Rice Residue-Based Biochar Mitigates N2O Emission from Acid Red Soil

Biochar application is considered an effective approach to mitigating nitrous oxide (N2O) emissions from agricultural soils. However, the mechanisms of biochar to mitigate N2O emissions from acidic red soils are still unclear. Therefore, the present study aims to underpin mechanisms associated with rice residue-based biochar in mitigating N2O emissions from acid soils. Soil treated with different rates of biochar control, from 1%, 2%, and 3%, and different soil properties, including soil pH, microbial biomass carbon (MBC), NH4+-N, NO3−-N, genes abundance (nosZ, nirK, AOA, and AOB), and enzymatic activities ((nitrate reductase (NR) and urease (UR)) were studied. The application of 3% biochar increased the soil pH (5.21–6.48), MBC (565–685 mg/kg), NO3−-N contents (24.23–44.5 mg/kg), genes abundance (nosZ, nirK, AOA, and AOB) and UR activity. The highest N2O emission (43.60 μg kg−1) was recorded and compared with the application of 1% (26.3 μg kg−1), 2% (18.33 μg kg−1), and 3% biochar (8.13 μg kg−1). Applying 3% biochar effectively reduced the N2O emission due to increased soil pH, MBC, NO3−-N contents, genes abundance (nosZ, nirK, AOA, and AOB), and weakened NH4+-N and NR activities. Therefore, increasing soil pH, genes abundance, and weakened nitrification following the addition of rice residue-based biochar can effectively reduce the N2O emissions from acidic red soils.


Introduction
Nitrous oxide (N 2 O) is a potent greenhouse gas (GHG) that has persisted in the atmosphere for over 120 years, accelerating ozone layer depletion [1]. Agriculture is a major source of N 2 O emissions, and it has contributed 60% to global anthropogenic N 2 O emissions [2]. In the agriculture sector, excessive use of nitrogenous fertilizer is a major reason for this increase in N 2 O emissions [3]. Similarly, soil organic matter, poor crop residue management, tillage practices, and crop rotations also affect N 2 O from agriculture

Experimental Details
The study was performed at Jiangxi Agricultural University in 2019. Soil samples from the experimental station were collected oven-dried and subjected to determine the diverse soil characteristics. The soil was recognized as silt loam (sand, silt, and clay: 24.7%, 57.8%, and 17.5%) with pH 5.21, organic carbon 1.61 g kg −1 , total nitrogen 1.0 g kg −1 , and cation exchange capacity (CEC) 7.10 cmol kg −1 . The soil collected for the experimental purpose was subjected to incubation at 40% water-filled pore spaces (WFPS) for seven days at 25 ± 1 • C to establish the microbial activities. The study was comprised of different rates of rice residues-based biochar, i.e., control 0% (no biochar), 1%, 2%, and 3% biochar application rates. In control, no biochar was applied, whereas, in 1%, 2%, and 3%, biochar was applied at the rate of 22.4, 44.8, and 67.2 t ha −1 . Biochar used for the study was prepared from rice residues by pyrolysis (600 • C) for 8 h. The prepared biochar was sieved (2 mm) prior to the start of the experiment. After that, 500 mL glass beakers were taken, 100 g incubated soil was added in each beaker, with biochar added according to treatments in each beaker and carefully mixed, and moisture was brought to 60% WFPS. The biochar had pH 9.95, carbon content 640 g kg −1 , nitrogen content 4.71 g kg −1 , and CEC 11.05 cmol kg −1 . The experiment was performed for 50 days at 25 ± 1 • C in the control chamber. The experimental beakers were regularly visited, and moisture content was maintained at 60% WFPS by the addition of distilled water [23]. The study was performed in a complete randomized design, with each treatment comprising of 3 replicates.

