Biochar produced at high temperature mitigates N2O emission and promotes nitrogen retention in subtropical forest soils

Biochar is produced by burning biomass under oxygen‐limited conditions, and it has been widely used as a soil amendment to improve soil functions such as nutrient retention. However, whether the impact of biochar application on soil nitrogen (N) transformation and N2O emission varies with the pyrolysis temperature remains unclear, especially in different forest types in subtropical regions. In this study, a 60‐day laboratory incubation experiment was conducted to evaluate the impact of biochar with different pyrolysis temperatures (300°C [BC300], 500°C [BC500], and 800°C [BC800]) on net N transformation rates and N2O emission in soils collected from Castanopsis kawakamii dominated natural forest (NF) and Chinese fir (Cunninghamia lanceolate, CF) plantation in subtropical China. The results showed that the application of biochar significantly increased soil ammonium (NH4+) content (p < 0.001) but reduced nitrate (NO3−) content (p < 0.001) compared with the control. The soil NH4+ content of the BC800 treatment was significantly higher than that of other treatments (p < 0.001). Biochar application significantly reduced soil net N mineralization (NRmin) and nitrification (NRnit) rate (p < 0.001), but increased net ammonification (NRamm) rate (p < 0.001). The application of biochar led to a remarkable decrease in cumulative N2O emission compared to the control (p < 0.001). In particular, soils treated with high‐temperature biochar emitted significantly lower N2O compared to other treatments (p < 0.001). The partial least squares path model demonstrated that biochar influenced N2O emission through a direct effect in NF soil and an indirect effect in CF soil. This study highlights the distinct role of biochar, particularly that produced under high pyrolysis temperatures as a soil amendment to mitigate N2O emission and promote N retention in both subtropical natural and planted forests.

+ content of the BC800 treatment was significantly higher than that of other treatments (p < 0.001).Biochar application significantly reduced soil net N mineralization (NR min ) and nitrification (NR nit ) rate (p < 0.001), but increased net ammonification (NR amm ) rate (p < 0.001).The application of biochar led to a remarkable decrease in cumulative N 2 O emission compared to the control (p < 0.001).In particular, soils treated with high-temperature biochar emitted significantly lower N 2 O compared to other treatments (p < 0.001).
The partial least squares path model demonstrated that biochar influenced N 2 O emission through a direct effect in NF soil and an indirect effect in CF soil.This study highlights the distinct role of biochar, particularly that produced under high pyrolysis temperatures as a soil amendment to mitigate N 2 O emission and promote N retention in both subtropical natural and planted forests.

K E Y W O R D S
biochar, Cunninghamia lanceolate, N 2 O emission, nitrogen retention, nitrogen transformation, pyrolysis temperature

