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Article

Effects of Three Years of Biochar Application on Soil Organic Nitrogen Fraction in Tropical Soil

by
Longwei Meng
1,2,3,†,
Chunlan Jiang
2,3,†,
Meirong Huang
2,
Qiqian Lu
1,2,
Yunxing Wan
1,2,
Anfu Yang
3,
Shuirong Tang
1,
Yanzheng Wu
2,
Xiaoqian Dan
2,
Qilin Zhu
1,*,
Lei Meng
1,
Ahmed S. Elrys
2 and
Jinbo Zhang
1
1
School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya 572025, China
2
School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China
3
Hainan Research Academy of Environmental Sciences, Haikou 571100, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(6), 1357; https://doi.org/10.3390/agronomy15061357
Submission received: 6 April 2025 / Revised: 29 May 2025 / Accepted: 30 May 2025 / Published: 31 May 2025
(This article belongs to the Special Issue Advances in Application Effects and Mechanisms of Fertilizer Products)
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Abstract

Biochar application can increase soil carbon (C) and nitrogen (N) storage. However, the short- and long-term effects of biochar application on soil organic N fractions remain poorly understood in tropical areas. Hence, an in situ combined laboratory incubation study was investigated to determine changes in soil organic N fractions and related chemical biological properties under 1 month (BF) and 3 years (BA) of different biochar application rates (1% and 2%). The results showed that biochar application significantly increased the hydrolysable ammonium N (HAN) by 8.67–18.90% (BF) and 9.45–17.32% (BA). Amino acid N (AAN) significantly increased by 6.08–12.90% (BF) and 5.32–12.16% (BA) compared to CK. The hydrolysable unknown N decreased significantly in BF. The HAN and AAN under the higher biochar application rate were significantly higher than those under the lower application rate. Total N, HAN, and AAN contents were positively correlated with MBN. The structural equation model results showed that soil pH directly promoted AAN, and indirectly promoted soil MBC and MBN. Our results implied that the 3-year biochar application had a more stable effect on the organic N fraction. Therefore, it is possible to increase and maintain soil organic N fractions by appropriate amounts of long-term biochar application in tropical areas.

