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Article

Olive Tree (Olea europaea) Biochar Differentially Affects N2O and CO2 Emissions in Neutral and Alkaline Olive Orchard Soils

by
Georgios Giannopoulos
1,2,*,
Ioannis Anastopoulos
1,
Vasileios A. Tzanakakis
3,
Eduardo Vázquez
4,
Pantelis E. Barouchas
5,
Anne Boos
6,
Dimitrios Kalderis
7,
Fotis Sgouridis
8,
Vassilis Aschonitis
2 and
George Arampatzis
2
1
Department of Agriculture, University of Ioannina, Kostakii Campus, 47100 Arta, Greece
2
Soil Water Resources Institute (SWRI), Hellenic Agricultural Organization (HAO)—DIMITRA, Thermi Campus, 57001 Thessaloniki, Greece
3
Department of Agriculture, Hellenic Mediterranean University, 71410 Heraklion, Greece
4
Departamento de Producción Agraria, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas (ETSIAAB), Universidad Politécnica de Madrid (UPM), 28040 Madrid, Spain
5
Department of Agriculture, University of Patras, 26504 Patras, Greece
6
Plateforme Analytique des Inorganiques (PAI), Centre National de la Recherche Scientifique (CNRS)/Université de Strasbourg, 67087 Strasbourg, France
7
Department of Electronics Engineering, Hellenic Mediterranean University, 73100 Chania, Greece
8
Biogeochemistry, School of Geographical Sciences, University of Bristol, Bristol BS8 1SS, UK
*
Author to whom correspondence should be addressed.
Nitrogen 2026, 7(2), 35; https://doi.org/10.3390/nitrogen7020035
Submission received: 19 February 2026 / Revised: 18 March 2026 / Accepted: 19 March 2026 / Published: 24 March 2026
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Abstract

Despite a growing interest in biochar for olive orchard fertility management, little is known about its effects on nitrogen (N) dynamics and greenhouse gas (GHG) emissions in Mediterranean soils, particularly when comparing neutral (pH 6.7) and alkaline (pH 8.2) soils using farmer-accessible flame-curtain pyrolysis biochar. In this 60-day soil mesocosm study, we hypothesized that biochar amendments in fertilized soils would enhance soil N availability and potentially reduce N2O emissions, with effects modulated by soil pH. Treatments included: control, urea fertilizer, and urea plus biochar (5% w/w). Urea fertilization significantly increased soil ammonium (NH4+) and total oxidized nitrogen (NO3− + NO2−) in both soils, and co-application of biochar further increased these pools, particularly in the neutral soil (NH4+: + 91% and + 62% in neutral and alkaline soil, respectively). Biochar addition consistently reduced cumulative carbon dioxide (CO2) emissions in both soils, supporting its role in stabilizing soil organic carbon. However, impacts on nitrous oxide (N2O) emissions were soil-pH-dependent: biochar slightly reduced N2O emissions in neutral soil, though nearly doubled N2O emissions in alkaline soil, highlighting that biochar’s efficacy for GHG mitigation is context-specific. These findings underscore biochar’s potential to improve soil N availability and reduce carbon losses but reveal clear limitations for N2O mitigation in alkaline soils, necessitating site-specific application strategies that explicitly consider soil pH when targeting climate benefits in Mediterranean olive production.

1. Introduction

Mediterranean olive orchards represent a unique agroecosystem that evolved following the early domestication of the olive tree [1], characterized by a complex mosaic of cultural, socio-economic, agricultural, and landscape factors. These rainfed and traditionally managed low-input orchards support regional agricultural economies and livelihoods, particularly in countries such as Greece, Spain, and Italy [2]. In recent decades, nitrogen (N) fertilization in olive orchards has increased, especially in new plantations to boost yields and profitability [3,4]. This intensification impacts N cycling in olive orchards by reducing N use efficiency, increasing N loss, and increasing the carbon (C) footprint of olive oil production compared to traditionally managed orchards [5,6]. Consequently, strategies are needed to mitigate these potential negative impacts.
Management of olive pruning residues, a key by-product, strongly influences the C footprint and GHG balance of olive orchards [7]. Typically, pruning residues were removed and burned to prevent pathogen overwintering or dispersal, contributing to environmental damage [8,9]. While modern mulching practices reduce soil erosion and enhance C sequestration [8,10], the labile C content of residues can lead to substantial CO2 emissions (up to 90% mineralization) [11], and their high C:N ratio causes net N immobilization, limiting crop N availability [12]. Biochar produced via flame-curtain pyrolysis offers an alternative that addresses these limitations by simultaneously valorizing pruning biomass within a circular bioeconomy framework [13] and enhancing soil C storage, nutrient retention, and GHG mitigation in agricultural soils [14]. Flame-curtain kilns provide cost-effective, low-emission methods for producing high-quality biochar at an on-farm scale, making biochar production accessible to rural areas [15,16,17], although flame-curtain biochar performance in Mediterranean olive orchards remains poorly characterized [16,18].
The interaction of biochar with the soil N cycle has been extensively studied in various agroecosystems, excluding Mediterranean orchards [19,20,21]. Biochar’s physicochemical properties, including high surface area, porosity, and cation exchange capacity, enable nutrient absorption, particularly ammonium (NH4+) and nitrate (NO3−), with effects varying by biochar feedstock type, pyrolysis conditions, and biochar modifications [22,23,24]. Biochar may supply potassium—a vital nutrient for olive production [6,25]. It can also improve soil aggregation, thereby enhancing water retention and availability, which are key factors for drought-resilient olive production in the Mediterranean region [26,27,28].
Soil type modulates these responses, particularly in Mediterranean agrosystems, characterized by high soil pH, distinct textures, and depleted soil organic matter (SOM), and where nitrous oxide (N2O) emissions arise mainly from nitrification and denitrification processes driven by N fertilization and irrigation [29,30,31,32,33,34]. In acidic to neutral soils, biochar may raise pH to favour N2O reductase and complete denitrification to N2 [34,35,36,37]; in alkaline soils, it risks amplifying ammonia volatilization (pKa = 9.25), AOB-driven nitrification, and thus enhanced N2O emissions [36,37]. Biochar combined with synthetic fertilizers can further mitigate N2O emissions via retarded nitrification and electron shuttling, thereby decreasing net emissions [38,39,40].
Despite the growing body of literature on biochar in agricultural soils, gaps persist for understanding the biochar’s effects on N dynamics (NH4+, NO3− + NO2−) and GHG fluxes (CO2, N2O) under the unique soils and management in the Mediterranean agrosystems. The present study investigated the effects of biochar addition into urea-fertilized soils on N availability (NH4+, NO3− + NO2−) and greenhouse gas emissions (CO2 and N2O) in two contrasting soils: neutral (pH 6.7) and alkaline (pH 8.2). We hypothesized that biochar amendments in fertilized soils would enhance soil N availability and potentially reduce N2O emissions, with effects modulated by soil pH-mediated nitrification/denitrification responses.

