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

Unlabelled: This study explores the genetic landscape of nitrous oxide (NO) 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 () genes and their association with other nitrogen metabolism pathways. A custom pipeline for clade-specific gene identification with higher sensitivity revealed 503 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 genes, with clade II being predominant, accounting for 93.7% of these genes. Taxonomic profiling highlighted the prevalence of -containing taxa within and exhibited unexpected affiliations with both the and secretory pathways, and all were found to contain the accessory gene, underscoring the importance of investigating the secretory pathway. The majority of non-denitrifying NO reducers were found within and . Additionally, HQ MAGs with genes for dissimilatory nitrate reduction to ammonium and assimilatory nitrate reduction frequently co-occurred with the gene. Traditional primers targeting often focus on short-length amplicons. Therefore, we introduced custom-designed primer sets targeting near-full-length 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 NO-reducing organisms in WWTPs, highlighting their potential as NO sinks with the potential for optimizing wastewater treatment processes and mitigating greenhouse gas emissions.

Importance: This study provides critical insights into the genetic diversity of nitrous oxide reductase (nosZ) genes and the microorganisms harboring them in wastewater treatment plants (WWTPs) by exploring 1,083 high-quality metagenome-assembled genomes (MAGs) from 23 Danish full-scale WWTPs. Despite the pivotal role of nosZ-containing organisms, their diversity remains largely unexplored in WWTPs. Our custom pipeline for detecting nosZ provides near-full-length genes with detailed information on secretory pathways and accessory nos genes. Using these genes as templates, we developed taxonomically diverse clade-specific primers that generate nosZ amplicons for phylogenetic annotation and gene-to-MAG linkage. This approach improves detection and expands the discovery of novel sequences, highlighting the prevalence of non-denitrifying NO reducers and their potential as NO sinks. These findings have the potential to optimize nitrogen removal processes and mitigate greenhouse gas emissions from WWTPs by fully harnessing the capabilities of the microbial communities.