Persistence of Biochar Effects on Soil and Nitrous Oxide Emissions: Evaluating Single vs. Repeated Applications in Multi-Year Field Trial

Abstract

Biochar has been widely recognized for its potential to improve soil quality and mitigate greenhouse gas (GHG) emissions. A field experiment was conducted in a temperate climate zone of Slovakia on Haplic Luvisol and evaluated the long-term impact of biochar on soil properties, nitrous oxide (N₂O) emissions, and winter wheat (Triticum aestivum L.) yield. Biochar was applied in 2014 at rates of 0, 10, and 20 t ha⁻¹ and reapplied in 2018 at the same rates, combined with nitrogen (N) fertilization (0, 140, and 210 kg N ha⁻¹). Measurements, conducted from March to October 2021, showed that biochar improved soil water content, increased soil pH, and enhanced soil organic carbon content. However, the concentrations of NH₄⁺-N and NO₃⁻-N generally decreased across all the treatments compared to their respective controls. Biochar reapplication rate at 20 t ha⁻¹, especially combined with second level of N-fertilization, led to a significant reduction in cumulative N₂O emissions by 38.40%. Winter wheat yield was positively correlated with both biochar application (10 and 20 t ha⁻¹) and N levels (140 and 210 kg N ha⁻¹), but these differences were not statistically significant (p > 0.05). The positive effects of biochar on soil properties and yield declined over time, with no significant yield differences observed 7 years after the initial application and 3 years after reapplication. These findings suggest that while biochar can enhance soil conditions and reduce GHG emissions in the short term, its long-term effectiveness remains uncertain. Further research is needed to explore alternative biochar feedstocks, application methods, and strategies to sustain its benefits in agricultural systems.

1. Introduction

The Industrial Revolution triggered a profound transformation in global socioeconomic conditions [1]. Rapid population growth has driven substantial shifts in land use, with projections suggesting that nearly one billion hectares may be converted to agricultural land by 2050 to sustain rising food demands. To meet these needs, agricultural practices have intensified, relying heavily on chemical fertilizers to enhance crop productivity [2]. According to Khampuang et al. [3], the use of N-fertilizer is expected to triple by 2050 to enhance crop productivity. While N-fertilization has greatly boosted global crop production, its excessive use results in substantial GHG emissions, which contribute to climate change [4]. Farmland is a major source of GHG primarily N₂O, CH₄, and CO₂, accounting for 10–12% of global GHG emissions annually [5]. Around 60% of N₂O emissions originate from agricultural practices [6]. In recent years, biochar has gained increasing popularity as a strategy to mitigate GHG emissions [7]. Enhancing soil C and N content through different methods (such as biochar application) is effective in reducing soil N₂O emissions and mitigating N loss from agricultural soil [8]. Biochar, a carbon-rich material, can be produced from diverse organic waste sources such as agricultural residues or municipal sewage sludge through the pyrolysis process [9]. The characteristics of the raw biomass and the conditions during pyrolysis determine the properties of the resulting biochar. Factors such as reaction temperature, heating rate, and residence time are critical parameters that affect the outcome of the pyrolysis process [10]. Variability in biochar key physicochemical properties, such as surface area, microporosity and pH, affects its efficiency for targeted applications [11]. Due to its distinctive features, including high C content, significant cation exchange capacity, large specific surface area, and stable structure, biochar is gaining attention as a valuable soil amendment [9]. Since biochar is an alkaline material with a high proportion of carbon highly aromatic structures, and functional groups such as COO⁻ on its surface, it can directly influence soil chemistry [12,13]. Biochar can improve several soil characteristics, including nutrient content, water-holding capacity, pH balance, and carbon sequestration, while simultaneously mitigating GHG emissions and increasing crop productivity [14]. The reduction in N₂O emissions is linked to enhanced soil aeration, elevated soil pH, adsorption of NO₃⁻, NH₄⁺, or even N₂O, as well as the inhibitory effects of biochar-derived organic compounds on nitrifying and denitrifying microbial communities.

Biochar can mitigate N₂O emissions through multiple interconnected mechanisms. It acts as an “electron shuttle” facilitating electron transfer to denitrifying soil microorganisms and combined with its liming effect, can promote the complete reduction of N₂O to inert N₂ gas [15]. This is supported by microbial evidence; for instance, Harter et al. [16] found that biochar improved microbial N₂O reduction and increased the abundance of N-fixing microorganisms. The relative gene and transcript copy counts of the nosZ-encoded bacterial N₂O reductase increased with biochar addition to soil, indicating a potential mechanism for reduced N₂O emissions. Field studies in temperate climates provide evidence for this mitigation potential, though the magnitude of the effect varies. Felber et al. [17] observed a 21.5% reduction in cumulative N₂O emissions, while Schimmelpfennig et al. [18] reported a more substantial 46% decrease in cumulative N₂O emissions in treatments with biochar application. Similarly, in humid warm-temperate, subtropical monsoon climate, Wu et al. [19] demonstrated that both single and repeated biochar applications effectively reduced CH₄, N₂O, and NO emissions by 14.5–27.1%, 31.5–43.4%, and 22.4–33.6%, respectively, compared to untreated soils. However, the response is not always consistent; a field study by Angst et al. [20] showed that while a high biochar application rate (18.8 t ha⁻¹) combined with manure resulted in the lowest cumulative N₂O and CH₄ emissions, the differences between treatments were not significant. This underscores a critical point: the impact of biochar on GHG emissions often differs between controlled laboratory and complex field environments, highlighting the value of multiple year-long field experiments like the present study. Biochar is also able to increase soil pH due to its “liming effect” [21,22,23]. By enhancing soil pH, carbon storage, water retention, and nutrient retention, biochar has the potential to boost crop productivity [24].