2. Materials and Methods

2.1. Sampling Location and Soil Properties

Two soils with distinct characteristics were selected: one sandy loam neutral (pH 6.7) soil from the Chania region of Crete, and one loam alkaline (pH 8.2) soil from the Thessaloniki region of Central Macedonia, Greece. Composite topsoil (0–25 cm) samples were collected from an existing rainfed olive orchard, Olea europaea cv. Koroneiki and O. europaea cv. Chontroelia Chalkidikis, for the neutral and alkaline soil, respectively. Soils were gently sieved (2 mm) and air-dried after sampling, and their basic soil properties were determined (Table 1).

2.2. Biochar Preparation and Properties

Biochar was prepared at the Hellenic Mediterranean University in Chania, Crete, Greece, by flame-curtain pyrolysis (open-flame pyrolysis). Olive tree pruning biomass was collected from olive orchards in Chania, Crete, Greece, then washed and air-dried to remove impurities. Biomass was subjected to flame-curtain pyrolysis in a portable conical kiln (0.75 m3 capacity; Hellenic Mediterranean University, Chania, Greece) based on Tsubota, Tsuchiya, Kusumoto, and Kalderis [17]. The process temperature was monitored through external thermocouples attached to the kiln and recorded at 540 ± 50 °C throughout the process. After pyrolysis, the biochar was quenched with water and air-dried. The dried biochar was ground using a rod mill (VWR, Darmstadt, Germany) to reduce large particles and make it suitable for further applications (particle size was between 100 and 1000 μm). A representative composite biochar sample was sent to Plateforme Analytique des Inorganiques (PAI), at the Centre National de la Recherche Scientifique (CNRS)/Université de Strasbourg, Strasbourg, France, for physicochemical characterization (Table 2).

2.3. Soil Mesocosms and Analytical Techniques

Three treatments per soil (alkaline and neutral) were prepared in a 720 mL glass jar (mesocosm), a control treatment of 400 g air-dried soil without any amendment, soil (400 g) amended with urea-N (46-0-0, Ziko SA, Thessaloniki, Greece; 55 mg N kg−1), and soil (400 g) amended with urea-N (46-0-0, Ziko SA, Greece; 55 mg N kg−1) and 5% per weight biochar (20 g). In total, 18 mesocosms were prepared, including three replicates per treatment, see Table 3 for experimental design. Mesocosms were rewetted to 20% per weight with DI-H2O and were allowed to equilibrate for 7 days at 25 °C in the dark. Then, the appropriate amount of urea was applied, and this event initiated the start of a 60-day incubation. Soil mesocosms were incubated open in darkness with constant room temperature (25 °C). Soil moisture was maintained weekly at 20% by weight. These incubation conditions are considered optimal for soil microbial activity. Repeated soil and gas samples were collected at regular intervals from each jar, as indicated in the figures, while minimizing disturbance. The incubation period and sampling procedures were designed based on the existing scientific literature to specifically capture the typical fertilizer response period, first, and second, the early pulses of N2O after urea fertilization [41,42,43,44].
Soil samples were analyzed by KCl extraction (2 M; 1:5) for extractable ΝH4+, and ΝO3− + ΝO2− were determined by absorption spectroscopy, following standard protocols (SSSA, 1994). The residence of the available soil ΝO3− + ΝO2−, and NH4+ was calculated as the area under the curve (AUC) of the determined concentration (mg N kg−1 d). Gas samples were analyzed for CO2 and Ν2O as in Giannopoulos et al. [45]. Briefly, headspace CO2 concentration was determined by attaching an infrared gas monitor (Li800, Li-Cor, Lincoln, NE, USA) to headspace fittings attached to an air-tight closed mesocosm. The accumulated CO2 (ppm), measured after approx. 30 min of mesocosm closure was converted to an hourly rate per soil mass (CO2 mg C kg−1 h−1) and interpolated to a cumulative flux per soil mass (CO2 mg C kg−1) assuming a linear relationship between measurements. Similarly, after approx. 40 min of sealing the mesocosms, gas samples for N2O detection were withdrawn with a gas-tight syringe (Hamilton, Reno, NV, USA) and injected directly (500 μL) into a GC system for N2O determination. The GC system consisted of a base unit (GC-2014, Shimadzu, Kyoto, Japan) equipped with an electron capture detector (ECD-2014, Shimadzu, Japan) and a 13-ft Porapak Q plot column (Restek GmbH, Bad Homburg, Germany). Ultrapure N2 (99.999%; Air Liquide Hellas SA, Athens, Greece) was used as a carrier and make-up gas at 35 mL min−1. The oven and ECD temperatures were set at 45 and 320 °C, respectively. The retention time for N2O was observed at 3.9 min, and any gas compound (including air, O2, CO2, and CH4) before this was vented out using an actuated valve (Valco, Vici A.G., Switzerland). The sample N2O concentration (ppm) was converted to an hourly rate (N2O mg N kg−1 h−1) and interpolated to a cumulative flux (N2O mg N kg−1) assuming a linear relationship between measurements.

