was not been changed after freezing which may result from adaptation of microorganisms inhabiting the soil to temperature fluctuations due to seasonal changes and the increasingly rare snow cover in recent years.However, other studies also showed a decline of C mic after freezing-thawing, especially in C-rich soils, as an effect of dry state of cells from altered osmotic potentials and the killing effect (Koponen and Bååth, 2016;Rosinger et al., 2022;Yanai et al., 2004).Similar to our results, the meta-analysis by Zhou et al. (2017a) revealed higher soil microbial biomass C in soil with biochar and the meta-analysis by Song et al. (2017) revealed no significant response of microbial biomass to freeze-thaw treatments.We observed a positive relationship between soil microbial biomass C and soil pH (Fig. 4).This may suggest a different composition of the microbial community, since soil pH has been reported as a strong regulator of bacterial taxonomic diversity, composition, richness and biomass (Bahram et al., 2018;Gangwar et al., 2022;Szafranek-Nakonieczna et al., 2018).An important indicator of microbial health in soils is its metabolic quotient, which relates to the microbial use of carbon for their energy consumption (Anderson and Domsch, 1978) and which is sensitive to short-term soil changes (Zhou et al., 2017a).Lower qCO 2 values after the addition of biochar indicate improved soil biophysical conditions (Zhou et al., 2017a), which was not significantly changed in thawed soil.
The meta-analysis by Shakoor et al. (2021) summarized that the biochar effect on soil GHG emissions across global croplands is GHG-specific and determined by many factors such as feedstock, temperature pyrolysis, experimental method, soil texture, biochar dose and the soil and biochar pH and C:N ratio.The authors concluded that biochar shows the mitigation potential of GHGs and may decrease emissions from agriculture.In our study, we tested the rapid response of GHG emissions after the freezing-thawing event, which is important part of the annual GHG output.
Agricultural soils are a source of CO 2 , although its emission is regulated by practices used and nitrogen fertilization results in stimulation of soil respiration and an enhancement in soil C mineralization (Lu et al., 2011).Soil parameters may be also modified by the application of biochar, being a commonly investigated practice with the potential of C sequestration, improving soil conditions and crop yield (Lehmann 2011;Liu et al., 2016b).In our study, biochar enrichment did not significantly change soil CO 2 emission, which concurs with other studies on agricultural soils (Castaldi et al., 2011;Huang et al., 2023;Liu et al., 2016b).However, a reduction (Sarkhot et al., 2012) or an increase in CO 2 emission after the addition of biochar (Sagrilo et al., 2014;Walkiewicz et al., 2020) have also been reported.The meta-analysis by Sagrilo et al. (2014), which showed a positive effect of biochar on soil respiration, included doses of biochar that were many times higher than those used in our study, which may explain the different effect.The authors found no effect of biochar on soil respiration when the pyrolysis temperature was above 350°C.Our biochar was produced at 650°C, which may also explain the lack of effect on soil respiration.A previous study on different soils, but with the same biochar, revealed a decrease in CO 2 emission in saturated forest soil and an increase in CO 2 emission in saturated orchard soil (Walkiewicz et al., 2020).No effect was observed under 55% WHC of fertilized orchard soil, which was confirmed in our experiment as the same soil moisture condition was used.
