Mechanism of Modified Biochar in Mitigating Carbon and Nitrogen Loss in Drought Soil with Green Manure Application

Abstract

With the frequent occurrence of global droughts, modified biochar has demonstrated the potential to be an efficient soil amendment, which could affect carbon and nitrogen sequestration in arid soil. Therefore, this study investigated the co-application of pristine biochar (BC), Fe-modified biochar (FB) and H₂O₂-modified biochar (HB) with green manure during a 70-day laboratory incubation under drought conditions and normal moisture conditions. The emissions were quantified using gas chromatography, while microbial necromass carbon and nitrogen were measured by quantifying the amino sugar content by gas chromatography–mass spectrometry, and other soil carbon and nitrogen fractions were determined through chemical analysis. The results revealed that under drought conditions, compared to BC co-application with green manure, the total carbon loss of FB and HB co-application with green manure was reduced from 24.38% to 13.14% and 14.27%, respectively, and the total nitrogen loss was also reduced from 14.61% to 7.23% and 7.27%, respectively. This reduction occurred because FB and HB protected soil organic matter through iron oxide binding and micropore adsorption, thereby increasing the content of soil total humus acid (>5%) and microbial necromass nitrogen (>16%). In addition, under normal moisture conditions, BC, FB and HB co-application with green manure enhanced microbial activity and promoted the formation of stable total humus acid, thereby enhancing carbon and nitrogen sequestration. In conclusion, this study provides crucial theoretical support for the optimization of the green manure return via modified biochar co-application in arid environments.

1. Introduction

Green manure is defined as a fertilizer derived from green plants [1]. Alfalfa (Medicago sativa L.) serves as both one of the most important and widely cultivated forage crops and a commonly used green manure [2]. To utilize alfalfa as green manure, farmers typically harvest it during the flowering stage when nutrient content peaks, then plow it directly into fields. This practice offers a natural alternative to chemical fertilizers while improving soil fertility and soil structure [3]. These benefits collectively promote both ecological sustainability and economic advantages within agricultural systems. However, studies have demonstrated that during green manure decomposition, the ammonification process releases NH₄⁺-N which gradually accumulates in poorly aerated soil, resulting in a large amount of nitrogen loss through ammonification [4]. Moreover, higher soil pH and drought increase the concentration of NH₃ in soil, which further promotes the release of NH₃ [5]. If the soil aeration is sufficient, green manure returns to the field stimulate nitrification and trigger high denitrification rates within the soil, which could intensify the emission of soil N₂O [6]. Furthermore, Wu et al. have indicated that this agricultural management leads to a significant increase in soil unstable carbon content, which may lead to a sharp increase in CO₂ emissions [6]. Consequently, enhancing soil carbon and nitrogen sequestration through the optimization of green manure return techniques, and realizing the synergistic effect of soil fertility improvement and carbon sequestration are pivotal scientific issues that must be addressed for the sustainable development of agriculture at present.

The application of biochar and green manure alone or in combination can significantly impact the soil nitrogen and carbon turnover. Regarding the nitrogen cycle, biochar promotes nitrification by increasing soil pH, thereby enhancing total soil nitrogen mineralization and substrate availability for nitrifying bacteria [7]. Moreover, biochar contains labile organic compounds and abundant pores. These properties provide favorable conditions for microorganisms: the labile carbon serves as a vital carbon source, while the porous structure offers habitats and attachment sites. Additionally, the electrical conductivity of biochar facilitates interspecific or intraspecific electron transfer among microorganisms. These combined characteristics profoundly influence nitrogen turnover. Regarding the carbon cycle in soil, studies have demonstrated that biochar can also induce negative priming effects in soil, which promote the sequestration of soil carbon. Lei et al. have demonstrated through a 13-year field experiment that biochar increases soil organic carbon (SOC) content by 85% through stabilizing exogenous carbon inputs and suppressing soil respiration. In comparison, straw return enhances microbial necromass carbon accumulation through the stimulation of microbial activity, which promotes the formation of organo-mineral complexes, consequently improving SOC protection by 43% [8]. Meanwhile, biochar exhibits a high surface charge density, a large specific surface area, and internal porosity. Furthermore, its surface contains both polar and non-polar functional sites, which collectively contribute to its efficient adsorption of organic molecules and nutrients in soil. Therefore, Wang and Lu have confirmed that the combined application of biochar and green manure significantly increases the content of total carbon, total nitrogen, heavy component organic nitrogen, microbial biomass carbon (MBC), and nitrogen (MBN) in the soil, thus markedly improving soil fertility [9].

