Multitechnique Characterization of Dissolved Organic Matter from Naturally Acidic and Circumneutral Waters Reveals Molecular Controls on Organic Nitrogen Speciation

Abstract The molecular characteristics of dissolved organic matter (DOM) vary across seasons, catchments, and land uses, highlighting the need for generalized structure–function relationships that can predict intrinsic DOM bioavailability. This study characterized DOM from naturally acidic (pH 4.3–5.7) and circumneutral (pH 6.8–8.3) freshwaters over two seasons: winter-spring (WS) and summer-autumn (SA) using multiple characterization techniques (optical, fluorescence, targeted LC-MS/MS, 13C NMR, and FTIR). DOM from acidic environments was higher in aromaticity, more humic-like, with a higher C:N ratio but with a greater percentage of nitrogen in the form of amino acids (i.e., DCAA-N%). Seasonality was only observed for neutral samples, with differences between WS and SA dominated by a decrease in nominal molecular weight (MWn) in SA, likely driven by photochemical disaggregation. FTIR analysis indicates that neutral DOM has a higher amide group abundance than acidic DOM despite the lower DCAA-N%, suggesting the dominance of nonproteinaceous amide-N. Very strong dependencies were observed for dissolved organic nitrogen (DON) speciation against aromaticity (SUVA254) and an FTIR-derived ratio of deprotonated carboxylic:aromatic groups, describing the relative hydrophilicity–hydrophobicity of the DOM environment. These parameters largely explain the DCAA distribution and may provide a more generalized way of characterizing DOM that could be applied across different aquatic ecosystems.