| INTRODUCTION
Nitrous oxide (N 2 O) is a potent greenhouse gas with a global warming potential about 298 times greater than that of an equivalent concentration of carbon dioxide (Shen et al., 2021).Forests, farmlands, wetlands, and grasslands are the major sources of global N 2 O emission, and N 2 O emission from forest soils with a mean annual rate of 3.62 Tg N, accounting for 38% of the total emission from soils (Zhang et al., 2022).Previous studies have shown that subtropical and tropical forests emit N 2 O at higher rates than boreal forests (Tian et al., 2019).Chinese fir [Cunninghamia lanceolate (Lamb.)Hook.] is the primary timber species in southern China, with the largest plantation area and standing volume (Liu et al., 2020).Due to its rapid growth and good timber quality, millions of hectares of Chinese fir have been established during the past decades (Selvaraj et al., 2017;Zhang et al., 2004).Reforestation of C. lanceolate plantations is usually established on the cutover land of broadleaf forest, with a short rotation time (20-25 years).Land used for Chinese fir plantation is usually retained for several rotations, as a result, forest conversion and successive rotation lead to degradation in soil physical, chemical, and biological properties, and thus decrease soil quality and forest productivity (Liu et al., 2010(Liu et al., , 2020)).
Biochar is produced through the pyrolysis of biomass under anoxic conditions and can be utilized as a soil amendment to enhance soil properties (Hagemann et al., 2017), such as improve soil aeration (Kaur et al., 2023), increase soil pH (Aamer et al., 2020), enhance soil water and nutrient retention (Lu et al., 2019;Villagra-Mendoza & Horn, 2018;Yu et al., 2021), improve soil Cto N-acquiring enzyme activities including βglucosidase, N-acetyl-βglucosaminidase and leucine aminopeptidase (Guo et al., 2020), and reduce soil N 2 O emission (Feng et al., 2022).Previous studies have shown that NH 4 + , rather than NO 3 − , is the dominant inorganic N form in soil N retention strategies in subtropical and tropical regions, which due to NO 3 − is vulnerable to ecosystem N loss through leaching, runoff, or gaseous N emission (Xie et al., 2018).Moreover, soil organic N is mineralized into NH 4 + , which is then converted into NO 3 − through nitrification.An increase in soil N retention capacity is indicated by the decrease in net nitrification rate (NR nit ) and the increase in net ammonification rate (NR amm ;Chen et al., 2020).Nevertheless, the effects of biochar on soil N transformation remain inconsistent among studies.For example, previous studies have shown that biochar applied to the Willow (Salix viminalis genotype Q83) forest significantly reduced soil net N mineralization (Prayogo et al., 2014).Furthermore, the application of high-temperature biochar (pyrolysis at 450 and 600°C) to moso bamboo forest inhibited soil net nitrogen mineralization rate (NR min ) and net nitrification rate (NR nit ;Deng et al., 2020).However, other studies reported that biochar produced by pyrolysis at 400°C increased N mineralization and nitrification in a subtropical forest (Yu et al., 2021).Biochar has the potential to regulate N 2 O emission by physical, chemical, and/or microbial mechanisms.For instance, biochar is typically alkaline, thereby reducing soil N 2 O emissions by increasing soil pH and enhancing N 2 O-reducing activities (Guo et al., 2020).Secondly, high porosity of biochar tends to decrease soil N 2 O emission by enhancing soil aeration and inhibiting denitrification, since N 2 O is produced by nitrate reduction under anoxic conditions (Li et al., 2021;Song et al., 2019).Thirdly, biochar possesses a substantial specific surface area, enabling it to absorb soil nutrients and serve as shelter for microorganisms, ultimately impacting soil N 2 O emissions (Lan et al., 2018).Finally, the presence of persistent free radicals (PFRs) on the surface of biochar can trigger the generation of reactive oxygen species, thwarting its ability to mitigate N 2 O emissions (Wu et al., 2023).Nevertheless, some studies found that adding various types of biochar to the red pine (Pinus resinosa) forest had no significant impact on N 2 O emission (Toczydlowski et al., 2023).Other studies have shown that biochar application significantly increases soil N 2 O emissions in the coastal Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco var.menziesii) forest (Hawthorne et al., 2017).Thus, the effect of biochar on N 2 O emissions in forest soil is inconsistent and requires further research.
Pyrolysis temperature is a crucial factor affecting the biochar properties, including pH, bioavailable C and N contents, specific surface area, PFRs, and porosity (Lee et al., 2021;Toczydlowski et al., 2023).The C:N ratio of biochar has a significant impact on soil N 2 O emissions.Lee et al. (2021) found that the C:N ratio of biochar increased as the pyrolysis temperature increased, resulting in a greater influence in reducing soil N 2 O emissions.Liu et al. (2018) discovered that biochar produced at lower temperatures (<400°C) would have shrunken surface area and weaker aromatic structure resulting in a diminished capacity to reduce N 2 O emission.Deng et al. (2020) added biochar produced by pyrolysis of spent mushroom substrate at different temperatures (300, 450, and 600°C) into the soil of a moso bamboo forest, and there was no significant difference in the effects of different biochar types on soil N 2 O emission.Moreover, Deng et al. (2021) found that higher soil N 2 O emission rates were observed with the application of biochar pyrolyzed at 300°C than those pyrolyzed at 450 or 600°C in Camellia oleifera plantation soil.Currently, most research on the impact of biochar on soil physical, and chemical properties and nitrogen transformation were conducted in farmland (Kaur et al., 2023).
However, it remains unclear whether biochar can improve forest soils since they are typically more heterogeneous and less fertile compared to agricultural soils.Besides, there is no consensus on the effect of biochar produced at different pyrolysis temperatures on soil N 2 O emission.Therefore, it is crucial to comprehend the impact of biochar generated at different pyrolysis temperatures on N transformation and N 2 O emission in forest soils.
The objective of this study was to investigate the effects of biochar with different pyrolysis temperatures on soil N transformation and N 2 O emission in natural and planted forests in subtropical regions.Specifically, we aim to achieve these objectives (1) to identify the N-retention ability of biochar under different pyrolysis temperatures; (2) to evaluate the mitigation effect of biochar at different pyrolysis temperatures on soil N 2 O emission; (3) to explore the mechanism that biochar with different pyrolysis temperatures affecting soil N 2 O emission from natural and planted forests.