Nevertheless, the existing literature on the application of biochar or green manure predominantly derives conclusions under conditions of sufficient soil moisture [10]. These findings may therefore have limited applicability to arid soils, and offer insufficient reference for addressing the impacts of future greenhouse gas-induced extreme climate events. The co-application of biochar and fertilizers under arid conditions may potentially increase the risk of carbon and nitrogen loss. Specifically, the lower soil moisture content weakens the dilution effect of NH₄⁺-N, while biochar raises soil pH, leading to an increase in NH₃ volatilization [11]. In addition, in controlled irrigation paddy fields, the application of biochar elevated the peak NH₃ volatilization flux [12]. Furthermore, under drought conditions, the application of biochar enhances the soil water-holding capacity and promotes microbial activity [13], which may also lead to increased N₂O and CO₂ emissions. Biochar disrupts the nitrification process, thereby facilitating the release of more by-products (N₂O) from incomplete nitrification [14]. On the other hand, biochar combined with inorganic fertilizers may also promote CO₂ emissions from acidic drought soils [15], particularly in soils with C/N ≤ 10, where liable carbon fractions in biochar could become an additional carbon source for microorganisms, thereby further increasing CO₂ emissions. These studies suggest that although the application of biochar can alleviate drought, the biochar co-application with fertilizers may paradoxically exacerbate the loss of carbon and nitrogen from the soil.

Modified biochar has been found to exhibit excellent nitrogen and carbon adsorption and fixation capacity, which could change the nitrogen and carbon dynamics in soil, thereby potentially mitigating their loss. Mandal et al. demonstrated that KOH modification enhanced the cation exchange capacity (CEC) of biochar, thereby increasing the amount of NH₄⁺-N exchanged and adsorbed onto the biochar. This improvement significantly reduced NH₃ volatilization [16]. Therefore, introducing oxygen-containing functional groups onto the surface of biochar to enhance its cation exchange capacity is particularly crucial for improving the fixation of nitrogen compounds in arid soils. H₂O₂ modification is a common and environmentally preferable method and significantly increases the carboxyl and sulfoxide groups on the surface of biochar [17]. However, the incorporation of oxygen-containing functional groups into H₂O₂-modified biochar disrupted the π-bonds, thereby diminishing the electron shuttling capacity. This, in turn, reduced the facilitating effect of biochar on electron transfer by denitrifying microorganisms, resulting in increased soil N₂O emissions [18]. Thus, further specific and in-depth research is needed on the application of H₂O₂-modified biochar in nitrogen fixation in arid soils. On the other hand, research indicates that iron oxides in soil contribute to the formation of soil organic/inorganic complexes [19]. Therefore, Fe-modified biochar may facilitate the fixation of carbon and nitrogen. In addition, Liao et al. found that the Fe²⁺/Fe³⁺ redox cycle on FeSO₄/FeCl₃-modified biochar denitrification reaction reduced N₂O to N₂, thereby decreasing N₂O emissions. By contrast, NO₃⁻ on Fe(NO₃)₃-modified biochar as a substrate for denitrification significantly increased soil N₂O emissions [20]. Consequently, post green manure application, soil nitrogen exists in multiple chemical species, and the mechanisms by which modified biochars retain specific nitrogen species or influence transformation pathways under drought conditions require further investigation.

This study investigated the effects of biochar co-application with alfalfa green manure under both drought and normal moisture conditions, with the aim of elucidating the mechanisms behind carbon and nitrogen stabilization facilitated by modified biochar. A particular focus was placed on identifying optimal application strategies for modified biochar in arid regions to enhance soil carbon sequestration and nitrogen retention. The objective of this research is to provide theoretical foundations and technical support for addressing future extreme climate changes. Therefore, the following two hypothesizes will be tested: (1) under drought, FB and HB mitigate carbon and nitrogen loss by protecting organic matter via iron oxide binding and microporous adsorption; (2) under normal moisture conditions, BC, FB and HB promote the conversion of microbial necromass and other organic matter into stable humic acid through enhanced microbial activity and abiotic stabilization processes, thereby enhancing the stability of soil carbon and nitrogen.

2. Materials and Methods

2.1. Overview of Experimental Area

The incubation experiment started on 27 April 2024 and ended on 5 July 2024. The experimental area was located in the research plots of the Faculty of Environmental Science and Engineering, Kunming University of Science and Technology (24°50′53″ N, 102°51′46″ E, elevation 1953 m). The study region has a multi-year average temperature of 14.7 °C, a relative humidity ranging from 58% to 83%, and annual sunshine hours exceeding 2400 h. The mean annual precipitation is 789 mm, with a frost-free period of 227 days. Precipitation exhibits uneven intra-annual distribution and considerable inter-annual variability. Soil samples were collected from the experimental field of Kunming University of Science and Technology at a depth of 10 cm. The samples were air-dried after being passed through a 2 mm sieve to remove gravel and plant residues, and subsequently sealed and stored under dry conditions. The soil is classified as Hapli-Udic Ferralosol (Chinese Soil Taxonomy, CST), which is characterized by its high acidity and low organic matter content. The soil texture was relatively heavy, composed of 21.8% clay, 58.5% silt, and 19.7% sand. The soil pH was measured as 5.50. The soil dry bulk density is 1.22 g/cm³. The total carbon content and total nitrogen content of the soil are 3.91 g/kg and 1.60 g/kg, respectively, while the total phosphorus content and available potassium content are 0.43 g/kg and 0.13 g/kg, respectively.

