Unraveling the genetic potential of nitrous oxide reduction in wastewater treatment: insights from metagenome-assembled genomes

This study explores the genetic landscape of nitrous oxide (N ₂O) reduction in wastewater treatment plants (WWTPs) by profiling 1,083 high-quality metagenome-assembled genomes (HQ MAGs) from 23 Danish full-scale WWTPs. The focus is on the distribution and diversity of nitrous oxide reductase (nosZ) genes and their association with other nitrogen metabolism pathways. A custom pipeline for clade-specific nosZ gene identification with higher sensitivity revealed 503 nosZ sequences in 489 of these HQ MAGs, outperforming existing Kyoto Encyclopedia of Genes and Genomes (KEGG) module-based methods. Notably, 48.7% of the total 1,083 HQ MAGs harbored nosZ genes, with clade II being predominant, accounting for 93.7% of these genes. Taxonomic profiling highlighted the prevalence of nosZ-containing taxa within Bacteroidota and Pseudomonadota. Chloroflexota exhibited unexpected affiliations with both the sec and tat secretory pathways, and all were found to contain the accessory nosB gene, underscoring the importance of investigating the secretory pathway. The majority of non-denitrifying N ₂O reducers were found within Bacteroidota and Chloroflexota. Additionally, HQ MAGs with genes for dissimilatory nitrate reduction to ammonium and assimilatory nitrate reduction frequently co-occurred with the nosZ gene. Traditional primers targeting nosZ often focus on short-length amplicons. Therefore, we introduced custom-designed primer sets targeting near-full-length nosZ sequences. These new primers demonstrate efficacy in capturing diverse and well-characterized sequences, providing a valuable tool with higher resolution for future research. In conclusion, this comprehensive analysis enhances our understanding of N ₂O-reducing organisms in WWTPs, highlighting their potential as N ₂O sinks with the potential for optimizing wastewater treatment processes and mitigating greenhouse gas emissions.