In-situ preparation of a urea-based product with urease-inhibiting properties from industrial integrated humic acid-enhanced urea production: A comparative study on the key role of functional group structures

Ammonia (NH 3 ) volatilization from urea fertilization causes substantial nitrogen (N) loss and environmental pollution risks. Humic acid-enhanced urea (HAU), produced by directly incorporating low-cost humic acid (HA) into molten urea during the production process, has been shown to significantly inhibit urease activity and reduce ammonia volatilization losses. However, the key functional components and underlying mechanisms remain unclear. Here, we demonstrate that HA complex formation with urea (HACU), generated in-situ during integrated production of HAU, serves as an effective urea-based product with urease inhibiting functionality. Specifically, using HA enriched with distinct functional groups (phenol-rich, carboxyl-rich, and aromatic- and oxygen-rich), we show that the resulting HACUs differ markedly in N speciation and molecular architecture, which directly affect their inhibition performance. Carboxyl-rich HA and aromatic- and oxygen-rich HA favor heterocyclic N structures that likely interact with urease thiol groups, whereas phenol-rich HA yields amide-N-dominated complexes that likely inhibit through steric hindrance. The corresponding urea products decrease urease‑mediated urea hydrolysis by up to 80.28% and NH 3 volatilization by 3.88%–19.45%, with performance comparable to the commercial urease inhibitor NBPT (which remains a genuine inhibitor). Random forest analysis identifies N content and speciation as key determinants of inhibition efficacy. These findings establish a mechanistic framework for designing economically viable, next‑generation HAU with in‑situ urease inhibition, offering a pathway toward sustainable agriculture.