2.2. Preparation and Characterization of Biochar and Green Manure

The bagasse was sourced from the Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences, and was naturally air-dried and crushed into pieces measuring 2–3 cm. It was then pyrolyzed in a continuous carbonization furnace at 350 °C to produce biochar (BC). Once the furnace had been heated to 350 °C, the biomass was added. The biomass underwent continuous pyrolysis for approximately 1 h, after which the biochar was cooled to room temperature in the closed furnace. The carbonization furnace was equipped with inflation and exhaust devices to maintain an oxygen-limited state in the pyrolysis chamber. The biochar yield was approximately 40%. The Fe-modified biochar was prepared using impregnation pyrolysis [21]. The mass ratio of biochar and iron (in the form of FeCl₃) was 20:1. The biochar/iron mixture was mixed with ultrapure water and ultrasonicated for 1 h at 25 °C. The mixture was then dried at 60 °C and subsequently pyrolyzed at 350 °C for 1 h. It was washed with deionized water until the pH of the filtrate stabilized. It was then dried in an oven, stored under dry conditions, and labeled as FB. The Fe content on the surface of FB is approximately 2.04%. The H₂O₂-modified biochar was prepared by the heating method [22]. The biochar was thoroughly mixed with H₂O₂ using a mass-to-volume ratio of 1:30 (g:mL), with H₂O₂ at a concentration of 10%. After mixing, the mixture was heated in an oven at 80 °C for 4 h. Following filtration, the biochar was washed with deionized water until the pH of the filtrate stabilized. It was then dried in an oven, stored under dry conditions, and labeled as HB. Green manure was air-dried and crushed into 2–3 cm. The elemental content (C, H, O, N) of the biochar and green manure were measured by an Elemental Analyzer (Microcube, Elementar, Hanau, Germany). The specific surface area of the biochar was analyzed using the N₂ adsorption/desorption method at 77 K by a BET Surface Area Analyzer (ASAP 2020, Mike Model, Karlsruhe, Germany). The electrical conductivity of biochar was measured at a powder compaction pressure of 6 MPa by a Powder Electrical Resistivity Meter (ST2722, Suzhou Lattice Electronics Co., Ltd., Suzhou, China). The surface functional groups of biochar were characterized by a Fourier Infrared Spectrometer (FTIR, Varian 640-IR, Thermo Fisher, Waltham, MA, USA). The surface crystal structure of biochar was examined using an X-Ray Diffractometer (XRD, D8 advance, Bruker, Billerica, MA, USA).

2.3. Experimental Design and Incubation Setup

2.3.1. Determination of Soil Water Holding Capacity (WHC)

The soil sample is collected using a core cutter and placed into a flat-bottom container, followed by the addition of water and immersion for 24 h. The core cutter containing the fully water-saturated soil sample is then placed on air-dried soil covered with filter paper. After 8 h of water infiltration, the soil sample in the upper core cutter is immediately weighed. Subsequently, it is oven-dried at 105 °C until a constant weight is achieved (12 h), cooled to room temperature, and weighed again to calculate the water content.

2.3.2. Experimental Treatments and Incubation Setup

The experimental design employed a two-factor treatment arrangement. The factorial structure included three types of biochar and two soil moisture conditions, resulting in a total of 6 treatment combinations plus controls with soil alone and green manure addition alone. Three replications were established for each treatment. The three types of biochar, green manure and soil were thoroughly mixed in a mass ratio of 2:1.6:100 (totaling 100 g on an air-dried weight basis) and transferred into 250 mL screw-cap glass vials, which were labeled as CK (soil alone), GM (green manure + soil), BG (green manure + BC + soil), FG (green manure + FB + soil) and HG (green manure + HB + soil). Based on the aforementioned conversion ratio, the application rate of biochar is 24.4 t/ha, and the application rate of green manure is 19.5 t/ha. The experiment involved two distinct moisture groups: a control group (labeled the N group) representing normal moisture conditions, and a treatment group (labeled the D group) simulating drought conditions. The soil moisture content in the N group was maintained at 65% of the water holding capacity (WHC), whereas the D group was maintained at 45% of the WHC. Each treatment in the drought and normal moisture groups was separately labeled as DCK, DGM, DBG, DFG, DHG, NCK, NGM, NBG, NFG and NHG. The incubator is maintained at a relative air humidity of around 60% and a temperature of 25 °C, with a photoperiod regime of 12 h of light exposure and 12 h of darkness. The duration of the incubation period was 70 days. The water content was maintained by daily weighing the entire vial and replenishing the weight loss with ultrapure water via spraying. During routine cultivation, all vial caps were kept loose to allow gas exchange. Caps were only tightened during gas sampling. Prior to gas collection, zero-grade air was introduced for 10 min, after which all vials were sealed for 2 h. The gas sample was then collected into a gas bag. Gas samples were collected on days 1, 2, 3, 5, 7, 10, 13, 16, 20, 25, 30, 35, 40, 50, 60 and 70, with N₂O and CO₂ concentrations determined using gas chromatography (8890 GC System, Agilent, Santa Clara, CA, USA). Subsequent to the cultivation period (70 days), soil samples were collected, and fresh soil was immediately placed in a −80 °C freezer for storage. Air-dried soil was distributed for natural desiccation and subsequently stored.