Biochar’s impact on the soil environment is complex and varies across different climatic regions [25,26]. As a significant source of stable C [27] with a high C:N ratio [28], biochar application can alter soil organic matter (SOM) dynamics and overall soil properties [29]. However, it can sometimes lead to nutrient immobilization, particularly N, which can negatively affect crop production in agroecosystems [30]. Mineral N-fertilization is often recommended to mitigate the negative effects of a high C:N ratio and optimize soil conditions (water, air, biological regimes) [28]. The long-term efficacy of biochar, its optimal application rates, and the necessity for reapplication remain critical areas of investigations. Most field studies last around 3 to 5 years, but this study has lasted 8 continuous years, which is rare for this type of research. This makes the observed results particularly valuable for other researchers.

This study aims to assess the impact of different biochar application rates, evaluated seven years after the initial application and three years after reapplication, in combination with varying N-fertilization levels. The research was conducted on Luvisols, a prevalent and intensively cultivated arable soil in Central Europe [31]. The study focuses on the soil properties, N₂O emissions, and crop yields, with the following hypotheses: (H1) the effects of biochar application and reapplication will be more pronounced in fertilized treatments compared to unfertilized treatments; however, its overall influence may decline over time due to biochar aging. (H2) N-fertilization, rather than biochar, will play a more dominant role in shaping soil properties, N₂O emissions, and crop yield. (H3) Biochar application and reapplication will mitigate N₂O emissions by lowering available N content and increasing soil pH.

2. Materials and Methods

2.1. Experimental Site, Soil and Biochar

The field experiment site is located in Dolná Malanta, Slovakia (48°19′23.41″ N, 18°09′0.7″ E) and it is maintained by the Slovak University of Agriculture in Nitra, Slovakia. This site is situated in a temperate climate zone, with an average total precipitation of 540.9 mm, (including 396.5 mm from March to October 2021). The average annual air temperature in the year the data were collected was 9.9 °C (30-year climate normal, 1961–1990). Prior to the experiment establishment, the agricultural field had been conventionally maintained for several decades. The main properties of the topsoil (0–0.1 m) are listed in

Table 1. Initial soil characteristics [33].

Soil Properties	Value	Units
sand	15.2	%
silt	59.9	%
clay	24.9	%
pH(KCl)	5.71	-
SOC	9.13	g kg−1

. The soil is classified as Haplic Luvisol [32] with silty loam texture.

The biochar used in this study was produced from cereal husks and paper industry sludge (1:1 ratio, sourced from Sonnenerde, Riedlingsdorf, Austria) by pyrolysis at 550 °C for 30 min using a Pyreg reactor (manufactured by Pyreg GmbH, Dörth, Germany). We chose this type of biochar because it has the desired properties, is of premium quality, and is also EBC-certified. The physicochemical properties of the biochar were determined based on the EBC certificate and are detailed in

Table 2. Biochar physical and chemical properties [34].

Biochar Properties	Value	Units
pH(KCl)	8.80	-
Ash content	38.3	%
Bulk density	0.21	g cm−3
SSA	21.7	m2 g−1
Ca	57.0	g kg−1
Mg	3.9	g kg−1
K	15.0	g kg−1
Na	0.77	g kg−1
TC	53.1	g kg−1
TN	14.0	g kg−1
C/N	37.9	-

Note: SSA-specific surface area, TC–total carbon content, TN–total N content.

.

2.2. Experimental Design

The field experiment was established in 2014 using a randomized complete block design (RCBD) with two experimental factors. The first factor was the application of biochar at rates of 0, 10, and 20 t ha⁻¹ (B0, B10, and B20, respectively), which was applied in 2014. In 2018, the original biochar plots were divided in half, and biochar was reapplied at the same rates to one half (reapB10, reapB20). The second factor was mineral N-fertilizer with three application levels: N0 (no fertilizer), N1 (based on the requirements of the cultivated crops in the experiment), and N2 (50% more fertilizer than the N1). In 2021, the year of this study, the following whole N-fertilizer rates of 0, 140, and 210 kg N ha⁻¹ were applied in three separate dressings. The first application was done at the beginning of March with an NPK-fertilizer (containing N, phosphorus, and potassium) applied at doses of 0, 30, and 45 kg N ha⁻¹. The second application was done at the end of March and consisted of calcium ammonium nitrate with dolomite (LAV 27), applied at doses of 0, 70, and 105 kg N ha⁻¹. The third application was done at the end of April with DASA 26/13 N-fertilizer applied at doses of 0, 40, and 60 kg N ha⁻¹. For each level of N-fertilization (N0, N1, and N2), plots without biochar application (B0) served as reference controls. These reference controls were used to evaluate the effects of increasing biochar doses (B10, B20, reapB10, and reapB20) under the respective N-fertilization level. All treatments were arranged in three replicates. Winter wheat (Triticum aestivum L.) variety GENIUS was planted on 9 December 2020, for the 2021 growing season, at a seeding rate of 268 kg ha⁻¹.