1. Introduction

Soil nitrogen (N) is an essential nutrient in terrestrial ecosystems [1], essential for the synthesis of nucleic acids, proteins, and other biomolecules in soil microbes and plants, and it also plays a significant role in regulating biogeochemical carbon (C) cycling [2]. Approximately 95% of soil N mainly exists in organic forms (e.g., proteins, amino sugars, nucleic acids, and alkaloids) [3]. The traditional model of N nutrition predominantly emphasizes inorganic N dynamics, but plants have a wide range of soil N sources [4]. For example, although most of the N absorbed by plants needs to be mineralized from organic into inorganic forms by microorganisms, a small amount of low-molecular organic N can be directly taken up by plants [5]. The utilization of organic N sources for absorption by plants has been demonstrated to stimulate root system growth while simultaneously enhancing N use efficiency (NUE) [6,7]. However, soil N status is typically assessed based solely on inorganic N content, often ignoring the role of organic N in agricultural production [8]. Therefore, addressing soil organic N fractions is essential for assessing the soil N supply capacity of crop systems and promoting agricultural sustainability.
Biochar is a C-rich, highly stable material formed by pyrolysis of organic matter at high temperatures under anaerobic or oxygen-limited conditions [9]. Biochar is widely used in agricultural systems to improve soil quality and reduce N losses [10,11]. The soil application of biochar can improve soil structure, increase organic N sequestration, and enhance microbial activity [12,13]. Furthermore, the co-application of biochar and chemical fertilizers has demonstrated significant potential in optimizing soil properties and reducing reliance on chemical fertilizers [10,14]. Numerous studies have indicated that biochar application can significantly increase soil total N content and enhance soil N supply capacity by regulating soil metabolites and increasing the abundance of N-cycling microbial communities [15,16,17]. Additionally, with an increase in biochar addition rate, its influence on other soil properties becomes more pronounced. Notably, the soil total N, acidolysis of total organic N, alkaline dissolved N, and microbial biomass N (MBN) increased significantly with increasing biochar application rate [18,19]. By comparing fresh and aged biochar, Victor et al. found that the influence of biochar on nutrient cycling changed over time [20]. The aged biochar was less effective than fresh biochar in protecting soil organic carbon (SOC) [21], and its ability to ameliorate the soil environment was weaker than that of fresh biochar, which in turn led to a reduction in soil nutrient supply [22]. The effect of biochar on soil N dynamics may vary depending on the duration of its application. Moreover, the ability of biochar to hold soil inorganic N also decreased with aging [23,24]. However, some studies have shown that aging biochar can still promote the transformation of N into soil organic N pool and thus increase soil total N [25,26]. Conversely, a field experiment revealed that after three years of biochar application, the SOC and total N decreased, accompanied by N loss [27]. These different results may stem from the neglect of soil N fractions in previous studies. Hence, evaluating soil fertility and N availability only through total N and neglecting the contents and roles of organic N fractions usually leads to over- or underestimates. This consideration is essential for a comprehensive understanding of the complex dynamics and functions within the soil ecosystem. Therefore, it is of crucial significance to meticulously consider the individual fraction of soil organic N to measure the N supply and turnover.
The chemical form and existence of organic N fractions are important for maintaining soil N supply and ecosystem functions [28]. Previous studies found that soil organic N pools had a significant effect on the soil N cycle [29,30,31], suggesting that changes in the soil content of organic N pools may have a significant impact on the overall N dynamics. Parts of soil organic N fractions (e.g., amino acid N (AAN), amino sugar N (ASN), hydrolysable ammonium N (HAN), and hydrolysable unknown N (HUN)) are directly taken up by plants, resulting in them being highly sensitive to agricultural practice and effective indicators of soil N pool balance and soil fertility. HAN, a type of ammonia produced by the hydrolysis of acids, has complex sources, including soil inorganic N, soil acid digestion products, and amide compounds, which account for about 20 to 35% of soil total N. Generally, HAN acts as a temporary N reservoir to conserve and release exogenous fertilizer N for a short period of time. This provides available N for plant absorption when external available N is insufficient, indicating HAN’s direct impact on soil N supply potential [32,33,34]. AAN is easily oxidized and is one of the most abundant organic N compounds, accounting for about 30–50% of soil total N. AAN mainly exists in the form of organic–inorganic compounds derived from soil microorganisms, plant and animal residues, and proteins and peptides from their decomposition products [35,36]. AAN is the main organic N form that reacts with microorganisms [37], which is easy to mineralize and produces inorganic N. In addition, some studies suggest that plants can uptake small molecular organic N pools such as amino acids [38], indicating that AAN is also a direct N source for plant demand. ASN exists in two forms in soil: one is a complex-state macromolecular compound and the other is tightly bound to inorganic colloids. The content of ASN accounts for about 5 to 10% of soil organic N. It is derived primarily from bacterial and fungal cell walls, rather than plant cells [39,40], making ASN closely related to microbial population and activity. Consequently, ASN content is commonly used as an indicator of microbial contribution to organic matter and soil N mineralization [41,42]. HUN, another N-containing compound not initially identified during soil acid digestion, accounts for about 10–20% of soil total N. The mineralization of HUN has a long duration, and the prolonged application of fertilizers can have a cumulative effect on soil total N, indirectly increasing the HUN content [43]. Thus, each soil organic N fraction has its irreplaceable role in soil N supply. Biochar application has been shown to significantly increase soil AAN and ASN, with both fractions increasing substantially as biochar application rates increased [44]. Furthermore, studies have indicated that biochar application can significantly increase ASN and HUN [45]. Based on this, we hypothesize that increasing biochar application will lead to a corresponding increase in soil organic N fractions.
Yet, it is still unclear how the content of soil organic N and its fractions change under long-term biochar application in the tropical region of China. In order to evaluate changes in soil organic N fractions induced by the duration of biochar application, the present study analyzed soil total N, soil organic N fraction, and their changes under biochar additions at two time points—1 month and 3 years—along with both low and high biochar application rates. This study aims to provide a theoretical basis for understanding how soil organic N fractions respond to biochar amendments in tropical China. Specifically, it seeks to clarify the mechanisms driving changes in soil organic N fractions as biochar age and application rate increase.

2. Materials and Methods

2.1. Soil Sample Site

The experiment began in October 2019 and the study site was located in Sangeng Village, southeast Hainan Province, southern China (18°23′ N,109°03′ E). It has a tropical monsoon climate, with a mean annual precipitation and temperature of 1664 mm and 23.8 °C, respectively. The soil type is acrisol (https://soilgrids.org/ accessed on 28 April 2025) [46] developed from shallow marine sedimentary rocks, consisting of 18.3% sand, 54.7% silt, and 27% clay. The main cropping plants in this area are rice. Urea (46% N), superphosphate (16% P2O5), and potassium sulphate (52% K2O) were applied at approximately 140–210, 30–100, and 130–200 kg ha−1 yr−1. The fertilizer application time was divided into two stages, namely base fertilizer (before rice seedlings were transplanted into the soil) and topdressing (from the jointing stage to the filling stage).

2.2. Experimental Design

Samples were taken at depths of 0–20 cm, and the soil was mixed and brought back to the laboratory for air-drying, during which the plant roots were removed and the air-dried soil was passed through a 2 mm sieve. Basic soil chemical properties: pH 6.40, organic matter 14.8 g kg−1, total N 3.49 g kg−1, available P 126 mg kg−1, available K 213 mg kg−1. The tested biochar was formed by cracking corn stover under anaerobic conditions at 400 °C for 24 h. It was provided by the Institute of Soil Science, Chinese Academy of Sciences, and had a pH of 7.58, organic C content of 49.5%, total N content of 1.79%, total P content of 0.48%, total K content of 2.29%, CEC of 46.9 cmol kg−1, pecific surface area of 3.75 m2 g−1, and Zeta-potential of 23.1 mV.
Biochar was mixed well with the soil at rates of 1% and 2% of the air-dried soil mass, added to the PVC tube (length: 20 cm, diameter: 15 cm), and then buried in its original location in October 2019 (BA). The CK without added biochar was treated at the same time. In September 2022, a portion of the soil from CK was taken and biochar was added at 1% and 2% of the air-dried soil mass (BF). To eliminate the systematic errors caused by the cultivation process, the BA treatment was also subjected to a one-month incubation period. The test soil (as dry soil) was weighed at 100.00 g from CK, BA, and BF, placed in 250 mL conical flasks, and placed together in a constant-temperature incubator at 25 °C. The soil moisture was maintained at 60% of the soil water holding capacity (WHC). It was sealed with plastic wrap with several small holes punctured with a needle to maintain ventilation, and determined after one month. Soil moisture was maintained at a constant level throughout the process by weighing. The experiment was divided into five treatments: unadded biochar (CK), 1% (BF1) and 2% (BF2) biochar incubated for 1 month, and 1% (BA1) and 2% (BA2) biochar incubated for 3 years, with three replications for each treatment (a total of 15 samples).

