Synergistic nitrogen removal by robust heterotrophic nitrification-aerobic denitrification and substantial...

The discovery of heterotrophic nitrification and aerobic denitrification (HN-AD) strains has challenged the traditional understanding that microbial nitrogen removal relies on collaborative metabolism between diverse nitrifiers and denitrifiers. Nevertheless, their nitrogen metabolic pathways and application potential remain unclear. Here, we isolated a novel HN-AD strain, Zobellella sp. An-6, capable of simultaneously removing ammonium and nitrate under both oxic and anoxic conditions. Genomic analysis revealed genetic potential for ammonium assimilation, nitrification, denitrification, assimilatory nitrate reduction (ANR), and dissimilatory nitrate reduction to ammonium (DNRA). ¹⁵N-isotopic tracing and kinetics experiments validated its HN-AD capability, demonstrating complete nitrogen removal pathways that integrates nitrification and denitrification [NH₄⁺ ↔ glutamine → NH₂OH → NO₂⁻ → NO₃⁻, NO₃⁻ → NO₂⁻ → NO → N₂O → N₂]. Amino acid isotope and nitrogen balance analyses indicated that nitrate reduction pathways are condition-dependent: denitrification (N-loss) dominates at low C/N ratios, whereas assimilatory/dissimilatory nitrate reduction (N-retention) prevails at high C/N ratios. Transcriptomic profiling revealed preferential ammonium utilization over nitrate by repression of nitrate transport and reduction, suggesting a nitrogen utilization hierarchy and niche adaptation. In aquaculture wastewater treatment, An-6 achieved 96.1 % NH₄⁺ and 93.3 % NO₃⁻ removal within 6 h, highlighting its potential for treating high nitrogen-containing wastewater. Microbial community analysis confirmed its competitive dominance over indigenous consortia. This study extends the understanding of HN-AD bacteria from pure cultures to microbial consortia. It provides the first direct evidence that a single bacterium can perform complete denitrification, ANR, DNRA, nitrification, and ammonium assimilation, offering an effective and streamlined strategy for nitrogen removal.