The experiment consisted of 27 original test plots (4 m × 6 m) arranged in a random pattern, with 1.2 m wide access pathways in the intermediate rows and 0.5 m wide protection strips between the plots to prevent cross-contamination and treatment effects. As was already mentioned above, the plots treated with biochar were split into two sections in 2018 (two 3 × 4 m subplots), and biochar was reapplied only to one subplot from the two. As a result, there were 15 treatments during the study period of 2018 (27 original plots plus 18 reapplication subplots, totaling 45 plots), as shown in Figure 1. In total, after the reapplication in 2018 the field experiment included 15 treatments in three replications (

Table 3. Overview of the treatments and specific amount of applied biochar and mineral N-fertilizer in the field experiment.

Treatments	Biochar Application in 2014 (t ha−1)	Biochar Reapplication in 2018 (t ha−1)	N-Fertilization Application in 2021 (kg N ha−1)
	N0 Level–Unfertilized Treatments
B0N0	0	0	0
B10N0	10	0	0
B20N0	20	0	0
reapB10N0	10	10	0
reapB20N0	20	20	0
	N1 Level–Fertilized Treatments
B0N1	0	0	140
B10N1	10	0	140
B20N1	20	0	140
reapB10N1	10	10	140
reapB20N1	20	20	140
	N2 Level–Fertilized Treatments
B0N2	0	0	210
B10N2	10	0	210
B20N2	20	0	210
reapB10N2	10	10	210
reapB20N2	20	20	210

).

Biochar was applied manually to the soil and incorporated into the upper layer of the soil (0 to 0.1 m) using a tractor-mounted combinator before sowing.

Throughout the experiment, non-inversion tillage methods were utilized. Weed control was achieved by applying Mustang Forte (1 L ha⁻¹) and Retacel Extra (1 L ha⁻¹) sprays in mid-April, followed by Lontrel (0.4 L ha⁻¹) sprayed with water (200 L ha⁻¹) at the end of May. The wheat crop was mechanically harvested on 23 July 2021. Throughout the experiment, natural rainfall served as the sole water supply. After the harvest the plots were fallow until April the 13th of the following year (2022). Post-harvest disking took place on 28 July 2021, to a depth of 0.10–0.12 m; to incorporate crop residues and reduce soil moisture evaporation. A second disking operation, for seedbed preparation, occurred on 13 December 2021, also to a depth of 0.10–0.12 m.

2.3. Soil Sampling and Analysis

After applying N-fertilizer to the soil surface and subsequently incorporating it into the soil at the beginning of March, soil sampling commenced at two-week intervals. Two sampling methods were employed: ‘main’ sampling, which involved collecting soil samples from 3 randomly selected locations within each plot and mixing them into an average sample, and ‘additional’ sampling, which entailed collecting soil samples from 1 random location in each plot. These methods differ in the type of analyses conducted on the soil samples. For ‘main’ samples, soil chemical properties including soil pH, inorganic N content (NO₃⁻-N, NH₄⁺-N), and soil water content (SWC) were measured. For ‘additional’ samples, only SWC was measured using the gravimetric method. Each disturbed soil sample was collected from a depth of 0 to 0.1 m. Simultaneously, soil temperature was measured up to a depth of 0.05 m using a Volcraft DET3R thermometer. Soil samples for SWC analysis were weighed straight after collection, dried in an oven at 105 °C for 24 h and then weighed again the following day. If ‘main’ sampling was performed, these samples were also weighed for inorganic N analyses. The remaining soil samples were dried under laboratory conditions for 5 to 6 days before soil pH analysis were conducted. Soil pH was measured potentiometrically in 1 M KCl at a soil:KCl ratio of 1:2.5 (w/w). The colorimetric method and a spectrometer (WTW SPECTROFLEX 6100, Weilheim, Germany) were used to measure the concentrations of N-NH₄⁺ and N-NO₃⁻ in soil filtrates [35]. Twice a year, undisturbed soil samples were taken using steel cores with a volume of 100 cm³ for the bulk density (BD) measurements. Three samples were randomly collected from each individual plot at a depth of 0.02–0.07 m, resulting in 9 representative soil samples per treatment and a total of 135 soil samples per sampling event. SWC and BD data were used to calculate the soil water filled pore space (WFPS).