2.4. Statistical Analyses

Differences in variance of dependent variables (AUC ΝO3− + ΝO2−, AUC NH4+, cum. N2O, and cum CO2) were tested with a two-way analysis of variance (ANOVA; Y ∼ Soil + Treatment + Soil × Treatment), and Bonferroni post hoc adjustment was used for evaluating differences between the treatments at p ≤ 0.05 level. For temporal measurements, we calculated area under the curve (AUC) values to integrate concentration changes over time, avoiding issues of temporal pseudoreplication inherent in repeated-measures designs. Residuals were inspected for normality (Shapiro–Wilk) and homoscedasticity (Levene). Where necessary, data were log-transformed before ANOVA. All statistical tests were performed, and illustrations were prepared in R Studio (v. 2024.12.1+563; RStudio Inc., Boston, MA, USA). Non-transformed means are presented in figures. Results of treatment replicates (n = 3) are shown as means ± standard errors (SE), unless otherwise indicated.

3. Results and Discussion

3.1. Temporal Nitrogen Kinetics and Soil Nitrogen Availability

Soil available ammonium (NH4+) concentrations varied significantly with soil type, treatment, and time (Figure 1a,b and Figure 2a,b). In both soils, the unamended Control showed a gradual decline in NH4+ from day 5 to 60 (Figure 1a,b), consistent with ongoing mineralization and nitrification processes [29,34,46]. Urea sharply increased NH4+, with early peaks around days 5–14, followed by a decline, while Urea + BC maintained higher early-stage concentrations (days 5–14) before converging toward Control levels later. Early peaks are consistent with rapid urea hydrolysis [47]. The combined treatment (Urea + BC) generally maintained higher NH4+ concentrations at the early stages (5–14) of the incubation compared to the Urea treatment (Figure 1a,b). Subsequently, the soil-available NH4+ concentration declined towards the latter stages of the incubation, rendering this effect transient. Neutral soils exhibited lower initial NH4+ in Controls but higher NH4+ retention under amendments (Urea, and Urea + BC) than alkaline soils (Figure 1a,b), aligning with pH effects on NH4+ retention and nitrogen cycling as assumed due to alkaline biochar addition [21,22]. The observed NH4+ patterns highlight biochar’s potential to modulate N availability and transformation in the short term (5–14 days), likely through combined effects of pH-induced nitrification and surface adsorption.
Soil available nitrate and nitrite (NO3− + NO2−) temporal concentrations varied significantly with soil type, treatment, and time (Figure 1c,d and Figure 2c,d). Urea application markedly increased NO3− + NO2− in both soils, with peaks between days 14 and 28, whereas Controls showed only a gradual increase (Figure 1c,d), consistent with basal mineralization and nitrification, with a slower nitrification rate in the neutral soil [47]. In alkaline soil, Urea + BC initially produced higher NO3− + NO2− than Urea at day 14, likely due to enhanced nitrification from increased NH4+ availability, but subsequently showed lower concentrations, suggesting biochar-mediated adsorption or microbial immobilization [40]. Neutral soil exhibited a similar but less pronounced pattern, with Urea + BC showing delayed NO3− peaks at day 21 (Figure 1c).
There is no indication of widespread anoxic, reductive conditions for denitrification, with water-filled pore space (WFPS) of approx. 50 and 60% for both soil [48], thus enhanced nitrification/nitrifier denitrification is the most plausible dominant source of soil available NO3− + NO2− in the biochar amended soils (Urea + BC). The NO3− depletion pattern may indicate, firstly, increased nitrification rates compared to Urea addition, due to higher NH4+ availability, secondly, biochar-induced NO3− reduction in anaerobic microsites (classical heterotrophic denitrification), potentially contributing to gaseous N loss, and thirdly, NO3− + NO2− adsorption to biochar and perhaps biochar-mediated microbial N immobilization.
These patterns align with previous findings that biochar can mitigate nitrate leaching and modulate N cycling dynamics [49]. Biochar’s porous structure and significant surface area can adsorb NH4+, reducing leaching losses, enhancing nitrogen retention in the soil matrix, and inducing microbial N transformations [19,50,51,52]. Despite aerobic incubation conditions, the NO3− depletion pattern may suggest biochar-induced nitrate reduction, potentially via denitrification (greater N2O production in biochar-amended soils; Figure 3) in anaerobic microsites within biochar pores, leading to gaseous N loss [22,53]. This hypothesis requires confirmation through isotopic tracing or functional gene analysis in future studies.
Moreover, biochar’s liming effect can influence N transformations by altering soil pH and microbial activity, which may explain the differential responses observed between neutral and alkaline soils. Post-incubation soil analyses confirmed pH increases in both soils following biochar addition (Table S1). It is well known that soil pH is a principal determinant of soil bacterial and fungal communities, exerting a significant effect on microbial populations and community composition and structure of the soils [54,55]. In urea-fertilized neutral soils, biochar addition may improve cation exchange capacity and microbial habitat without drastically altering pH, thereby enhancing soil nitrogen availability more effectively [39]. In urea-fertilized alkaline soils, where pH is already high, the effect of biochar on soil nitrogen availability may be less pronounced due to a limited pH modification and different microbial community dynamics, as observed previously in Mediterranean soils [46,56].
Our results demonstrated a clear and consistent increase in soil N availability, expressed as an integrated value (AUC; mg kg−1 d) of temporal concentrations of soil NH4+, following urea fertilization in both soil types due to the direct input of mineral N (urea-N; Figure 2a,b). The NH4+ availability (AUC NH4+) for the Urea treatment was approx. 2185 and 3414 mg N kg−1 d (Figure 2a,b), and approx. 4894 and 6687 mg N kg−1 d AUC NO3- + NO2- (Figure 2c,d) for the neutral and alkaline soil, respectively, significantly greater than the Control (Figure 2a–d). In the neutral soil, biochar (Urea + BC) increased NH4+ availability (AUC NH4+) by approximately 91% relative to the Control, while in alkaline soil, the increase was approximately 62% (Figure 2a,b). In alkaline soils, the addition of biochar to urea-fertilized soils (Urea + BC) reduced AUC NH4+ by approximately 30% compared to Urea treatment. Ventura et al. [57] and Martos et al. [58] postulated that ammonia volatilization is the primary mechanism in the strongly alkaline microenvironment created around biochar particles, as it competes with nitrate production via nitrification. Given the alkaline pH of the soil (8.2) and of the biochar (9.3), ammonia volatilization likely represents a significant N loss pathway in the Urea + BC treatment in alkaline soil, potentially explaining the observed reduction in mineral N availability despite urea application. Future studies should employ acid traps or dynamic chamber systems to quantify NH3 fluxes. Elsewhere, studies have observed greater variability in the effects of biochar addition on neutral and alkaline soils compared to acidic ones, attributing this to the diminished liming effect of biochar in neutral and alkaline soils [49,57,59,60]. Post-incubation soil analyses confirmed pH increases in biochar-amended treatments (Table S1), supporting the liming effect interpretation.

