Nitrogen-Enriched Nanobiochar Enhances Spinach Growth via Improved Nitrogen Retention and Uptake Mechanisms

Abstract

The increasing demand for sustainable agriculture requires innovative strategies to enhance nitrogen use efficiency while minimizing environmental losses associated with conventional fertilizers. This study aimed to develop and compare ammonium chloride- and ammonium nitrate-modified nanobiochar as controlled-release nitrogen carriers and to elucidate their effects on nitrogen retention, soil properties, and physiological nitrogen utilization in spinach (Spinacia oleracea L.). Nitrogen-modified nanobiochar was synthesized using ammonium chloride (NB-AC) and ammonium nitrate (NB-AN) at three nitrogen rates (0.03, 0.06, and 0.12 g N g⁻¹ NB) and applied to soil at 1% (w/w). Soil properties, nutrient dynamics, and plant growth and physiological traits were analyzed after 15 and 30 days. Nitrogen modification significantly improved soil nitrogen retention and nutrient availability compared with unmodified nanobiochar. The highest nitrogen loading treatments (NB-AC3 and NB-AN3) notably improved spinach growth, photosynthetic efficiency, pigment content, nitrogen metabolism enzymatic activities, and accumulation of key metabolites (soluble sugars, flavonoids). Nitrogen-release assessments indicated a pronounced controlled-release with reduced nitrogen leaching and greater retention, particularly under NB-AN3. Overall, this study demonstrates that nitrogen-modified nanobiochar functions as an effective nitrogen carrier that enhances nitrogen utilization and growth. These findings provide mechanistic insights into its potential as a sustainable alternative to conventional nitrogen fertilizers.

1. Introduction

Urbanization and industrialization have substantially contributed to global economic growth; however, they have intensified challenges related to sustainable resource management, soil fertility, and food security [1]. Among the essential plant nutrients, nitrogen plays a vital role in plant growth and productivity, yet its inefficient utilization remains a major problem in terrestrial ecosystems. Although nitrogen fertilizers sustain nearly half of the global food supply, their use efficiency is typically limited to 30–50%, leading to substantial nutrient losses and the need for higher application rates to meet agricultural demands [2,3]. These inefficiencies not only increase production costs but also contribute to environmental problems such as greenhouse gas emissions, nitrate leaching, and eutrophication [4,5]. These challenges are particularly pronounced in agriculture-dependent regions such as Pakistan, where intensive farming practices combined with declining soil fertility pose significant challenges to sustainable crop production. Given that soil stores nearly three times more carbon than the atmosphere, maintaining and enhancing soil organic matter is critical for improving nitrogen retention, carbon sequestration, and climate change mitigation [6]. Consequently, innovative strategies to enhance nitrogen use efficiency while reducing environmental impacts are urgently required.

Biochar, a carbon-rich material produced through the pyrolysis of organic biomass, has emerged as a promising soil amendment due to its porous structure, high surface area, and abundant functional groups, which enable it to enhance soil carbon storage, nutrient retention, and agricultural productivity [7,8,9]. Nanobiochar (NB), a nanoscale derivative of biochar, further amplifies these properties by providing higher cation exchange capacity, improved nutrient adsorption, and better interaction with soil components due to its enriched functional groups [9]. These characteristics make NB a versatile material for pollutant adsorption, heavy metal sequestration, and improving soil health and nutrient management [10,11]. Previous studies have shown that NB soil application can reduce nutrient losses, enhance water retention, and improve nutrient bioavailability, thereby addressing challenges such as nutrient leaching and low fertilizer efficiency [12,13]. In addition, NB has been reported to enhance crop performance significantly, increasing dry matter yield by 32% and improving soil microbial biomass and nutrient availability [14]. Despite these advantages, the potential of NB as a carrier for slow-release nitrogen fertilizers remains comparatively underexplored. Nitrogen-enriched nanobiochar, therefore, represents a promising strategy to mitigate nitrogen losses through leaching, volatilization, and denitrification.

Compared with conventional biochar, which requires high application rates (20–50 tons ha⁻¹), which are often characterized by broader particle-size distribution [15], NB offers greater flexibility as a fertilizer carrier through approaches such as impregnation, pelletizing, and encapsulation. Nanobiochar-based slow-release fertilizers can supply nutrients over extended periods, reduce nutrient losses, minimize root zone toxicity, and enhance crop performance [15,16,17]. Moreover, such formulations provide synergistic benefits, including improved soil structure, enhanced nutrient retention, higher crop yields, and greater carbon sequestration potential [16,17]. Recent advances in nitrogen-modified nanobiochar, achieved through chemical adsorption or physical modification using ammonium salts, have shown encouraging improvements in nitrogen retention and controlled-release behavior [18,19]. However, despite these advances, a systematic comparison of different inorganic nitrogen forms immobilized on nanobiochar under identical modification levels, and their subsequent plant–soil responses remains limited.

In the present study, we address this knowledge gap by directly comparing nanobiochar modified with two chemically distinct nitrogen sources [ammonium chloride (NH₄Cl) and ammonium nitrate (NH₄NO₃)] at equivalent nitrogen loading rates. This approach enables a mechanistic assessment of how nitrogen form influences release kinetics, soil nitrogen availability, and plant physiological responses when delivered via a nanobiochar carrier.

Spinach (Spinacia oleracea L.) was selected as the test crop because it is a fast-growing leafy vegetable of high nutritional and economic importance [20,21]. Agronomically, spinach is characterized by rapid biomass accumulation and a strong dependence on nitrogen availability, which directly influences leaf expansion, chlorophyll synthesis, and yield formation. Its shallow root system and short growth cycle make it particularly sensitive to nitrogen management strategies [22]. Consequently, spinach is widely used as a model crop for evaluating nitrogen fertilizers and controlled-release formulations [23]. In this context, spinach provides an effective system for assessing the performance of nitrogen-modified nanobiochar in regulating nitrogen release, soil nitrogen dynamics, and plant physiological responses.

The specific objectives of this study were to (i) synthesize nitrogen-enriched nanobiochar using ammonium-based compounds at defined loading concentrations, (ii) characterize nitrogen release and retention behavior in soil, and (iii) evaluate the effects of these formulations on spinach growth, nutrient uptake, and physiological performance. By integrating material characterization with soil and plant responses, this study provides a focused and comparative framework for optimizing nanobiochar-based nitrogen fertilizers, contributing to more efficient nitrogen management strategies and sustainable crop production in resource-limited agricultural systems.

2. Materials and Methods

2.1. Synthesis of Nitrogen-Modified Nanobiochar

Nitrogen-modified nanobiochar was synthesized based on the method outlined by Ramesh and Raghavan [7], with slight modifications. For the N-functionalization of NB, 15 g of NB was dispersed in 75 mL of distilled water in a conical flask, followed by the addition of ammonium salts (NH₄Cl and NH₄NO₃) to achieve final nitrogen concentrations of 0.03, 0.06, and 0.12 g N/g NB. The mixture was placed on a shaker (speed 120 rpm) at room temperature and then incubated for 8 h to allow adsorption to take place. Finally, the mixture was filtered, and the residue was dried in an oven at 105 °C for 24 h. The dried material was stored in airtight containers to prevent moisture absorption and ensure stability until use.

2.2. Characterization of Nanobiochar and Nitrogen-Modified Nanobiochar

2.2.1. Surface Morphology and Elemental Mapping

The surface morphology and functional groups of nanobiochar (NB) and nitrogen-modified nanobiochar (NB-AC3 and NB-AN3) were characterized using FTIR and SEM–EDX. Particle size distribution and detailed elemental composition of nanobiochar are provided in the Supplementary Materials (Supplementary Methods S1 and Table S1).

2.2.2. Swelling Ratio and Equilibrium Water Content

The swelling ratio (SR) and equilibrium water content (EWC%) were determined by mixing 2.5 g of unmodified and modified nanobiochar with 25 mL of deionized water, followed by shaking at 120 rpm for 1 h at room temperature [7]. The mixtures were then incubated for 24 h, and the filtrates were used to determine SR and EWC% using Equations (1) and (2):

$$\mathrm{S}\mathrm{R}={(\mathrm{W}}_{\mathrm{S}}-{\mathrm{W}}_{\mathrm{D}})/{\mathrm{W}}_{\mathrm{D}}$$

(1)

$$\mathrm{E}\mathrm{W}\mathrm{C} (\mathrm{\%})={\displaystyle \frac{{\mathrm{W}}_{\mathrm{S}}-{\mathrm{W}}_{\mathrm{D}}}{{\mathrm{W}}_{\mathrm{S}}}}\times 100$$

(2)

where W_S is the swollen weight, and W_D is the dry weight of the sample.

2.2.3. Nitrate, Ammonium, and Total Nitrogen Content

For nitrogen analyses, samples consisted of unmodified and nitrogen-modified nanobiochar obtained after the synthesis process. The materials were oven-dried at 105 °C for 24 h, gently ground using a mortar and pestle, and passed through a 0.25 mm sieve to ensure homogeneity. The prepared nanobiochar powders were stored in airtight containers before chemical analyses.

i.

Determination of nitrate content

Nitrate content was analyzed using the salicylic acid colorimetric method [24]. Briefly, 200 mg of the nanobiochar sample was dispersed in 50 mL of distilled water. Then, 1 mL of salicylic acid reagent (prepared in 5% sulfuric acid) was added and incubated for 20 min at room temperature. After that, 20 mL of 2 N sodium hydroxide (NaOH) was added, and absorbance was recorded at 410 nm using a UV–Vis spectrophotometer. Nitrate concentration was quantified using a calibration curve prepared with potassium nitrate standards (1–100 mg L⁻¹).

ii.

Determination of ammonium content

Ammonium content was determined using the indophenol blue method [25]. For this, 0.2 g NB sample was extracted in 50 mL of 2% potassium chloride (KCl) by shaking at room temperature. The extract was reacted with 1 mL phenol solution and 1 mL sodium hypochlorite and incubated at 37 °C for 20 min. Absorbance was measured at 640 nm. The ammonium concentration was calculated from a standard curve prepared with ammonium sulfate solutions (1–100 mg L⁻¹).

iii.