2.3. Determination of Soil Chemical Indicators

The basic analyses of soil properties were conducted based on the methods of Agrochemical Analysis of Soil [47]. Soil pH was determined using a pH meter at a soil–water ratio of 1:2.5 (w/w). Soil organic C content was determined using a potassium dichromate–sulphuric acid–external heating method, while soil total N content was determined using semi-micro Kjeldahl. The available phosphorus in soil was extracted with 1 M NH4F and determined by molybdenum blue spectrophotometry. Soil available K content was determined using 1 M NH4OAc (pH = 7.0) and a flame photometric assay.

2.4. Determination of Soil Active Organic N

Soil microbial biomass of C and N was determined by the chloroform fumigation method [48]. Soil N fractions were measured by Bremner’s method [49]. Organic N fraction was determined in hydrolysates prepared by refluxing with 6 mol L−1 HCl in an oil bath at 120 °C. Total hydrolyzed acidified organic N (AHN) was determined by steam distillation, after Kjeldahl digestion with 10 mol L−1 NaOH. The HAN was determined by steam distillation at 3.5% of MgO (w/v). ASN was calculated as the difference between hydrolysates obtained by steam distillation with phosphate borate buffer at pH 11.2. AAN was determined using phosphate borate buffer, decomposing hexosamine by treatment with 0.5 mol L−1 NaOH at 100 °C to remove NH3-N, and the addition of ninhydrin powder to convert amino-N to NH3-N. The amount of HUN was calculated by the following equation: HUN = AHN − (HAN + AAN + ASN).

2.5. Soil Extracellular Enzyme Determination

Four extracellular enzymes were measured using the microplate fluorescence method, namely C- (β−1, 4−glucosidase, BG), N- (leucine aminopeptidase, LAP; β−N−acetylglucosaminidase, NAG), and P-acquiring enzymes (acid phosphatase, AP). Briefly, 2.75 g of fresh soil was taken, 91 mL of 50 mmol L−1 sodium acetate buffer (pH 5.5) was homogenized in a warming mixer at 25 °C for 1 min, and subsequently, 800 μL of the slurry was pipetted into a black 96-deep-well flat-bottomed microplate. Next, 200 µL of 200 µmol L−1 appropriate fluorometric substrate was added into the corresponding wells of the sample plate (4−MUB−β−D−glucoside as a substrate for BG, 4–MUB–N–acetyl–β–D–glucosaminide as a substrate for NAG, L–Leucine–7–amino–4–methylcoumarin as a substrate for LAP, and 4–MUB–phosphate as a substrate for AP). The soil suspension was obtained by shaking at 200 rpm min-1 for 30 min. Subsequently, 200 μL aliquots of 4−methylunbelliferone standard solution at different concentrations (0, 5, 10, 20, 50, 100, 150, and 200 μmol L−1) were added to each sample hole of the marking plate. After addition, all samples were sealed with self-adhesive sealing film, shaken and mixed, and incubated for 4 h in an incubator at 25 °C in the dark. After incubation, the deep-hole plates were centrifuged at 4800 rpm min−1 for 3 min at 4 °C, and 250 μL of the supernatant was obtained and read with a microplate reader at a wavelength of 450 nm. The matrix conversion rate per gram of sample per hour represents the enzyme activity of the sample (nmol g−1 h−1).

2.6. Statistical Analysis

A one-way analysis of variance (ANOVA) was used to test the significant differences in soil properties and N fractions among treatments. The correlations between soil N fractions and soil properties were performed using bivariate correlation analyses and curve-fitting methods (Origin Pro 2021). Structural equation models (SEMs) were constructed using AMOS software (version 25) to analyze the direct and indirect effects of soil properties and N fractions on soil microbial biomass.

