Molecular determinants of humic and fulvic acids in enhancing micronutrient availability in calcareous soils: a comparative study on Fe, Mn, Zn, and Cu dynamics

Micronutrient deficiencies in calcareous soils, exacerbated by high pH and low organic matter, necessitate sustainable solutions. This study investigated the efficacy of humic (HA) and fulvic acids (FA) in enhancing the availability of Fe, Mn, Zn, and Cu across six calcareous soils, focusing on their molecular properties, application rates, and soil interactions. Purified commercial HA and FA samples were characterized via elemental analysis, FTIR, NMR, and functional group quantification. Incubation experiments assessed DTPA-extractable micronutrients after applying low (12.5 mg/kg) and high (1250 mg/kg) humic substances (HS) rates. Results revealed that conventional application rates of HA and FA had negligible effects (≤ 4–10%). Crucially, high FA application significantly increased Mn availability (up to 3061% in soil 6), with structural analyses establishing a novel mechanistic link: efficacy was driven by hydrophobic lignin-derived domains (alkyl/O-alkyl ratio > 1.1) rather than acidic functional group abundance—a paradigm shift in humic–metal interaction understanding. HA treatments reduced Cu availability (up to 35.5%), likely due to stable Cu-HA complexes. Zinc and Fe availability remained largely unaffected. Practically, these findings demonstrate FA’s unique potential as a targeted Mn-mobilizing agent in Mn-deficient calcareous soils when applied at elevated rates, particularly for sandy, carbonate-rich systems. Soil-specific responses underscored the role of mineralogy, advocating precision-humic management based on structural architecture and soil context.