Variation of dissolved organic nitrogen in full-scale wastewater treatment revealed by HRMS coupled with hydrogen/deuterium isotope labeling

Dissolved organic nitrogen (DON) is an important nitrogenous fraction of wastewater effluent dissolved organic matter (DOM), with implications for eutrophication and nitrogenous disinfection byproduct formation. However, the molecular variation of DON, including amino-containing compounds, remains insufficiently elucidated across treatment trains. Herein, DOM and DON were characterized along a full-scale domestic wastewater treatment train using high-resolution mass spectrometry (HRMS). At the same time, amino-containing compounds were analyzed using HRMS coupled with hydrogen/deuterium isotope labeling. The results revealed that DON was a major and chemically distinct fraction of DOM, with lower molecular weight but greater unsaturation and aromaticity, indicating a more refractory molecular signature. Treatment evidently redistributed the occurrence patterns of DOM and DON, mainly through attenuation of lipid-like components and the increasing prominence of lignin/CRAM-like components. Adjacent-unit analysis showed that DON was jointly reshaped by attenuation and regeneration rather than monotonically removed. The front anaerobic-anoxic-oxic (AAO) biological treatment constrained DON but left a more recalcitrant residual profile. Membrane bioreactor (MBR)-moving bed biofilm reactor (MBBR) ultimately extended the AAO trend toward greater carbon-skeleton unsaturation and aromaticity, driven mainly by MBBR. Terminal disinfection only partially moderated this trend. Isotope labeling showed that amino-containing features persisted throughout treatment and were reshaped mainly by MBR-MBBR. Noticeably, MBBR increased their level and altered primary/secondary amine-like distributions before disinfection. These findings demonstrate that effective DON control should be evaluated by considering not only bulk parameters, but also the internal structure and functional-group characteristics of residual DON.