2.4. Gas Sampling and Analysis

Gas sampling was carried out from March to October 2021 using the closed chamber technique [36]. A galvanized metal collar was inserted 0.1 m into the soil within each plot and left undisturbed throughout the experiment. During soil management activities, the collars were temporarily removed and repositioned in the same location to maintain consistency. During each sampling event, PVC chambers, measuring 0.25 m in height and 0.3 m in diameter, were affixed to the bottom collars and sealed with water. Gas samples were collected at fortnight intervals (along with the soil samples) using a 60 mL plastic syringe from 20 mL tube fittings sealed with a septum at 0, 30, and 60 min after chamber enclosure. These gas samples were transferred to pre-evacuated 12 mL glass vials (Labco Exetainer, Wales, UK). N₂O concentrations in the gas samples were measured using gas chromatography (GC-2010 Plus, Shimadzu, Kyoto, Japan) equipped with an electron capture detector (ECD). Daily average N₂O emissions are reported in g ha⁻¹ day⁻¹, while cumulative N₂O fluxes (from March to October) were calculated by interpolating emissions between each sampling day and are expressed in g ha⁻¹.

2.5. Plant Sampling and Analysis of Yield Parameters

Prior to harvesting, wheat was manually sampled from each plot on 21 July 2021. Samples were taken from randomly selected areas, each consisting of 2 adjacent rows of 1.5-m long. The sampling involved counting the plants in the selected rows, cutting the aboveground biomass at soil level and transporting the material to the laboratory. In the laboratory, the ears were separated from the stems and counted. The grains were then manually separated from the ears and counted using a laboratory grain counter. The dry weight of the grains was determined after drying them in an oven at 60 °C until a constant weight was achieved. Grain yield was characterized by the total number of ears per m², number of grains per ear and average dry grain weight [26].

2.6. Statistical Analysis

Statistical analyses were conducted using Statgraphics Centurion XV.I software (Statpoint Technologies, Inc., Washington, DC, USA). Mean values were compared between the treatments within the same fertilization level at p < 0.05 using one-way analysis of variance (ANOVA) and the least significant difference (LSD) test. Pearson’s r correlation coefficient was used to assess correlations between wheat yield, soil chemical and physical properties (0–0.1 m depth) and N₂O emissions (both cumulative and flux).

3. Results and Discussion

3.1. Biochar Effect on Soil Physical Properties

Soil physical properties (

Table 4. Effect of biochar application on soil physical properties. The data are presented as the mean (n = 3) ± standard deviation.

Treatment	SWC (% Mass)	WFPS (% Vol.)	Soil T (°C)	BD (Spring) (g cm−3)	BD (Autumn) (g cm−3)
N0 Level–Unfertilized Treatments
B0N0 B10N0 B20N0 reapB10N0 reapB20N0	14.66 a ± 1.1 14.14 a ± 0.7 15.46 a ± 0.8 14.60 a ± 0.6 15.71 a ± 1.2	50.38 a ± 3.5 54.04 a ± 3.0 56.41 a ± 2.9 56.08 a ± 2.6 53.64 a ± 3.8	16.20 a ± 0.4 16.30 a ± 0.3 15.95 a ± 0.3 16.28 a ± 0.3 16.05 a ± 0.4	1.50 a ± 0.04 1.57 a ± 0.03 1.53 a ± 0.03 1.57 a ± 0.03 1.47 a ± 0.06	1.59 ab ± 0.06 1.71 b ± 0.01 1.55 ab ± 0.07 1.70 b ± 0.03 1.51 a ± 0.07
N1 Level–Fertilized Treatments
B0N1 B10N1 B20N1 reapB10N1 reapB20N1	14.54 a ± 0.7	47.42 a ± 2.4	15.54 a ± 0.4	1.46 a ± 0.04	1.63 a ± 0.07
	14.65 a ± 1.0	47.25 a ± 3.6	15.36 a ± 0.2	1.45 a ± 0.01	1.52 a ± 0.11
	15.89 a ± 0.8	47.74 a ± 1.8	15.31 a ± 0.4	1.41 a ± 0.04	1.42 a ± 0.01
	14.64 a ± 0.9 15.56 a ± 0.7	51.90 a ± 3.3 46.71 a ± 2.4	15.46 a ± 0.3 15.24 a ± 0.3	1.51 a ± 0.02 1.41 a ± 0.06	1.58 a ± 0.08 1.52 a ± 0.03
N2 Level–Fertilized Treatments
B0N2 B10N2 B20N2 reapB10N2	14.92 a ± 0.9 15.04 a ± 0.7 15.75 a ± 0.8 15.12 a ± 0.8	49.82 ab ± 3.5 49.21 ab ± 2.1 50.51 ab ± 2.8 46.40 a ± 2.4	15.28 a ± 0.2 15.68 a ± 0.4 15.42 a ± 0.2 15.58 a ± 0.3	1.48 a ± 0.03 1.47 a ± 0.05 1.45 a ± 0.03 1.42 a ± 0.03	1.56 a ± 0.05 1.60 a ± 0.11 1.74 a ± 0.02 1.58 a ± 0.08
reapB20N2	15.92 a ± 0.7	55.36 b ± 3.1	15.31 a ± 0.3	1.50 a ± 0.04	1.63 a ± 0.02

Note: different letters within a column indicate significant differences between treatments at the same fertilization level (p < 0.05) using LSD test; SWC-soil water content; WFPS–water-filled pore space; Soil T–soil temperature; BD–dry bulk density.