3. Results

3.1. Soil Chemical and Biological Properties

The 1-month biochar application significantly increased soil pH compared to the control, but the opposite was recorded under the 3-year application of biochar (Table 1). Compared with CK, the pH increased by 3.07–4.91% (BF) and decreased by 3.48–5.92% (BA), respectively. The SOC, total N, available K, MBC, MBN and C/N in the biochar addition treatments were significantly higher than those in the CK treatment. Compared to the CK, the AP content increased significantly under 1 month, but not 3 years, of biochar application (BF1 and BF2). The increase in the available P content amounted to 42.88–57.02% (BF) and 6.13–22.15% (BA) compared to CK. The SOC, total N, C/N ratio, MBC, MBN, and available K under the higher application rate of biochar were significantly higher than those under the low application rate of biochar under both 1 month and 3 years of biochar application. Under the low application rate of biochar, the C/N ratio, MBC, available P, and available K were significantly higher in the 1-month than in the 3-year biochar application, and no significant differences under the 1-month and 3-year biochar applications were recorded for SOC, C/N ratio, and MBN. Under the high application rate of biochar, the SOC, total N, MBC, available P, and available K were significantly higher in the 1-month than in the 3-year biochar applications, but no significant differences were observed for the C/N ratio.

3.2. Soil N Fractions

The ASN, HAN, and AAN contents were significantly higher in the biochar addition treatments than those without biochar addition (Figure 1). Compared to the CK, the ASN content increased significantly under the one-month and three-year biochar applications (BF and BA). The increase in the ASN content amounted to 70.97–74.19% (BF) and 77.42–119.35% (BA) compared to CK. Under the 1-month and 3-year biochar applications, the HAN was significantly higher in the high biochar application rate. The increase in HAN content amounted to 8.67–18.90% (BF) and 9.45–17.32% (BA) compared to CK. Compared to CK, the HUN content decreased significantly under the 1-month biochar applications (BF1 and BF2), and was lowest under the low application rate. Under the 3-year biochar applications, the HUN was also significantly lower compared to CK in the low biochar application treatment (BA1), but the opposite was observed in the high biochar application treatment (BA2). The AAN under the high biochar application rate was significantly higher than that under the low application rate under both the 1-month and 3-year biochar applications. The increase in AAN content amounted to 6.08–12.90% (BF) and 5.32–12.16% (BA) compared to CK.
HAN was the main contributor to total N content across all treatments (Figure 2). The content of HAN in soil total N was not significant. The HUN proportion was significantly higher in BA2, and the lowest was recorded in BF1 and BF2. The ASN proportion was significantly higher in BA1, and the lowest was recorded in CK. The ASN proportion was significantly higher in CK, and the lowest was recorded in BA1. There was no significant difference between the 1% and 2% biochar applications for each soil organic N fraction in the soil total N in BF.

3.3. Soil Enzyme Activity

Biochar application significantly affected the activities of soil C, N, and P enzymes (Figure 3). Compared with CK, soil BG activity was markedly higher in the biochar application treatments. Moreover, the BG activities were significantly greater in soils treated with high-concentration biochar compared to those treated with low-concentration biochar. At the same biochar application, the BG activity when treated with the 1-month biochar application was significantly higher than those treated with the 3-year biochar application. The activity of soil NAG enzymes was highest in soils treated with high-concentration biochar, followed by soils with low-concentration, 3-year and 1-month biochar application. The CK treatment exhibited the lowest NAG enzyme activity. Compared with CK, the soil LAP significantly increased in the biochar application treatments. High-concentration biochar application led to significantly greater LAP activity than in the low-concentration biochar application treatments. However, no significant difference was observed between the 1-month and 3-year biochar treatments at the same concentration. Under the same biochar application, the AP enzyme activity in the soil with the 1-month biochar treatment was higher than that in the soil with the 3-year biochar application.

3.4. Fitting Analysis of TN, N Fractions, and Chemical Properties

The SOC was positively associated with total N, MBC, MBN, AAN, and HAN (p < 0.01; Figure 4a–e). The soil MBN, MBC, AAN, and HAN levels increased significantly with increasing soil total N content (p < 0.01; Figure 4f–i). The contents of MBC and MBN increased with increasing ANN (p < 0.01; Figure 4j,k). The contents of MBC and MBN showed a significant positive correlation with increasing HAN (p < 0.01; Figure 4l,m). The content of HAN was positively correlated with AAN (p < 0.01; Figure 4n).
The soil organic nitrogen fractions were significantly correlated with the chemical and biological characteristics of soil (Figure 5). HAN is significantly positively correlated with SOC, TN, AP, and AK (p < 0.05). AAN is significantly positively correlated with SOC, TN, AK, and HAN (p < 0.05). In addition, both HAN and AAN show significant positive correlations with MBC, MBN, BG, NAG, LAP, and PHOS (p < 0.05). The soil pH was significantly correlated with AP and AK (p < 0.05). A positive correlation was found to exist between the SOC and TN, AP, AK, HAN, AAN, MBC, MBN, BG, NAG, LAP, and PHOS (p < 0.05). TN demonstrated a positive correlation with AP, AK, HAN, AAN, MBC, MBN, BG, NAG, LAP, and PHOS (p < 0.05). AP exhibited a positive correlation with AK, HAN, MBC, MBN, BG, LAP, and PHOS (p < 0.05). AK was positively correlated with HAN, AAN, MBC, MBN, BG, NAG, LAP, and PHOS (p < 0.05).