3.2. Temporal Patterns of Soil CO2 and N2O Flux Emissions

In both soils, CO2 fluxes increased from day 5, peaked between days 14 and 35, and declined by day 60, reflecting microbial response to substrate addition and subsequent depletion (Figure 3). Neutral soil (higher SOM: 5.4%) showed equal or greater CO2 fluxes than alkaline soil (3.2% SOM) across all treatments. Control, Urea, and Urea + BC treatments ranged approximately 149–435, 145–386, and 93–477 µg C kg−1 h−1 in neutral soil (Figure 3a,b), and 71–425, 64–385, and 75–397 µg C kg−1 h−1 in alkaline soil (Figure 3c,d), respectively. Urea + BC showed delayed peaks (days 21–28), indicating delayed biochar stimulation in fertilized soils. N2O fluxes followed similar temporal dynamics to CO2 (peak days 14–35), with alkaline soil consistently showing higher emissions than neutral soil across all treatments (Figure 4c,d). Controls exhibited low, stable emissions (0.01–0.05 μg N kg−1 h−1 neutral; 0.07–0.15 μg N kg−1 h−1 alkaline). Urea increased fluxes (0.06–0.41 neutral; 0.04–0.54 alkaline), while Urea + BC produced the highest initial pulse in alkaline soil (1.3 μg N kg−1 h−1 at day 2; Figure 4c) with persistently elevated emissions throughout incubation, contrasting with neutral soil where Urea + BC showed initial elevation (0.6 μg N kg−1 h−1 day 2, Figure 4a) followed by moderate sustained fluxes. These patterns reflect the stimulation of nitrification and denitrification processes, known to produce N2O, following mineral N fertilization, and are intrinsically linked to microbial activity and carbon respiration. Alkaline soils consistently showed higher N2O fluxes compared to the neutral soils under all treatments, indicating enhanced microbial nitrogen transformations in alkaline pH environments. Overall, urea addition (Urea) substantially increased N2O flux compared to Control in both soil types, while biochar amendment in fertilized soils (Urea + BC) modulated the magnitude and temporal dynamics of emissions differently depending on soil pH. This attenuation likely results from biochar’s capacity to improve soil aeration, adsorb ammonium and nitrate, and alter microbial community structure, thereby reducing denitrification hotspots and N2O production.