) showed some variation based on biochar application rates and N-fertilization levels. While there was a non-significant trend towards increased SWC with higher biochar application rates, this was not statistically significant in either fertilized or unfertilized treatments. This contrasts with some studies [37,38] that have reported positive effects of biochar on soil water availability. However, our findings align with the results of study by Karhu et al. [39], who found no significant differences in SWC (p = 0.334) or soil temperature (p = 0.838) with biochar application at 9 t ha⁻¹ in a silt loam soil. The insignificant differences in SWC in our study could be due to the weather conditions during the 2021 growing season, the timing of sampling and the aging of biochar in the soil environment (7 years post-initial application and 3 years post-reapplication).

Biochar application and reapplication generally led to insignificant increases in water-filled pore space (WFPS) (ranging from 0.67% to 11.97%) compared to the individual control treatments (B0N0, B0N1, B0N2) across all the N-fertilization levels. In the treatments without N-fertilization, WFPS increased by 6.47% in reapB20N0 and by 11.97% in B20N0 compared to the control (B0N0). At the first level of N-fertilization, WFPS increased by 0.67% (B20N1) and 9.45% (reapB10N1) while on the second N-fertilization level, it increased by 1.38% (B20N2) and 11.12% (reapB20N2) in comparison to respective controls B0N1 and B0N2, respectively. The only statistically significant difference (p < 0.05) in WFPS was observed between the reapB20N2 and reapB10N2 treatments (Table 4). This insignificant effect on WFPS is consistent with findings by Han et al. [40]. Biochar application did not significantly affect soil temperature (Soil T) (p > 0.05). Applying biochar to the soil surface can enhance solar radiation absorption and improve soil properties through changes in soil color. Several studies [41,42,43,44,45,46] indicate that biochar can increase the soil water content and mitigate temperature fluctuations at the soil surface. The complex interplay of biochar’s effects on both chemical and physical soil properties makes it difficult to isolate a single cause for temperature variations [47]. Bulk density (BD) values were generally lower in all treatments in spring than in autumn (Table 4) which is consistent with the natural soil settling processes [48]. Values above 1.45 t m⁻³ are considered critical for loamy soils. Higher values indicate soil compaction and deteriorated physical conditions. BD above 1.5 t m⁻³ is unsuitable for growing crops such as wheat. Under such conditions, root growth and development is restricted and yields can be significantly reduced [49]. Biochar application and reapplication did not significantly change the soil BD in either spring or autumn, regardless of fertilization status. This contrasts with expectations, as biochar typically has lower BD, and is often observed to decrease soil BD, especially in sandy soils [50,51,52]. The lack of a significant effect in the studied loamy soil 7 years after application and 3 years after reapplication suggests that biochar’s impact on the soil BD may have diminished, which confirms H1. This might be due to factors like biochar mineralization [53], accelerated mineralization in N-fertilized treatments [54], filling of biochar pores with finer soil particles [51] and overall biochar aging.

3.2. Soil Physico-Chemical Properties

Most of the biochar are usually alkaline (our biochar had a pH of 8.8) and can have liming effect, particularly in acidic soils [55], which has been confirmed by several studies in this experiment [56,57]. Interestingly, however, a statistically significant that such liming effect was not observed in unfertilized treatments 7 years after application or 3 years after reapplication (

Table 5. Effect of biochar application on soil chemical properties. The data are presented as the mean (n = 3) ± standard deviation.

Treatment	pH(KCl)	NH4+-N (mg kg−1)	NO3−-N (mg kg−1)
	N0 Level–Unfertilized Treatments
B0N0 B10N0 B20N0 reapB10N0 reapB20N0	5.32 a ± 0.2 5.46 a ± 0.2 5.46 a ± 0.1 5.49 a ± 0.1 5.50 a ± 0.1	12.21 ab ± 1.9 10.58 ab ± 1.7 11.65 ab ± 2.0 10.25 a ± 1.5 12.93 b ± 2.3	6.54 a ± 1.1 5.93 a ± 0.5 5.88 a ± 0.5 5.46 a ± 13.4 5.77 a ± 0.5
	N1 Level–Fertilized Treatments
B0N1 B10N1 B20N1 reapB10N1 reapB20N1	4.97 a ± 0.2 5.40 b ± 0.3 5.31 b ± 0.1 5.40 b ± 0.3 5.39 b ± 0.1	30.90 a ± 8.8 24.17 a ± 5.2 25.66 a ± 7.1 26.33 a ± 4.2 24.08 a ± 6.6	16.80 a ± 4.2 16.26 a ± 2.4 13.99 a ± 3.5 16.42 a ± 3.1 13.34 a ± 2.3
	N2 Level–Fertilized Treatments
B0N2 B10N2 B20N2 reapB10N2 reapB20N2	4.63 a ± 0.1 5.01 b ± 0.2 5.14 b ± 0.2 5.07 b ± 0.3 5.28 b ± 0.2	54.11 a ± 12.1 44.48 a ± 12.0 42.69 a ± 9.8 38.90 a ± 15.3 36.43 a ± 6.8	16.50 a ± 5.3 20.14 a ± 4.4 21.14 a ± 3.2 18.27 a ± 3.2 18.69 a ± 3.9

Notes: different letters within a column indicate significant differences between the treatments at the same fertilization level (p < 0.05) using LSD test.

