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 <i>CT573071</i>, coding a porin-cytochrome c protein complex associated with EET, co-occurred with <i>hao</i>, <i>hzsABC</i>, and <i>hdh</i> genes in <i>Candidatus Kuenenia</i>, suggesting its role in Feammox. Furthermore, four high-quality metagenome-assembled genomes (MAGs) affiliated with <i>Kuenenia stuttgartiensis_A</i> harbored <i>CT573071</i>, <i>hao-like</i>, <i>hzsABC</i>, and <i>hdh</i> genes, along with the <i>hao-cluster</i>, which catalyzes the oxidation of NH₄⁺-N to hydroxylamine. This genomic evidence further supports their dual metabolic capacity. Metatranscriptomic analysis confirmed <i>CT573071</i> upregulation and its coexpression with the <i>hao</i>, <i>hzsABC</i>, and <i>hdh</i> genes. These findings establish the potential role of <i>K. stuttgartiensis_A</i> <i>in Feammox</i>, providing novel insights into nitrogen removal in low-strength wastewater treatment systems.