2.4. Characterization of Soil Indicators

2.4.1. Soil Physicochemical Characteristics

The determination of total carbon and total nitrogen was carried out using air-dried soil, while all other indicators were determined using fresh soil. The pH level of the soil was measured using a pH meter. Soil total nitrogen and carbon were determined by an Elemental Analyzer (Microcube, Elementar, Germany). NH₄⁺-N and NO₃⁻-N in the soil were leached using a 2 M KCl solution at a soil-to-solution ratio of 1:2.5 (g:mL). After 2 h of shaking, the solution was centrifuged and filtered for subsequent measurement using a continuous flow chemical analyzer (SKALAR San++Classic, SKALAR ANALYTICAL B.V., Ridderkerk, The Netherlands). Total humus acid was extracted using 0.1 M (NaOH + Na₄P₂O₇) [23]. Briefly, 5 g of soil was extracted several times until the extract was nearly colorless. A portion of the leachate was acidified with HCl (6 M, 1.1 g/mL) to pH = 1 and allowed to stand for 24 h at 25 °C. The precipitate was collected as humic acid. Humic acid was dissolved in 0.1 M NaOH and analyzed using a total organic carbon analyzer (vario TOC cube, Elementar, Germany). In addition, prior to total humus acid nitrogen extraction, the soil was washed with 2 M KCl. Total humus acid nitrogen was determined by alkaline potassium persulfate oxidation combined with an Ultraviolet–visible Spectrophotometer (UV-2600, Shimadzu, Kyoto, Japan).

2.4.2. Soil Microbiological Analysis

Soil microorganism sequencing was then performed by Personal Biotechnology Company (Shanghai, China) by using the Pacific Biosciences platform and the Illumina NovaSeq platform [24]. Soil MBC and MBN were determined using the chloroform fumigation extraction method. Briefly, 2.5 g of soil was placed in a beaker and transferred to a vacuum desiccator containing chloroform. A vacuum was applied for 2 min, and the sample was then incubated at 25 °C for 24 h in the dark. After the incubation, the sample was extracted with 10 mL of 0.5 M K₂SO₄ by shaking for 30 min. The mixture was centrifuged and filtered. The nonfumigated soil was used as a control. Total organic carbon in filtrate was determined using a total organic carbon analyzer (vario TOC cube, Elementar, Germany), and total nitrogen was determined by the alkaline potassium persulfate oxidation method combined with the Ultraviolet–visible Spectrophotometer (UV-2600, Shimadzu, Japan). The soil amino sugar content was determined by GC-MS (7890A GC equipped with 5975C quadrupole mass selective detector, Agilent, USA). The process of amino sugar extraction is outlined in full in Text S1. The estimation of the carbon and nitrogen content of fungi, bacteria and microbial necromass was conducted using the following equations. The molar ratio of glucosamine (GlcN) and muramic acid (MurN) in bacterial cells was hypothesized to be 2:1. Moreover, 179.17 and 251.23 in the Equations (1) and (4) represent the molecular weights of GlcN and MurN, respectively, where 9 is the conversion value from GlcN to fungal necromass carbon and 45 is the conversion value from MurN to bacterial necromass carbon. The conversion factor of GlcN to fungal necromass nitrogen is 1.4, and the conversion factor of MurN to bacterial necromass nitrogen is 6.67 [25].

$$\mathrm{F}\mathrm{u}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{C}=(\mathrm{G}\mathrm{l}\mathrm{c}\mathrm{N}/179.17-2\times \mathrm{M}\mathrm{u}\mathrm{r}\mathrm{N}/251.23)\times 179.17\times 9$$

(1)

$$\mathrm{B}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{C}=\mathrm{M}\mathrm{u}\mathrm{r}\mathrm{N}\times 45$$

(2)

$$\mathrm{M}\mathrm{i}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{b}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{C}=\mathrm{F}\mathrm{u}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{C}+\mathrm{B}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{C}$$

(3)

$$\mathrm{F}\mathrm{u}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{N}=(\mathrm{G}\mathrm{l}\mathrm{c}\mathrm{N}/179.17-2\times \mathrm{M}\mathrm{u}\mathrm{r}\mathrm{N}/251.23)\times 179.17\times 1.4$$

(4)

$$\mathrm{B}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{N}=\mathrm{M}\mathrm{u}\mathrm{r}\mathrm{N}\times 6.67$$

(5)

$$\mathrm{M}\mathrm{i}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{b}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{N}=\mathrm{f}\mathrm{u}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{N}+\mathrm{B}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s} \mathrm{N}$$

(6)

2.5. Data Analysis

The data were analyzed using IBM SPSS Statistics (v.25.0, IBM Corp., Armonk, NY, USA). The Shapiro–Wilk test and Q-Q plot were employed to assess the normal distribution of data. Additionally, a univariate analysis of variance (ANOVA) was conducted to investigate the variations within drought conditions and normal moisture conditions. All significant differences are reported at the 0.05 probability level. Capital letters represent the statistical significance of the index values under normal moisture conditions, while lowercase letters represent the statistical significance of the index values under drought conditions. To facilitate an analysis of the differences between drought and normal moisture conditions within the same treatment, an independent samples t-test was conducted. “*” indicates significant differences between the same treatment under normal moisture and drought conditions.

