To conduct the incubations, areas of similar soil characteristics were selected in the municipality of Nova Esperança do Piriá, Pará, Brazil (2°15′S, 46°58′W; altitude 73 m). The local climate is classified as a tropical monsoon (Am) type according to the Köppen classification, with a short dry season and heavy rains during the rest of the year (Alvares et al., 2013). In the year of the collect the total precipitation is 3,000 mm and mean temperature is 27.7°C, with maximum and minimum temperature of 33.8°C and 22.6°C, respectively. The soil was collected at a depth of 20 cm from three land-use systems (forest, pasture, and agriculture), approximately 2 km from each other. The soil characteristics are presented in Table 1. The incubations were conducted in the foraging laboratory at the State University of São Paulo, Jaboticabal, São Paulo, Brazil.

The systems (Figure 1) were chosen based on a diagnosis conducted in the municipality of Nova Esperança do Piriá, Pará, Brazil, because the region’s economy revolves around black pepper production. Pasture systems (2°19′34″S; 46°56′22″W) invest little in the system’s intensification. The native forest (2°19′14″S; 46°56′26″W) area represents a reference of the original ecosystem of the region. The pasture used for soil collection was formed in 2007 after burning with the implantation of Urochloa brizantha cv. Marandú. A swidden process is performed annually to remove invasive plants. The production system is extensive, with cattle production using a continuous stocking system at various stocking rates. The last fertilization was carried out in 2018 using natural reactive phosphate (90 kg P₂O₅ ha⁻¹). The area corresponding to agriculture (2°19′38″S; 46°55′40″W) has cultivated black pepper (Piper nigrum), which was opened in 2012 through burning. The planting was 5 years old and in the third collection cycle. At the time of collection, the crop was in its fifth year of cultivation and received annual fertilization of 130 kg ha^–1 of nitrogen, 80 kg ha^–1 of phosphorus (P₂O₅), and 200 kg ha^–1 of potassium (KCl).

Soil samples were collected, air-dried for 72 h, homogenized, and mechanically macerated using a roller to break the clods.

Three incubations were carried out to determine the CH₄ emissions and flux in each land-use system, as well as their interactions with temperature variations and soil nitrogen input.

a) Incubation 1: A completely randomized design consisting of three treatments corresponding to land use (forest, pasture, and agriculture) with five replicates, totaling 15 experimental units.

The study was conducted under controlled conditions: 100 g of dry soil (forest, pasture, or agriculture) was added to flask (500 mL), and the soil moisture was maintained at 33% of the water retention capacity in all soil systems. In incubations where the effect of temperature was not tested, 25.0°C ± 1.0°C was maintained. In incubation 1 and 2, 0.018 mg g⁻¹ soil, corresponding to 50 kg of N ha⁻¹, was added in each soil in the form of a liquid solution to stimulate the soil’s microbial population, wheres in incubation 3, the addition of nitrogen was an evaluating factor.

a) Soil temperature elevation treatment

Greenhouses with forced air circulation were maintained at a constant temperature corresponding to the defined values. The vials were kept at 25°C, 30°C, 35°C, and 40°C and withdrawn only for gas collection.

b) Soil nitrogen input treatment

Urea solutions with predefined nitrogen concentrations were prepared. To nitrogen concentrations of 90, 180, and 270 kg of N ha⁻¹, 0.032, 0.064, and 0.096 mg g⁻¹ soil, respectively, were added. For the treatment of 0 kg of N ha⁻¹, only water was added to the vials to maintain the desired humidity.

To quantify the emissions of CH₄, the technique of a closed static chamber consisting of 400 mL of free space was used. The sampling routine was between 9 a.m. and 10 a.m., as described by Cardoso et al. (2017). The flux of CH₄ emissions was monitored for 25 days in incubation 1 and 28 days in incubations 2 and 3. Samples were collected every day during the first week and at 2-day until the end of the incubation period.

To measure CH₄ production, bottle caps were sealed for 30 min and the change in free-space concentration was quantified. At the end of the incubation period (T30), air samples were collected using 50 mL polypropylene syringes. Additionally, ambient gas samples (T0) were collected from the five chambers before sealing to measure the initial CH₄ concentration. The chambers used for the initial sample collection were rotated for each collection.