| Soil collection and biochar preparation
Soils used in the laboratory incubation experiment were collected from natural and planted forests at Xinkou Forest Farm of Fujian Agriculture and Forestry University (26° 100 E, 117° 280 N) in Sanming city, Fujian Province in southeast China.The climate is subtropical humid monsoon, with an annual precipitation of 1749 mm and a mean annual temperature of 19.1°C.The mean evapotranspiration is 1585 mm, and the annual mean relative humidity is 81.0%.The altitude of this region ranges from 175 to 264 m above sea level.The soil is classified as Silty Oxisol based on the United States Department of Agriculture (USDA) soil taxonomy, which was derived from parent sandstone and shale (Liu et al., 2020).The natural forest (NF) was dominated by Castanopsis kawakamii, while the understory vegetation mainly comprised shrubs and herbs (such as Dicranopteris dichotoma (Thunb.)Berhn, Gahnia tristis Nees and Alpinina chinensis (Retz.)Rosc).Part of the native forest was cleared, and residues were slashed and burned before planting C. lanceolate.In 2022, four experimental plots (20 m × 20 m each) were established in each forest, with a buffer zone >1 km among each plot, giving a total of 8 plots in this study.In April 2022, five soil cores from a depth of 0-20 cm were randomly selected in each plot to make a composite soil sample.The collected soils were stored in an ice box and transported to the lab, then passed through a 2 mm sieve for incubation experiments.Biochar was produced from corn straw through a slow pyrolysis process at 300, 500, and 800°C under an atmosphere of N 2 (The Qinfeng Biochar Co., Nanjing, China).The basic properties of soil and biochar are shown in Table 1.

| Soil microcosm incubation experiment
The incubation experiment was in a 2 × 3 factorial design with four replicates.The treatments include forest type (CF or NF) and application of biochar with different pyrolysis temperatures (300, 500, or 800°C).A total of 120 g of fresh soil was added to a 350 mL incubation flask placed in an incubator set to 25°C and incubated for 1 week under dark conditions.Biochar produced at varying pyrolysis temperatures was added, in an amount equal to 5% of the soil weight.A total of 32 samples were incubated for 60 days, and soil water content was maintained at 60% of the field capacity during the whole incubation period by adding distilled water.

| Sampling and analytical procedures
Samples of the incubated soil and/or soil-biochar mixtures were collected at the 1st and 60th days of incubation.Soil pH was measured in water (water-soil ratio 1:2.5) with a pH meter.Soil moisture was determined gravimetrically after drying at 105°C for 24 h, and all results were expressed on an oven-dry basis.Microbial biomass C (MBC) and N (MBN) were determined by the chloroform fumigation extraction method (Inubushi et al., 1991).Soil mineral N (NH 4 + , NO 3 − ) was extracted using 2 M KCl and measured by Westco Smart Chem Discrete Wet Chemistry Analyzer (Westco Scientific Instruments, United States).TC and TN were measured using an elemental analyzer (Vario MICRO cube, Elementar, Germany).Dissolved organic C (DOC) and N (DON) were extracted by 2 M KCl using a soil-to-water ratio of 1:5 and then measured by a TOC-VCPH/CPN analyzer fitted with a TN unit (Shimadzu Scientific Instruments, Japan).Gas samples for N 2 O analysis were taken from the headspace of the glass jars after adding the biochar at 1, 8, 13,15,22,29,43,50,53,56, and 60 days.The incubation bottles were covered with plastic to prevent water loss.To ensure proper ventilation conditions, a breathable film was placed on the lid (Aamer et al., 2020).Before collecting gas, open the culture bottle cap in advance and let it sit for 1 h.To take a sample, seal the culture bottle with a rubber stopper, and use a syringe to extract 5 mL of gas from the bottle, then immediately close the three-way valve on the rubber stopper after pumping.Allow 1-h interval before extracting another 5 mL of gas with a syringe.The gas samples were collected and the nitrous oxide concentration was measured via GC-2014 Gas Chromatograph (Shimadzu, Japan) equipped with an electron capture detector (ECD).

