Genome-Resolved Metagenomic and Metatranscriptomics Reveal Feammox Metabolism of Anaerobic Ammonia Oxidation Bacteria in Microaerobic Granular Sludge

Anammox is an energy-efficient nitrogen removal process in which anammox bacteria (AnAOB) oxidize NH₄⁺-N to N₂ using NO₂^--N as the electron acceptor. Recent evidence suggests that AnAOB can perform extracellular electron transfer (EET), potentially coupling Fe(III) reduction with NH₄⁺-N oxidation (Feammox). However, whether AnAOB directly participate in Feammox within complex wastewater treatment systems remains unclear. Here, we investigated the iron-mediated nitrogen metabolism pathways in a microaerobic granular sludge (MGS) reactor by integrating enzyme inhibition assays with analyses of gene dynamics and co-occurrence patterns of nitrogen- and iron-cycling genes. Results demonstrate that AnAOB contributed to Feammox activity. The iron reduction gene CT573071, coding a porin-cytochrome c protein complex associated with EET, co-occurred with hao, hzsABC, and hdh genes in Candidatus Kuenenia, suggesting its role in Feammox. Furthermore, four high-quality metagenome-assembled genomes (MAGs) affiliated with Kuenenia stuttgartiensis_A harbored CT573071, hao-like, hzsABC, and hdh genes, along with the hao-cluster, which catalyzes the oxidation of NH₄⁺-N to hydroxylamine. This genomic evidence further supports their dual metabolic capacity. Metatranscriptomic analysis confirmed CT573071 upregulation and its coexpression with the hao, hzsABC, and hdh genes. These findings establish the potential role of K. stuttgartiensis_A in Feammox, providing novel insights into nitrogen removal in low-strength wastewater treatment systems.