The gas samples were transferred to 20 mL pre-evacuated vials (Shimadzu vials) for quantitative analysis using gas chromatography (Shimadzu Green House Gas Analyzer GC-2014; Kyoto, Japan) to measure CH₄. The analysis was performed under the following conditions: injector temperature of 250°C, column temperature of 80°C, N₂ carrier gas flow rate of 30 mL min⁻¹, and flame ionization detector (FID) temperature set at 280 °C.

The CH₄ flux (μg CH₄ m⁻² h⁻¹) was determined by calculating the change in concentration over the sampling period, assuming a linear increase in CH₄ concentration. The calculations were performed using the following equations (Cardoso et al., 2019): C H 4 f l u x = P R × T × V A × Δ C Δ t

Where, P is the air pressure at the sampling site, R denotes the gas constant, T is the temperature inside the chamber, V is the volume of the chamber, A is the surface area of the sampling soil, and ΔC/Δt is the linear slope of the concentration change during the sampling period.

Daily flux data were plotted over time to generate cumulative emissions by applying a linear interpolation between sampling days. This approach allowed for data integration. To determine the CH₄ flux attributed to the treatments, the cumulative emissions were subtracted from the ambient gas collected on the same day.

Soil mineral nitrogen content was assessed at the end of the incubation period by extracting it with 2 M KCl and conducting colorimetric analysis to determine the levels of ammonium (Forster, 1995) and nitrate (Doane and Horwath, 2003).

To verify the normality of the residuals and homogeneity of variances, the data were analyzed using the Shapiro-Wilk and Levene tests, respectively. When the presence of outliers was detected, they were excluded from the analysis. Analysis of variance (ANOVA) was performed for the three incubation tests. When ANOVA was significant for land use, the means were compared using Tukey’s test, considering p < 0.05 as a significant difference. When the temperature and nitrogen variations were significant; regression adjustment was performed to identify the effects of the treatments. All statistical analyses were performed using the statistical program R (version 4.0.2; R Core Team, 2014).

The soil with agricultural use of 0 and 90 kg N ha⁻¹ exhibited the lowest ammonium content (7.50 mg N-NH₄ kg⁻¹ of dry soil), whereas the forest treatment at 25°C showed the highest ammonium content (30.0 mg N-NH₄ kg⁻¹ dry soil). Conversely, the lowest nitrate content was observed in the forest treatment (0.30 mg N-NO₃ kg⁻¹ of dry soil), whereas the highest nitrate content was found in the pasture treatment at 180 kg N ha⁻¹ (12.5 mg N-NO₃ kg⁻¹ of dry soil). Table 2 presents the results of soil mineral nitrogen analysis.

When evaluating the effect of land use, higher total emissions of CH₄ (p < 0.001) were observed in pasture soil (470 μg CH₄ g⁻¹ of dry soil; Figure 2) compared the soils of the other two systems. In contrast, there was a process of oxidation of CH₄ in tropical rainforest soils (−303 μg CH₄ g⁻¹ of dry soil) and agriculture (−267 μg CH₄ g⁻¹ of dry soil), which did not differ (p > 0.05) between them. Therefore, these values are considered negative. Throughout the 25 days of incubation, the highest CH₄ yields occurred between days 2 and 3 in the pasture soils, ranging from 263 to 275 ng CH₄ g⁻¹ of dry soil day⁻¹. After this period, the emissions remained at zero until the end of the experimental period. CH₄ emissions from forest soils and agricultural land resulted in an almost undetectable flux over the 25 days of incubation (Figure 3).

An interaction effect between the land-use system and temperature (p < 0.001) was observed. The forest and agriculture treatments presented the lowest (p < 0.001) emissions of CH₄ among the land uses, except at the temperature of 40°C, where the lowest production was observed only in the agricultural soil (p > 0.05). In the pasture soil, a negative quadratic effect (p < 0.001) was observed in gas emissions as a function of temperature, where the lowest gas production was observed at temperatures of 35°C. Forest and agricultural soils did not vary (p > 0.05) in the emissions of CH₄ with the increase in incubation temperature. Pasture soils showed the highest methane emissions, regardless of the temperature applied, while Forest and agricultural soils showed no statistical difference (p > 0.05; Table 3).