| Data analysis
The N 2 O flux was calculated using the following equation (Shaaban et al., 2015): where F is the flux rate of each N 2 O (μg•kg −1 •h −1 ); ρ is the density of the N 2 O at standard conditions (1.25 kg•m −3 ); V is effective space for gas in culture bottle (m 3 ); W is the weight of dry soil (kg); Δc is the gas production over a period of 1 h (μg•kg −1 ); Δt is the time interval between two samplings (h); T indicates the incubation temperature (°C).
The cumulative N 2 O fluxes (μg•kg −1 ) were determined by the equation given by Xu et al. (2020) using the following equation: where

| RESULTS
3.1 | Soil pH, soil water content, total carbon, total nitrogen, and dissolved organic C content Biochar pyrolysis temperature (BT), forest type (FT), and their interactions had significant impacts on soil pH, DOC, and SWC (p < 0.01, Table S1).Biochar application led to a significant increase in soil pH but a decrease in DOC in comparison with control (p < 0.001, Table 2).Biochar application had no significant impact on SWC in CF soil, while it considerably reduced SWC in NF soil (p < 0.001, Table 2).Biochar application significantly increased TC and TN compared to the control (p < 0.001, Table 2).However, no significant difference in soil TC and TN among the BC300, BC500, and BC800 treatments in both forests was observed (p > 0.05, Table 2).
Biochar application significantly decreased NO 3 − content (p < 0.001, Figure 1b), while significantly increasing soil NH 4 + and DON content (p < 0.001, Figure 1c,d) compared to control regardless of forest type.Similar patterns of soil NO 3 − and DIN were observed among different biochar treatments in CF and NF soils, with higher NO 3 − and DIN of BC 500 and BC800 treatments (1) (3) compared with that of BC300 treatment (Figure 1b,e).Soil DTN content showed a similar trend under biocharamended treatments in both forest types, with slightly higher DTN content in high-temperature pyrolyzed biochar treatment (Figure 1a).

| Soil microbial biomass carbon and nitrogen content
Biochar pyrolysis temperature, forest type, and their interactions significantly influenced soil MBC and MBN content (p < 0.001, Figure 2).For CF soil, the application of BC300 and BC500 significantly decreased soil MBC compared to control, while no significant effect was observed for BC800 amendment (p < 0.001, Figure 2a).For NF soil, MBC content of BC500 and BC800 treatments were significantly lower than those of control and BC300 treatment (p < 0.001, Figure 2a), while no significant difference was observed between the control and BC300 treatment.Application of biochar in CF soils resulted in a significant increase of MBN compared to the control, and a greater increase was observed with the increase of pyrolysis temperature (p < 0.001, Figure 2b).Soil MBN content of BC800 treatment was significantly lower than those of control, BC300 and BC500 treatments (p < 0.001, Figure 2b).

| Soil net N mineralization and nitrification rates
Regardless of forest type, biochar application led to a significant reduction in soil net N mineralization (NR min ) and nitrification (NR nit ) rates (p < 0.001, Figure 3a,c, Table S2), but a significant increase in net N ammonification (NR amm ) rates (p < 0.001, Figure 3b, Table S2).In CF soil, BC500 amendment resulted in the lowest NR min compared to other treatments, while BC300 amended soils showed the lowest NR nit compared to other treatments (p < 0.001, Figure 3).

| Soil nitrous oxide reductase activity and N 2 O emission fluxes
The interaction of biochar pyrolysis temperature and forest type had a significant impact on soil nitrous oxide reductase (N 2 OR) activity (p < 0.01, Figure 4).Hightemperature biochar (BC500 and BC800) significantly increased soil N 2 OR activity, while there was no significant difference between control and BC300 treatment in both forest soils (Figure 4).Biochar pyrolysis temperature, forest type and their interactions had significant impacts on soil N 2 O emission (p < 0.001, Figure 5).Generally, biochar addition  ), and dissolved organic N (DON) content.decreased the N 2 O fluxes and cumulative N 2 O emission compared to control in both forest soils, except for BC500 treatment in CF soil and BC300 treatment in NF soil (Figure 5).BC800 amendment significantly reduced the cumulative N 2 O emission in both forest soils (p < 0.001, Figure 5b).

