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Abstract

Iron-dependent denitrification is a safe and promising technology for nitrogen removal in freshwaters. However, the understanding of microbial physiology and interactions during the process was limited. Denitrifying systems inoculated with freshwater samples were operated with and without iron(II) at a low C/N ratio for 54 days. Iron addition improved nitrogen removal. Batch experiments confirmed that microbially mediated reaction rather than abiotic reaction dominated during the process. Metagenomics recovered genomes of the five most abundant microorganisms, which accounted for over 99% of the community in every triplicate of the iron-based system. Based on codon usage bias, all of them were fast-growing organisms. The total abundance of fast-growing organisms was 38% higher in the system with iron than in the system without iron. Notably, the most abundant organism Diaphorobacter did not have enzymes for asparagine and aspartate biosynthesis, whereas Rhodanobacter could not produce serine and cobalamin. Algoriphagus and Areminomonas lost synthesis enzymes for more amino acids and vitamins. However, they could always obtain these growth-required substances from another microorganism in the community. The two-partner relationship minimized the limitation on microbial reproduction and increased community stability. Our results indicated that iron addition improved nitrogen removal by supplying electron donors, promoting microbial growth, and building up syntrophic interactions among microorganisms with timely communications. The findings provided new insights into the process, with implications for freshwater remediation.