Temperature variation altered the flux of CH₄ in forest soils (Figure 4A). At temperatures of 30°C, 35°C, and 40°C, the flux began on the first day of incubation, whereas at 25°C, it began only on the third day. At each studied temperature, peak production occurred at different times. At 25°C, the peak was observed on the seventh day, producing ∼103 ng CH₄ g⁻¹ day⁻¹. At 30°C, the peak was on day 21, with ∼109 ng CH₄ g⁻¹ day⁻¹. At 35°C on the ninth day, with ∼62 ng CH₄ g⁻¹ day⁻¹, and at 40°C, two peaks were observed on the ninth day (∼135 ng CH₄ g⁻¹ day⁻¹) and 19th day (∼146 ng CH₄ g⁻¹ day⁻¹). Production began on the first day in pasture soil (Figure 4B). However, a higher flux was observed at 25°C, with peak production reaching ∼12,160 ng CH₄ g⁻¹ day⁻¹ on the 25th day of incubation. In the agricultural soil (Figure 4C), higher fluxes were observed at two temperatures of 25°C, with two production peaks on the seventh day (165 ng CH₄ g⁻¹ day⁻¹) and on the 11th day (205 ng CH₄ g⁻¹ day⁻¹) and at 40°C, a peak on the sixth day (148 ng CH₄ g⁻¹ day⁻¹). The CH₄ fluxes at 30°C and 35°C remained unchanged throughout the incubation period.

An interaction (p < 0.001) between land use and soil nitrogen on the total emission of CH₄ was observed (Table 4). The addition of nitrogen to the soil affected (p < 0.05) the total emission of CH₄ in all soils. A quadratic effect was observed for total emissions of CH₄ in forest soils (p = 0.005), with higher values observed in soils that received 180 kg N ha⁻¹. In pasture soils, a decreasing linear effect was observed (p = 0.01), whereas agricultural soils showed an increasing linear effect (p = 0.04) as the nitrogen concentration in the soil increased. Pasture soils showed higher total emissions of CH₄, regardless of soil nitrogen concentration, with values ranging from 129.2 to 52.8 μg CH₄ g⁻¹.

Nitrogen rates did little to alter the flux of CH₄ in forest soils (Figure 5A) or in agricultural soils (Figure 5C) with incubation time. In forest soils, only one large production peak was observed on the third day of incubation (∼390 ng CH₄ g⁻¹ day⁻¹), whereas agricultural soils showed peak production on the sixth day of incubation (∼1,285 ng CH₄ g⁻¹ day⁻¹). In contrast, the pasture soil (Figure 5B) presented greater variation between the applied doses, with higher (>600 ng CH₄ g⁻¹) fluxes observed when urea was not applied to the soil. When nitrogen fertilizer was applied, production peaks decreased as the dose increased.

When different land use types were evaluated, higher emissions of CH₄ in pasture soils were found (Figure 1). According to several studies (Potter et al., 1996; Azevedo et al., 2018; Machacova et al., 2020), forest soils are sinks for CH₄ due to the vast methanotrophic communities present in the soil. Land-use change tends to cause changes in the soil microbial community, stimulating methanogenic Archaea (Kroeger et al., 2021). These changes were due to changes in the botanical community, soil pH, nutrient cycling, and allocation of carbon in the soil. This set of factors alters the oxidation of CH₄ in the soil, causing the area to emit greater amounts of gas (Tate, 2015).

CH₄ is produced in the soil by the decomposition of organic matter (Ye et al., 2015). Pasture soils are in a constant cycle of tiller appearance and senescence, thus increasing the rate of litter deposition in the soil, which enters the decomposition process (Chianese et al., 2009). The agricultural soil studied was characterized by a large spacing between lines (2 m × 2 m), being a well-ventilated soil, providing greater oxidation of CH₄ (Kirschke et al., 2013; Broucek, 2014).