N 2 O Sampling and Analysis
The sample of N 2 O gas was collected from the headspace of the glass beakers after adding the biochar at different intervals (1,3,5,7,10,13,16,19,23,26,29,31,32,35,38,41,44,47, and 50 days). The glass jars were covered with a thin plastic sheet to prevent moisture loss. Nonetheless, 40 pinholes in each sheet were made for proper airing conditions. N 2 O produced in each glass jar was measured by the procedures of Shaaban et al. [24]. After that, jars were closed for 60 min with an airtight lid with a rubber septum for gas sampling. The quantity of gas produced in head-spaces was measured twice. Once when jars were closed (T 0 ) and later after 60 min (T 60 ) of closing glass jars. We used an airtight three-way stop-cock syringe to collect the gas samples from headspaces. After that, a gas-chromatograph (GC) (Agilent 7890B Santa Clara, CA, USA) was used to measure N 2 O from the gas collected from headspace, and flux of N 2 O gas was determined by methods Shaaban et al. [24] with given below equation: In this equation, F indicates the N 2 O emission rate (µg kg −1 h −1 ), and P indicates the N 2 O density at standard conditions. Furthermore, V and W indicate the volume of the jar (500 mL) and soil weight. ∆c is N 2 O production over 60 min and ∆t is sealing time for N 2 O production, which was 60 min, and T indicating the temperature of incubation (25 • C). Additionally, cumulative N 2 O flux (µg kg −1 ) was measured using daily N 2 O emission over a 50-day period with an equation given by Jin et al. [25].
Here, Ri is N 2 O gas emission (µg kg −1 h −1 ) at sampling dates, whereas n shows the number of sampling while Di is days in sampling interval.

Determination of Soil Characteristic
A total of 600 g soil was taken in 1000 mL beakers to determine different soil properties and placed in the same conditions where the glass beakers for N 2 O determination were placed. Similarly, in these glass beakers, biochar was also applied as per treatment discussed above for gas jars. The soil samples were taken after 2,9,15,22,28,35,42, and 50 days to determine soil pH, MBC, NH 4 + -N, and NO 3 − -N contents. Soil pH was determined by pH meter in a (soil: distilled water (1:2.5)) mixture Shaaban et al. [26]. Likewise, soil extract was prepared using 1 M KCL solution in 1:5 ratios of soil and KCL. After that, the soil solution was shaken for 1 h, and the filtrate was obtained using Whatman filter paper (No. 40). The NH 4 + -N and NO 3 − -N contents in the extract were determined with a discrete chemistry analyzer (Smart Chem 200, Italy) [27]. Soil MBC was estimated by the procedures of Vance et al. [28] and Xiao et al. [29]. We took 20 g soil and fumigated it with chloroform for 24 h. After that, we took both the fumigated and the non-fumigated soil and extracted these with K 2 SO 4 (0.5 M), which were shaken for 30 min. The filtrates were obtained with filter paper, and soil carbon content was measured by total organic carbon (TOC) analyzer.

Analysis of the Genes and Soil Enzymes
The genes abundance (nosZ, nirK, AOA, and AOB) in soil samples was determined before applying the biochar and 1, 15, 30, and 50 days after applying biochar. The soil DNA was extracted with a special DNA kit for soils ((Nohe Zhiyuan Science and Technology Co. Ltd., Beijing, China). After that, extracted DNA was dissolved in 100 µL sterile water and stored (−80 • C). After that, q-PCR was performed on a CFX96 Real-Time PCR System (Bio-Rad, USA) to quantity the AOA, AOB, nosZ, and nirK genes. For the q-PCR amplification, the reaction system consisted of 20 µL, which included 10 µL SYBR Green (TaKaRa, Japan), 0.2 µL Rox DYEII, 1 µL DNA template, 0.4 µL each primer (10 µmol/L), and 8.0 µL sterilized water. The copy number of the AOA, AOB, nosZ, and nirK genes per g/dry soil was determined by normalizing the extraction yield. The activity of soil nitrate reductase (NR) was measured by the phenol disulfonic acid procedure, whereas the activity of urease (UR) was determined by the sodium phenate-sodium hypochlorite colorimetric method [30].

Statistical Analysis
ANOVA was implemented using the Statistix-8.1 to compare the significant difference in soil properties, N 2 O emissions, enzymatic activities, and genes abundance caused due to input of different amounts of biochar application. The percent increase and decrease in each parameter were calculated by subtracting the maximum value from the minimum value. Then, the minimum and last obtained value was multiplied with 100 to calculate the percent increase and decrease [31]. The soft origin-10 was used to prepare the different graphs [32].