3. Results and Discussion

3.1. Properties of Biochar

The polarity (0.45), aromaticity (0.86) and specific surface area (3.81 m²/g) of FB showed no significant differences compared to BC; however, the electrical conductivity increased significantly by approximately two times. In contrast, HB exhibited the highest polarity and specific surface area (161.90 m²/g). The FTIR signal peaks of HB at 1708 cm⁻¹ (-COOH) and 2357 cm⁻¹ (-CH₂-) exhibited marked enhancement, indicating that the H₂O₂ modification successfully introduced numerous carboxyl functional groups and carbon chain structures on the biochar surface [26]. Notably, this modification also led to a marked decrease in its electrical conductivity by approximately 4.6 times when compared to BC (

Table 1. Basic properties of biochar and green manure (air-drying matter). BC, FB, HB and GM represent pristine biochar, Fe-modified biochar, H₂O₂-modified biochar and green manure, respectively. C%, O%, N% and H% denote mass percentages, while (O+N)/C and H/C represent atomic ratios. “-” indicates that the value was not detected.

Sample	C%	O%	N%	H%	(O+N)/C	H/C	pH	Electrical Conductivity (nS/cm)	Specific Surface Area (m2/g)
BC	38.41	20.99	2.42	2.99	0.46	0.93	7.12	0.45	3.15
FB	34.79	18.46	2.26	2.49	0.45	0.86	3.04	1.34	3.81
HB	35.89	25.69	2.25	3.09	0.59	1.03	3.90	0.08	161.90
GM	23.47	-	8.49	5.02	-	-	-	-	-

). In contrast, the FTIR spectra of FB and BC showed minimal differences and maintained comparable profiles (Figure 1a). The XRD analysis revealed that, in comparison with BC, FB incorporated iron oxides (Fe₂O₃ and Fe₃O₄), as evidenced by the characteristic peaks of iron oxides, indicating that Fe was successfully loaded onto the biochar (Figure 1b). The adsorption test (Figure S1) demonstrated that the adsorption capacity of HB for NH₄⁺-N was increased by 69%compared to that of BC. This may be due to the interaction between oxygen-containing functional groups (carboxyl, hydroxyl, etc.) on the biochar and NH₄⁺-N, such as through electrostatic interactions or complexation [27]. The enhanced adsorption capacity of FB for NO₃⁻-N is likely attributable to the iron oxides or iron ions (Fe²⁺/Fe³⁺), which have the capacity to adsorb NO₃⁻-N via mechanisms like ligand exchange or chemical bonding [28].

3.2. Soil Emissions of N₂O and CO₂

3.2.1. The Properties of Modified Biochar Determined N₂O Emissions

There were no significant N₂O emissions from the drought group (Figure 2a). This was likely due to drought conditions reducing organic nitrogen mineralization and diminishing the effectiveness of soil carbon and nitrogen cycle. Furthermore, a decline in soil microbial activity and a decrease in NO₃⁻-N content in the soil were observed (Figure S2). These changes impaired the denitrification process, thereby significantly suppressing N₂O emissions [29]. Moreover, the abundance of assimilatory nitrate reduction functional genes increased under drought conditions compared to normal moisture conditions (Figure 2b). Assimilatory nitrate reduction, which belongs to an anabolic pathway [30], exhibits lower energy demands compared to dissimilatory reduction, making it more adaptable to resource-scarce arid environments. A higher abundance of assimilatory nitrate reduction was also observed for HB and FB co-application with green manure under drought conditions. The assimilatory nitrate reduction process relies on the electron transport chain [30], and therefore, HB and FB could provide electrons that are critical for promoting this assimilatory nitrate reduction pathway, potentially enhancing enzymatic catalytic activity and improving the efficiency of the reduction reactions. In addition, the elevated microbial necromass nitrogen content associated with HB and FB co-application with green manure under drought conditions further supports this hypothesis (Section 3.3.2).

Under normal moisture conditions, HB co-application with green manure exhibited the highest cumulative N₂O emissions of 0.012 µg/g, which was approximately 68% higher than those of GM. BC and FB co-application with green manure resulted in a substantial reduction in N₂O emissions, showing 20% and 62% decreases relative to GM, respectively (Figure 2a). The observed N₂O reduction in BC co-application with green manure is likely due to the BC adsorption capacity for NH₄⁺-N (Figure S1) [31]. Furthermore, BC and FB, which have better electrical conductivity (Table 1), can act as an electron shuttle, thereby promoting complete denitrification and reducing N₂O emissions [32]. In addition, the presence of iron oxides on FB promotes complete denitrification, further reducing N₂O emissions [33]. However, HB co-application with green manure showed significantly higher cumulative N₂O emissions, likely attributable to the lower electrical conductivity (Table 1). The increased C=O groups on the biochar surface compete for electrons with N₂O in the denitrification process, thus increasing soil N₂O emissions [18]. The introduction of oxygenated functional groups disrupts the conjugated π-bonds of the biochar, weakening the biochar’s ability to shuttle electrons and promoting incomplete denitrification, thus increasing soil N₂O emissions [34]. Furthermore, the abundance of functional genes for denitrification and dissimilatory nitrate reduction were elevated under normal moisture conditions in comparison with those observed under drought conditions (Figure 2b). Consequently, the N₂O emissions in the normal moisture conditions were predominantly attributable to denitrification and dissimilatory nitrate reduction (Figure 2b).