3.3. Complex Effects on Cumulative CO2 and N2O Soil Emissions

Cumulative CO2 emissions were slightly higher in neutral than in alkaline soils for most of the incubation, consistent with greater microbial activity under neutral pH conditions (Figure 3b,d). Control-treated neutral soils consistently exhibited high cumulative CO2 emissions, reaching approximately 369 mg C kg−1 by the end of the incubation (day 60). The addition of urea to neutral soils resulted in a modest reduction, with final cumulative emissions of 354 mg C kg−1, i.e., about 15 mg C kg−1 lower than the Control. The Urea + BC treatment produced the lowest cumulative CO2 emissions in neutral soils, with a final value of 317 mg C kg−1, representing a reduction of more than 50 mg C kg−1 relative to the Control. In alkaline soils, CO2 emission patterns were initially slightly lower than in neutral soils, but the alkaline Control ultimately reached 398 mg C kg−1 by day 60, surpassing the neutral Control. In alkaline soils, Urea reduced cumulative CO2 emissions to 340 mg C kg−1, and Urea + BC further reduced them to 337 mg C kg−1. Comparing treatments across both soil types, the highest cumulative CO2 emissions were observed in the Control for both alkaline and neutral soils (398 and 369 mg C kg−1, respectively), while the lowest were observed under Urea + BC (317 mg C kg−1 in neutral soil (Figure 3b) and 337 mg C kg−1 in alkaline soil; Figure 3d). Statistical analysis showed that only the treatment factor significantly affected cumulative CO2 emission variability (p < 0.001), and for both soils, Urea and especially Urea + BC significantly reduced cumulative CO2 emissions compared to the Control (Figure 3b,d).
The consistent reduction in cumulative CO2 emissions under Urea and particularly Urea + BC across both soils indicates a strong control of fertilization and biochar amendment on microbial respiration dynamics. The greater decrease in neutral soil under Urea + BC (over 50 mg C kg−1 relative to Control) suggests that biochar may have enhanced carbon use efficiency and reduced decomposition of native soil organic matter, in line with the concept of negative priming, whereby N addition and sorption of labile C reduce the need for microbes to mineralize existing SOM [61]. The magnitude of this reduction in cumulative CO2 emissions should not be overlooked because urea hydrolysis contributes stoichiometrically up to 23.6 mg C kg−1 soil. Meta-analyses have also reported up to approx. 25% lower CO2 emissions when biochar is combined with N fertilizers such as urea [62], supporting the observed mitigation. Although this study did not directly quantify changes in SOC pools during the incubation, the decrease in cumulative CO2 emissions, together with the documented increase in total soil C in biochar-amended neutral soil (Table S1), is consistent with biochar acting as a relatively stable carbon input and partially suppressing microbial respiration [20,52].
Across all time points, cumulative N2O emissions were substantially higher in amended soils (Urea and Urea + BC) than in the Control, with the most pronounced effects in alkaline soil (Figure 4d). In neutral soil, the Control reached 48 μg N kg−1 by day 60 (Figure 4b). Urea increased cumulative N2O emissions to 233 μg N kg−1, a 384% rise relative to the Control. The Urea + BC treatment in neutral soil resulted in 186 μg N kg−1, about 20% lower than Urea alone, although still 287% higher than the Control by day 60 (Figure 4b). In alkaline soil (Figure 4d), the Control reached 154 μg N kg−1 by day 60. Urea amendment increased cumulative N2O emissions to 215 μg N kg−1 (approx. 40% above the Control). The highest emissions were observed in Urea + BC, with 380 μg N kg−1, representing a 146% increase compared to Urea alone and a 147% increase compared to the Control. Overall, urea application markedly increased cumulative N2O emissions in both soils relative to the Control, while biochar co-application reduced cumulative N2O in neutral soil (Figure 4b) but strongly amplified it in alkaline soil (Figure 4d). Soil type, treatment, and their interaction exerted a strong control over cumulative N2O emission variability (p < 0.001; Figure 4b,d).
The strong N2O response to urea is consistent with the well-documented stimulation of nitrification and denitrification by mineral N fertilization, modulated by soil physicochemical properties and microbial communities [63]. Biochar co-application (Urea + BC) produced divergent N2O responses, approx. 20% reduction relative to Urea in neutral soil versus a 146% increase relative to Urea in alkaline soil. This pattern indicates that the efficacy of biochar as a N2O mitigation strategy is strongly soil type-dependent, with soil pH acting as a critical modulator of biochar effects on nitrogen transformations, as also highlighted by Chen [40].
In neutral soil, the non-significant mitigation of N2O under Urea + BC may be associated with several mechanisms, including altered microbial N transformations and reduced substrate availability for denitrifiers and nitrifiers due to adsorption of ammonium and nitrate onto biochar surfaces [20,38,39,53]. Conversely, the strong increase in N2O emissions in alkaline soil amended with biochar parallels reports where biochar raised N2O production under higher pH conditions [37,45]. Alkaline conditions can favour nitrification pathways that produce N2O, and biochar’s influence on pH, aeration, and substrate availability may enhance these processes. Under the aerobic incubation conditions used here (WFPS 50–60%), nitrification likely contributed substantially to N2O emissions, and the stronger response in the alkaline soil is compatible with known shifts towards ammonia-oxidizing bacteria (AOB) dominance and higher N2O yields in high-pH, high-NH4+ environments [64,65,66,67,68]. While our results strongly suggest pH-dependent shifts in microbial nitrogen transformations, direct molecular confirmation of microbial community shifts was beyond the scope of this study.
As cumulative N2O was comparatively lower than the observed decreases in available nitrate, additional processes likely regulated nitrate availability in biochar-amended treatments, including enhanced microbial N immobilization and/or nitrate adsorption onto biochar, as indicated in previous work [50,51]. The modest reduction in measured CEC in the biochar-amended alkaline soil (from 22.4 to 17.0 cmol kg−1; Table S1) may reflect a redistribution or complexation of exchangeable cations on biochar surfaces; however, this hypothesis requires further investigation. Although biochar is generally expected to increase soil CEC owing to its porous structure and abundance of surface functional groups, the 24% decrease observed here is plausibly attributed to complex interactions among the low-CEC (5 cmol kg−1), high-ash content (36%) of biochar, the calcareous soil matrix (5.2% CaCO3), and the absence of any post-activation or surface oxidation biochar treatment. Overall, the contrasting N2O responses between neutral and alkaline soils underscore that initial soil pH and associated microbial communities critically determine whether biochar co-applied with urea will mitigate or exacerbate N2O emissions.