Effect of Rice Residue-Based Biochar on Soil pH
Biochar application significantly (p ≤ 0.01) affected the soil pH during the study period. However, the application of 3% biochar significantly increased the soil pH by 13.78% compared to the control ( Figure 1). The biochar application (3%) increased the soil pH from 5.21 to 5.82 on day two, and afterward, pH showed an increasing trend up to 28 days, where pH reached the maximum value (6.48). Moreover, after 28 days, soil pH in biochar treated and control showed a decreasing trend until the end of the study (Figure 1).

Effect of Rice Residue-Based Biochar on Soil Microbial Biomass Carbon
Biochar application significantly (p ≤ 0.01) affected the microbial biomass carbon (MBC) contents ( Figure 2). A significant increase of 11.08% in MBC was noted with biochar addition compared to control ( Figure 2). The increase in biochar application rate significantly increased the soil MBC ( Figure 2). Soil MBC showed an increasing trend until day 22, with a maximum MBC (685 mg kg −1 ) recorded on day 22 with 3% biochar rate; after that, 2% biochar rate (669 mg kg −1 ), and a lowest MBC (639 mg kg −1 ) was recorded

Effect of Rice Residue-Based Biochar on Soil Microbial Biomass Carbon
Biochar application significantly (p ≤ 0.01) affected the microbial biomass carbon (MBC) contents ( Figure 2). A significant increase of 11.08% in MBC was noted with biochar addition compared to control ( Figure 2). The increase in biochar application rate significantly increased the soil MBC ( Figure 2). Soil MBC showed an increasing trend until day 22, with a maximum MBC (685 mg kg −1 ) recorded on day 22 with 3% biochar rate; after that, 2% biochar rate (669 mg kg −1 ), and a lowest MBC (639 mg kg −1 ) was recorded in control ( Figure 2). After 22 days, soil MBC showed a decrease until the end of the study period ( Figure 2).

Effect of Rice Residue-Based Biochar on Soil Microbial Biomass Carbon
Biochar application significantly (p ≤ 0.01) affected the microbial biomass carbon (MBC) contents ( Figure 2). A significant increase of 11.08% in MBC was noted with biochar addition compared to control ( Figure 2). The increase in biochar application rate significantly increased the soil MBC ( Figure 2). Soil MBC showed an increasing trend until day 22, with a maximum MBC (685 mg kg −1 ) recorded on day 22 with 3% biochar rate; after that, 2% biochar rate (669 mg kg −1 ), and a lowest MBC (639 mg kg −1 ) was recorded in control ( Figure 2). After 22 days, soil MBC showed a decrease until the end of the study period ( Figure 2).