The bacterial community is also responsible for CH₄ cycling and several factors affect the methanogenic community. The conversion from areas by forest to pasture changes the soil microbial community, increasing the abundance and action of methanogenic archaea, as a consequence of this, pasture tends to emit more methane into the atmosphere (Alves et al., 2022). Among the factors, soil moisture and the addition of nutrients may be the largest contributors to CH₄ emissions (Knief, 2019). CH₄ production in pasture soils and oxidation in forest and agricultural soils occur during the same period because of the addition of nitrogen and water to the soil.

Temperature variations are important in studies of greenhouse gas emissions, as global warming tends to increase the temperatures of the globe over the years. Therefore, understanding how these variations alter CH₄ emission patterns today will help in the research for alternatives in the future.

CH₄ emissions from forest soils and agriculture are independent of temperature because forest soils are considered resilient. Therefore, they are less affected by adverse conditions, such as temperature variations, thereby reducing CH₄ emissions in the system (Peri et al., 2017). A study that evaluated 41 peer-reviewed publications observed that a temperature rise did not much affect CH₄ emissions from forested areas (Dijkstra et al., 2012). In turn, the agricultural soils studied are black pepper, a crop that stands out for its growth habit, of the creeper type. The spacing used in the study was large, about 2 m × 2 m, thus decreasing the content of organic matter present in the soil and keeping the soil more aerated, which causes the methanogenic archaea communities to decrease (Abduh et al., 2020).

In pasture soils, the highest emissions at 25°C are due to the ability of the soil to maintain its moisture for longer than at temperatures of 30°C and 35°C, causing methanogenic bacteria to carry out methanogenesis (Conrad, 2009; Dijkstra et al., 2012). In pasture soils, significant variation in the levels of ammonium before incubation was observed, showing an increase as the temperature increased (except at 40°C). Another important point to be observed is the presence of bovine feces in the area, in which authors show that greater emissions occur above 20°C, however, increasing this temperature beyond 25°C decreases the activities of methanogenic bacteria (Cárdenas et al., 2021; Rennie et al., 2021).

As seen in the previous study, land-use changes directly impact CH₄ emissions, as at all temperatures studied, CH₄ emissions were higher in pasture soils. Moreover, the change in land use alters the patterns of the soil, whether microbiological, structural, or biodiversity, present in the environment (Ball, 2013; Kroeger et al., 2021). In addition, the lower emissions of CH₄ from agricultural soil (black pepper) concerning pastures show that more aerated soils with low litter deposition emit less CH₄ into the atmosphere (Kirschke et al., 2013; Valenzuela et al., 2017).

The flux in the pasture soil can be well observed because the variations over time in the N-NH₄ ⁺ contents are greater, thus favoring the oxidation of soil CH₄. However, the peaks observed in the forest and agricultural soils can be explained precisely by the moments at which the soil presented an environment conducive to gas production.

Agriculture and livestock contribute significantly to greenhouse gas emissions owing to practices that alter soil carbon and nitrogen dynamics (Bento et al., 2018). Fertilization can affect these emissions. Nitrogen fertilization contributes greatly to soil quality and increases crop yield and nutritional value (Delevatti et al., 2019; Santos et al., 2020). However, the source, dose, and application form must be considered because inappropriate use increases greenhouse gas emissions (Raposo et al., 2020; Corrêa et al., 2021; Cardoso et al., 2022).

The fertilizer used in the treatment was urea, an ammoniated fertilizer that increases ammonium levels in pasture soils as the dose increases. Ammonium directly affects methanotrophy, thereby increasing CH₄ oxidation in the system (He et al., 2019). The same pattern was not observed in the agricultural soils. However, emissions matched only at higher nitrogen doses.

The variation found in the forest soils was due to the peak observed at a dose of 180 kg N ha⁻¹ on the third day of evaluation. However, the soil quickly returned to the emission patterns, indicating the resilience of forest soils (Peri et al., 2017). A similar pattern was observed in agricultural soils at a dose of 270 kg N ha⁻¹.

The peak observed in pasture soils at all doses was due to urea being converted to ammonia only between days 4 and 6 in the soil (He et al., 2019), which explains the drop in CH₄ yields after the sixth day of evaluation.