3.5. Interactions Between Soil Organic Nitrogen Fractions and Environmental Factors

Structural modeling (SEM) data showed (Figure 6) that soil pH was negatively correlated with AAN after biochar application (p < 0.01). The SOC was positively correlated with ASN (p < 0.05) and negatively correlated with AAN. Total N was positively correlated with HAN (p < 0.001). AAN was positively correlated with MBC and MBN (p < 0.001). HAN was positively correlated with MBN (p < 0.05) and MBC was negatively correlated with HUN (p < 0.001).

4. Discussion

4.1. Effect of Biochar Addition on Soil Chemical and Biological Properties

This study found that biochar application significantly increased the soil total N and SOC content, which was in line with previous studies [15]. Biochar contains large amounts of C and N, which directly lead to higher soil total N and SOC when added to the soil [50]. The biochar production process produces alkaline substances such as ash, resulting in an increase in pH when applying acidic soil [51]. Our results also revealed that 1-month biochar application significantly increased soil pH (Table 1). However, the effect of promoting soil pH decreased (BA) with the extension of biochar application time (three years; Table 1). Soil pH decreasing with long-term biochar addition can potentially be attributed to the weakening cation exchange capacity (CEC) of aging biochar [52]. The decrease in soil pH may be related to microbial activity, as certain microorganisms (e.g., phosphorus-solubilizing microorganisms) excrete organic acids during metabolic processes [53]. Recent studies have demonstrated that both chemically and naturally aged biochar lead to a reduction in soil pH, with naturally aged biochar causing a more moderate decrease [54]. This may be due to a more gradual alteration in the chemistry of naturally aged biochar, along with an increase in acidity associated with the formation of acidic functional groups over time. Previous studies have shown that biochar additions can significantly increase soil MBC and MBN [55]. This increase is attributed to the positive correlation between MBC and MBN with SOC and total N, which was the energy substrate for microbes. Furthermore, biochar addition has been found to significantly enhance soil SOC and total N content, thereby promoting microbial abundance and diversity [56]. Biochar application significantly enhanced the activity of soil C, N, and P extracellular enzymes (Figure 3). This is primarily due to the presence of biochar, which serves as a readily available carbon source that can be easily absorbed and utilized by soil microorganisms. The incorporation of biochar into the soil leads to an increase in microbial populations, subsequently stimulating the secretion of extracellular enzymes.

4.2. Effect of Biochar Addition on Soil Organic N Fractions

Biochar, as a C source, can significantly affect soil N fractions and the quantity and abundance of soil microorganisms [57]. ASN has a close relationship with microorganisms. The residues of death of microorganisms after decomposition favor the accumulation of soil amino acid and ASN. In addition, the macropores of biochar provide favorable habitats for soil microbial habitation and reproduction [58], and adequate C and N sources promote microbial life and death cycles, thereby increasing soil AAN accumulation. Soil organic N fractions are closely related to total N [59]. The SEM results showed a positive relationship between total N and soil organic N fraction (Figure 5). Moreover, biochar application significantly increased soil HAN, ASN, and AAN content (Figure 1), likely due to the enhancement of SOC and total N, both of which are positively correlated with soil organic N fractions (Table 1 and Figure 3, Figure 4 and Figure 5). Previous studies have demonstrated that different preparation conditions have a significant effect on both the N retention and the N form in biochar [60]. High temperature can effectively promote the formation of the microporous structure of biochar [61,62], which may lead to significant differences in the content of soil organic N fractions after application of biochar with different pyrolysis conditions. Moreover, ASN content significantly increases with the application of N fertilizer [63], suggesting that biochar application could potentially replace a portion of N fertilizer in agricultural systems. Biochar application can promote inorganic N transformation to organic N pools, and improve the N stability in the soil [64]. The presence of readily available soluble substances in biochar can increase microbial activity and abundance [65], which is conducive to enhancing the conversion of soil inorganic N to organic N, ultimately resulting in an increase in soil HAN, ASN, and AAN content.
Microorganisms play a pivotal role in soil N dynamics [66]. The SOC determines the structure of the soil microbial community [67,68], while soil total N serves as the foundation for determining soil N turnover. Our study corroborated the influence of SOC and total N on microbial communities. Moreover, our study further found that AAN and HAN were significantly positively correlated with MBC and MBN (Figure 3, Figure 4 and Figure 5). AAN is one soil organic N reservoir, which is an effective N source for plants and microbials. Our study found that the percentage of AAN was low in tropical soils and the addition of biochar contributed to an increase in the content of AAN. Previous studies have found an increase in AAN for ploughing [69], suggesting that agricultural measures can increase soil AAN content. As an important strategy for increasing C sinks in agriculture, biochar also makes a significant contribution to soil AAN. This is due to AAN mainly originating from proteins and peptides produced during the decomposition of soil, plant, and animal residues. In this study, the relatively low proportion of AAN to total N in the absence of agricultural practices (Figure 2) further supports this notion. HAN is the most active N pool in soil, which can be directly available for uptake and utilization by crops in the current season, and can be used as a method of characterizing the soil potential N supply [18]. Biochar application has been shown to significantly increase HAN content [69], and our results are consistent with this finding. HAN has a substantial impact on soil available N, and it was the highest among all N fractions following biochar application [32].
The effect of biochar on soil active C fractions is usually influenced by the application rate. Our results found that HAN and AAN increased significantly with increasing biochar addition (Figure 1). This is due to the higher C/N with the addition of biochar, which relieves microbial C limitation and promotes microbial proliferation and N assimilation [12,70]. Moreover, soil MBC and MBN contents significantly increased with the increase in biochar application, due to the immobilization of N and phosphorus by microorganisms, which in turn enhanced MBC and MBN levels (Table 1). The high quantity and activity of microorganisms contribute to the accumulation of AAN and HAN. However, in practical applications, economic considerations are paramount. Recent studies have demonstrated that applying a moderate amount of biochar (8 t ha−1) has the highest economic effect on the crop compared to applying a small amount or a large amount of biochar (4 t ha−1 and 28 t ha−1) [71].