Determination of total nitrogen

The total nitrogen content of unmodified and nitrogen-modified nanobiochar samples was measured using the Kjeldahl digestion method [26]. A 0.5 g sample was digested in 5 mL of concentrated sulfuric acid (H₂SO₄) with a catalyst mixture of potassium sulfate and copper sulfate in a digestion tube. The mixture was heated at 420 °C for 3 h. After cooling, the digest was neutralized with 50% sodium hydroxide, followed by steam distillation. The released ammonia was collected in 4% boric acid containing methyl red indicator and titrated with 0.01 N hydrochloric acid (HCl) to determine total nitrogen content.

2.2.4. Nitrogen Slow-Release Efficiency

Nitrogen slow-release efficiency of unmodified (NB) and six nitrogen-modified nanobiochar treatments was evaluated in aqueous media by monitoring ammonium (NH₄⁺) and nitrate (NO₃⁻) release over time (3, 6, 9, and 12 h). The corresponding nitrogen salts, ammonium chloride (NH₄Cl) and ammonium nitrate (NH₄NO₃), applied at nitrogen concentrations equivalent to those in NB-AC and NB-AN treatments, respectively, were included for comparison. Briefly, 1 g of NB, modified nanobiochar, or nitrogen source was mixed with 100 mL of distilled water and incubated. Aliquots (20 mL) were collected after 3, 6, 9, and 12 h. Ammonium, nitrate, and total nitrogen contents were analyzed using standard analytical procedures, including colorimetric assays for NH₄⁺ and NO₃⁻ and Kjeldahl digestion for total nitrogen [27]. Nitrogen slow-release efficiency was calculated as the percentage reduction in nitrogen release over time relative to the corresponding mineral nitrogen source, allowing for an assessment of the controlled-release behavior of nitrogen-modified nanobiochar.

2.3. Experimental Setup and Treatments

A pot experiment was conducted in a glasshouse to evaluate the effects of nanobiochar and nitrogen-modified nanobiochar on spinach (Spinacia oleracea L., cv. Desi-Palak). Seeds were procured from the Ayub Agriculture Research Institute, Faisalabad, Pakistan. Ten seeds were sown per pot filled with 1 kg of loamy soil. The physicochemical properties of the loamy soil used in the experiment were: EC 1.6 dS m⁻¹, pH 6.8, 3.3% organic matter, 30 mg kg⁻¹ available P, 70 mg kg⁻¹ extractable K, 2.0 g kg⁻¹ total N, 1.3 g kg⁻¹ available N, 600 mg kg⁻¹ N-NH₄⁺, and 120 mg kg⁻¹ N-NO₃⁻.

The experiment was arranged in a completely randomized design. Soil treatments were applied before sowing and consisted of the following: control without nanobiochar (no-NB), unmodified nanobiochar (NB), nanobiochar modified with ammonium chloride at three nitrogen loading rates, 0.03, 0.06, and 0.12 g N g⁻¹ nanobiochar (NB-AC1, NB-AC2, and NB-AC3), and nanobiochar modified with ammonium nitrate at the same nitrogen loading rates (NB-AN1, NB-AN2, and NB-AN3). All nanobiochar treatments were applied at a rate of 1% (w/w).

2.4. Soil Physicochemical Characteristics

Soil samples were collected from the pots after 30 days of crop growth. The samples were analyzed for pH, EC, organic matter, available phosphorus, extractable potassium, saturation percentage, total nitrogen, ammonium, and nitrate content. The characterization methods are described below.

2.4.1. Electrical Conductivity and pH

The EC and pH of soil samples were determined using a 1:2.5 soil-to-water suspension mixture. A 10 g soil sample was mixed with 25 mL of distilled water and stirred occasionally for 30 min to allow equilibrium. Measurements were performed at room temperature using an AD8000 pH/mV/EC meter (Adwa Instruments Inc., Szeged, Hungary).

2.4.2. Organic Matter

Organic matter content was analyzed using the Walkley and Black method [28]. Soil sample (1 g) was mixed with 10 mL of 1 N potassium dichromate (K₂Cr₂O₇) and 20 mL of concentrated sulfuric acid (H₂SO₄). The mixture was heated and titrated with 0.5 N ferrous sulfate (FeSO₄) until the endpoint, indicated by a color change. Organic carbon content was calculated and multiplied by 1.72 to obtain the organic matter content.

2.4.3. Primary Macronutrient Analyses from Soil

i.

Available phosphorus

The availability of phosphorus in soil was determined using the Bray and Kurtz [29] method. Briefly, a 10 g air-dried soil sample was extracted with 100 mL of Bray-1 extraction solution (0.03 N HCl + 0.025 N NH₄F) at a 1:10 soil-to-solution ratio by shaking for 30 min on a shaker. The phosphorus concentration in the extract was measured using a UV–visible spectrophotometer (Shimadzu Corporation, Kyoto, Japan) at 880 nm.

ii.

Available potassium

Extractable potassium in soil was measured following the method of Hanway and Heidel (1952) as described in the Soil Science Society of America method [30]. A 10 g soil sample was extracted with 100 mL of 1 N ammonium acetate (pH 7.0) at a 1:10 soil-to-solution ratio and filtered through Whatman filter paper. Potassium concentration was measured using a flame photometer (Jenway Ltd., Stone, UK).

iii.

Total nitrogen

Total nitrogen content was determined using the Kjeldahl digestion method [31] (Kjeldahl, 1883). A 0.5 g soil sample was digested with 10 mL concentrated H₂SO₄ and a catalyst mixture (potassium sulfate and copper sulfate) using a Kjeldahl digestion unit (Gerhardt Analytical Systems, Königswinter, Germany). After digestion, the samples were neutralized with 50% NaOH and subjected to steam distillation (Gerhardt Analytical Systems, Königswinter, Germany). The released ammonia was collected in 4% boric acid containing a mixed indicator and titrated with 0.1 N HCl. The total nitrogen content was calculated based on the HCl volume used.

iv.

Ammonium and nitrate contents

Soil ammonium and nitrate contents were extracted using a 2 M potassium chloride (KCl) solution. A 10 g soil sample was shaken with 100 mL of KCl solution and filtered. Ammonium content was determined spectrophotometrically at 425 nm using Nessler’s reagent with a UV–visible spectrophotometer (Shimadzu Corporation, Kyoto, Japan) following Bremner [27]. Nitrate content was determined after reduction with cadmium and subsequent reaction with sulfanilamide and N-1-naphthyl ethylenediamine dihydrochloride, and absorbance was recorded using a UV–visible spectrophotometer (Shimadzu Corporation, Kyoto, Japan) following Nelson and Sommers [32].

2.4.4. Soil Saturation Percentage and Water Retention

The saturation percentage and water retention were determined using the method described by Richards [33]. To measure the saturation percentage, a 50 g soil sample was placed in a container and saturated with distilled water until the surface appeared visibly wet. Excess water was allowed to drain for 24 h. The volume of retained water was calculated as a percentage of the total soil volume. The water retention (WR) of soil was calculated as a percentage relative to the control (no-NB), which was set to 100% in the formula. The results represent the comparative water retention capabilities of NB, NB-AC3, and NB-AN3 treatments over 12 days. Each treatment comprised 30 g of soil mixed with 12 mL of distilled water, and 0.3 g of the respective amendment was added to the treated samples. For the control (no-NB), 30 g of soil was mixed with 12 mL of distilled water without any amendment. The NB treatment consisted of 30 g of soil mixed with 0.3 g of nanobiochar and 12 mL of distilled water. Similarly, for modified nanobiochar treatments, 30 g of soil was combined with 0.3 g of NB-AC3 and NB-AN3, along with 12 mL of distilled water. Each mixture was prepared in labeled cups, and the initial weight of each cup was recorded after the additions. The prepared samples were placed in a container to protect them from external environmental factors such as airflow or excessive evaporation. For 12 days, the cups were weighed daily using a precision balance to monitor weight changes caused by water loss or retention. Water retention was calculated using Formula (3):

$$\mathrm{W}\mathrm{R}\left(\%\right)={\displaystyle \frac{{\mathrm{W}}_{\mathrm{X}}}{{\mathrm{W}}_{\mathrm{C}}}}\times 100$$

(3)

where W_C represents the final weight of the control cup (no-NB), and Wx denotes the final weight of the treatment cups (for NB, NB-AC3, and NB-AN3).

2.5. Growth, Physiological, and Ion Attributes of Spinach

2.5.1. Growth Characteristics

The growth parameters of spinach were recorded at 15 and 30 days after sowing. Briefly, plant height, dry and fresh weight of shoot and root, shoot leaf weight ratio (LWR), specific leaf area (SLA), leaf area index (LAI), total leaf area (LA), and leaf area ratio (LAR) were recorded as described earlier [34]. The dry weights were recorded after oven-drying the leaves at 70 °C to a constant weight. Data were collected from three replicates per treatment, and standard formulas were applied:

$$\mathrm{L}\mathrm{W}\mathrm{R} (\mathrm{m}\mathrm{g} {\mathrm{m}\mathrm{g}}^{-1})={\displaystyle \frac{\mathrm{D}\mathrm{r}\mathrm{y} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{l}\mathrm{e}\mathrm{a}\mathrm{v}\mathrm{e}\mathrm{s}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{d}\mathrm{r}\mathrm{y} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}}}$$

(4)

$$\mathrm{S}\mathrm{L}\mathrm{A} ({\mathrm{c}\mathrm{m}}^2{\mathrm{m}\mathrm{g}}^{-1})={\displaystyle \frac{\mathrm{L}\mathrm{e}\mathrm{a}\mathrm{f} \mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}}{\mathrm{L}\mathrm{e}\mathrm{a}\mathrm{f} \mathrm{d}\mathrm{r}\mathrm{y} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}}$$

(5)

Individual leaf areas were determined using graph paper by counting the squares covered by each leaf, and LA per plant was calculated by summing the areas of all leaves.

$$\mathrm{L}\mathrm{A}\mathrm{I}={\displaystyle \frac{\mathrm{L}\mathrm{e}\mathrm{a}\mathrm{f} \mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}}{\mathrm{G}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d} \mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}}}$$

(6)

$$\mathrm{L}\mathrm{A}\mathrm{R} ({\mathrm{c}\mathrm{m}}^2{\mathrm{m}\mathrm{g}}^{-1})={\displaystyle \frac{\mathrm{L}\mathrm{e}\mathrm{a}\mathrm{f} \mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}}{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t} \mathrm{d}\mathrm{r}\mathrm{y} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}}$$

(7)

