Nanobiochar and Copper Oxide Nanoparticles Mixture Synergistically Increases Soil Nutrient Availability and Improves Wheat Production

**Background:** Nanomaterials have attracted attention in agriculture as nanofertilizers due to their small size, high surface area, and charged surfaces, which can improve nutrient delivery and reduce environmental losses. However, metallic nanoparticles such as copper oxide (nanoCu) at concentrations ≥100 mg kg⁻¹ soil have been shown to be toxic to soil organisms, reduce microbial biomass, and impair plant growth. Nanobiochar (nanoB), an organic nanomaterial produced from pyrolyzed organic matter, may mitigate this toxicity while retaining the beneficial properties of nanomaterials. This study aimed to synthesize nanoB from goat manure and investigate the effects of nanoCu, nanoB, and their mixture on soil microbial biomass, nutrient availability, and wheat productivity.

**Methods:** NanoB was produced by pyrolyzing goat manure at 500°C for 5 hours, followed by ball milling for 24 hours at 300 rpm. Characterization was performed using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Commercial nanoCu (<50 nm, 23 m² g⁻¹) was purchased from Sigma-Aldrich. A pot experiment was conducted with four treatments: control (no fertilization), nanoCu (1000 mg kg⁻¹ soil), nanoB (1000 mg kg⁻¹ soil), and nanoB+nanoCu mixture (each at 1000 mg kg⁻¹ soil), each replicated three times. Wheat was grown from November 2020 to April 2021 in 13 kg pots filled with clayey loam soil (pH not reported, EC 26.7 µS cm⁻¹). Soil samples were analyzed for pH, EC, dissolved organic carbon (DOC), microbial biomass C and N (fumigation-extraction), mineral N (2M KCl extraction), plant-available P and K, and total Cu. Wheat growth parameters and N and Cu uptake were measured at harvest. Nonparametric Kruskal-Wallis ANOVA with Bonferroni correction was used for statistical analysis.

**Key Results:** XRD confirmed nanoB synthesis with a mean crystal size of 20.3 ± 3.4 nm and a distinct carbon peak at 2θ = 42.9°. FTIR revealed functional groups including hydroxyl, carboxyl, carbonyl, amine, nitrile, and sulfonyl groups. SEM showed cubical, pentagonal, needle, and spherical shapes (<1 µm). Soil pH was unaffected by treatments. EC was 28% higher in nanoB than control (34.0 vs. 26.7 µS cm⁻¹). DOC was 94% higher in nanoB+nanoCu than control (not significant for nanoB alone). Microbial biomass C was 129% higher in nanoB+nanoCu than control, and 97% and 74% higher than nanoCu and nanoB alone, respectively. Microbial biomass N was 57% higher in nanoB than control, and 61% higher in nanoB+nanoCu than nanoB alone (84 vs. 52 mg kg⁻¹). NanoCu alone did not affect any macronutrient. NanoB increased soil mineral N by 28%, plant-available P by 64%, and K by 13% versus control. The nanoB+nanoCu mixture further increased N, P, and K by 18%, 41%, and 38% compared to nanoB alone. Soil Cu was 146% higher in nanoCu than control. Wheat grain yield was 62% higher in nanoB+nanoCu (3483 kg ha⁻¹) than control (2153 kg ha⁻¹). Biological yield was 35% higher in nanoB+nanoCu than control. Grains per spike increased by 28% (36 vs. 33) and 100-grain weight by 28% (5.6 vs. 4.4 g) in the mixture treatment. Wheat N uptake was 80% higher in nanoB+nanoCu than control, and 40% higher than nanoB alone. Wheat Cu uptake was 91% higher in nanoCu than control (23.2 vs. 12.1 g ha⁻¹), and 37% higher in nanoB+nanoCu (31.8 g ha⁻¹) than nanoCu alone.

**Clinical Implications:** This study demonstrates that co-application of nanobiochar with copper oxide nanoparticles can synergistically improve soil microbial activity, nutrient availability, and wheat productivity without the toxicity typically associated with high concentrations of metallic nanoparticles. The clayey loam soil texture likely protected microbes from nanoCu toxicity. The findings support the use of nanoB+nanoCu mixtures as nanofertilizers to address micronutrient deficiencies and improve crop yields, particularly in clayey soils. However, the results are limited to a single soil type, crop, and pot conditions; long-term field studies across diverse soils and crops are needed before practical recommendations can be made.