4.3. Changes in Soil Organic Nitrogen Fractions Due to Years of Biochar Addition

Previous studies found that an increase in the duration of biochar application can still significantly increase soil total N [72]. This is consistent with the results of this study; however, we further found that the duration of biochar application influenced soil N fractions. Specifically, HUN increased with increasing years of biochar application (Figure 1). The effect of biochar on bacterial communities is highly time-dependent [73]. Long-term application of biochar creates a relatively stable and suitable living environment for soil microorganisms, which leads to the deep adjustment of microbial community structure [74]. Some microbial populations that can efficiently utilize the nutrients and pore structure on the surface of the biochar gradually took the lead, and these microbes produced more types and quantities of N-containing compounds during metabolism, which in turn increased the content of HUN. HAN and AAN may be related to soil enzyme activity. Biochar can increase soil enzyme activity in the initial phase after application, but its positive effect on enzyme activity is limited and diminishes as the biochar ages [75]. Our study found that the HAN and AAN content remained stable with increasing years of biochar application, which may be because the relative abundance of Ascomycetes was stable and did not change significantly after years of biochar application [76]. Some fungi in the phylum Ascomycota have the ability to decompose nitrogenous organic matter [77]. For example, they can decompose nitrogenous compounds such as proteins and peptides and convert them into small molecules such as amino acids, and further reactions of these small molecules may affect the HAN and AAN content [44]. Soil N fractions have an indirect effect on maize dry matter accumulation [78]. After seven years of field aging, the application of biochar (15.75–31.50 t ha−1) continued to promote maize growth [79]. This study is based on a single tropical site with typical hotspot soil characteristics and agricultural production patterns. Although the findings cannot be directly generalized to all tropical regions, they can provide an important reference for regions with similar conditions. In addition, the research area is primarily rice-growing, and we adjusted the moisture content to 60% WHC in the incubation experiment, which differs from the actual moisture conditions. To improve realism, future studies could focus on in situ experiments. Given the presence of other diverse soil types in the tropics, it would be intriguing to investigate the changes in soil organic N fractions after longer-term biochar applications at different sites.

5. Conclusions

Our findings demonstrate that biochar application significantly enhanced soil N fractions relative to the control (no biochar). Notably, the N fractions exhibited remarkable stability over three years of biochar application. Specifically, soil total N, HAN, ASN, AAN, and MBN increased proportionally with rising biochar dosage. Among these, HAN, ASN, and AAN showed the most pronounced increases under biochar treatment. These results suggest that biochar can be strategically utilized to improve soil N supply capacity, thereby promoting long-term soil fertility and N cycling efficiency.

Author Contributions

Conceptualization, Q.L., Q.Z., Y.W. (Yunxing Wan), L.M. (Lei Meng), A.Y. and J.Z.; investigation, L.M. (Longwei Meng), C.J., M.H., A.S.E., A.Y. and S.T.; formal analysis, Q.L., Q.Z., Y.W. (Yunxing Wan), L.M. (Lei Meng), X.D., and Y.W. (Yanzheng Wu); software, Q.L., Q.Z., X.D., J.Z., and A.S.E.; supervision, S.T., L.M. (Lei Meng), J.Z., Y.W. (Yanzheng Wu), and X.D.; writing—original draft preparation, L.M. (Longwei Meng), C.J., M.H., A.S.E., and S.T.; writing—review and editing, Q.L., L.M. (Longwei Meng), C.J., M.H., Y.W. (Yunxing Wan), A.Y. and Y.W. (Yanzheng Wu). All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Technology Talent and Platform Plan (202405AM340004), the Graduate Innovation Research Project of Hainan Province, China (Grant No. Qhyb2024-89), and the Graduate Innovation Research Project of Hainan Province, China (Grant No. Qhys2024-115).