3.2.2. The Properties of Modified Biochar Reduce CO₂ Emissions

Under drought conditions, BC and HB co-application with green manure significantly increased CO₂ emissions by 76% and 34% compared to GM (Figure 3). These two biochar additions significantly increased the CO₂ emissions, probably due to the increase in available carbon and moisture [13], promoting microbial respiration. The exogenous organic carbon could stimulate CO₂ emissions when added to sandy arid soil, which has been linked to the adaptation of microorganisms to limited nutrients in poor soils and the more efficient utilization of available unstable compounds [35]. Conversely, under normal moisture conditions, the CO₂ emissions of BC, FB and HB co-application with green manure were reduced by 8%, 16% and 11%, respectively, in comparison with GM (Figure 3). This may be attributed to the capacity of biochar to provide locations for the co-localization of soil organic matter and nutrients, thereby enhancing the carbon sequestration [36]. However, under both normal moisture and drought conditions, the co-application of both FB and HB with green manure was able to further reduce the CO₂ emissions relative to BC, and the CO₂ emissions were lower under FB addition. This may be because the iron oxides in FB have the capacity to combine with organic carbon, thereby forming stable organic/inorganic complexes and reducing the mineralization of organic carbon. Cooper et al. demonstrated that biochar can form organo-minerals with SOC, thus providing physical protection from mineralization, which is in agreement with this study [37]. The “mineral carbon pump” hypothesis further emphasizes the pivotal role of biochar in stabilizing organic carbon through organic/mineral interactions, whereby iron oxides from biochar can promote aggregation and convert plant or microorganism-derived labile organic carbon into a more stable form [38], thereby reducing their mineralization. In addition, the large specific surface area and pore volume of HB (Table 1) facilitate the adsorption of organic carbon released from green manure, thereby inhibiting the mineralization of organic carbon [39].

3.3. Changes in Soil Nitrogen

3.3.1. Modified Biochar Reduces Nitrogen Loss from Drought Soil by Minimizing N₂O Leaching and NH₃ Volatilization

Figure 4a shows that, after 70 days of GM application, the total nitrogen content in soil decreased by 18.31% under drought conditions and 7.97% under normal moisture conditions. Under normal moisture conditions, no significant loss of total nitrogen was observed in soil after 70 days of with BC, FB or HB co-application with green manure. However, under drought conditions, the total nitrogen content in soil decreased by 14.61% after 70 days of BC co-application with green manure. In comparison, the total nitrogen loss was reduced by 7% when FB and HB were co-applied with green manure (Figure 4a). These results demonstrated that modified biochar can effectively reduce total nitrogen loss in soil. This is consistent with our adsorption test (Figure S1), which showed that FB and HB have an enhanced capacity to adsorb NH₄⁺-N and NO₃⁻-N, respectively, thereby retaining more nitrogen in the soil. In addition, the Fe-modified biochar can also enhance the assimilation of NO₃⁻-N by microorganisms, which facilitated the nitrogen fixation. Furthermore, the oxygen-containing functional group on the H₂O₂-modified biochar surface, particularly carboxyl groups, led to an augmentation in NH₃ adsorption [17]. Under drought conditions, the minimal N₂O emissions were observed in GM and BC, and FB and HB co-application with green manure (Figure 2a). However, a decline in total nitrogen content was observed in the soil, and we speculated that this nitrogen loss under drought conditions likely occurred through alternative pathways, primarily involving the emission of NH₃ and N₂ [40]. The NH₄⁺-N content in the soil after BC co-application with green manure was lower than in the FB and HB co-application with green manure (Figure S2), suggesting that the nitrogen loss might be due to NH₃ volatilization. The results of the investigation of soil functional gene abundance (Figure 2b) further demonstrated that the loss of nitrogen may also be attributable to the complete denitrification process.