The tested soil showed weak methanotrophic activity since low CH 4 uptake was observed in the control sample without biochar.The reduction of methanotrophy in fertilized soils has been reported previously as an effect of N fertilization, often explained by the competitive inhibition of monooxygenase by NH 4 + (Bodelier and Steenbergh, 2014).Interactions between C and N are still not fully recognized and there are also contrary studies showing stimulation of methanotrophy by N fertilization (Bodelier and Steenbergh, 2014).The biochar addition in our experiment resulted in a shift of soils from weak sink into weak source of CH 4 , but the differences were not statistically significant.An experiment on different soils fertilized but enriched with the same biochar showed a stimulation of methanotrophy, especially under saturated conditions, which may have resulted from improved soil aeration and increased O 2 availability for methanotrophs (Walkiewicz et al., 2020).In our experiment, the soil was unsaturated and microorganisms were exposed to O 2 which may lead to an insignificant impact of biochar on soil methanotrophy.However, the current experiment investigated ambient CH 4 uptake over a short incubation period, while the previous study was conducted over a longer time period and under conditions of elevated (2% v/v) CH 4 concentration.Different types of methanotrophs, referred to as low-and high-affinity methanotrophs, are responsible for the methanotrophic process under such different CH 4 concentrations, and their populations may respond differently to environmental conditions (Tate et al., 2012).In fertilized soils, both methanotrophs and nitrifiers may be responsible for CH 4 oxidation as an effect of activity of a similar enzyme, particulate methane monooxygenase (MMO) vs. ammonia monooxygenase (AMO) (Hyman and Wood, 1983).The long-term nitrogen fertilization of the soil investigated may have favoured the nitrifier community and a microbial test would be needed to identify the microbes inhabiting it.It is difficult to assess the longer lasting effect of biochar on methanotrophy in a rapid soil response experiment.
The tested soil was a source of N 2 O which is typical for fertilized soils.Similarly to CO 2 and CH 4 , the rapid response of soil N 2 O emission to the application of biochar was not significantly changed over the short incubation period.A longer term incubation period of another soil fertilized with the same biochar showed a reduction in N 2 O emissions as an effect of suppression of N 2 O production and stimulation of reduction of N 2 O to N 2 (Dong et al., 2020).This N 2 O mitigation has also been reported in various meta-analyses (e.g.Huang et al., 2023;Kaur et al., 2022;Shakoor et al., 2021).Different results can be found in the literature which underlines that the response of soil to biochar is determined by N fertilizer dose, cation exchange capacity, pH and biochar application rate (Huang et al., 2023;Sun et al., 2017).The mitigation potential of biochar has been reported for pH 8-9, while our biochar has lower pH.The highest reduction of N 2 O emission has been reported for soil organic C content in the range from 10-20 mg g -1 (Kaur et al., 2022), while our soil has a higher C content.These parameters may explain the lack of effect of biochar on rapid N 2 O emission in our study.Another explanation may be the amount applied, since 10-20 t ha -1 of biochar did not significantly change N 2 O emissions (Kaur et al., 2022).
When interpreting our results, it is important to consider the procedure used of frequent measurements over a short period of time, which is dictated by the course of the soil thawing process, discussed below.Measurements over a longer time period would provide more accurate information on the impact of biochar on GHG fluxes.
Biochemical soil processes in the non-growing season are notably affected by soil thawing because of an increase in the availability of soil water and mobilization of nutrients which results in rehydrate cells, increased microbial metabolism and improved gas diffusion (Kim et al., 2012).Our study found that the frozen soil emitted all the key GHGs during the thawing period, but the magnitude of emissions was dependent on the ambient temperature dynamic.The rapid ambient temperature growth typically lasted up to 2 h after the soil vessels were moved from freezing to a controlled temperature of 20°C.There then occurred a rapid pulse of emissions of CO 2 and CH 4 (and N 2 O in soil with biochar) which reached the unfrozen level after the ambient temperature was stabilized at 20°C.Similar results were obtained in thawing forest soils but the N 2 O and CH 4 fluxes reached the maximum after 6 h, while the CO 2 flux maximum was observed after 12 h and emissions stabilized within about 48 to 96 h (Wu et al., 2020).The study on alpine meadow and alpine peatland also showed analogous trends, although of longer duration and with GHG fluxes specific to the soils studied (Gao et al., 2015).A similar trend of changes in GHG fluxes during soil thawing was confirmed in our experiment, but the differences in moment and duration of the GHG peaks and next fluxes stabilization may have been due to various lab procedures and soil parameters, particularly the moisture content.Both physi-cal and biological mechanisms could be responsible for GHG emissions in frozen soils (Kim et al., 2012).When interpreting our results, it should be considered that laboratory studies may show a different effect to the practical application of biochar in the field (Huang et al., 2023;Kaur et al., 2022).An important element justifying the different responses of GHG emissions to the addition of biochar is the incubation time of the soils, since the procedure we used was driven by the rate of soil thawing.Therefore, our experiment included the rapid response of the soil by frequent measurements over a short time period.