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Organic nitrogen components under different biochar treatments. CK represents treatment without biochar, BF1 represents 1-month incubation of 1% biochar, BF2 represents 1-month incubation of 2% biochar, BA1 represents 1% biochar cultivated for 3 years, and BA2 represents 2% biochar cultivated for 3 years. ASN: amino sugar N, HAN: hydrolysable ammonium N, HUN: hydrolysable unknown N, ANN: amino acid N. Different lowercase letters in the figure represent significant differences between treatments (p < 0.05). In the results of two-factor ANOVA, T represents different time treatment and C represents different biochar content. T × C represents the interaction effect of different time treatments and different biochar content. NS: no significant difference; **: p < 0.05. (a–d) indicate the subgraph number in the caption.
Figure 1. Organic nitrogen components under different biochar treatments. CK represents treatment without biochar, BF1 represents 1-month incubation of 1% biochar, BF2 represents 1-month incubation of 2% biochar, BA1 represents 1% biochar cultivated for 3 years, and BA2 represents 2% biochar cultivated for 3 years. ASN: amino sugar N, HAN: hydrolysable ammonium N, HUN: hydrolysable unknown N, ANN: amino acid N. Different lowercase letters in the figure represent significant differences between treatments (p < 0.05). In the results of two-factor ANOVA, T represents different time treatment and C represents different biochar content. T × C represents the interaction effect of different time treatments and different biochar content. NS: no significant difference; **: p < 0.05. (a–d) indicate the subgraph number in the caption.
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Figure 2. The proportion of each component of soil acidolysis organic nitrogen to total nitrogen. CK represents without biochar, BF1 represents 1-month incubation of 1% biochar, BF2 represents 1-month incubation of 2% biochar, BA1 represents 1% biochar cultivated for 3 years, and BA2 represents 2% biochar cultivated for 3 years. ASN: amino sugar N, HAN: hydrolysable ammonium N, HUN: hydrolysable unknown N, ANN: amino acid N. Different lowercase letters in the figure represent significant differences between treatments (p < 0.05).
Figure 2. The proportion of each component of soil acidolysis organic nitrogen to total nitrogen. CK represents without biochar, BF1 represents 1-month incubation of 1% biochar, BF2 represents 1-month incubation of 2% biochar, BA1 represents 1% biochar cultivated for 3 years, and BA2 represents 2% biochar cultivated for 3 years. ASN: amino sugar N, HAN: hydrolysable ammonium N, HUN: hydrolysable unknown N, ANN: amino acid N. Different lowercase letters in the figure represent significant differences between treatments (p < 0.05).
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Figure 3. Extracellular enzyme activities under different treatments. CK represents without biochar, BF1 represents 1-month incubation of 1% biochar, BF2 represents 1-month incubation of 2% biochar, BA1 represents 1% biochar cultivated for 3 years, and BA2 represents 2% biochar cultivated for 3 years. BG: β-Glucosidase, NAG: β-N-Acetylglucosaminidase, LAP: Leucine Aminopeptidase, AP: Acid Phosphatase. (a–d) indicate the subgraph number in the caption. Different lowercase letters in the figure represent significant differences between treatments (p < 0.05).
Figure 3. Extracellular enzyme activities under different treatments. CK represents without biochar, BF1 represents 1-month incubation of 1% biochar, BF2 represents 1-month incubation of 2% biochar, BA1 represents 1% biochar cultivated for 3 years, and BA2 represents 2% biochar cultivated for 3 years. BG: β-Glucosidase, NAG: β-N-Acetylglucosaminidase, LAP: Leucine Aminopeptidase, AP: Acid Phosphatase. (a–d) indicate the subgraph number in the caption. Different lowercase letters in the figure represent significant differences between treatments (p < 0.05).
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Figure 4. Correlation analysis of soil total nitrogen and its components with environmental factors. SOC: soil organic carbon, TN: soil total nitrogen, MBC: soil microbial carbon, MBN: soil microbial nitrogen, ASN: amino sugar N, HAN: hydrolysable ammonium N, ANN: amino acid N. (a–n) indicate the subgraph number in the caption.
Figure 4. Correlation analysis of soil total nitrogen and its components with environmental factors. SOC: soil organic carbon, TN: soil total nitrogen, MBC: soil microbial carbon, MBN: soil microbial nitrogen, ASN: amino sugar N, HAN: hydrolysable ammonium N, ANN: amino acid N. (a–n) indicate the subgraph number in the caption.
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Figure 5. Correlation analysis of soil organic N and its fractions with soil properties. * Significant correlation at 0.05; white * indicates a correlation coefficient |r| ≥ 0.7, while black * indicates a correlation coefficient |r| < 0.7. SOC: soil organic carbon, TN: soil total nitrogen, Available P: soil available phosphorus, Available K: soil available potassium. ASN: amino sugar N, HAN: hydrolysable ammonium N, HUN: hydrolysable unknown N, ANN: amino acid N, MBC: soil microbial carbon, MBN: soil microbial nitrogen, BG: β-Glucosidase, NAG: β-N-Acetylglucosaminidase, LAP: Leucine Aminopeptidase, AP: Acid Phosphatase.
Figure 5. Correlation analysis of soil organic N and its fractions with soil properties. * Significant correlation at 0.05; white * indicates a correlation coefficient |r| ≥ 0.7, while black * indicates a correlation coefficient |r| < 0.7. SOC: soil organic carbon, TN: soil total nitrogen, Available P: soil available phosphorus, Available K: soil available potassium. ASN: amino sugar N, HAN: hydrolysable ammonium N, HUN: hydrolysable unknown N, ANN: amino acid N, MBC: soil microbial carbon, MBN: soil microbial nitrogen, BG: β-Glucosidase, NAG: β-N-Acetylglucosaminidase, LAP: Leucine Aminopeptidase, AP: Acid Phosphatase.
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Figure 6. Direct and indirect effects of soil properties and soil N fractions on soil N pool when adding biochar. SOC: soil organic carbon, TN: soil total nitrogen, ASN: amino sugar N, ANN: amino acid N, HAN: hydrolysable ammonium N, HUN: hydrolysable unknown N, MBC: soil microbial carbon, and MBN: soil microbial nitrogen. Black arrows and red arrows indicate positive and negative significant relationships. χ2/df, chi-square/degrees of freedom; P, probability level; NFI, normalized fitting index; RMSEA, root mean squared error of approximation. Significance levels of each predictor are p < 0.01. *** significant correlation at 0.001.
Figure 6. Direct and indirect effects of soil properties and soil N fractions on soil N pool when adding biochar. SOC: soil organic carbon, TN: soil total nitrogen, ASN: amino sugar N, ANN: amino acid N, HAN: hydrolysable ammonium N, HUN: hydrolysable unknown N, MBC: soil microbial carbon, and MBN: soil microbial nitrogen. Black arrows and red arrows indicate positive and negative significant relationships. χ2/df, chi-square/degrees of freedom; P, probability level; NFI, normalized fitting index; RMSEA, root mean squared error of approximation. Significance levels of each predictor are p < 0.01. *** significant correlation at 0.001.
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Table 1. Soil basic properties of studied soils under different biochar treatments.
Table 1. Soil basic properties of studied soils under different biochar treatments.
ParameterCKBF1BF2BA1BA2
pH6.51 ± 0.07 bc6.71 ± 0.08 ab6.83 ± 0.06 a6.13 ± 0.17 d6.23 ± 0.07 c
SOC (g kg−1)11.78 ± 0.45 d21.99 ± 0.70 c29.68 ± 0.54 a21.04 ± 0.78 c27.42 ± 0.14 b
TN (g kg−1)0.97 ± 0.02 d1.54 ± 0.22 c1.89 ± 0.04 a1.56 ± 0.02 c1.78 ± 0.05 b
C/N12.11 ± 0.48 d14.32 ± 0.48 b15.69 ± 0.57 a13.45 ± 0.18 c15.44 ± 0.47 a
Available P (mg kg−1)106.06 ± 7.78 b151.54 ± 24.03 a166.53 ± 55.13 a112.55 ± 16.96 b129.55 ± 1.41 ab
Available K (mg kg−1)173.53 ± 26.07 e394.76 ± 10.20 b645.65 ± 13.60 a222.42 ± 24.94 d282.54 ± 3.40 c
MBC (mg kg−1)113.16 ± 13.42 d252.63 ± 23.24 b339.47 ± 6.44 a207.89 ± 9.85 c265.79 ± 7.44 b
MBN (mg kg−1)12.17 ± 0.81 c14.99 ± 0.89 b17.88 ± 0.53 a13.82 ± 0.59 b17.28 ± 0.28 a
BF1 represents 1-month incubation of 1% biochar, BF2 represents 1-month incubation of 2% biochar, BA1 represents 1% biochar cultivated for 3 years, and BA2 represents 2% biochar cultivated for 3 years. SOC: soil organic carbon, TN: soil total nitrogen, C/N: carbon-to-nitrogen ratio, Available P: soil available phosphorus, Available K: soil available potassium, MBC: soil microbial carbon, and MBN: soil microbial nitrogen. Values are mean ± standard error (n = 3). Different letters indicate significant differences in different soils (p < 0.05).
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MDPI and ACS Style