Biochar co-application with green manure enhances soil MBN content, showing significantly greater enhancement under normal moisture conditions compared to drought conditions (Figure S3). Xu et al. have shown that MBN content decreased by 42.2%, 46.2% and 53.8% when soil moisture content decreased from 25.7% to 23.8%, 23.7% and 24.4%, which is in agreement with this study [41]. However, no significant difference was observed in the soil total humus acid nitrogen content between BC co-application with green manure and green manure application alone under drought and normal moisture conditions. This indicated that pristine biochar does not have a significant effect in promoting humification. This is consistent with the finding of Xie et al. [42] that biochar addition alone has a relatively weak effect on enhancing soil humification. The reduction in soil moisture content weakened microbial metabolism and had a detrimental effect on the soil humification process, so the total humus acid nitrogen content was lower in arid soils [43]. However, under drought conditions, FB and HB co-application with green manure application still significantly increased the total humus acid nitrogen content by 16% and 19%, relative to BC co-application with green manure (Figure 4b). Moreover, under normal moisture conditions, the total humus acid nitrogen content also increased by 9% and 14%, respectively, relative to BC. Iron oxides in FB could complex with humus through ligand coordination, forming stable organic iron complexes that facilitate soil humus polycondensation [44]. The incorporation of porous biochar enhances the stability and activity of microorganisms, thereby increasing the degree of humification [45].

3.3.2. Modified Biochar Retains Nitrogen in Microbial Necromass to Reduce Nitrogen Loss Under Drought Conditions

Microbial necromass nitrogen refers to the accumulation of nitrogen from microbial biomass following the death of microorganisms [46]. Microbial necromass nitrogen was included in the total humus acid nitrogen content, and the reuse of microbial necromass may be converted to more stable fractions of humus acids. Green manure returns to the field enhanced the retention of fungal and bacterial necromass [47]. The soil microbial necromass nitrogen content was predominantly influenced by the fungal necromass nitrogen content (Figure 5a). This was due to the fact that fungi compete more effectively than bacteria in decomposing green manure, making them more conducive to nitrogen accumulation in fungal necromass [48]. Under drought conditions, the fungal necromass nitrogen content in the soil was 89%, 123%, 156% and 152% higher in GM, BC, FB and HB co-application with green manure, respectively, compared to CK (Figure 5a). Moreover, under normal moisture conditions, the soil fungal necromass nitrogen content also increased by 48%, 567%, 525% and 313%, respectively, compared to CK. Therefore, fungal necromass nitrogen is involved in the retention and supply of nitrogen, and plays the role of “capacitor” in controlling the availability of soil nitrogen [49]. Under drought conditions, FB and HB co-application with green manure resulted in a significant increase in microbial necromass nitrogen content. The functional groups and high specific surface area of modified biochar facilitated the adsorption and immobilization of necromass nitrogen [50], thus further enhancing nitrogen stabilization.

3.3.3. Moisture Promotes the Humification Process of Microbial Necromass

Under normal moisture conditions, the soil microbial necromass nitrogen content is lower than that under drought conditions, which may be related to higher soil humification. HB co-application with green manure exhibited a lower necromass nitrogen content than that of BC and FB co-application with green manure. Furthermore, the ratio of soil microbial necromass nitrogen to total humus acid nitrogen under FB and HB co-application with green manure was lower than that of BC (Figure S4). These phenomena may be attributed to the larger specific surface area of HB with active sites (-OH, -COOH, etc.), which could polymerize soil nitrogen (including microbial necromass nitrogen) into total humus acid nitrogen (Figure 4b) [51]. Jia et al. also showed that the decline in microbial necromass nitrogen content could be attributed to the utilization of microbial necromass by the microorganisms as a source of their own nitrogen, or for the conversion process into a more stable humus fraction [52]. It is evident from the proportion of soil nitrogen fractions in the total nitrogen that FB and HB co-application with green manure under normal moisture conditions resulted in an increase in the organic nitrogen (total humus acid nitrogen) content relative to BC (Figure 5b), which increased the nitrogen fixation capacity of the soil.

3.4. Changes in Soil Carbon

3.4.1. Modified Biochar Reduces Soil Carbon Losses

After 70 days of incubation, the total carbon content of GM exhibited a substantial decline, reaching 24.92% and 24.17% under drought and normal moisture conditions, respectively (Figure 6a). Under normal moisture conditions, there was no significant difference in total carbon loss among BC, FB and HB after 70 days of incubation. Under drought conditions, the loss of total carbon when BC was co-applied with green manure was approximately 24%. However, FB and HB co-application with green manure reduced the total soil carbon loss by approximately 10% compared to BC co-application with green manure. Under drought conditions, BC and FB co-application with green manure increased soil MBC content by 17% and 35%, respectively, compared to GM. In contrast, HB exhibited no statistically significant impact on soil MBC (Figure 6b). Under normal moisture conditions, BC co-application with green manure had no substantial impact on soil MBC content. However, FB and HB co-application with green manure significantly increased soil MBC content by 13% and 19%, respectively. The porosity of biochar is conducive to the retention of elements such as carbon and nitrogen, and is more beneficial for microbial colonization and growth. Biochar containing iron oxides may enhance microbial extracellular electron transfer and carbon utilization efficiency. Additionally, the physical protection provided by minerals also contributes to soil MBC accumulation [53].