Effect of Rice Residue-Based Biochar on Soil Mineral Nitrogen (NH4 + -N and NO3 − -N)
Soil NH4 + -N content was markedly (p ≤ 0.01) influenced by diverse rates of biochar ( Figure 3). The application of biochar increased the NH + 4-N contents by 17.48% compared Soil NH 4 + -N content was markedly (p ≤ 0.01) influenced by diverse rates of biochar ( Figure 3). The application of biochar increased the NH + 4 -N contents by 17.48% compared to control (Figure 3). The NH + 4 -N concentration at day 2 in control, 1%, 2%, and 3% biochar remained as 35.4, 34.5, 33.4, and 30.2 mg kg −1 ; however, after day 2 biochar application favored the significant reduction in NH 4 + -N concentration until day 28. On day 28, maximum NH 4 + -N concentration (46.5 mg kg −1 ) was noted in no-biochar addition; after that, 1% biochar and lowest NH 4 + -N concentration (42.4 mg kg −1 ) was noted in 3% biochar rate ( Figure 3). Moreover, after 28 days, soil NH 4 + -N showed a declining trend in control and biochar-treated soils. Biochar application significantly (p ≤ 0.01) increased the soil NO 3 − -N concentration. However, this increase was the maximum with a higher Agronomy 2021, 11, 2462 6 of 13 rate of biochar application (3%) compared to control and 1% and 2% biochar rate ( Figure 4). In control soil, NO 3 − -N concentration showed a decreasing trend from start to end. While in biochar application, NO 3 − -N concentration showed an increasing trend from day 2 until day 28, and after soil NO 3 − -N concentration was consistently decreased in biochar-treated soil until the end of the study (Figure 4). The overall trend of soil NO 3 − -N concentration for biochar application was recorded as: 3% > 2% > 1% > control (Figure 4). to control (Figure 3). The NH 4-N concentration at day 2 in control, 1%, 2%, and 3% biochar remained as 35.4, 34.5, 33.4, and 30.2 mg kg −1 ; however, after day 2 biochar application favored the significant reduction in NH4 + -N concentration until day 28. On day 28, maximum NH4 + -N concentration (46.5 mg kg −1 ) was noted in no-biochar addition; after that, 1% biochar and lowest NH4 + -N concentration (42.4 mg kg −1 ) was noted in 3% biochar rate ( Figure 3). Moreover, after 28 days, soil NH4 + -N showed a declining trend in control and biochar-treated soils. Biochar application significantly (p ≤ 0.01) increased the soil NO3 − -N concentration. However, this increase was the maximum with a higher rate of biochar application (3%) compared to control and 1% and 2% biochar rate ( Figure 4). In control soil, NO3 − -N concentration showed a decreasing trend from start to end. While in biochar application, NO3 − -N concentration showed an increasing trend from day 2 until day 28, and after soil NO3 − -N concentration was consistently decreased in biochar-treated soil until the end of the study (Figure 4). The overall trend of soil NO3 − -N concentration for biochar application was recorded as: 3% > 2% > 1% > control (Figure 4).   to control (Figure 3). The NH 4-N concentration at day 2 in control, 1%, 2%, and 3% biochar remained as 35.4, 34.5, 33.4, and 30.2 mg kg −1 ; however, after day 2 biochar application favored the significant reduction in NH4 + -N concentration until day 28. On day 28, maximum NH4 + -N concentration (46.5 mg kg −1 ) was noted in no-biochar addition; after that, 1% biochar and lowest NH4 + -N concentration (42.4 mg kg −1 ) was noted in 3% biochar rate ( Figure 3). Moreover, after 28 days, soil NH4 + -N showed a declining trend in control and biochar-treated soils. Biochar application significantly (p ≤ 0.01) increased the soil NO3 − -N concentration. However, this increase was the maximum with a higher rate of biochar application (3%) compared to control and 1% and 2% biochar rate (Figure 4). In control soil, NO3 − -N concentration showed a decreasing trend from start to end. While in biochar application, NO3 − -N concentration showed an increasing trend from day 2 until day 28, and after soil NO3 − -N concentration was consistently decreased in biochar-treated soil until the end of the study (Figure 4). The overall trend of soil NO3 − -N concentration for biochar application was recorded as: 3% > 2% > 1% > control (Figure 4).