) of biochar in our experiment. Biochar can contain carbonates and organic anions, which cause their alkalinity [58], and therefore the absence of a significant effect on soil pH in our experiment, especially after biochar reapplication, was rather unexpected. The explanation may lie in the change in ΔpH and zero charge, which cause cation or anion sorption in the soil [59]. As Zołotajkin et al. [60] stated, ΔpH depends on the content of SOM, then it is obvious that the application of biochar can influence the ΔpH values and zero charges [61], and in this way also the soil sorption properties [61,62], and final soil pH. However, soil pH is dynamic, and the mechanisms by which biochar influences it are still not fully understood [63]. As expected, N-fertilization significantly acidified the soil [64] with lower pH values observed at higher N application rates (N2) (Table 5). However, despite the soil acidification, biochar application and reapplication still showed a tendency to increase soil pH. At the first level of N-fertilization, pH increased by 6.84–8.65% compared to the control (B0N1), and at the second level of N-fertilization, it increased by 8.21–14.04% compared to the control (B0N2). Overall, biochar application consistently increased soil pH, however reapplication treatments (reapB10N2 and reapB20N2) showed a more pronounced pH increase compared to (Table 5).

N-fertilization in the control treatments increased the content of NH₄⁺-N by 18.69 mg kg⁻¹ (153.07%) in B0N1 and by 41.90 mg kg⁻¹ (243.16%) in B0N2, and increased the content of NO₃⁻-N by 10.26 mg kg⁻¹ (156.88%) in B0N1 and by 9.96 mg kg⁻¹ (152.29%) in B0N2, compared to the unfertilized control (B0N0). Biochar application generally showed a tendency to decrease NH₄⁺-N content, particularly in reapplication treatments and at higher N-fertilization levels (Table 5). This reduction in the NH₄⁺-N content is consistent with the findings of Wang et al. [65], who attributed it to an increased surface area leading to increased cation exchange capacity (CEC) and surface sorption capacity, resulting in direct binding of NH₄⁺-N [66]. Biochar can alter N cycling in soils by reducing mineral N availability through direct adsorption (NH₄⁺-N) or microbial immobilization (NO₃⁻-N) [67,68,69]. Biochar generally decreased (non-significantly) NO₃⁻-N content at first level of N-fertilization (Table 5). These findings are consistent with other studies that demonstrate a decrease in NO₃⁻-N extraction from soil following biochar addition [68,69]. This decrease is likely due to factors other than direct adsorption (due to biochar’s limited anion exchange capacity), such as direct chemical interactions with biochar [70,71,72] or microbial immobilization following biochar addition to the soils [69,70], and N uptake by plants [64]. However, at the second level of N-fertilization (N2), initial biochar application (and to a lesser extent, reapplication) tended to increase NO₃⁻-N content, suggesting a promotion of nitrification. The results partially support H1, as the effects on mineral N were more pronounced in N-fertilized treatments. However, the persistence of a pH increasing effect, even after extended time periods, indicates that biochar’s influence on soil chemistry is more complex than initially hypothesized.

3.3. Biochar Effect on Crop Yields

Crop yields (Figure 2) clearly demonstrate the importance of N-fertilization [64,73]. Biochar can act as a nutrient regulator [74]. Grain yields in the unfertilized treatments (2–4 t ha⁻¹) were lower than in N-fertilized treatments (3 to 6 t ha⁻¹). The importance of nitrogen, a major factor for sustainable and profitable crop production [75], was also evident in our study in combination with the initial and repeated biochar applications at doses of 10 and 20 t ha⁻¹. We assume that this was not observed in our study due to the initial and repeated application of biochar in doses of 10 and 20 t ha⁻¹. Biochar application and reapplication had no statistically significant impact on grain yield in 2021, neither 7 years after initial application, nor 3 years after reapplication. While there was a slight tendency for lower yields in some N1 treatments, this was not statistically significant. A slight decrease in the crop yield might be partly related to soil chemistry (Table 5), but also to soil physical properties (Table 4; Table S1). Biochar tended to reduce NH₄⁺-N and worsen the physical condition of the soil, even 3 years after reapplication of biochar, this effect was neutral and completely consistent with the control. In N1, biochar application and its reapplication had a non-significant tendency to reduce the yield at 10 t ha⁻¹, while at 20 t ha⁻¹ there was no such an effect. At B0N2, the yield was slightly lower than at B0N1. Overall, even at the second fertilization level, biochar did not have a statistically significant effect on wheat grain yield in 2021. These findings support both H1 and H2, demonstrating the dominance of N-fertilization and the diminished effect of biochar on the crop yields over time.