3.4. Synthesis: Soil pH as a Critical Modulator

The hypothesis that biochar addition to fertilized soils enhances soil nitrogen availability, specifically soil ammonium (NH4+) and nitrates (NO3− + NO2−), while concurrently reducing greenhouse gas emissions (CO2 and N2O), is partially supported by our findings. Biochar significantly increased soil nitrogen availability in both neutral and alkaline soils during the early stages of the incubation period, indicating an initial stimulation of nitrogen retention or mineralization processes. However, this enhancement was transient, as a notable depletion of soil available NH4+ and NO3− + NO2− occurred in later stages, likely reflecting increased microbial uptake, adsorption, and/or transformation. The effects on greenhouse gas emissions were more complex: biochar amendments consistently reduced CO2 emissions across both urea-fertilized soil types, demonstrating a clear mitigation potential for soil respiration-related carbon release. In contrast, biochar’s impact on N2O emissions was soil pH-dependent; it effectively mitigated N2O fluxes in neutral soils but unexpectedly elevated emissions in alkaline soils. Whether the observed pH-dependent N2O responses persist, diminish, or intensify over longer timescales remains unknown, as this study captured the short-term response of agricultural biochar addition to soils. Ageing processes may alter its physicochemical properties over months to years, and this represents a critical knowledge gap for long-term field management.
Our findings align with the growing body of literature emphasizing the context-dependent nature of biochar effects [20,52]. It highlights the need for site-specific assessments before recommending biochar as a climate-smart agricultural tool. This contrasting response suggests that biochar influences nitrification and denitrification pathways differently depending on soil chemical environment, potentially through alterations in microbial communities or substrate availability. Consequently, while biochar shows promise as a soil amendment for improving nitrogen retention and reducing CO2 emissions, its effects on N2O emissions require careful consideration within specific soil contexts, particularly alkaline conditions where it may exacerbate N2O release. These results underscore the nuanced role of biochar in soil nitrogen cycling and greenhouse gas dynamics, emphasizing the need for tailored management approaches based on soil and biochar properties.

3.5. Challenges and Implications for Wider Adoption in Olive Orchard Agroecosystems

Despite biochar’s potential to enhance N availability and reduce CO2 emissions in olive orchards, adoption faces challenges related to variable responses in calcareous, alkaline Mediterranean soils [25,39]. In this study, biochar increased N2O emissions in alkaline soil, while field studies in Spanish olive orchards (Calsisol) reported negligible effects on NH4+ retention [37,46,53]. Such unpredictability necessitates site-specific evaluation of biochar–soil–fertilizer interactions. Whether the pronounced N2O increase observed at 5% biochar application would occur at agronomically feasible rates (10–20 t ha−1) requires field-scale validation.
Economic barriers include biomass feedstock availability, pyrolysis costs, and transport, particularly for resource-limited farmers [19]. On-site open-flame pyrolysis has several advantages, including low cost, accessibility, and portability to produce certified biochar [69], which can effectively close the carbon cycle in olive orchards by converting pruning waste into biochar for direct soil application, sequestering carbon, and preventing open burning emissions [16,17]. Long-term studies on olives are scarce, yet essential for assessing persistence, soil health, yields, and gas fluxes.
Key findings indicate biochar improves soil N availability and cuts down CO2 emissions in olive soils, aiding climate mitigation. However, N2O reduction is reliable mainly in neutral or acidic soils. Agronomists and growers should tailor applications to soil pH, texture, and nutrients, optimizing biochar type, method, and rates. A systematic investigation across a pH gradient using multiple Mediterranean soils would allow optimization of fertilization combined with biochar addition, thus maximizing benefits. Our study did not directly measure changes in the soil microbial community or N2 emissions, limiting mechanistic interpretation. Future research must clarify N2O and N2 mechanisms by integrating isotopic approaches, developing targeted biochars, and conducting long-term trials and life cycle assessments for sustainable Mediterranean olive production.

4. Conclusions

Biochar addition to urea-fertilized soils enhances total soil N by approximately 20% and consistently reduces CO2 emissions in both neutral and alkaline soils. However, its effect on N2O emissions is soil-dependent, reducing emissions in neutral soil but nearly doubling them in alkaline soil. Thus, this study provides evidence that the climate mitigation potential of biochar is strongly soil pH-dependent, reinforcing the need for site-specific management in Mediterranean olive systems.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nitrogen7020035/s1, Table S1: Selected physicochemical properties of soils measured at the end of the 60-day incubation period (mean ± SE, n = 3).