Effect of Rice Residue-Based Biochar on Soil N 2 O Emissions
Biochar application had a significant (p ≤ 0.01) impact on soil N 2 O emissions ( Figure 5). N 2 O emissions were significantly higher in control; after that, 1% and 2% biochar application and the lowest N 2 O emission was recorded in 3% biochar application ( Figure 5). The application of 3% biochar markedly reduced the N 2 O emission by 462% compared to control. Moreover, the highest N 2 O emission (0.34 µ g kg −1 ha −1 ) was recorded on day 2 in the control soil; after that, 1% biochar and 2% and 3% application ( Figure 5). After Agronomy 2021, 11, 2462 7 of 13 day 2 N 2 O emissions showed an inconsistent trend until the end of the study period. The 3% biochar application remained the most effective, and it significantly reduced the N 2 O emissions compared to the 1% and 2% biochar rate and control ( Figure 5). The biochar application also reduced the cumulative N 2 O emission compared to control ( Figure 6). The highest cumulative N 2 O emission was recorded in control (no biochar), while the lowest N 2 O emission was recorded in soil treated with 3% biochar (Figure 6). The application of 3% biochar reduced N 2 O emissions by 462%, whereas 2% biochar and 1% biochar application reduced the N 2 O emissions by 225% and 113% compared to control ( Figure 6).
Biochar application had a significant (p ≤ 0.01) impact on soil N2O emissions ( Figure  5). N2O emissions were significantly higher in control; after that, 1% and 2% biochar application and the lowest N2O emission was recorded in 3% biochar application ( Figure 5). The application of 3% biochar markedly reduced the N2O emission by 462% compared to control. Moreover, the highest N2O emission (0.34 μ g kg −1 ha −1 ) was recorded on day 2 in the control soil; after that, 1% biochar and 2% and 3% application ( Figure 5). After day 2 N2O emissions showed an inconsistent trend until the end of the study period. The 3% biochar application remained the most effective, and it significantly reduced the N2O emissions compared to the 1% and 2% biochar rate and control ( Figure 5). The biochar application also reduced the cumulative N2O emission compared to control ( Figure 6). The highest cumulative N2O emission was recorded in control (no biochar), while the lowest N2O emission was recorded in soil treated with 3% biochar (Figure 6). The application of 3% biochar reduced N2O emissions by 462%, whereas 2% biochar and 1% biochar application reduced the N2O emissions by 225% and 113% compared to control ( Figure 6).  Biochar application had a significant (p ≤ 0.01) impact on soil N2O emissions ( Figure  5). N2O emissions were significantly higher in control; after that, 1% and 2% biochar application and the lowest N2O emission was recorded in 3% biochar application ( Figure 5). The application of 3% biochar markedly reduced the N2O emission by 462% compared to control. Moreover, the highest N2O emission (0.34 μ g kg −1 ha −1 ) was recorded on day 2 in the control soil; after that, 1% biochar and 2% and 3% application ( Figure 5). After day 2 N2O emissions showed an inconsistent trend until the end of the study period. The 3% biochar application remained the most effective, and it significantly reduced the N2O emissions compared to the 1% and 2% biochar rate and control ( Figure 5). The biochar application also reduced the cumulative N2O emission compared to control ( Figure 6). The highest cumulative N2O emission was recorded in control (no biochar), while the lowest N2O emission was recorded in soil treated with 3% biochar (Figure 6). The application of 3% biochar reduced N2O emissions by 462%, whereas 2% biochar and 1% biochar application reduced the N2O emissions by 225% and 113% compared to control ( Figure 6).

Effect of Rice Residue-Based Biochar on Gene's Abundance
Biochar application showed a significant (p ≤ 0.01) positive influence on nitrous oxide reductase (nosZ), nitrite reductase (nirK), ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (AOB) abundance during the study period (Figure 7). At the start of the study, there was no significant difference in genes abundance for control and biochar treated soils (Figure 7). Overall, maximum nosZ and nirK genes during the study period were recorded in soil treated with 3% biochar; after that, 2% biochar and the lowest number of both nosZ and nirK genes were recorded in control (Figure 7a,b).
AOA and AOB were similar in control and biochar treated soils at the start of the study; however, both genes showed an increasing trend until the end of the study and reached a maximum value at 45 days (Figure 7c,d). On day 45, the maximum number of AOA genes (4.75 × 10 6 g −1 dry soil) was recorded in soil treated with 3% biochar; after that, 2% biochar (4.35 × 10 6 g −1 dry soil) and 1% biochar and the lowest number of AOA genes (3.95 × 10 6 g −1 dry soil) was recorded in control soil (Figure 7c). Likewise, a higher number of AOB genes (3.94 × 10 6 g −1 dry soil) at day 45 was recorded in 3% biochar amended soil followed closely with 2% biochar amended soil (3.65 × 10 6 g −1 dry soil) and a minimum number of AOA genes (3.23 × 10 6 g −1 dry soil) was recorded in control un-amended soil (Figure 7d).
Biochar application showed a significant (p ≤ 0.01) positive influence on nitrous oxide reductase (nosZ), nitrite reductase (nirK), ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (AOB) abundance during the study period (Figure 7). At the start of the study, there was no significant difference in genes abundance for control and biochar treated soils (Figure 7). Overall, maximum nosZ and nirK genes during the study period were recorded in soil treated with 3% biochar; after that, 2% biochar and the lowest number of both nosZ and nirK genes were recorded in control (Figure 7 a, b).
AOA and AOB were similar in control and biochar treated soils at the start of the study; however, both genes showed an increasing trend until the end of the study and reached a maximum value at 45 days (Figure 7 c, d). On day 45, the maximum number of AOA genes (4.75 × 10 6 g −1 dry soil) was recorded in soil treated with 3% biochar; after that, 2% biochar (4.35 × 10 6 g −1 dry soil) and 1% biochar and the lowest number of AOA genes (3.95 × 10 6 g −1 dry soil) was recorded in control soil (Figure 7c). Likewise, a higher number of AOB genes (3.94 × 10 6 g −1 dry soil) at day 45 was recorded in 3% biochar amended soil followed closely with 2% biochar amended soil (3.65 × 10 6 g −1 dry soil) and a minimum number of AOA genes (3.23 × 10 6 g −1 dry soil) was recorded in control un-amended soil (Figure 7d).