| Relationship between N 2 O emissions and soil physiochemical factors
For CF soil, cumulative N 2 O emission showed a significantly negative correlation with pH, MBN, DON, NH 4 + , and NR amm (p < 0.05, Figure S1).For NF soil, cumulative N 2 O emission showed a significantly negative correlation with pH, DON, NH 4 + , and DTN, but a significantly positive correlation with MBC and MBN (p < 0.05, Figure S1).Soil NR min and NR nit were significantly positively correlated with DOC (p < 0.01, Figure S1) and negatively correlated with NH 4 + content in both forest soils (p < 0.001, Figure S1).SEM analysis suggested a stronger direct effect of biochar on N 2 O emission in NF soil than CF soil, while biochar mainly impacted N 2 O emission through indirect pathway in CF soil (Figure 6).Specifically, soil N availability (NH 4 + , NO 3 − , MBN, DON) appeared to play a direct role in affecting soil N 2 O emissions in CF soil (Figure 6).

| Effects of biochar application on soil N contents and N transformation rates
Our study revealed a significant reduction of soil NO 3 − contents after biochar application in subtropical forest soils (Figure 1b).Similarly, Deng et al. (2020) reported that biochar addition decreased soil NO 3 − contents in moso bamboo forests, which might be due to biochar directly adsorbs nitrate nitrogen, or biochar effectively enhances soil C:N ratio and inhibit N nitrification, ultimately reducing soil NO 3 − contents.In this study, the soil C:N ratio increased significantly and the net nitrification rate decreased significantly with biochar application.Hence, our study found that biochar reduced soil NO 3 − content through the above two mechanisms.Additionally, soil NO 3 − content of the BC300 treatment was significantly lower than those of BC500 and BC800 treatments (Figure 1b).A study revealed that low-temperatures biochar (≤400°C) improved the soil adsorption of NH 4 + , compared to high-temperatures biochar (≥600°C), possible explanation may be that reducing available substrate for nitrification and decreasing NO 3 − production (Lin et al., 2019).Indeed, lower soil NH 4 + content was observed under BC300 than under BC800 treatment.In our study, biochar application resulted in a significant increase in soil NH 4 + content (Figure 1c).Similarly, previous studies showed that biochar increased soil NH 4 + content as the result of high biochar C:N ratio (Gao et al., 2019;Liao et al., 2022).Moreover, the higher soil NH 4 + content after the addition of biochar produced at low temperatures might also be due to its higher capability for soil NH 4 + adsorption (Gai et al., 2014).
Previous studies have indicated that soil microbial N immobilization increased with increasing soil microbial biomass (Li et al., 2020).Tahovská et al. (2013) employed the 15 N tracer technique to investigate soil N transformation in the acidified soils of the Bohemian Forest and found that microbial N immobilization had a significant effect on soil N retention.We found soil NH 4 + in CF soils was significantly positively correlated with MBN, while NO 3 − was significantly negatively correlated with MBN (Figure S1).In our study, MBN in soils treated with high-temperature biochar increased significantly compared to other treatments (Figure 2b), indicating that high-temperature biochar significantly increased soil microbial N immobilization.Besides, biochar with a large specific surface area and porous structure, which provided potential habitats for soil microorganisms (Jaafar et al., 2015).Previous studies have shown that the specific surface areas of pine needles biochar produced at 300 and 700°C were 5.61 and 420.33 m 2 g −1 (Ahmad et al., 2016).Thus, high-temperature biochar provided greater access for microbial colonization and promoted soil microbial N immobilization.
In this study, biochar application reduced N mineralization and nitrification in subtropical forest soils, which was consistent with Liao et al. (2022), who reported that the application of biochar (pyrolyzed at 600°C) to poplar plantations resulted in decreased soil N mineralization and nitrification.Similar findings were reported in other studies (Deng et al., 2020;Luo et al., 2016).Biochar may absorb inorganic N and provide C sources for microorganisms.In our study, both soil N mineralization and nitrification rates showed a significant positive correlation with DOC content (Figure S1).It is widely recognized that the process of soil nitrification leads to the production of NO 3 − , which reduces soil pH (Qian et al., 2023;Zhou et al., 2023).In our study, soil net nitrification rates were significantly decreased after the incorporation of biochar, especially those produced at low temperatures (Figure 3c).Similar findings were observed in previous studies (Liu et al., 2018).It is worth noting that there was originally higher NO 3 − but lower NH 4 + content in CF than NF soil, indicating a higher risk of N leaching in Chinese fir plantations than NFs.In this case, biochar applied to soil, particularly in Chinese fir plantation, is favorable to soil N retention, due to its effects on increasing soil microbial N immobilization and NH 4 + content, as well as decreasing soil NO 3 − content.

