Ammonification Process Is Limited by Transformation of Medium Molecular Weight Nitrogenous Organics but Not Hydrolysis of Proteins

Ammonification is a critical step in nitrogen cycling framework, but the role of molecular heterogeneity within dissolved organic nitrogen (DON) remains elusive. A controlled laboratory experiment across redox regimes resolved stepwise conversion of DON from proteins to small nitrogenous intermediates. Integrated datasets from macromolecules to monomers from a steady reactor overcame challenging in impossible quantification of all DON compounds in the kinematic research. Data-independent based proteomic analysis revealed that over 85% of proteins remained stable under anaerobic and microaerobic conditions. Non-targeted profiling and paired-mass-difference analysis revealed that partial transformations of DON did not cover nitrogen-containing functional groups. Peptide-level results identified methionine- and tryptophan-rich sequences as the persistent constituents, as well as theoretical analysis of frontier molecular orbital. Targeted quantification of DON monomers presented low concentrations of amino acids and purine derivatives, reflecting rapid and nearly complete transformation thereof. Thereby, ammonification was constrained by the formation and subsequent transformation of medium-molecular-weight nitrogenous intermediates instead of protein hydrolysis process. These findings underscore the structural constraints governing nitrogen mineralization as a new mechanism of DON transformation.