Author Contributions

G.G.: Conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing—original draft preparation, writing—review and editing, project administration, funding acquisition. I.A.: Conceptualization, validation, formal analysis, writing—review and editing. V.A.T.: Conceptualization, validation, formal analysis, writing—original draft preparation, writing—review and editing. E.V.: validation, formal analysis, writing—review and editing. P.E.B.: Conceptualization, methodology, validation, writing—review and editing. A.B.: methodology, validation. D.K.: Conceptualization, validation, formal analysis, writing—review and editing. F.S.: methodology, validation, writing—review and editing. V.A. and G.A.: methodology, validation, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This work was partly supported by the EPICUR European University Alliance—EPICUR Research—EPIClusters Research Mobility (EU Horizon 2020; Grant Agreement No. 101016926), hosted by the University of Strasbourg (Université de Strasbourg, Strasbourg, France), by the Plateforme Analytique des Inorganiques (PAI) at the Centre National de la Recherche Scientifique (CNRS; Strasbourg, France), and by the Aristotle University of Thessaloniki (Thessaloniki, Greece). This study was partly supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the ‘2nd Call for H.F.R.I.’ Research Projects to support Post-Doctoral Researchers ‘Nitro-Ag: Towards greater nitrogen use efficiency in agroecosystems’ (Project Number: 01053), awarded to Dr. Georgios Giannopoulos. Corteva Agriscience Hellas S.A. supported Dr. Georgios Giannopoulos with a personal travel grant.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors wish to thank the technical personnel of the Hellenic Agricultural Organization (HAO)—DIMITRA for facility access and soil analyses support. We thank Fotis Bilias for determining CEC in soil and biochar samples.

Conflicts of Interest

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Temporal concentrations of soil available ammonium (NH4+), nitrate and nitrite (NO3− + NO2−) from the neutral and alkaline soil under different treatments over a 60-day incubation. Panels show soil available ammonium (NH4+) (a) in neutral, and (b) in alkaline soil, available soil nitrate and nitrite (NO3− + NO2−) (c) in neutral, and (d) in alkaline soil. Treatments include unamended soil (Control; grey circles), soil amended with urea (Urea; blue squares), and soil amended with urea plus biochar (Urea + BC; orange diamonds). Error bars denote standard error (mean ± SE, n = 3).
Figure 1. Temporal concentrations of soil available ammonium (NH4+), nitrate and nitrite (NO3− + NO2−) from the neutral and alkaline soil under different treatments over a 60-day incubation. Panels show soil available ammonium (NH4+) (a) in neutral, and (b) in alkaline soil, available soil nitrate and nitrite (NO3− + NO2−) (c) in neutral, and (d) in alkaline soil. Treatments include unamended soil (Control; grey circles), soil amended with urea (Urea; blue squares), and soil amended with urea plus biochar (Urea + BC; orange diamonds). Error bars denote standard error (mean ± SE, n = 3).
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Figure 2. Integrated soil ammonium (NH4+) and combined nitrate and nitrite (NO3− + NO2−) availability, expressed as the area under the curve (AUC) of their temporal concentrations, for the two studied soils. Panels (a,b) depict the integrated values of NH4+ availability for the neutral and alkaline soil, respectively. Panels (c,d) depict the integrated values of NO3− + NO2− availability for the neutral and alkaline soil, respectively. The treatments are: unamended soil (Control), urea-amended soil (Urea), and urea plus biochar-amended soil (Urea + BC). Data are presented as mean ± SE (n = 3). Different letters indicate significant differences between treatments (p < 0.05).
Figure 2. Integrated soil ammonium (NH4+) and combined nitrate and nitrite (NO3− + NO2−) availability, expressed as the area under the curve (AUC) of their temporal concentrations, for the two studied soils. Panels (a,b) depict the integrated values of NH4+ availability for the neutral and alkaline soil, respectively. Panels (c,d) depict the integrated values of NO3− + NO2− availability for the neutral and alkaline soil, respectively. The treatments are: unamended soil (Control), urea-amended soil (Urea), and urea plus biochar-amended soil (Urea + BC). Data are presented as mean ± SE (n = 3). Different letters indicate significant differences between treatments (p < 0.05).
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Figure 3. Cumulative CO2 emissions from two soils under different treatments over a 60-day incubation (mean ± SE, n = 3). Panels show cumulative CO2 emissions (mg C kg−1) in neutral soil (a) throughout the incubation and (b) at the end of the incubation, and cumulative CO2 emissions (mg C kg−1) in alkaline soil (c) throughout the incubation and (d) at the end of the incubation. Treatments include unamended soil (Control; grey circles), soil amended with urea (Urea; blue squares), and soil amended with urea plus biochar (Urea + BC; orange diamonds). Bars represent mean ± SE (n = 3). Different letters indicate significant differences among treatments within each soil type (p < 0.05).
Figure 3. Cumulative CO2 emissions from two soils under different treatments over a 60-day incubation (mean ± SE, n = 3). Panels show cumulative CO2 emissions (mg C kg−1) in neutral soil (a) throughout the incubation and (b) at the end of the incubation, and cumulative CO2 emissions (mg C kg−1) in alkaline soil (c) throughout the incubation and (d) at the end of the incubation. Treatments include unamended soil (Control; grey circles), soil amended with urea (Urea; blue squares), and soil amended with urea plus biochar (Urea + BC; orange diamonds). Bars represent mean ± SE (n = 3). Different letters indicate significant differences among treatments within each soil type (p < 0.05).
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Figure 4. Cumulative N2O emissions from two soils under different treatments over a 60-day incubation (mean ± SE, n = 3). Panels show cumulative N2O emissions (μg N kg−1) in neutral soil (a) throughout the incubation and (b) at the end of the incubation, and cumulative N2O emissions (μg N kg−1) in alkaline soil (c) throughout the incubation and (d) at the end of the incubation. Treatments include unamended soil (Control; grey circles), soil amended with urea (Urea; blue squares), and soil amended with urea plus biochar (Urea + BC; orange diamonds). Bars represent mean ± SE (n = 3). Different letters indicate significant differences among treatments within each soil type (p < 0.05).
Figure 4. Cumulative N2O emissions from two soils under different treatments over a 60-day incubation (mean ± SE, n = 3). Panels show cumulative N2O emissions (μg N kg−1) in neutral soil (a) throughout the incubation and (b) at the end of the incubation, and cumulative N2O emissions (μg N kg−1) in alkaline soil (c) throughout the incubation and (d) at the end of the incubation. Treatments include unamended soil (Control; grey circles), soil amended with urea (Urea; blue squares), and soil amended with urea plus biochar (Urea + BC; orange diamonds). Bars represent mean ± SE (n = 3). Different letters indicate significant differences among treatments within each soil type (p < 0.05).
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Table 1. Selected soil properties of the neutral and alkaline soils used in this study (mean ± SE, n = 3).
Table 1. Selected soil properties of the neutral and alkaline soils used in this study (mean ± SE, n = 3).
Soil Property (Units)Neutral SoilAlkaline Soil
RegionChania, GreeceThessaloniki, Greece
TextureSandy LoamLoam
Sand (%)77 ± 1.834 ± 1
Silt (%)13 ± 1.845 ± 1
Clay (%)10 ± 0.721 ± 1
pH 1:106.4 ± 0.28.2 ± 0.2
EC (μS cm−1)450 ± 10.0965 ± 15
CaCO3 (%)0.2 ± 0.05.2 ± 0.5
Soil organic matter (SOM; %)5.4 ± 0.13.2 ± 0.1
total N (mg kg−1)1.8 ± 0.10.2 ± 0.05
NH4+ (mg kg−1)26.5 ± 2.518.3 ± 1.5
NO3− (mg kg−1)85.7 ± 565.5 ± 4
NO2− (mg kg−1)2.1 ± 0.51.5± 0.5
P (mg kg−1)24.1 ± 317.5 ± 2
K (mg kg−1)132 ± 12120 ± 15
Cation Exchange Capacity (CEC; cmol kg−1)12 ± 0.622.4 ± 0.5
Table 2. Biochar properties *.
Table 2. Biochar properties *.
Biochar Property (Units)Value
FeedstockOlive tree pruning without leaves
Pyrolysis Typeflame-curtain (open-flame) pyrolysis
Pyrolysis Temperature (°C)540 ± 50
C (%)79.2
N (%)0.4
H (%)1.9
O (%)18.5
pH9.3
Ash content (%)36
Bulk density (g cm−3)0.54
P (mg kg−1)22.6
K (mg kg−1)28.8
Na (mg kg−1)3.7
Ca (mg kg−1)8.9
Mg (mg kg−1)3.4
Cation Exchange Capacity (CEC; cmol kg−1)5
Polycyclic Aromatic Hydrocarbons
(PAHs; mg kg−1)		<6
* Values represent analyses of a composite biochar sample.
Table 3. Experimental design of this study (mean ± SE, n = 3).
Table 3. Experimental design of this study (mean ± SE, n = 3).
SoilTreatmentDescription
NeutralControlUnamended soil (400 g)
UreaSoil (400 g) amended with urea-N at 55 mg N kg−1
Urea + BCSoil (400 g) amended with urea-N at 55 mg N kg−1 and 5% biochar (20 g).
AlkalineControlUnamended soil (400 g)
UreaSoil (400 g) amended with urea-N at 55 mg N kg−1
Urea + BCSoil (400 g) amended with urea-N at 55 mg N kg−1 and 5% biochar (20 g).
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MDPI and ACS Style