Effect of Rice Residue-Based Biochar on Soil Enzymatic Activities
The activities of both enzymes nitrate reductase (NR) and urease (UR) were significantly (p ≤ 0.01) different among control and biochar amended soils (Figure 8). Biochar application significantly inhibited the activity of NR by 32% during the study period compared to control (Figure 8a). However, it was dose-dependent, and a higher rate of biochar (3%) considerably inhibited the NR activity compared with the 1% and 2% biochar rate and control (Figure 8a). The NR activity was found maximum at the start of the study control soil, and after that, 1%, 2%, and 3% biochar rate; after that, NR activity showed a consistent decrease until day 45, whereas again, the maximum NR activity was noted in control and the lowest NR activity was noted in 3% biochar rate (Figure 8a). Conversely, the addition of different biochar rates improved the UR activity by 18.24% compared to control (Figure 8b). The maximum UR activity (0.48 NH 3 -N g −1 d −1 ) was recorded at the start of the study in soil treated with 3% biochar; after that, 2% biochar treated soil, and the lowest UR activity (0.4 NH 3 -N g −1 d −1 ) was recorded in control. After that, UR activity showed a decreasing trend in both biochar amended soils and control (Figure 8b).
The activities of both enzymes nitrate reductase (NR) and urease (UR) were significantly (p ≤ 0.01) different among control and biochar amended soils (Figure 8). Biochar application significantly inhibited the activity of NR by 32% during the study period compared to control (Figure 8a). However, it was dose-dependent, and a higher rate of biochar (3%) considerably inhibited the NR activity compared with the 1% and 2% biochar rate and control (Figure 8a). The NR activity was found maximum at the start of the study control soil, and after that, 1%, 2%, and 3% biochar rate; after that, NR activity showed a consistent decrease until day 45, whereas again, the maximum NR activity was noted in control and the lowest NR activity was noted in 3% biochar rate (Figure 8a). Conversely, the addition of different biochar rates improved the UR activity by 18.24% compared to control (Figure 8b). The maximum UR activity (0.48 NH3-N g −1 d −1 ) was recorded at the start of the study in soil treated with 3% biochar; after that, 2% biochar treated soil, and the lowest UR activity (0.4 NH3-N g −1 d −1 ) was recorded in control. After that, UR activity showed a decreasing trend in both biochar amended soils and control (Figure 8b).

Correlation Analysis
The data were subjected to Pearson's correlation analysis to determine the relationship between different studied traits ( Figure 9). Soil N2O emission was positively linked with NH4 + -N contents; however, N2O emissions were negatively linked with soil pH, NO3 − -N, genes abundance, and NR activity. Moreover, a strong positive correlation was noted between N2O emissions and NH4 contents, whereas a strong negative association was recorded between N2O emission and soil pH, NO3, genes abundance, and NR activity ( Figure 9).

Correlation Analysis
The data were subjected to Pearson's correlation analysis to determine the relationship between different studied traits ( Figure 9). Soil N 2 O emission was positively linked with NH 4 + -N contents; however, N 2 O emissions were negatively linked with soil pH, NO 3 − -N, genes abundance, and NR activity. Moreover, a strong positive correlation was noted between N 2 O emissions and NH 4 contents, whereas a strong negative association was recorded between N 2 O emission and soil pH, NO 3 , genes abundance, and NR activity ( Figure 9).