| Effects of biochar application on N 2 O emission
Our results showed that biochar application led to a significant decrease in N 2 O emission in subtropical forest soils (Figure 5).A previous study has shown that adding raw wood biochar (pyrolyzed at 500°C) reduced soil N 2 O emission by 58%-89% in poplar plantations, which is likely to due to that nitrification is the primary pathway for N 2 O production, contributing over 90% of cumulative N 2 O emission, and biochar application inhibits nitrification (Liao et al., 2022).In this study, cumulative N 2 O emission had a significant negative correlation with soil NH 4 + content, which increased after biochar application due to the reduced consumption of NH 4 + via nitrification (Figure 1c, Figure S1).These findings suggested that biochar application had the potential to reduce N 2 O emission by inhibiting nitrification.Furthermore, biochar application significantly increased soil pH (Table 2).A previous study showed that increasing pH in acidic soils enhanced microbial biomass and diversity (Aciego Pietri & Brookes, 2008).For instance, Aamer et al. (2020) found that biochar application increased the pH of acidic soil, increasing N 2 O-reducing microbial activities.Although this study showed that biochar decreased N 2 O emission primarily through the inhibition of nitrification, the effect of biochar on the conversion of N 2 O to N 2 cannot be disregarded.It has been shown that biochar affects microbial nitrification and denitrification by changing the physical and chemical properties of soil, eventually inhibiting N 2 O emission (Liao et al., 2021).Our study also found that soil N 2 OR activity was enhanced after biochar application (Figure 4), indicating that biochar application promoted the conversion of N 2 O to N 2 by increasing the N 2 OR activity, thereby inhibiting N 2 O emission.
In this study, the soil NR nit of BC800 was significantly higher than that of BC300, while the cumulative N 2 O emission of BC800 was significantly lower than that of BC300 (Figures 3 and 5).Previous studies also reported that high-temperature biochar application enhanced the conversion of N 2 O to N 2 by increasing the N 2 OR activity (Deng et al., 2020;Tan et al., 2018).Our results also showed that the soil N 2 OR activity of BC800 treatment was significantly higher than that of BC300.Additionally, the application of low-temperature biochar to NF soil resulted in considerably higher N 2 O emission compared with high-temperature biochar treatments (Figure 5).A significant positive correlation between the cumulative N 2 O emission and MBN for NF soil (p < 0.05, Figure S1).The PLS-PM indicated that the primary influence in the CF was the indirect effect (Figure 6a).To be more precise, biochar application lessened the N 2 O emissions from the soil by increasing soil pH and altering soil N availability, whereas, biochar application has a direct impact on N 2 O emission in the NF soils (Figure 6b).This likely explained by the lower pH and C:N in CF soil than those in NF soil (Table 1), resulting in greater effect of biochar on N 2 O emission in CF than NF soil.Moreover, NO 3 − is the major form of inorganic N in CF soil while total inorganic N in NF soil is dominated by NH 4 + , indicating that nitrification activity in CF soil is higher than that in NF soil.Therefore, biochar primarily reduces N 2 O emissions by inhibiting nitrification in CF soil.However, other mechanisms might play a more important role in mitigating N 2 O emissions from NF soil, and further research is needed.Collectively, the incorporation of high-temperature biochar could be effective in reducing N 2 O emission and increasing the soil N retention capacity in both Chinese fir plantations and C. kawakamii-dominated NFs.

| CONCLUSIONS
Soil N 2 O emission and nitrogen transformation in subtropical natural and planted forests responded similarly to the application of biochar.Most importantly, high-temperature biochar had a better inhibitory effect on N 2 O emission, which may be mainly attributed to nitrification inhibition.Moreover, biochar application appeared to enhance soil N retention by inhibiting N mineralization and nitrification, leading to increased NH 4 + and decreased NO 3 − content in soil.Although laboratory incubation cannot completely reflect the field conditions, it allows us to investigate the underlying mechanisms that biochar interacting with nutrient-poor subtropical forest soils.To better predict the biochar effects on soil nitrogen cycling in Chinese fir plantations and C. kawakamii-dominated NFs in future studies, it is important to apply biochar in field conditions and combine it with 15 N tracer technology.Collectively, our results provide insight into the potential role biochar could play in mitigating global climate change as well as improving soil fertility in subtropical forests.