3.4. Biochar Effect on Soil N₂O Flux

N₂O emissions (Figure 3a) from March to October 2021 were significantly influenced by N-fertilization, with much higher fluxes observed in the N-fertilized treatments compared to unfertilized treatments. This confirms H2 and aligns with the results of previous research [76,77,78]. These studies have reported an overall positive effect of biochar application on reducing N₂O emissions, although with varying effectiveness, which is limited by different factors such as biochar properties, biochar combination with mineral fertilizers, or by soil-climatic conditions [28,79]. Our results suggest that long-term biochar application could be a useful strategy for reducing N₂O emissions in temperate agricultural soils, providing guidance for sustainable fertilization practices and supporting policy development promoting biochar use as part of climate-smart agriculture initiatives. Horák et al. [76]; Li et al. [80]; Zhang et al. [78] reported that the increase in N₂O soil flux was primarily due to mineral N-fertilization.

N₂O emissions from the soil with non-fertilized treatments (Figure 3a) were low and relatively stable throughout the observation period (March–October 2021), with one exception: a peak after the harvest (on the 216th Julian day). At this day, significant differences in the N₂O flux were found between the soil with B0N0, B10N0, and the reapplication treatments. Available N from the soil reserves, products of microbial transformation of SOM and biochar were no longer taken up by the cultivated crop, which resulted in the increased N₂O emission from the soil into the atmosphere. Reapplied biochar, at both rates, showed a reduction in N₂O fluxes compared to the initial application and the unfertilized control (B0N0). However, overall daily or cumulative N₂O fluxes from the soil with these treatments did not differ significantly due to low variability (Figure 3b,c).

In both N-fertilization levels, three peaks in N₂O emissions were observed, corresponding to N-fertilization dates (at the beginning of March, end of March, and end of April) and the harvest (summer maximum). The application and re-application of biochar resulted in a significant reduction in the emissions, especially at the second peak, being more evident at the higher (20 t ha⁻¹) rather than the lower (10 t ha⁻¹) rate of biochar and at its re-application rather than just the initial application compared to the N-fertilized control (B0N1). During the summer maximum (3rd peak), the reduction in N₂O flux was the highest in the soil with both re-applied rates of biochar. The daily N₂O flux in treatments with the first level of N-fertilization (N1) increased significantly, depending on the rate of biochar applied initially and after reapplication. Significant differences, however, were found only between B10N1 and reapB20N1 (Figure 3b). Overall, the most effective reduction in cumulative N₂O emissions was found under the influence of the combination of the first level of fertilization with the re-application of biochar in both rates. A positive effect was also observed after the initial application of 20 t ha⁻¹ of biochar together with N1 compared to B0N1 and B10N1.

In the second level of N-fertilization (N2) at the beginning of March, the initial application of both rates of biochar resulted in a significant increase in the N₂O emissions from the soil into the atmosphere compared to the fertilized control (B0N2). The increase was more pronounced at the higher than the lower rate of biochar. The combination of N2 with re-application of biochar in both rates effectively eliminated the increase in N₂O emissions at the beginning of the growing season, as well as during the second and the third (summer) peak compared to the N-fertilized control (B0N2). Again, the differences were more pronounced at the higher than the lower re-applied rate of biochar. However, statistical analysis did not reveal statistically significant differences in the average daily N₂O emissions (Figure 3b) for these treatments. But according to one-way ANOVA there were statistically significant differences in the cumulative N₂O emissions for the period March-October 2021 between N-fertilized treatments. Cumulative N₂O emissions in reapB20N2 were statistically significantly lower by 460.82, 339.97, 458.50, and 185.31 g N₂O-N ha⁻¹, corresponding to reductions of 38, 31, 38, and 20% compared to B0N2, B10N2, B20N2, and reapB10N2, respectively (Figure 3c).

The content of mineral N plays a crucial role in plant nutrition [73]. On the other hand, high levels of mineral N in the soil pose ecological risks and can cause health problems [64]. Increasing levels of inorganic forms of N (NH₄⁺-N and NO₃⁻-N) in the soil linearly increase the content of N₂O, which can be emitted into the atmosphere [81] and cause issues related to climate change [78,80]. The results of this study also confirmed a statistically significant negative effect of both inorganic forms of N (NO₃⁻-N and NH₄⁺-N) on increasing cumulative N₂O emissions over the observed period. Higher contents of NO₃⁻-N and NH₄⁺-N did not have a direct impact on the N₂O flux, but the higher their content in the soil, the higher cumulative N₂O emissions were found (

Table 6. Pearson’s correlation coefficients and p-values (in parentheses) between yield components, soil properties, and N₂O emissions.

	N2O Flux (g N2O-N ha−1 day−1)	Cumulative Emissions of N2O (g N2O-N ha−1)
Above ground dry biomass (t ha−1) Grain yield (t ha−1) Average weight of 1 grain (mg) Number of ears per plant (m2) Number of grains per ear (m2) pH (units) NO3−-N (mg kg−1) NH4+-N (mg kg−1) Nan (mg kg−1) SWC (% mass) SWC (% vol.) WFPS (%) Soil T (°C) Bulk density (2 June 2021) (g cm−3) Bulk density (11 November 2021) (g cm−3)	0.256 (0.005) 0.048 (0.600) 0.059 (0.525) 0.054 (0.558) 0.065 (0.482) 0.429 (0.000) 0.115 (0.211) 0.008 (0.935) 0.042 (0.648) 0.587 (0.000) 0.567 (0.000) 0.533 (0.000) 0.343 (0.000) 0.205 (0.025) 0.011 (0.903)	0.418 (0.000) 0.074 (0.422) 0.105 (0.252) 0.076 (0.411) 0.124 (0.178) 0.626 (0.000) 0.288 (0.001) 0.251 (0.006) 0.270 (0.003) 0.293 (0.001) 0.003 (0.268) 0.230 (0.011) 0.207 (0.023) 0.292 (0.001) 0.037 (0.689)

Note: Nan inorganic nitrogen, it represents the sum of NO₃⁻-N and NH₄⁺-N contents.