2.5.2. Allometric Analyses

Growth indicators of spinach plants were measured 15 and 30 days after sowing to assess growth dynamics under different treatments. After that, relative growth rate (RGR), absolute growth rate (AGR), crop growth rate (CGR), leaf area duration (LAD), and NAR (net assimilation rate) were calculated as follows:

$$\mathrm{R}\mathrm{G}\mathrm{R} ({\mathrm{d}}^{-1})={\displaystyle \frac{{\mathrm{l}\mathrm{o}\mathrm{g}}_{\mathrm{e}} {\mathrm{W}}_2-{\mathrm{l}\mathrm{o}\mathrm{g}}_{\mathrm{e}} {\mathrm{W}}_1}{{\mathrm{t}}_2-{\mathrm{t}}_1}}$$

(8)

$$\mathrm{A}\mathrm{G}\mathrm{R} \left(\mathrm{m}\mathrm{g} {\mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}}^{-1} {\mathrm{d}}^{-1}\right)={\displaystyle \frac{{\mathrm{W}}_2-{\mathrm{W}}_1}{{\mathrm{t}}_2-{\mathrm{t}}_1}}$$

(9)

$$\mathrm{C}\mathrm{G}\mathrm{R} \left({\mathrm{m}\mathrm{g} \mathrm{c}\mathrm{m}}^{-2} {\mathrm{d}}^{-1}\right)={\displaystyle \frac{{\mathrm{W}}_2-{\mathrm{W}}_1}{\rho \left({\mathrm{t}}_2-{\mathrm{t}}_1\right)}}$$

(10)

where W₁ and W₂ are the whole plant dry weight at time t₁ and t₂, respectively, and $\rho$ is the ground area (cm²) corresponding to the pot surface area.

$$\mathrm{N}\mathrm{A}\mathrm{R} \left(\mathrm{m}\mathrm{g} {\mathrm{c}\mathrm{m}}^{-2} {\mathrm{d}}^{-1}\right)={\displaystyle \frac{{\mathrm{W}}_2-{\mathrm{W}}_1}{{\mathrm{t}}_2-{\mathrm{t}}_1}}\times {\displaystyle \frac{{\log }_{\mathrm{e}} {\mathrm{L}}_2-{\log }_{\mathrm{e}} {\mathrm{L}}_1}{{\mathrm{L}}_2-{\mathrm{L}}_1}}$$

(11)

$$\mathrm{L}\mathrm{A}\mathrm{D} ({\mathrm{c}\mathrm{m}}^2 \mathrm{d})={\displaystyle \frac{{\mathrm{L}}_1+{\mathrm{L}}_2}{2}}\times ({\mathrm{t}}_2-{\mathrm{t}}_1)$$

(12)

where W₁ and W₂ are the dry weights of the whole plant, and L₁ and L₂ are the leaf areas at t₁ and t₂, respectively. t₂ − t₁ is the time interval in days

2.5.3. Photosynthetic Activity

Gas exchange parameters were measured using a portable gas exchange system (LCi-T, ADC BioScientific Ltd., Hertfordshire, UK). Fully expanded leaves were selected and enclosed in the leaf cuvette. The system was calibrated according to the manufacturer’s instructions. Photosynthetic rate (µmol CO₂ m⁻² s⁻¹), transpiration rate (mmol H₂O m⁻² s⁻¹), stomatal conductance (mol H₂O m⁻² s⁻¹), and intercellular CO₂ concentration (µmol CO₂ mol⁻¹) were measured on a sunny day from 10 am to 1 pm.

2.5.4. Photosynthetic Pigments

Fresh leaf samples (0.5 g) were homogenized in 80% acetone, and a 10 mL final volume was maintained. The leaf extracts were incubated overnight at −20 °C, and absorbance was recorded at 663, 645, and 480 nm [35,36]. The following formulas were used to calculate photosynthetic pigments:

$$\mathrm{C}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l} \mathrm{a}(\mathrm{m}\mathrm{g} {\mathrm{g}}^{-1} \mathrm{F}\mathrm{W})=\left[12.7\left({\mathrm{O}\mathrm{D}}_{663}\right)-2.69\left({\mathrm{O}\mathrm{D}}_{645}\right)\right]\times {\displaystyle \frac{\mathrm{V}}{1000}}\times \mathrm{W}$$

(13)

$$\mathrm{C}\mathrm{h}\mathrm{l}\mathrm{o}\mathrm{r}\mathrm{o}\mathrm{p}\mathrm{h}\mathrm{y}\mathrm{l}\mathrm{l} \mathrm{b} (\mathrm{m}\mathrm{g} {\mathrm{g}}^{-1} \mathrm{F}\mathrm{W})=\left[22.9\left({\mathrm{O}\mathrm{D}}_{645}\right)-4.68\left({\mathrm{O}\mathrm{D}}_{663}\right)\right]\times {\displaystyle \frac{\mathrm{V}}{1000}}\times \mathrm{W}$$

(14)

$$\mathrm{C}\mathrm{a}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{o}\mathrm{i}\mathrm{d}\mathrm{s} (\mathrm{m}\mathrm{g} {\mathrm{g}}^{-1} \mathrm{F}\mathrm{W})={\mathrm{O}\mathrm{D}}_{480}+0.114\left({\mathrm{O}\mathrm{D}}_{663}\right)-0.638\left({\mathrm{O}\mathrm{D}}_{645}\right)\times {\displaystyle \frac{100}{2500}}$$

(15)

Here, V is the extract volume (mL), and W is the leaf weight (g).

2.5.5. Total Free Amino Acids and Total Soluble Sugars

Fresh leaf sample (1 g) was extracted with 10 mL potassium phosphate buffer (100 mM; pH 7) after grinding, centrifugation, and filtration. The samples were frozen at −20 °C for the subsequent determination of metabolites. For total soluble proteins (mg g⁻¹ FW), leaf extracts were reacted with Na₂CO₃, K₂C₄H₄O₆, and CuSO₄ solutions, followed by Folin–Ciocalteu reagent. The mixture was incubated for 30 min at room temperature, and absorbance was measured at 620 nm [37].

For total free amino acids (mg g⁻¹ FW), leaf extracts were reacted with ninhydrin and pyridine solutions and heated in a water bath for 30 min. The color change was measured at 570 nm using a spectrophotometer. Leucine was used as a standard [38].

For total soluble sugars (mg g⁻¹ FW), leaf extracts were treated with anthrone solution and heated at 90 °C for 10 min. The green color intensity was measured at 620 nm using a spectrophotometer [39]. A glucose standard curve was used for quantification. For total flavonoid content (mg quercetin g⁻¹ FW), extracts were reacted with 2% aluminum chloride and 1 M sodium acetate solutions. The absorbance of the resulting pink color was measured at 510 nm [40].

2.5.6. Nitrogen Metabolizing Enzymes

i.

Nitrite reductase activity (NiR)

NiR activity was measured following the colorimetric method described by Jaworski [41]. Plant extracts (1 mL) were incubated with 0.5 mL of 0.2 M sodium nitrite (NaNO₂) in aluminum foil-wrapped test tubes to prevent light. The reaction mixture was incubated in a water bath at 30 °C for 30 min with gentle shaking. The reaction was stopped by placing the test tubes in boiling water for 2 min and immediately cooling them on ice. Subsequently, sulfanilamide solution (0.5 mL, 1% in 2 N HCl) and 0.5 mL of 0.02% N-(1-naphthyl) ethylenediamine dichloride were added to each tube. After a 20 min incubation at room temperature, the final volume was adjusted to 10 mL with distilled water. Absorbance was measured at 540 nm using a UV–visible spectrophotometer. NiR activity was calculated using a NaNO₂ standard curve and expressed as µmol min⁻¹ g⁻¹ FW.

ii.

Nitrate reductase activity (NR)

NR activity was determined according to the Hageman and Reed [42] method with minor modifications, including the use of potassium nitrate (KNO₃) as the substrate, controlled incubation temperature, and colorimetric detection of nitrite produced during the reaction. Briefly, 1 mL of plant extract was incubated with 0.5 mL of 0.2 M KNO₃ in aluminum foil-wrapped test tubes in a water bath at 30 °C for 30 min with gentle shaking. The reaction was terminated by boiling the test tubes, followed by rapid cooling on ice. Nitrite produced during the reaction was quantified by adding 0.5 mL of 1% sulfanilamide in 2 N HCl and 0.5 mL of 0.02% N-(1-naphthyl) ethylenediamine dichloride. After 20 min of incubation at room temperature, the volume was adjusted to 10 mL with distilled water, and absorbance was recorded at 540 nm. NR activity was calculated using a KNO₃-derived standard curve and expressed as µmol min⁻¹ g⁻¹ FW.

2.5.7. Nutrient Analysis of Leaf

Leaf nitrogen content was determined using the acid digestion method following Kjeldahl nitrogen estimation [26]. Briefly, 1 g of oven-dried spinach leaves was cut into small pieces and transferred to a digestion flask. Then, 3 mL of sulfuric acid (H₂SO₄) was added, and the samples were allowed to pre-digest overnight at room temperature. The mixture was then heated on a hot plate at 300 °C for approximately 45 min. One mL of 30% hydrogen peroxide (H₂O₂) was added dropwise to each flask to complete oxidation, and digestion continued until the solution became colorless. The digested samples were used for total N determination by the Kjeldahl method. The nitrogen values were recorded and used for further analysis. The experiment consisted of four replications per treatment.

2.6. Statistical Analysis

Statistical analyses were performed using Statistix software (version 10.0.1.5, Analytical Software, Tallahassee, FL, USA). Data were first checked for normality and homogeneity of variances before analysis. For experiments involving multiple treatments measured at a single time point (e.g., soil properties, growth parameters, physiological traits, biochemical attributes, and gas exchange parameters), data were analyzed using one-way analysis of variance (ANOVA), and treatment means were compared using Tukey’s honestly significant difference (HSD) test at p ≤ 0.05. For experiments involving two independent factors, such as treatments and growth stages (15 and 30 days after sowing) in leaf morphological traits, data were analyzed using two-way ANOVA, and mean separation was performed using Tukey’s HSD test where appropriate.

For the water retention experiment, which involved repeated measurements over time but focused on comparing the average water retention over the 12 days among treatments, mean values were compared using an independent-samples t-test. The use of the t-test for this specific analysis is indicated in the corresponding figure caption.