Giannopoulos, G.; Anastopoulos, I.; Tzanakakis, V.A.; Vázquez, E.; Barouchas, P.E.; Boos, A.; Kalderis, D.; Sgouridis, F.; Aschonitis, V.; Arampatzis, G. Olive Tree (Olea europaea) Biochar Differentially Affects N2O and CO2 Emissions in Neutral and Alkaline Olive Orchard Soils. Nitrogen 2026, 7, 35. https://doi.org/10.3390/nitrogen7020035

AMA Style

Giannopoulos G, Anastopoulos I, Tzanakakis VA, Vázquez E, Barouchas PE, Boos A, Kalderis D, Sgouridis F, Aschonitis V, Arampatzis G. Olive Tree (Olea europaea) Biochar Differentially Affects N2O and CO2 Emissions in Neutral and Alkaline Olive Orchard Soils. Nitrogen. 2026; 7(2):35. https://doi.org/10.3390/nitrogen7020035

Chicago/Turabian Style

Giannopoulos, Georgios, Ioannis Anastopoulos, Vasileios A. Tzanakakis, Eduardo Vázquez, Pantelis E. Barouchas, Anne Boos, Dimitrios Kalderis, Fotis Sgouridis, Vassilis Aschonitis, and George Arampatzis. 2026. "Olive Tree (Olea europaea) Biochar Differentially Affects N2O and CO2 Emissions in Neutral and Alkaline Olive Orchard Soils" Nitrogen 7, no. 2: 35. https://doi.org/10.3390/nitrogen7020035

APA Style

Giannopoulos, G., Anastopoulos, I., Tzanakakis, V. A., Vázquez, E., Barouchas, P. E., Boos, A., Kalderis, D., Sgouridis, F., Aschonitis, V., & Arampatzis, G. (2026). Olive Tree (Olea europaea) Biochar Differentially Affects N2O and CO2 Emissions in Neutral and Alkaline Olive Orchard Soils. Nitrogen, 7(2), 35. https://doi.org/10.3390/nitrogen7020035

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