In our study, in the first stage of thawing, the CO 2 peak was almost five times higher than in unfrozen soil, regardless of biochar presence.A respiration pulse in freezing-thawing events has been reported previously in soils of different ecosystems, but studies have highlighted the dependence of CO 2 pulse on soil type, freeze-thaw cycle frequency, temperature and duration of freezing (Gao et al., 2015;Kim et al., 2012;Koponen and Bååth, 2016).Our results are in agreement with the study on different agricultural soils frozen at -18°C, where CO 2 peak occurred within 1-5 h and the CO 2 rate then returned to the same level as unfrozen soil (Koponen and Bååth, 2016).The soil respiration pulse has been explained by an increase in temperature, moisture content, amount of available C and enhanced microbial metabolism of microorganisms that survived in soil, and improved gas diffusions (Gao et al., 2015;Goldberg et al., 2008;Hou et al., 2020;Kim et al., 2012).The biological mechanism in our soil without biochar was confirmed by the positive and significant correlation between cumulative CO 2 and C mic (Fig. 5), although physical CO 2 release during thawing cannot be excluded either.
The application of biochar did not significantly affect CO 2 emissions in thawing soil, both in the initial and final stages.Similarly to non-amendment soil, a CO 2 pulse occurred during dynamic temperature increase, while CO 2 emission was at similar level to unfrozen soil after ambient temperature stabilization.In soil with biochar, we observed a significant and positive correlation of CO 2 flux with temperature, which also was reported by Hou et al. (2020).Research on the effect of biochar on soil CO 2 emissions during thawing is scarce.Although biochar may act as a C  source for soil microorganisms, a non-significant CO 2 pulse in soil without and with biochar in our study may suggest C stability during freeze-thaw cycles.The lack of a significant correlation between C mic and soil CO 2 emissions in biochar-amended soil may suggest a non-biological mechanism for CO 2 release (Fig. 5).Other authors have shown stimulation of CO 2 emissions in biochar-enriched soil under thawing, but in a longer experiment with a variable number of freeze-thaw cycles (Hou et al., 2020;Liu et al., 2017).
Frozen soil was a significant CH 4 source during the first stage of thawing, while CH 4 uptake was detected after temperature stabilization.This shift from a source to a sink may be an effect of reduced gas diffusion by freezing, lower redox potential and creation of anaerobic conditions in frozen soils that are favourable for CH 4 production by methanogens (Dutaur and Verchot, 2007;Wu and Mu, 2019;Yu et al., 2007).The study on alpine peatland soils (which act a CH 4 source) and alpine meadow soils (which act a CH 4 sink) revealed that freezing significantly increased the release of CH 4 from the peatland but reduced the CH 4 uptake in the meadow (Gao et al., 2015).The authors suggest that freezing and thawing accelerated the decomposition of soil organic matter and the C mineralization, providing substrates for methanogenesis.However, the study involved a longer duration of experiment, which may have allowed the methanogens to become active.Considering the short measurement time in our study, we assumed that CH 4 stored in soil could be released during the first stage of thawing as an effect of changes in pressure during freezing (Mastepanov et al., 2008) rather than methanogenesis.The presence of biochar in soil led to a four-fold increase in the CH 4 peak at the initial soil thawing, but the soil started to consume the gas once the ambient temperature was stabilized, regardless of biochar presence.The mechanism responsible for the observed trend may be connected with the porous structure of biochar.We suppose that the frozen soil with biochar may have stored more CH 4 than the soil without biochar addition, because of the sorption capacity of biochar and CH 4 storage in small pores (Memetova et al., 2022).Therefore a higher amount of stored CH 4 was released into the atmosphere at the first thawing stage of soil with biochar.Subsequently, the soil water thaws, moves deeper into the soil profile, the porosity and temperature of the soil increases, enabling methanotrophic bacteria activity (Hou et al., 2020;Fest et al., 2017) whose presence has been confirmed in control soil.We did not detected a significant effect of biochar on CH 4 uptake after ambient temperature stabilization, although a slight increasing trend occurred, which could be verified by a longer soil incubation time.In summary, a physical mechanism (gas diffusion) may have been involved in CH 4 emissions during the first thawing stage and a biological mechanism (CH 4 uptake by methanotrophs) after temperature stabilization.