3. Results

3.1. Effects of Nitrogen Modification on the Structural and Functional Properties of Nanobiochar

3.1.1. Structural Properties

The FTIR spectrum of unmodified nanobiochar (NB) exhibited characteristic peaks typically associated with its functional groups, such as a peak near 2300 cm⁻¹, which is attributed to the adsorption of CO₂ or C≡C stretching vibrations on the surface of nanobiochar. It is a common feature in biochar with a porous structure. Peaks near 2100 cm⁻¹ also indicate C≡C stretching, suggesting the presence of alkyne. A peak at 1990 cm⁻¹ represents C=O stretching, likely originating from carbonyl or carboxyl groups present on the surface of biochar. That at 1430 cm⁻¹ can be attributed to O–H bending in carboxylic acid or C–H bending in alkanes, aromatic, or phenolic structures., while 870 cm⁻¹ corresponds to the aromatic C–H out-of-plane bending, confirming the aromatic nature of biochar (Figure 1a). The FTIR spectrum of ammonium chloride-modified nanobiochar (NB-AC3) and ammonium nitrate-modified nanobiochar (NB-AN3) showed changes in intensities or shifts in peaks compared to the NB spectrum, such as the shifting of a peak from 1416 cm⁻¹ to 1450 cm⁻¹, which can indicate changes in the chemical environment or bonding of the functional groups present in the material, and verifying the interaction between ammonium chloride and the nanobiochar (Figure 1b,c).

The SEM images of unmodified nanobiochar at 3000× resolution showed a compact and irregular surface morphology with rough edges and some porous structures (Figure 2a). The surface mainly appeared unmodified, with visible cracks and an uneven distribution of pores. A slightly higher view at 1000× and 500× confirmed the irregular texture; larger structural features dominated, showing a non-uniform surface without significant particle deposition or coatings. The unmodified nanobiochar demonstrated a relatively intact structure typical of raw biochar, with evidence of inherent porosity and rough texture. The SEM images of ammonium chloride-modified nanobiochar (NB-AC3) displayed increased surface roughness compared to the unmodified nanobiochar at 3000× resolution (Figure 2b). Ammonium chloride treatment appeared to result in finer particles and enhanced surface area, with visible adhesion of small particles. At 1000× resolution, the surface showed a more pronounced aggregation of particles, likely due to the deposition of ammonium chloride. The texture appeared denser and more interconnected. At 500× resolution, larger-scale features showed evidence of material deposition, with the overall structure appearing denser and less porous compared to unmodified nanobiochar.

The SEM images of ammonium nitrate-modified nanobiochar (NB-AN3) at 3000× resolution revealed a highly textured surface with finer particles distributed across the biochar matrix (Figure 2c). The structure appeared more compact, with less exposed porosity than the unmodified version. At 1000× resolution, enhanced particle aggregation was observed, similar to the ammonium chloride-modified version but with finer structural modifications. Some pore fillings and surface deposition of ammonium nitrate were evident. At 500× resolution, larger features indicated significant material deposition, with an overall smoother appearance compared to the unmodified sample.

The EDX spectra of nanobiochar showed no significant changes in structural groups except for an increase in the Cl group in ammonium-chloride-modified nanobiochar (Figure S1). No N peak was evident in any EDX spectra, which might be possible because nitrogen has a relatively weak x-ray emission compared to heavier elements, and its signal can be overshadowed if the sample contains a high amount of other elements like carbon and oxygen in nanobiochar. Therefore, low-resolution limitations and overlapping signals might be the possible cause of no N peak.

3.1.2. Swelling Ratio and Equilibrium Water Content

The results indicate significant improvements in the properties of nanobiochar upon modification with ammonium chloride (NB-AC) and ammonium nitrate (NB-AN) (

Table 1. Swelling ratio, equilibrium water content, ammonium content, nitrate content, and available nitrogen content of nanobiochar and nitrogen-modified nanobiochar.

Treatments	Swelling Ratio	Equilibrium Water Content (%)	N-NH4+ (mg kg−1)	N-NO3− (mg kg−1)	Available N (g kg−1)
NB	0.80 d ± 0.03	47.65 e ± 1.4	4552 de ± 12.6	428 cd ± 10.6	4.98 cd ± 0.11
NB-AC1	1.32 a ± 0.01	56.89 a ± 1.3	5608 cd ± 25.8	608 c ± 16.5	6.22 cd ± 0.13
NB-AC2	1.16 a–c ± 0.01	53.70 cd ± 2.7	6952 c ± 60.1	667 c ± 10.7	7.62 c ± 0.52
NB-AC3	1.24 ab ± 0.05	55.35 ab ± 0.9	30,900 a ± 47.5	104 d ± 5.3	31.00 a ± 0.11
NB-AN1	0.96 cd ± 0.10	48.97 e ± 1.6	2472 e ± 19.3	720 c ± 10.5	3.19 d ± 0.10
NB-AN2	1.08 bc ± 0.03	51.92 d ± 2.6	4040 de ± 39.4	2080 b ± 12.3	6.12 cd ± 0.88
NB-AN3	1.24 ab ± 0.01	55.35 ab ± 2.7	14,400 b ± 50.5	4560 a ± 20.1	18.96 b ± 0.15
ANOVA (F-values)	45.5 ***	74.3 ***	105.1 ***	84.0 ***	37.0 ***

NB: nanobiochar; NB-AC1, NB-AC2, and NB-AC3 indicate nanobiochar modified with 0.03, 0.06, and 0.12 g N g⁻¹ nanobiochar using ammonium chloride; NB-AN1, NB-AN2, and NB-AN3 indicate nanobiochar modified with 0.03, 0.06, and 0.12 g N g⁻¹ nanobiochar using ammonium nitrate, respectively. Values represent means ± standard error (n = 4). Different letters within the same column indicate significant differences among treatments at p ≤ 0.05 according to the Tukey HSD test following one-way ANOVA. F-values are shown for each parameter, and asterisks indicate significance levels (*** p ≤ 0.001).

). The swelling ratio of NB was the lowest (0.80), significantly increasing with ammonium chloride modifications, particularly NB-AC1 (1.32) and NB-AC3 (1.24), showing enhanced water absorption and structural flexibility. Ammonium nitrate modifications also improved the swelling ratio, with NB-AN3 (1.24) performing similarly to NB-AC3. Equilibrium water content followed a similar trend, with NB-AC1 (56.89%) achieving the highest retention, significantly outperforming NB (47.65%). Among the NB-AN treatments, NB-AN3 (55.35%) exhibited comparable water retention to NB-AC3 (55.35%), indicating the beneficial impact of nitrate modification on water-holding capacity.

3.1.3. Nitrate, Ammonium, and Total Nitrogen Content

Ammonium content was significantly higher in modified treatments of NB-AC2, NB-AC3, and NB-AN3 than in NB (Table 1). Ammonium content was minimal for NB-AN1, which was significantly similar to NB and NB-AN1. Nitrate content was significantly increased for the treatments modified with ammonium nitrate, i.e., NB-AN2 and NB-AN3, which showed a higher nitrate content than NB, and nanobiochar modified with ammonium chloride (NB-AC). These findings demonstrate the enhanced ability of ammonium chloride-modified nanobiochar to retain ammonium and ammonium nitrate in retaining ammonium and nitrate species. Available nitrogen content was highest in NB-AC3 (31.0 g kg⁻¹), followed by NB-AN3 (18.96 g kg⁻¹), significantly exceeding NB (4.98 g kg⁻¹) and other treatments. Overall, higher levels of NB-AC3 and NB-AN3 showed higher nitrogen availability.

3.1.4. Nitrogen-Release Efficiency

The release dynamics of NH₄⁺ and nitrate NO₃⁻ from NB, NB-AC, NB-AN, and reference salts were evaluated in aqueous media over 12 h (

Table 2. Ammonium (NH₄⁺) release and nitrate (NO₃⁻) release from unmodified nanobiochar and nitrogen-modified nanobiochar in aqueous media over time (3, 6, 9, and 12 h).

Treatment	NH4+ Release (mg L−1)				NO3− Release (mg L−1)
	3 h	6 h	9 h	12 h	3 h	6 h	9 h	12 h
NB	140 m ± 1.0	140 m ± 3.9	140 m ± 3.1	168 m ± 2.0	672 ij ± 3.2	700 h–j ± 8.4	784 gh ± 1.1	784 gh ± 1.2
NB-AC1	224 lm ± 3.5	224 lm ± 2.2	280 kl ± 2.7	308 j–l ± 6.1	504 k ± 3.5	728 h–j ± 3.6	784 gh ± 2.5	868 g ± 7.0
NB-AC2	308 j–l ± 8.1	280 kl ± 3.1	364 i–k ± 11.4	364 i–k ± 3.4	508 k ± 3.0	980 f ± 2.3	780 gh ± 3.4	980 f ± 3.4
NB-AC3	392 ij ± 2.0	336 i-k ± 12.2	364 i-k ± 3.7	280 kl ± 6.9	712 h–j ± 4.8	648 j ± 3.2	760 hi ± 5.0	760 hi ± 5.3
NB-AN1	336 i–k ± 1.9	308 j–l ± 3.4	420 i ± 4.1	364 i–k ± 2.5	1204 c–e ± 3.7	1120 e ± 4.9	1204 c–e ± 19.2	1148 e ± 7.0
NB-AN2	336 i–k ± 3.2	280 kl ± 2.0	280 kl ± 3.6	336 i–k ± 4.2	1120 e ± 6.3	1120 e ± 1.2	1176 de ± 16.3	1204 c–e ± 4.2
NB-AN3	672 gh ± 3.6	588 h ± 5.2	644 gh ± 6.4	728 g ± 9.4	1204 c–e ± 8.1	1260 cd ± 11.0	1288 c ± 9.0	1288 c ± 6.1
NH4Cl	2688 e ± 10.4	3500 b ± 12.6	3360 c ± 15.7	4620 a ± 4.0	ND	ND	ND	ND
NH4NO3	2090 b ± 12.7	2976.4 d ± 24.5	3290 c ± 26.5	3318 c ± 12.1	1512 b ± 21.4	1204 c–e ± 7.1	1512 b ± 8.1	1568 a ± 11.0
ANOVA (F-values)	84.10 ***				69.20 ***

NB: nanobiochar; NB-AC1, NB-AC2, and NB-AC3 indicate nanobiochar modified with 0.03, 0.06, and 0.12 g N g⁻¹ nanobiochar using ammonium chloride; NB-AN1, NB-AN2, and NB-AN3 indicate nanobiochar modified with 0.03, 0.06, and 0.12 g N g⁻¹ nanobiochar using ammonium nitrate, respectively. Values represent means ± standard error (n = 4). Different letters within the same column indicate significant differences among treatments at p ≤ 0.05 according to the Tukey HSD test following one-way ANOVA. F-values are shown for each parameter, and asterisks indicate significance levels (*** p ≤ 0.001).