Discussion
Soil pH is the most important factor that affects the N 2 O emission from soils [5]. Thus, altering soil pH triggers not only the various soil biochemical processes but also affects soil health and N 2 O emission [33]. Biochar addition significantly augmented the soil pH ( Figure 1). However, the application of biochar (3%) significantly increased the soil pH compared to 1% and 2% biochar application and control (Figure 1). The increase in soil pH altered the soil microbe's activity and regulated the microbial N availability, therefore affecting the soil N 2 O emission [34]. The N 2 O emissions in the current study were negatively associated with soil pH, and the application of 3% substantially increased soil pH and resulted in minimum N 2 O emissions ( Figure 5). Higher soil pH suppresses NR activity that converts the NO 3 − into NO 2 [35]. Indeed, rice residue biochar expressively reduced NR activity, particularly at a 3% biochar rate, and enhanced the NO 3 − and decreased the NO 2 and consequently N 2 O emission. Therefore, biochar-induced augmentation in soil pH is a possible mechanism for reducing N 2 O emissions from acidic soils [1].
Mineral nitrogen plays a significant role in N 2 O production soils. Likewise, NH 4 acts as a reaction substrate of nitrification and plays an impervious role in N 2 O emissions from soils [36]. In the current study, the application of biochar reduced the NH 4 availability (Figure 3), which correlated with lower N 2 O emissions. Previously, some researchers also noted that biochar application reduced the NH 4 availability in soils [14,37]. These researchers recorded that biochar reduced the NH 4 availability due to oxidation of biochar surface and its ability to absorb the NH 4 due to the presence of large adsorption sites, thus resulting in a reduction in NH 4 availability and subsequent N 2 O emissions. The reduction in NH 4 content in biochar-treated soil showed a lack of nitrification substrate that resulted in a significant reduction in N 2 O emissions ( Figure 5). NO 3 − is a major reaction substrate for de-nitrification that involves the conversion of NO 3 − into NO 2 − and finally into N 2 O [13]. In the current study, the application of biochar increased the NO 3 − contents (Figure 4). This increase in NO 3 − contents was due to a decrease in NR activity following the addition of biochar, thus leading to the accumulation of N 3 O − [38] and resulting in a reduction in N 2 O emissions. Thus, in this study, we believe that denitrification is main process linked with a biochar-induced reduction in N 2 O emissions. Additionally, NO 3 − promoted microbial activity for N 2 O reduction and thus indirectly reduced N 2 O emissions [39]. We noted a more pronounced inhibitory effect on N 2 O emission at higher biochar application compared to lower rates ( Figure 5). The higher biochar application provides favorable conditions for reducing bacteria, and it increasing nosZ genes abundance, thus significantly reduces the N 2 O emissions [40].
AOA, AOB, nosZ, and nirK genes play a significant role in de-nitrification, and nosZ genes are mainly linked with the N 2 O reductase enzyme (N 2 OR), which involves in the conversion of N 2 O to N 2 [41]. In the current study, the application of biochar significantly increased the abundance of AOA, AOB, nosZ, and nirK genes (Figure 7). The variable rates of biochar (1%, 2%, and 3%) inhibited N 2 O emission because of the increase in abundance of nosZ genes that promote the N 2 O reduction into N 2 [42,43]. Nonetheless, the abundance of nosZ genes significantly increased with increasing soil pH (Figure 1). Biochar application enhanced the transcription of nosZ genes at higher pH [44] and led to a reduction in N 2 O emissions. Moreover, higher N 2 O emissions ( Figure 5) in control (no biochar soil) can be attributed to restricting the functioning of N 2 OR at low pH owing to less transcription of nosZ genes [45]. Thus, this investigation provides evidence that biochar increased the abundance of nosZ genes, which in turn increased the bacterial ability to synthesize the N 2 OR, consequently reducing the N 2 O emissions.

Conclusions
The rice residue-based biochar significantly inhibited the N 2 O emission compared to un-amended soil. The application of a higher biochar rate (3%) increased the soil pH, genes abundance (AOA, AOB, nosZ, and nirK), NO 3 , and decreased the NH 4 contents and reductase activity, and inhibited N 2 O emission. Thus, this study indicates that biocharinduced inhibition in N 2 O emission from acid soils is linked with increased soil pH, genes abundance, and decreased NH 4 + contents. Additionally, this study also indicates that the amount of biochar application is crucial to maximize the inhibitory effect of biochar on N 2 O emissions from acidic soils.