[
BC500], and 800°C [BC800]) on net N transformation rates and N 2 O emission in soils collected from Castanopsis kawakamii dominated natural forest (NF) and Chinese fir (Cunninghamia lanceolate, CF) plantation in subtropical China.The results showed that the application of biochar significantly increased soil ammonium (NH 4 + ) content (p < 0.001) but reduced nitrate (NO 3 − ) content (p < 0.001) compared with the control.The soil NH 4 Properties of soil and biochar used in this study.

+
the sample number, and d indicates the number of days in the sampling intervals.The net N mineralization (NR min ) during the incubation period was calculated as the changes in inorganic N (NH 4+ and NO 3 − ;Deng et al., 2020).whereNR min is soil net N mineralization rate, NR amm is soil net ammonification mineralization rate, NR nit is soil net nitrification rate.c(NH 4 inorganic N at the beginning and end of incubation, respectively.t 1 -t 0 is the incubation period (day).Two-way ANOVA [biochar (BC300, BC500, BC800) and forest type (CF, NF)] followed by least significant differences (LSD; p = 0.05) were applied to test the differences among treatments.Structural equation modeling (SEM) was used to investigate the direct and indirect effects of biochar application on N 2 O emission.SEM is an empirical model, which usually needs to be constructed under the support of theory and rule of thumb.First, we established an initial model based on known knowledge and relationships between the soil physicochemical properties and N 2 O emission.Then we parameterize the initial SEM model and use the partial least squares path modeling (PLS-PM) to test its goodness of fit.All statistical analyses were conducted in the R software (4.3.1), and Origin 2021 (OriginLab, United States).The data are expressed as mean ± SD.
Total carbon (TC), total nitrogen (TN), dissolved organic C (DOC), pH, and soil water content (SWC) in Chinese fir plantation (CF) and natural forest (NF) soils at the end of the incubation period.

F
, d), and dissolved inorganic N (DIN, e) content at the end of the 60-day incubation.Different letters indicate significant differences among the treatments for Chinese fir plantation (CF) or natural forest (NF) soils (p < 0.001).CK, control; BC300, BC500, and BC800 represents biochar produced at 300, 500, and 800°C, respectively.Error bars show SD.The same is below.F I G U R E 2 Soil microbial biomass carbon (MBC) (a) and nitrogen (MBN) (b) content at the end of the 60-day incubation.Different letters indicate significant differences among the treatments for Chinese fir plantation (CF) or natural forest (NF) soils (p < 0.001).

F
I G U R E 3 Soil net nitrogen mineralization (NR min , (a), ammonification (NR amm , (b), and nitrification (NR nit , (c) rates during the 60-day incubation period.Different letters indicate significant differences among the treatments for Chinese fir plantation (CF) or natural forest (NF) soils (p < 0.001).F I G U R E 4 Soil nitrous oxide reductase (N 2 OR) activity in two forest soils at the end of the 60-day incubation.Different letters indicate significant differences among the treatments for Chinese fir plantation (CF) or natural forest (NF) soils (p < 0.01).F I G U R E 5 Soil N 2 O fluxes (a) and cumulative N 2 O emissions (b) during the 60-day incubation period.Error bars show SD.Different letters indicate significant differences among the treatments for Chinese fir plantation (CF) or natural forest (NF) soils (p < 0.001).F I G U R E 6 A partial least squares path modeling (PLS-PM) showing the direct and indirect effects of biochar pyrolysis temperature (BT), soil pH, and soil N (NH 4 + , DTN, MBN, DON), and soil water content (SWC) on N 2 O emissions in Chinese fir plantation (CF, a) or natural forest (NF, b) soils.Greater path coefficients were shown by wider arrows, while red and blue colors indicated positive and negative effects, respectively.The GOF values of CF and NF soils were 0.68 and 0.69, respectively.Standardized direct and indirect effects on N 2 O emissions derived from the PLS-PMs were shown below the PLS-PM plot for CF (c) and NF (d) soils, respectively. ) Abbreviations: CF, Chinese fir plantation; NF, natural forest.