). NH₄⁺ release by ammonium chloride was higher than by ammonium nitrate after 3, 6, and 12 h. The highest NH₄⁺ was released by ammonium chloride at 12 h, reaching 4620 mg L⁻¹. NH₄⁺ released by ammonium nitrate was also increased with exposure time, reaching a maximum (3318 mg L⁻¹) at 12 h. Ammonium nitrate also showed nitrate release (1288 mg L⁻¹ at 9 and 12 h), demonstrating its dual nitrogen release capability. NB showed limited NH₄⁺ and NO₃⁻ release, with NH₄⁺ levels starting at 140 mg L⁻¹ at 3 h and increasing to 168 mg L⁻¹ at 12 h. Similarly, NO₃⁻ release from NB was relatively low, stabilizing at 784 mg L⁻¹ after 9 h.

The modified nanobiochar exhibited distinct release profiles depending on the level and type of modification (Table 2). Among the ammonium chloride-modified treatments, NB-AC1 and NB-AC2 showed moderate NH₄⁺ release, peaking at 308 mg L⁻¹ at 12 h for NB-AC1 and 364 mg L⁻¹ at 9 and 12 h for NB-AC2. NB-AC3 demonstrated a controlled release pattern, with NH₄⁺ release peaking at 392 mg L⁻¹ at 3 h but decreasing to 280 mg L⁻¹ at 12 h, indicating improved nitrogen retention. Ammonium nitrate-modified nanobiochars showed higher NH₄⁺ release compared to NB-AC treatments. NB-AN1 and NB-AN2 showed moderate NH₄⁺ release, with NB-AN2 releasing 336 mg L⁻¹ at 12 h, while NB-AN3 released the highest levels among the modified nanobiochars, starting at 672 mg L⁻¹ at 3 h and peaking at 728 mg L⁻¹ at 12 h. Similarly, NB-AN treatments demonstrated significantly higher NO₃⁻ release, with NB-AN3 releasing 1288 mg L⁻¹ by 12 h, closely matching the release profile of ammonium nitrate.

To evaluate the effect of nitrogen amendment on NH₄⁺ and NO₃⁻ release, percent reductions were calculated based on the total release from their respective nitrogen fertilizer sources. For NB-AC, the total release of NH₄⁺ from ammonium chloride was used as a reference (100%), while for NB-AN, the total release of NH₄⁺ and NO₃⁻ from ammonium nitrate served as the baseline. The results demonstrated that both NB-AC and NB-AN treatments significantly reduced NH₄ release compared to their respective fertilizer sources (Figure 3). NB-AC showed an 85–93% reduction in NH₄⁺ release, indicating its strong ability to retain ammonium ions and slow their release over time (Figure 3a). For NB-AN treatment, the reduction in NH₄⁺ release ranged from 67–89% relative to the NH₄⁺ released by NH₄Cl (Figure 3b). This demonstrates the effectiveness of NB-AN in controlling the release of ammonium ions while still maintaining bioavailability. The reduction in NO₃⁻ release by NB-AN treatments was also observed, but to a lesser extent than NH₄⁺. NB-AN exhibited a 14–27% reduction in NO₃⁻ release relative to the nitrate release from ammonium nitrate (Figure 3c). While this reduction was consistently lower than the reduction observed for NH₄⁺ release, it still highlights the role of NB-AN in mitigating nitrate losses, particularly through leaching. The performance of NB-AN3 was particularly noteworthy, as it achieved the highest reductions in both NH₄⁺ and NO₃⁻ release across all treatments. The superior nitrogen retention of NB-AN3 emphasizes its potential to minimize environmental nitrogen losses and improve nitrogen-use efficiency. These findings are critical for reducing nitrogen leaching and volatilization, which are major contributors to environmental pollution.

3.2. Effects of Nanobiochar and Modified Nanobiochar on Soil Properties

3.2.1. Soil Chemical and Nutrient Properties

The results showed significant differences in soil properties across treatments (

Table 3. Physicochemical characteristics of soil after 30 days of spinach growth following the application of nanobiochar and nitrogen-modified nanobiochar.

Treatments	EC (dS/m)	pH	Organic Matter (%)	Available P (mg kg−1)	Extractable K (mg kg−1)	Saturation (%)	Total N (g kg−1)	Available N (g kg−1)	N-NH4+ (mg kg−1)	N-NO3− (mg kg−1)
No-NB	1.80 bc ± 0.04	6.47 a ± 0.36	3.43 a ± 0.26	31.4 c ± 0.86	67.5 a ± 2.22	40.3 d ± 1.42	2.30 b ± 0.03	1.60 cd ± 0.01	1400 d ± 10.1	203.3 c ± 9.38
NB	3.60 a ± 0.21	7.88 a ± 0.12	3.28 a ± 0.17	65.2 b ± 3.71	63.6 a ± 5.40	41.1 b–d ± 0.97	2.10 bc ± 0.08	1.80 c ± 0.03	1700 c ± 11.1	115.3 d ± 4.67
NB-AC1	2.50 ab ± 0.40	6.05 a ± 0.24	3.28 a ± 0.17	31.0 c ± 0.37	69.0 a ± 5.88	40.6 cd ± 0.26	1.90 c ± 0.11	1.60 cd ± 0.02	1500 d ± 38.1	103.3 d ± 18.27
NB-AC2	1.71 bc ± 0.15	7.60 a ± 0.23	3.50 a ± 0.17	99.0 a ± 1.24	77.1 a ± 7.59	42.0 ab ± 0.31	2.41 b ± 0.03	2.20 b ± 0.03	2000 b ± 6.02	204.0 c ± 8.18
NB-AC3	1.00 c ± 0.22	6.55 a ± 0.58	3.43 a ± 0.26	57.7 bc ± 2.60	67.5 a ± 6.06	40.0 d ± 0.70	2.40 b ± 0.08	1.80 c ± 0.02	1400 d ± 26.5	403.3 a ± 18.27
NB-AN1	1.70 c ± 0.27	7.73 a ± 0.67	3.43 a ± 0.06	62.4 b ± 0.47	69.4 a ± 4.93	42.3 a ± 0.51	1.90 c ± 0.09	1.50 d ± 0.01	1100 e ± 55.9	402.0 a ± 15.04
NB-AN2	1.20 c ± 0.11	7.68 a ± 0.70	3.35 a ± 0.08	60.0 b ± 3.16	65.0 a ± 4.90	41.7 a–c ± 0.61	1.90 c ± 0.05	1.80 c ± 0.01	1500 d ± 47.6	302.3 b ± 14.31
NB-AN3	1.61 bc ± 0.31	7.76 a ± 0.57	3.28 a ± 0.17	31.6 c ± 4.97	67.0 a ± 5.19	42.7 a ± 0.50	3.20 a ± 0.06	2.70 a ± 0.01	2400 a ± 35.7	305.3 b ± 6.38
ANOVA (F-values)	11.5 ***	2.29 ns	0.22 ns	74.3 ***	0.55 ns	1.90 ns	35.1 ***	53.2 ***	145.1 ***	83.3 ***

No-NB: no nanobiochar; NB: nanobiochar; NB-AC1, NB-AC2, and NB-AC3 indicate nanobiochar modified with 0.03, 0.06, and 0.12 g N g⁻¹ nanobiochar using ammonium chloride, respectively; NB-AN1, NB-AN2, and NB-AN3 indicate nanobiochar modified with 0.03, 0.06, and 0.12 g N g⁻¹ nanobiochar using ammonium nitrate, respectively. Values represent means ± standard error (n = 3). Data were analyzed using one-way ANOVA. Different lowercase letters within the same column indicate significant differences among treatments at p ≤ 0.05 according to Tukey’s HSD test. F-values are shown for each parameter to indicate overall treatment effects; ns indicates non-significant differences, while asterisks denote significance levels (*** p ≤ 0.001).

). Electrical conductivity (EC) was highest in NB (3.6 dS m⁻¹), while NB-AC2, NB-AC3, and all NB-AN exhibited low EC (significantly similar). The pH ranged from 6.05 to 7.88 among treatments, with no significant differences observed. Organic matter content remained consistent across treatments, ranging from 3.28% to 3.50%. Available phosphorus (P) was significantly higher in NB-AC2 (99 mg kg⁻¹) compared to other treatments, while the lowest values were observed in NB-AC1 (31 mg kg⁻¹) and No-NB (31.4 mg kg⁻¹). Extractable potassium (K) levels showed no significant differences, ranging from 63.6 to 77.1 mg kg⁻¹.

Total nitrogen content was significantly higher in NB-AN3 (3.20 g kg⁻¹) compared to other treatments, while NB-AC1 and NB-AN1 showed the lowest levels (1.9 g kg⁻¹). Available nitrogen followed a similar trend, with the highest levels in NB-AN3 (2.70 g kg⁻¹) and the lowest in NB-AN1 (1.50 g kg⁻¹). Ammonium content was highest in NB-AN3 (2400 mg kg⁻¹), followed by NB-AC2 (2000 mg kg⁻¹), while NB-AN1 had the lowest ammonium content (1100 mg kg⁻¹). Nitrate content was significantly higher in NB-AC3 (400 mg kg⁻¹) and NB-AN1 (400 mg kg⁻¹) compared to the other treatments, with the lowest levels observed in NB (100 mg kg⁻¹). These results highlight the impact of different nanobiochar treatments on soil nutrient availability and chemical properties.

3.2.2. Water Retention and Saturation Percentage

The saturation percentage varied significantly across treatments, with the highest saturation percentage at NB-AN3 significantly similar to NB-AN1, NB-AN2, and NB-AC3. These treatments showed significantly higher saturation percentages than no-NB (Table 3). The water retention (WR) of soil was evaluated for soil samples with no-NB, NB, NB-AC3, and NB-AN3, using no-NB as the control (100%) (Figure 4). The WR for NB significantly increased compared to no-NB, with a mean value of 101.07%, indicating improved retention. Further enhancements were observed with NB-AC3 (mean WR of 101.4%) and NB-AN3 (mean WR of 101.23%), showing the impact of modifications. Statistical analysis revealed a highly significant difference between NB, NB-AC3, and NB-AN3 and no-NB, while the differences between NB, NB-AC3, and NB-AN3 were non-significant. NB-AC3 shows the highest WR among the treatments, followed by NB-AN3 and NB. Variability across days suggests consistent retention benefits for modified nanobiochar treatments compared to the no-NB.