3.4.2. Modified Biochar Enhanced the Conversion of Microbial Necromass to Humic Acid

Total humus acid consists of two primary components: humic acid and fulvic acid. Humic acid is predominantly composed of aromatic compounds, and its molecules contain abundant active functional groups such as carboxyl groups and phenolic hydroxyl groups. In contrast, fulvic acid has a relatively simple chemical structure [54]. As an important component of organic matter, humic acid has a significant impact on soil properties such as water retention, nutrient retention/availability and aeration, and serves as an important carbon source and indirect energy source for soil microorganisms [55]. Under drought conditions, BC, FB and HB co-application with green manure increased the total humus acid content by 5%, 11% and 31% relative to GM (Figure 7a). The trend of total humus acid content under normal moisture conditions was similar to that under drought conditions, and its content was higher under normal moisture conditions. Furthermore, soil humic acid content was significantly higher under normal moisture than under drought conditions. Under drought and normal moisture conditions, HB co-application with green manure resulted in a significant enhancement of humic acid content (Figure 7b). The FTIR spectrum of humic acid (Figure S5) indicated that HB co-application with green manure increased the aromaticity of soil humic acid, enhanced its stability and consequently improved the stability of soil organic matter. Zhang et al. showed that biochar increased the abundance of two bacterial genera involved in humus synthesis, which enhanced the stability of the trans-domain network between bacteria and fungi. In addition, microorganisms contribute to the humification by consuming more humus precursors and synthesizing more humus [56]. As a result, the content of humic acids was significantly increased, further enhancing the degree of soil humification.

The function of microorganisms in the formation and transformation of soil organic matter involves ex vivo modification and in vivo turnover [57]. Ex vivo modification is the process in which plant residues are converted by extracellular enzymes into recalcitrant components. In contrast, in vivo turnover refers to the organic components that are easily utilized by microorganisms and are subsequently converted into microbial organic matter through cellular uptake, growth and death. The rapid rate of turnover and the brief growth cycle of soil microorganisms are pivotal factors in the continuous accumulation of microbial necromass. Under drought conditions, BC, FB and HB co-application with green manure increased the fungal necromass carbon content by 52%, 36% and 34%, respectively, relative to GM (Figure 8a). In addition, under normal moisture conditions, the fungal necromass carbon content also increased by 351%, 322% and 255%, respectively, relative to GM. The soil bacterial necromass carbon content is comparatively low (below 200 mg/kg) compared to fungal necromass carbon. This might be because bacteria possess a relatively weak enzyme system, resulting in higher carbon emissions during respiration and lower carbon utilization for biomass synthesis. In contrast, fungi can synthesize a larger amount of biomass due to their higher efficiency in food utilization [58]. The above conclusion suggests that fungal necromass carbon dominates the changes in soil microbial necromass carbon. In addition, soil microbial necromass carbon contents were significantly higher under drought conditions compared to normal moisture conditions, which is consistent with the findings reported by Liu et al. [59]. The increase in microbial and fungal necromass carbon in arid soil can be attributed to the extensive aggregation of microorganisms near soil minerals as a survival strategy to avoid dehydration and nutrient limitation [59].

The soil microbial necromass carbon content of FB and HB co-application with green manure was lower than that of BC co-application with green manure under normal moisture conditions. This is probably because FB and HB enhanced microbial catabolic activity (e.g., amino acid and carbohydrate metabolism) by promoting microbial colonization, providing nutrients and mediating the process of electron transfer between microorganisms [60]. These processes accelerated the recirculation of necromass carbon and the chemical condensation reaction during humus synthesis, thereby reducing the soil microbial necromass carbon content. The ratio of soil microbial necromass carbon to total humus acid carbon was lower for FB and HB co-application with green manure than for BC under both drought and normal moisture conditions (Figure 8b), further illustrating the conversion of microbial necromass to humus acids. Additionally, the proportion of soil carbon components within the total carbon pool indicates that FB and HB co-application with green manure significantly increased the total humus acid content, with a more pronounced effect under normal moisture conditions (Figure 8c). Consequently, modified biochar co-application with green manure could be an effective strategy for mitigating soil carbon loss, primarily through the preservation of soil humusacid.

4. Conclusions

Given the increasingly frequent extreme drought events driven by global climate change, elucidating how modified biochar regulates carbon and nitrogen turnover during green manure decomposition is crucial for advancing sustainable agricultural development. In this study, we systematically investigated the mechanisms through which pristine biochar (BC), Fe-modified biochar (FB) and H₂O₂-modified biochar (HB) co-application with green manure affect the turnover of soil carbon and nitrogen under drought and normal moisture conditions. Our results identified that iron oxide on the FB surface can form organo-mineral complexes with biochar microbial necromass; the high specific surface area and abundant microporous structure enables the effective adsorption of soil organic matter, thereby significantly improving the efficiency of soil carbon and nitrogen retention. Once there is enough water in the soil, the active sites on the surface of the modified biochar can subsequently promote the humification process of microbial-derived carbon and nitrogen, forming more stable humus complexes.

However, in the absence of moisture, organic compounds retained on the surface of modified biochar cannot be further transformed into a more stable form of organic matter, such as humus. Therefore, the long-term stability of these compounds still remains unclear and needs further evaluation. In summary, optimizing carbon and nitrogen retention efficiency during green manure decomposition under drought conditions through precisely regulating biochar surface properties and implementing appropriate water management will be a critical strategy for agricultural carbon and nitrogen management in arid regions.