Meng, L.; Jiang, C.; Huang, M.; Lu, Q.; Wan, Y.; Yang, A.; Tang, S.; Wu, Y.; Dan, X.; Zhu, Q.; et al. Effects of Three Years of Biochar Application on Soil Organic Nitrogen Fraction in Tropical Soil. Agronomy 2025, 15, 1357. https://doi.org/10.3390/agronomy15061357

AMA Style

Meng L, Jiang C, Huang M, Lu Q, Wan Y, Yang A, Tang S, Wu Y, Dan X, Zhu Q, et al. Effects of Three Years of Biochar Application on Soil Organic Nitrogen Fraction in Tropical Soil. Agronomy. 2025; 15(6):1357. https://doi.org/10.3390/agronomy15061357

Chicago/Turabian Style

Meng, Longwei, Chunlan Jiang, Meirong Huang, Qiqian Lu, Yunxing Wan, Anfu Yang, Shuirong Tang, Yanzheng Wu, Xiaoqian Dan, Qilin Zhu, and et al. 2025. "Effects of Three Years of Biochar Application on Soil Organic Nitrogen Fraction in Tropical Soil" Agronomy 15, no. 6: 1357. https://doi.org/10.3390/agronomy15061357

APA Style

Meng, L., Jiang, C., Huang, M., Lu, Q., Wan, Y., Yang, A., Tang, S., Wu, Y., Dan, X., Zhu, Q., Meng, L., Elrys, A. S., & Zhang, J. (2025). Effects of Three Years of Biochar Application on Soil Organic Nitrogen Fraction in Tropical Soil. Agronomy, 15(6), 1357. https://doi.org/10.3390/agronomy15061357

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