Chemical Characterization and Antimicrobial Activity of Pyrolysis Liquids from Walnut Residue

Pyrolysis liquid (PL) derived from biomass pyrolysis exhibits biopesticidal properties and represents a promising value-added product within the sustainable circular economy framework. However, knowledge about the antimicrobial potential of PLs produced from walnut residue at different pyrolysis temperatures remains limited. We investigated the chemical composition and antimicrobial activity of PLs obtained from agricultural walnut residue (Juglans regia L.) against selected plant pathogenic bacteria and fungi. PLs were produced at four temperature ranges: 200-300 °C (W-1), 300-400 °C (W-2), 400-500 °C (W-3), and 500-600 °C (W-4). Chemical characterization was performed using Gas chromatography-mass spectrometry (GC-MS), High-performance liquid chromatography (HPLC), and Inductively coupled plasma optical emission spectrometry (ICP-OES), with determination of total phenolic and flavonoid contents. Pyrolysis temperature significantly influenced the chemical profile and bioactive compound content of the PLs, with W-4 showing the highest total phenolic and flavonoid levels. Heavy metal analysis indicated minimal contamination in all samples. Antibacterial activity was observed in stock solutions, whereas diluted applications showed limited effects. The W-4 fraction showed the strongest antibacterial activity and exhibited MIC values of 12.50 µL/mL against Clavibacter michiganensis subsp. michiganensis, Xanthomonas euvesicatoria, and Pseudomonas syringae pv. syringae, and 25.00 µL/mL against Erwinia amylovora. Antifungal activity differed markedly across temperature ranges, with W-3 and W-4 displaying superior activity against Fusarium oxysporum and Verticillium dahliae, achieving complete mycelial growth inhibition at 5%, compared to 10% for W-2 and 20% for W-1. Positive controls confirmed assay validity (ciprofloxacin for antibacterial assays and cycloheximide for antifungal assays), whereas negative controls showed no inhibitory effect. Overall, higher pyrolysis temperatures, particularly 400-600 °C, enhanced the antimicrobial potential of walnut residue-derived PLs, supporting their possible use as bio-based antifungal agents for sustainable crop protection.