Intra/extracellular electron transfer and metagenomic analysis elucidated the roles of magnetic iron powder (Fe₃O₄) on mixotrophic denitrification system

Elemental iron provides a viable strategy to improve the denitrification efficiency by expediting electron transport. However, the roles of magnetic iron powder (Fe₃O₄) on mixotrophic denitrification remains unknown. In this study, the intra/extracellular electron transfer (IET/EET) and microbial metabolism mechanisms were explored in a Fe₃O₄-mediated sulfide-autotrophic and heterotrophic denitrification system. The results showed that Fe₃O₄ promoted the formation of dense clump structure with filamentous cross-linking in activated sludge. Fe₃O₄ could increase the coenzyme Q activity in IET and the content of free riboflavin and cytochrome c in EET. Metagenomic analysis indicated that denitrification, sulfide oxidation and sulfate reduction were the main pathways of nitrogen and sulfur metabolism, and the enriched denitrifying bacteria (Halomonas and Hypobacterium) and sulfur-oxidizing bacteria (Marinicella) could stably support nitrate removal. This study expands our understanding of the IET/EET during Fe₃O₄-mediated mixotrophic denitrification process, providing a novel insight for nitrogen removal from marine recirculating aquaculture wastewater.