To date, CH 4 has received very little attention in terms of thawing, hence the uncertainties and the need for continued research in this area (Kim et al., 2012).
Nitrification and denitrification are the main processes responsible for soil N 2 O emission, which are sensitive to O 2 availability (Szarlip et al., 2010).In our study, biochar did not significantly change the rapid response of N 2 O emission in unfrozen soil, but an N 2 O pulse occurred during the first stage of thawing of amended soil.Other studies show a significant N 2 O pulse, a small increase in emissions or no N 2 O response following thawing.A high N 2 O peak may be an effect of enhanced microbial metabolism (because of higher temperature and improved substrate supply) and a physical mechanism increasing gas diffusion (summarized by Kim et al., 2012).The soil N 2 O mitigation potential of biochar has been often described, e.g., Liu et al. (2016a) reported that biochar application reduced soil N 2 O from decreased soil nitrification which was confirmed by lower NO 3 -concentrations.In our study, higher NO 3 -concentrations in soil with biochar may suggest lowering of denitrification through improving aeration.N 2 O peaks many magnitudes higher than in unfrozen soil occurred only in the first thawing stage in the biochar amended soil, similar to a field study on agricultural soil (Zhou et al., 2017b).The authors suggested that porous biochar improves microbial growth by increasing the supply of C and N after freezing and therefore stimulating N 2 O production.Frozen conditions in soil create anoxia from enhanced O 2 consumption and therefore promote denitrification (Mørkved et al., 2006, Yang et al., 2022).The application of biochar may improve soil aeration and enhance nitrification during thawing which has been confirmed by the highest extractable NO 3 concentrations.We may thus suppose that nitrification and gas diffusion could be a sources of N 2 O pulse in the frozen variant with biochar.CONCLUSIONS 1. Biochar addition did not significantly change the rapid response of GHG emissions in unfrozen soil.
2. In thawing soil, the GHG emission rate is determined by the dynamic of growth in ambient temperature.GHG pulses occurred in the period of the fast temperature growth and then reached the level as in the sample without freezing.
3. Biochar application showed negative effect on climate since significantly increased CH 4 and N 2 O peak in thawing soil, although did not affect CO 2 emission.
4. Biochar enrichment improve soil conditions as indicated by microbial parameters.
5. Both physical (stored gas diffusion) and biological mechanisms could be responsible for GHG emissions in frozen soils.

Conflict of interest.
The Authors declare they have no conflict of interest.

Fig. 2 .
Fig. 2. GHG emission rates (columns) and ambient temperature change (line) in soils without and with biochar including variants: (i) incubation at 20°C; (ii) freezing at -20°C and thawing at 20°C.The initial stage of thawing indicates the GHG emission rate during dynamic temperature growth; the final stage of thawing indicates the rate in constant temperature in the vessel.The same letters above the columns indicate no statistically significant differences between experiment variants, separately for each gas (one-way ANOVA, Tukey post-hoc test, p < 0.05).

Fig. 3 .
Fig. 3. Relationships between air temperature in the vessel and CO 2 , CH 4 and N 2 O concentrations during the first stage of soil thawing (significant differences at p < 0.05).

Fig. 4 .
Fig. 4. Relationships between C mic and pH in soil without and with biochar, including unfrozen and frozen variants (statistically significant at p < 0.05).

Fig. 5 .
Fig. 5. Relationships between C mic and cumulative CO 2 emissions in soil: a -without and b -with biochar (statistically significant at p < 0.05).