). For this reason, it is extremely important to pay increased attention to the process of nitrogen transformation to N₂O and strive to create a soil environment that is optimal for plant growth and production while not causing ecological burden. Various types of biochar have been tested recently, as several studies pointed to its positive effect on reducing emissions of N₂O and other GHGs in different climatic regions of the world [14,80,82], including soils of Slovakia [81,83,84]. Due to its properties, biochar can influence nutrients in the soil, including N [85,86,87,88], which, together with N uptake by plants, can contribute to reducing N₂O fluxes and overall emissions from the soil to the atmosphere. The particularly positive result of this study was that no statistically significant correlations were found between yield parameters (except above-ground dry biomass) and the average daily flux of N₂O and cumulative N₂O emissions (Table 6). It indicates that the set soil management, including applied and re-applied biochar and its combination with N-fertilization, is eco-friendly and sustainable in terms of eliminating N₂O emission from the soil to the atmosphere. The average daily flux and cumulative emissions of N₂O were directly influenced by soil pH, soil moisture, soil temperature, WFPS, and BD. The higher these variables were, the higher the effect of applied and re-applied biochar and its combination with N, and thus the higher the average daily flux and cumulative N₂O flux (Table 6). Soil pH plays a crucial role in eliminating N₂O emissions from the soil [47]. Alkaline biochar increases the pH of acidic soils, which positively affects the soil environment, including microbial activity, which plays a significant role in the processes of N₂O production in the soil [21,89]. The results of Xu et al. [28] also confirm the positive effect of higher soil pH values–the result of biochar application and its combination with mineral fertilization–on GHG emission reduction, including N₂O. It was also confirmed in this study 4 years after the initial application of biochar and its re-application in the current year [81]. However, newer data from this experiment point to the opposite trend, meaning higher pH can also cause higher N₂O emissions (Table 6) which does not confirm H3. The explanation may lie in the fact that 7 years after the initial biochar application and 3 years after its re-application the liming effect of biochar was no longer significant, as documented by the obtained results (Table 5). A lower or no liming effect disrupts the above-described mechanism of biochar’s influence on soil pH. Additionally, this may be contributed to the deteriorated physical environment of the soil (Table 4). In general, Luvisols (the soil on the experimental field) are fertile soils, provided that farmers pay increased attention to them through fertilization, liming, etc. [47]. In the first years after biochar application in this experiment, an improvement in soil structural condition [33] and in some other physical properties [90] was observed. The obtained results from the period March-November 2021, 7 and 3 years after the initial application and re-application of biochar and N-fertilizers, show that the treatments no longer have a significant effect on improving soil physical properties such as temperature, moisture, WFPS, and bulk density (Table 4) resulting in increasing N₂O fluxes and overall cumulative N₂O emissions (Table 6).

4. Conclusions

This study demonstrates a clear differentiation in the effects of biochar and nitrogen fertilization on soil properties and ecosystem functions. Seven years after initial application and three years post-reapplication, biochar’s influence on soil physical properties remained minimal. In contrast, soil chemistry was significantly altered, primarily driven by nitrogen fertilization, which had a more substantial impact on soil pH and mineral N content than biochar alone. This pattern extended to crop productivity, where grain yields in N-fertilized treatments were nearly double those of non-fertilized treatments, while biochar application and reapplication did not significantly affect yield. A key agronomic and environmental benefit of biochar was its capacity to mitigate N₂O emissions. Biochar, particularly at the higher rate (20 t ha⁻¹) and upon reapplication, was effective in reducing N₂O fluxes following N-fertilization, with a notable suppression of peak emissions observed from March to November 2021. A limitation of this study is that gas sampling on biweekly intervals may have missed short-term, high-intensity emission peaks immediately following fertilizer application. Despite this, our data robustly show that the primary drivers of cumulative N₂O emissions were elevated mineral N, soil pH, soil moisture, soil temperature and deteriorating soil physical conditions. Overall, these findings point to a diminishing effect of biochar on soil properties and crop yields over time. However, its role as a tool for N₂O mitigation remains significant. To sustain and optimize its agronomic and environmental benefits, future research should focus on how biochar aging, feedstock type, and pyrolysis temperature influence its long-term interactions with soil microbial and physicochemical processes. Ultimately, understanding and enhancing biochar’s capacity to reduce N₂O emissions is critical for improving the greenhouse gas balance of agricultural systems and advancing climate-smart land management strategies.