3.3. Effects of Nanobiochar and Modified Nanobiochar on Spinach Growth, Physiology, and Nitrogen Content

3.3.1. Growth and Growth Dynamics

Nanobiochar (NB) and nitrogen-modified NB treatments influenced spinach growth and biomass accumulation at both 15 and 30 days after sowing (DAS), with more substantial treatment effects observed at 30 DAS (Table S1). At 15 DAS, treatments significantly affected shoot length (SL) and shoot fresh weight (SFW), whereas root length (RL) did not differ significantly among treatments, and root fresh weight (RFW) showed a modest but significant treatment effect. The unmodified NB treatment significantly increased SL and SFW compared with no-NB. Among the modified NBs, NB-AN3 and NB-AN2 produced the greatest SL, while differences in RL remained statistically non-significant. RFW was highest under NB-AC2, whereas NB-AN1 recorded the lowest RFW. Shoot fresh weight was numerically reduced under NB-AN1 compared with other treatments, but was not statistically different from the no-NB (control).

At 30 DAS, treatment effects were highly significant for SL, RL, and SFW, while RFW did not differ significantly among treatments. NB-AN3 produced the maximum SL, RL, and SFW, followed by NB-AN2. The respective increase in SL, RL, and SFW was 53%, 35%, and 1.8 times under NB-AN3, compared to no-NB, whereas no-NB consistently recorded the lowest values for SL and SFW. The unmodified NB treatment increased SFW relative to no-NB but did not significantly affect SL or RL at this stage. Although numerical differences in RFW were observed among treatments, these differences were statistically non-significant.

NB application significantly improved the growth dynamics traits of spinach, as shown in Figure 5. Compared with no-NB (control) and unmodified NB, all nitrogen-modified treatments exhibited enhanced growth efficiency, with ammonium nitrate-modified nanobiochar (NB-AN) consistently outperforming ammonium chloride-modified nanobiochar (NB-AC). Relative growth rate (RGR) increased progressively with nitrogen loading (Figure 5a). Among all treatments, NB-AN3 exhibited the highest RGR value and was the only treatment significantly higher than NB-AC3, whereas NB-AN1 and NB-AN2 remained statistically similar to NB-AC.

Crop growth rate (CGR) demonstrated a marked increase under NB-AN2 and NB-AN3 treatments compared to the NB, no-NB, and NB-AC treatments (Figure 5b). NB-AC1 and NB-AC2 showed less CGR than no-NB, and NB-AC3 showed statistically equal CGR to no-NB. Leaf area duration (LAD) increased progressively with nitrogen loading in both NB-AC and NB-AN treatments (Figure 5c). Among NB-AC, only NB-AC3 showed significant improvement compared to no-NB, while it was significantly similar to NB. Only NB-AN3 showed higher LAD compared to all other treatments, including NB and no-NB.

For the absolute growth rate (AGR), NB-AN treatments showed a consistent advantage over no-NB and NB (Figure 5d). At the same time, all NB-AC treatments showed no significant increase in AGR in comparison to no-NB and NB. NB-AN3 recorded the highest AGR and was significantly superior to both NB-AC2 and NB-AC3, indicating faster whole-plant biomass accumulation. Net assimilation rate (NAR) showed a noticeable increase by nitrogen modification, with NB-AC2, NB-AC3, and all NB-AN treatments showing significantly higher values than no-NB and NB (Figure 5e). The highest NAR values were observed under NB-AN2 and NB-AN3, which were significantly similar to each other but greater than the NB-AC treatments, indicating improved photosynthetic efficiency per unit leaf area.

Overall, NB-AN3 significantly outperformed all treatments, exhibiting 2.3-fold higher RGR, 1.28-fold higher CGR, 3.2-fold higher NAR, 2-fold higher AGR, and 6-fold higher LAD compared to no-NB. These results underscore the superior performance of ammonium nitrate-modified nanobiochar in enhancing plant growth dynamics, with NB-AN3 demonstrating the most substantial improvements across all parameters.

3.3.2. Leaf Growth and Morphology

Leaf growth and morphological traits of spinach were significantly influenced by NB and nitrogen-modified nanobiochar treatments at both 15 and 30 days after sowing (Figure 6). Leaf weight ratio (LWR) showed apparent treatment effects (Figure 6a). At 15 days, no-NB and NB-AC3 exhibited the lowest LWR and were statistically similar, whereas NB-AC1, NB-AC2, and NB-AN1 recorded significantly higher LWR values, with NB-AN1 reaching the maximum. At 30 days, LWR increased across all treatments; no-NB remained significantly lower, while NB-AC1, NB-AN1, NB-AN2, and NB-AN3 formed a statistically similar high group, indicating enhanced biomass allocation to leaves under nitrogen-modified nanobiochar.

Specific leaf area (SLA) responded modestly at 15 days (Figure 6b). NB-AC2 and NB-AN2 showed significantly higher SLA, while no-NB, NB, NB-AC1, NB-AC3, and NB-AN1 remained statistically similar. At 30 days, treatment effects were more pronounced; NB-AN1, NB-AC3, and NB-AN2 exhibited the highest SLA values, forming a statistically similar group, whereas no-NB and NB recorded the lowest values, reflecting limited leaf expansion without nitrogen modification.

Leaf area index (LAI) increased progressively with nitrogen-modified nanobiochar application (Figure 6c). At 15 days, no-NB showed the lowest LAI, while NB-AN3 recorded the highest value, followed by NB-AN2 and NB-AN1, which were statistically similar. At 30 days, NB-AN3 exhibited a significantly higher LAI than all other treatments, with NB-AN2 and NB-AC3 forming an intermediate group.

Total leaf area (LA) followed trends similar to LAI (Figure 6d). At 15 days, no-NB recorded the smallest leaf area, whereas NB-AN3 produced the highest LA, followed by NB-AN2 and NB-AN1. At 30 days, NB-AN3 again showed the maximum leaf area, significantly exceeding all other treatments, while NB and no-NB remained the lowest.

Leaf area ratio (LAR) increased consistently with increasing nitrogen loading (Figure 6e). At 15 days, NB-AN3 exhibited the highest LAR, followed by NB-AN2 and NB-AN1, which were statistically similar. At 30 days, NB-AN3 maintained the highest LAR, significantly exceeding all other treatments, indicating a greater proportion of photosynthetic surface area relative to total biomass. Overall, NB-AN consistently enhanced leaf expansion, canopy development, and biomass allocation to leaves, with effects becoming more pronounced at 30 days after sowing.

3.3.3. Photosynthetic Pigments

The application of NB showed no effect on photosynthetic pigments compared to the no-NB (Figure 7). However, the modified nanobiochar treatments showed a dose-dependent increase in pigment content compared to those with no-NB. NB-AC and NB-AN treatments demonstrated progressive increases in chlorophyll a, b, and total chlorophyll with increasing nitrogen loading, with NB-AN3 consistently outperforming other treatments. Total chlorophyll was significantly higher under NB-AC3, NBAN1, NBAN2, and NB-AN3 than under no-NB and NB, while NB-AC1 and NB-AC2 were statistically similar to no-NB and NB (Figure 7a). Similarly, chlorophyll a was statistically higher under NB-AC3, NBAN1, NBAN2, and NB-AN3 than under no-NB and NB (Figure 7b).

Chlorophyll b was significantly higher under all modified nanobiochar than under no-NB and NB, with the highest chlorophyll b at NB-AN3 (Figure 7c). Carotenoid content was significantly higher for NB and all modified nanobiochar treatments (except NB-AN2) than for no-NB, while NB-AN3 showed the maximum carotenoid content (Figure 7d). Overall, NB-AN3 showed maximum improvement in pigments in comparison to no-NB. The NB-AN3 treatment demonstrated a 2.6-fold increase in total chlorophyll, 2.2-fold increase in chlorophyll a, 2.6-fold increase in chlorophyll b, and 3.1-fold increase in carotenoids compared to no-NB.

3.3.4. Metabolites

Total soluble sugar (TSS), total soluble protein (TSP), total free amino acid (TFAA), and total flavonoid content (TFC) were determined in spinach leaves in response to NB and modified nanobiochar treatment (Figure 8). NB treatment demonstrated significantly higher content of TSS than no-NB. At the same time, the effect on other metabolites was non-significant (Figure 8a). All levels of NB-AC showed significantly higher TSS than no-NB, and NB-AC2 and NB-AC3 showed higher TSS than NB. NB-AC2, NB-AC3, and all levels of NB-AN showed significantly higher TSP as compared to no-NB and NB (Figure 8b). TFAA was significantly higher under NB-AC1, NB-AN1, and NB-AN3 than under no-NB. In contrast, most of the modified nanobiochar showed significantly similar TFAA to NB (Figure 8c). The TFC of NB and all modified nanobiochar treatments were significantly higher than that of the no-NB (Figure 8d). NB modification with ammonium nitrate resulted in a significant enhancement in all metabolites compared to no-NB and NB. NB-AC2 showed 232% higher TSS compared to no-NB. Similarly, a 37% increase in TSP, 125% increase in TFAA, and 145% increase in TFC were observed at NB-AN3 in comparison to no-NB.

3.3.5. Photosynthetic Activity

The photosynthetic performance of spinach plants was significantly improved with the application of NB and nitrogen-modified nanobiochar (NB-AC and NB-AN) (

Table 4. Photosynthetic gas exchange parameters of spinach leaves at 30 days after sowing, following the application of nanobiochar and nitrogen-modified nanobiochar.

