Deciphering dual effects of humic substrates on anaerobic ammonium oxidation: from metabolic facilitation to systemic...

Anaerobic Ammonium Oxidation (Anammox) is a promising strategy for nitrogen removal from landfill leachate due to its energy efficiency and independence from external carbon sources. However, continuous changes in recalcitrant organic compounds (i.e., fulvic acid (FA) and humic acid (HA)) at different landfill stages have potential effect on stability of anammox process. The present study systematically investigated the successive change of FA/HA concentrations and ratio that induced by different landfill age on anammox process in a laboratory-scale moving bed biofilm reactor operated over 200 days. Results showed a distinct dual-effect transition of FA and HA on the anammox process. 50-100 mg/L FA significantly improved nitrogen removal, achieving a peak total nitrogen removal efficiency (TNRE) of 91%. In contrast, the introduction of HA (100-200 mg/L) triggered a decline in TNRE to 80% and caused substantial nitrate (NO₃^--N) accumulation. Microbial analysis revealed that FA-driven carbon abundance stimulated Candidatus Kuenenia and DNRA-functional Paracoccus and Bacillus, whereas HA suppressed DNRA bacteria in favor of nitrification. Metagenomic analysis showed that the electron shuttle and carbon source characteristics of FA enriched the relative abundance of energy metabolism genes (i.e., F-type ATPase) and quorum sensing genes (i.e., lamB), accelerating the anammox and denitrification process. However, HA interfered with the nitrogen metabolic flux via suppressing denitrification process (i.e., napA/B and nirS) and promoting nitrification genes (i.e., amoA and nxrA/B), leading to the accumulation of NO₃^--N. These findings provide a mechanistic basis for optimizing anammox processes under varying humic substance conditions in landfill leachate treatment.