Treatments	Pn (µmol CO2 m−2 s−1)	E (mmol H2O m−2 s−1)	gs (mol H2O m−2 s−1)	Ci (µmol CO2 mol−1)
No-NB	9.99 e ± 0.33	7.33 d ± 0.34	0.17 b ± 0.07	103 f ± 8.50
NB	10.55 e ± 0.41	10.38 c ± 0.51	0.19 b ± 0.08	118 ef ± 3.51
NB-AC1	14.41 d ± 0.06	11.41 c ±0.58	0.23 b ± 0.07	131.33 def ± 5.21
NB-AC2	14.99 d ± 0.01	14.32 b ± 0.33	0.27 ab ± 0.05	146 cde ± 3.60
NB-AC3	15.41 d ± 0.24	14.41 b ± 0.24	0.29 ab ± 0.09	148 cd ± 8.50
NB-AN1	20.92 c ± 0.38	14.92 b ± 0.38	0.30 ab ± 0.06	173 bc ± 3.21
NB-AN2	23.76 b ± 0.44	14.76 b ± 0.61	0.33 ab ± 0.05	186 ab ± 6.11
NB-AN3	25.73 a ± 0.53	17.06 a ± 0.27	0.46 a ± 0.14	209 a ± 5.68
ANOVA (F-value)	284 ***	68.4 ***	3.69 ***	36.6 ***

No-NB: no nanobiochar; NB: nanobiochar; NB-AC1, NB-AC2, and NB-AC3 indicate nanobiochar modified with 0.03, 0.06, and 0.12 g N g⁻¹ nanobiochar using ammonium chloride, respectively; NB-AN1, NB-AN2, and NB-AN3 indicate nanobiochar modified with 0.03, 0.06, and 0.12 g N g⁻¹ nanobiochar using ammonium nitrate, respectively. Values represent means ± standard error (n = 4). Data were analyzed using one-way ANOVA. Different lowercase letters within the same column indicate significant differences among treatments at p ≤ 0.05 according to Tukey’s HSD test. F-values indicate overall treatment effects, and asterisks denote significance levels (*** p ≤ 0.001).

). Compared to no-NB, nitrogen-modified nanobiochar exhibited a marked enhancement in net photosynthesis (Pn). The order of improvement was no-NB < NB < NB-AC < NB-AN, with NB-AN treatments showing the highest increase of 157% compared to no-NB. The transpiration rate (E) followed a similar trend, with nitrogen-modified nanobiochar showing significant increases compared to no-NB and NB alone. NB-AN treatments consistently outperformed NB-AC treatments in enhancing transpiration rates with a 132% increase compared to no-NB.

The stomatal conductance (gs) showed no significant differences among the no-NB, NB, and NB-AC treatments or lower NB-AN levels, except for NB-AN3, which displayed significantly higher values compared to all other treatments and was 170% higher than no-NB (Table 4). Intercellular CO₂ concentration (Ci) increased significantly with all modified nanobiochar treatments compared to no-NB, with NB-AN3 achieving the highest values, with 102% higher than no-NB. These findings suggest that NB-AN and NB-AC improved spinach plant photosynthesis, highlighting their potential as sustainable fertilizers.

3.3.6. Enzyme Activities

Nitrate reductase (NR) activity was significantly higher in nitrogen-modified nanobiochar compared to no-NB and NB (Figure 9a). NB-AC and NB-AN treatments showed a progressive increase in activity with increasing nitrogen rate for modification. NB-AC showed a higher NR than NB-AC. Overall, maximum NR was observed at NB-AN3, which was 52% higher than that of no-NB. Nitrite reductase (NiR) activity was significantly higher in NB, NB-AC, and NB-AN compared to no-NB (Figure 9b). Furthermore, NiR activity showed a progressive increase with an increase in the level of NB-AC. In contrast, the differences in the levels of NB-AN were non-significant.

3.3.7. Nitrogen Content in Leaves

The leaf nitrogen content varied significantly across the different treatments (Figure 10). The control plants (no-NB) exhibited the lowest nitrogen content, averaging around 14.4 g kg⁻¹ DW. Treatments with NB-AC1, NB-AC2, and NB-AC3 showed a progressive increase in nitrogen content, with NB-AC2 and NB-AC3 showing a significant increase of 1.8 and 2-fold higher, respectively, compared to no-NB. Among the ammonium chloride-modified nanobiochars, NB-AC3 reached the highest nitrogen content of approximately 28.8 g kg⁻¹ DW. Among the ammonium nitrate-modified nanobiochars, only NB-AN3 demonstrated a significant increase by 2-fold in nitrogen content compared to the no-NB.

4. Discussion

This study involved the synthesis and application of nitrogen-modified nanobiochar (NB) and evaluated its effects on spinach growth, physiological processes, enzymatic activities, and nutrient release dynamics. Our findings demonstrate that structural modifications in NB could contribute to enhanced nutrient release, which is discussed in detail here.

4.1. Structural Modifications and Nutrient Retention in Nitrogen-Modified Nanobiochar

The FTIR results presented in Figure 1 highlighted significant structural modifications in nanobiochar (NB) following treatment with ammonium chloride (NB-AC3) and ammonium nitrate (NB-AN3). In unmodified NB (Figure 1a), absorption peaks at 2653, 2300, 2100, 1800, and 870 cm⁻¹ indicated carbon-rich, aromatic structures with oxygen-containing functional groups. The peak at 2653 cm⁻¹ suggests the presence of a carboxyl group, which is known to enhance adsorption capacity for metal ions and polar molecules. In this context, carbonyl and hydroxyl groups indicate reactive sites, improving biochar effectiveness in adsorption-based applications [43].

Peaks at 2300 and 2100 cm⁻¹, observed in Figure 1a, likely reflect structural imperfections and unsaturated carbon bonds, which may increase adsorption sites [44]. The peak at 870 cm⁻¹ confirms the aromatic backbone of biochar, contributing to its structural stability and persistence in soil environments [45]. Such porous, aromatic structures are also capable of physically adsorbing gases such as CO₂ through micropore-driven mechanisms, as reported previously [46,47].

In contrast, FTIR spectra of NB-AN3 (Figure 1c) showed the appearance of a new peak at 3195 cm⁻¹, which can be attributed to N-H stretching vibrations, indicating incorporation of nitrogen during modification [48]. Additionally, nitrate-associated peaks around 1350–1450 cm⁻¹ (asymmetric stretching) and 800–850 cm⁻¹ (symmetric stretching) were more pronounced after ammonium nitrate modifications, consistent with NO₃⁻ interactions on the nanobiochar surface [49]. The modification process might lead to the formation of new functional groups, such as the amine group (-NH₂) from the interaction of NH₄⁺ with the nanobiochar surface and nitro group (-N₂O) if nitrate ions react with the nanobiochar surface [50].

A noticeable shift in the peak from 2328 cm⁻¹ in NB to 2359 cm⁻¹ in modified nanobiochar (Figure 1b,c) further indicates alterations in the chemical environment of surface functional groups, likely due to ionic interactions and hydrogen bonding involving NH₄⁺ and NO₃⁻ ions [51]. In nanobiochar, the peak at 1416 cm⁻¹ might initially correspond to C-H bending in aliphatic chains or O-H bending in hydroxyl groups [52]. After modification with ammonium chloride or ammonium nitrate, the shift to 1450 cm⁻¹ could indicate the increased hydrogen bonding due to the presence of ammonium ions or the changes in the electronic environment of C-H or O-H groups due to interactions with NH₄⁺ or NO₃⁻ ion-formation of new functional groups (e.g., amine or nitro groups) that can alter the vibrational frequencies [53,54].

Despite these changes, the SEM images presented in Figure 2 confirmed that the core structure of nanobiochar remained largely intact following modification. Unmodified NB (Figure 2a) displayed a porous, rough surface with irregular morphology typical of raw biochar, whereas NB-AC3 and NB-AN3 (Figure 2b,c) showed increased surface roughness, particle aggregation, and partial pore filling. These morphological changes are consistent with improved adsorption and retention capacity without extensive structural degradation. Importantly, these structural modifications translated into functional improvements. As shown in Table 1, NB-AC and NB-AN treatments exhibited significantly higher swelling ratios and equilibrium water content than unmodified NB, which can be attributed to increased hydrogen bonding between modified functional groups and water molecules [55]. Moreover, the enhanced nitrogen retention and controlled-release behavior observed in Table 2 and Figure 3 directly reflect these surface and structural changes.

Specifically, NB-AC3 reduced NH₄⁺ release by 85–93%, while NB-AN3 reduced NH₄⁺ release by 67–89% and NO₃⁻ release by 14–27% relative to their respective fertilizer sources (Figure 3). These results demonstrate the strong capacity of modified nanobiochar to mitigate nitrogen losses through leaching and volatilization while maintaining nutrient availability. The superior ammonium retention of NB-AC3 likely results from stronger electrostatic interactions between NH₄⁺ and oxygen-containing functional groups, whereas the dual ammonium–nitrate retention capacity of NB-AN3 highlights its versatility for nitrogen management [56].

Overall, the combined FTIR (Figure 1), SEM (Figure 2), swelling behavior (Table 1), and nitrogen release data (Table 2; Figure 3) collectively confirm that ammonium chloride and ammonium nitrate modifications enhance surface functionalization, adsorption capacity, and controlled-release performance of nanobiochar. While these modifications significantly improve nutrient retention, it is also important to note that retention efficiency may decline at high nutrient loading due to surface saturation. Future studies should explore strategies to increase functional group density further and assess long-term stability under field conditions.

4.2. Transforming Soil: Enhanced Fertility and Water Retention with Modified Nanobiochar

The application of nanobiochar treatments resulted in notable improvements in the soil properties, particularly in nutrient dynamics and water retention (Table 3; Figure 4). Nitrogen-modified NB significantly influenced nutrient availability compared with unmodified NB and the no-NB control. For instance, NB-AC2 enhanced phosphorus content (Table 3), indicating its role in improving phosphorus retention and availability in soil [57,58]. In contrast, the low phosphorus levels observed under NB-AC1 and no-NB (Table 3) further support the importance of nanobiochar modifications in enhancing nutrient availability. Nitrogen retention, a key focus of this study, was most prominent in NB-AN3, which exhibited the highest total nitrogen content, available nitrogen, and ammonium content (Table 3). These findings highlight the capacity of ammonium nitrate-modified nanobiochar to retain nitrogen in both ammonium and nitrate forms. The dual retention of these nitrogen species ensures a steady nutrient supply for crops while minimizing environmental losses [57,58]. Similarly, the higher ammonium and nitrate retention observed in NB-AC3 and elevated nitrate levels in NB-AN1 also underscore the versatility of different nitrogen modifications in addressing different nutrient demands and soil conditions.

The swelling ratio and water-retaining capacity of nanobiochar significantly influence soil characteristics, as reflected in the observed variations in saturation percentage and water retention (Table 3). NB-AN3 demonstrated the highest saturation percentage, statistically comparable to NB-AN1, NB-AN2, and NB-AC3, all of which showed significantly higher values than the no-NB (control). These improvements are consistent with the enhanced porosity and surface area of nanobiochar, which facilitate greater water absorption and retention [9,59]. The acidic (NB-AC) and neutral (NB-AN) modifications appear to optimize the swelling capacity of nanobiochar by improving its interaction with soil particles and water molecules.

Soil water retention dynamics further supported these observations. The soil amended with NB, NB-AC3, and NB-AN3 showed significantly higher water retention than no-NB throughout the 12-day evaluation period (Figure 4). The highest mean water retention was recorded for NB-AC3 (101.40%), followed by NB-AN3 (101.23%) and NB (101.07%), indicating that both unmodified and modified nanobiochars improve soil water holding ability. Although differences among NB, NB-AC3, and NB-AN3 were statistically non-significant (Figure 4), the consistently higher values for NB-AC3 suggest that acidic modification may confer a slight advantage in enhancing soil hydrophilicity and water retention [60].

4.3. Nitrogen Retention and Slow-Release Efficiency of Ammonium and Nitrate in Nitrogen-Modified Nanobiochar

On a comparative basis, the NB-AC2, NB-AC3, and NB-AN3 exhibited significantly higher ammonium content than nanobiochar (NB) (Table 1), confirming the effectiveness of nitrogen modification in enhancing NH₄⁺ retention. Likewise, nitrate retention was highest in NB-AN3 (Table 1), indicating the superior ability of ammonium nitrate-modified nanobiochar to retain NO₃⁻ ions. Overall, the higher ammonium retention and higher total nitrogen content observed in NB-AC3 indicate that ammonium chloride is more effective in enhancing nitrogen retention, likely due to stronger interactions of NH₄⁺ with oxygen-containing surface functional groups (e.g., carboxyl or hydroxyl groups) in biochar [61]. In contrast, NB-AN3, while slightly less effective than NB-AC3 in total nitrogen retention, shows a dual capacity to retain both ammonium and nitrate ions (Table 1), making it a versatile option for controlled nutrient release.

The nitrogen retention and slow-release efficiency of ammonium (NH₄⁺) and nitrate (NO₃⁻) in nitrogen-modified nanobiochar are critically important for sustainable agriculture, environmental protection, and enhanced soil health [62,63]. In conventional nitrogen fertilizers, a significant portion of nitrogen is lost through leaching (NO₃⁻), volatilization (NH₃), or denitrification (N₂O). In this context, nanobiochar represents a sustainable option as its high surface area, porosity, and functional groups enhance nitrogen retention in the soil and delay release [8,9].

The nitrogen release experiments conducted in aqueous media indicated that both ammonium chloride- and ammonium nitrate-modified nanobiochar significantly reduced NH₄⁺ release compared with their respective fertilizer source (Figure 3). NB-AC treatments achieved an 85–93% reduction in NH₄⁺ release relative to NH₄Cl, whereas NB-AN treatments reduced NH₄⁺ release by 67–89% compared with NH₄NO₃ (Figure 3a,b). These findings demonstrate the strong capacity of modified nanobiochar to slow ammonium release. As expected, NO₃⁻ retention was more challenging due to its higher mobility [64,65]; nevertheless, NB-AN treatments reduced NO₃⁻ release by 14–27% relative to ammonium nitrate (Figure 3c), highlighting their potential to mitigate nitrate leaching losses.

The controlled-release behavior of NB-AC3 and NB-AN3 (Table 2; Figure 3) can be attributed to enhanced surface functionalization, which improves ion exchange capacity and electrostatic adsorption of NH₄⁺ and NO₃⁻ [56,61]. This gradual nitrogen release ensures sustained nutrient availability, minimizes leaching losses, and ultimately improves fertilizer-use efficiency [65,66].

It should be noted that nitrogen release was initially evaluated under distilled water conditions (Table 2; Figure 3), providing a controlled baseline for comparing the intrinsic release behavior of modified nanobiochar. Although this setup does not fully capture the complexity of soil environments, the consistency between release data and subsequent soil nitrogen availability (Table 3) and plant nitrogen uptake (Figure 10) supports the agronomic relevance of the observed patterns. Future studies under different soil types and field conditions are required to further validate nitrogen dynamics and long-term performance.

4.4. N-Modified Nanobiochar Promotes Spinach Growth and Metabolic Efficiency

The application of N-modified nanobiochar, particularly NB-AC3 and NB-AN3, promoted spinach shoot and root growth more effectively than unmodified NB, as evidenced by the significant increases in shoot length, root length, and shoot fresh weight at 30 DAS (Table S1). This increased biomass accumulation can be attributed to enhanced soil aeration, improved water retention, and greater nutrient availability, which collectively optimize root proliferation and shoot development [14,57,58,65]. These improvements are consistent with the enhanced soil nitrogen availability and water retention observed under NB-AC3 and NB-AN3 treatments (Table 3; Figure 4). Furthermore, nitrogen-modified NB, particularly NB-AC3 and NB-AN3, maximized nitrogen use efficiency (NUE) and nutrient uptake, thereby contributing to increased net primary productivity [9]. Likewise, we previously reported that NB significantly improved spinach biomass under control and saline conditions [34].

Spinach plants treated with nanobiochar also exhibited pronounced increases in leaf structural traits, including leaf weight ratio, specific leaf area, leaf area index, total leaf area, and leaf area ratio, as shown in Figure 6. The substantial increases observed in LAI and SLA (up to 7.3- and 5.3-fold, respectively) reflect nitrogen-driven leaf morphological plasticity, a well-recognized physiological response in leafy vegetables grown under improved nitrogen availability. Nitrogen plays a central role in regulating cell division, cell expansion, and leaf thickness; therefore, enhanced nitrogen supply favors the development of larger, thinner leaves with expanded mesophyll surface area, resulting in disproportionate increases in SLA and canopy coverage [67]. Such amplification of leaf area indices is particularly pronounced during early canopy establishment, where baseline values are low and relative changes are therefore magnified.

The increase in leaf area directly enhanced light interception and canopy photosynthetic capacity, thereby supporting higher photosynthetic activity, as reflected by increased net photosynthesis, transpiration rate, and intercellular CO₂ concentrations under NB-AN3 (Table 4). Improved nitrogen availability facilitates chlorophyll biosynthesis and the formation of photosynthetic proteins, which together enhance carbon assimilation efficiency [67]. Given that crops differ in their preference for NH₄⁺ or NO₃⁻ forms of nitrogen [68], the higher growth performance of spinach under NB-AN3 treatment suggests an efficient utilization of ammonium-dominated nitrogen sources, consistent with previous reports on leafy vegetables [69]. Additionally, the dual retention of NH₄⁺ and NO₃⁻ in NB-AN treatments (Table 1) enhances nutrient availability and provides an agronomic advantage for fast-growing leafy crops.

Improved physiological growth was further reflected in higher relative growth rate, crop growth rate, leaf area duration, absolute growth rate, and net assimilation rate, as illustrated in Figure 5. These responses indicate a coordinated adjustment between canopy structure and physiological function, whereby expanded leaf area and prolonged leaf lifespan increase cumulative carbon gain over time. Among the treatments, NB-AC3 and NB-AN3 provided sustained nitrogen release, as demonstrated by nitrogen release dynamics and reduction percentages (Table 2; Figure 3), ensuring continuous chlorophyll synthesis and efficient carbon assimilation. Increased chlorophyll content in spinach leaves under these treatments directly supported higher photosynthetic rates, leading to enhanced biomass accumulation [12,34]. Nanobiochar also contributed to improved water retention, maintaining stable stomatal conductance and gas exchange (Table 4; Figure 4), which further enhanced photosynthetic efficiency and overall plant metabolism [70].

The enhanced nitrogen availability from nitrogen modification of nanobiochar played a crucial role in increasing total soluble sugars, total soluble proteins, total free amino acids, and total flavonoids in spinach leaves, as illustrated in Figure 8. Nitrogen is a core constituent of enzymes involved in carbon and nitrogen metabolism; thus, sustained nitrogen supply supports active biosynthetic pathways and prevents premature metabolic downregulation. These metabolic changes were driven by the consistent nitrogen supply from NB-AC3 and NB-AN3, which minimized losses through leaching and volatilization, thereby sustaining carbohydrate and protein metabolism [19,59,71]. Enhanced nitrate and nitrite reductase activities under these treatments (Figure 9) further indicate improved nitrogen assimilation capacity.

Consequently, plants treated with NB-AC3 and NB-AN3 exhibited significantly higher leaf nitrogen content, as shown in Figure 10, reflecting efficient nitrogen uptake and assimilation rather than excessive nitrogen accumulation [72]. Overall, the significant percentage increases in growth and leaf structural parameters represent a physiologically coherent cascade, beginning with improved nitrogen retention in soil (Table 1, Table 2 and Table 3), followed by enhanced leaf expansion (Figure 6), increased photosynthetic surface area and activity (Figure 5; Table 4), and ultimately amplified biomass production (Table S1).

5. Conclusions

This study demonstrates that nitrogen modification of nanobiochar with ammonium chloride and ammonium nitrate substantially enhances the functional performance of nanobiochar as a controlled nitrogen carrier. Structural analyses confirmed that these modifications altered surface functional groups and morphology without compromising the core structure of nanobiochar, resulting in improved swelling behavior and water-holding capacity. Nitrogen-modified nanobiochar, particularly at higher loading rates, exhibited markedly enhanced ammonium and nitrate retention and significantly reduced nitrogen release compared with conventional fertilizer sources, indicating strong slow-release potential. These improvements translated into the soil–plant interface, including increased soil nitrogen availability, improved water retention, and favorable nutrient dynamics. In spinach, nitrogen-modified nanobiochar, especially ammonium nitrate-modified nanobiochar, consistently promoted growth, canopy development, photosynthetic capacity, metabolic activity, and nitrogen assimilation. The coordinated increases in growth rates, leaf traits, photosynthetic pigments, enzyme activities, and leaf nitrogen content highlight the physiological relevance of controlled nitrogen delivery via nanobiochar. These findings highlight the potential of nitrogen-modified nanobiochar to improve nitrogen use efficiency and metabolic performance in leafy vegetables. Future studies incorporating field conditions and isonitrogenous mineral fertilizer controls will further refine the relative contributions of nanobiochar carrier effects and nitrogen availability under practical agricultural systems.