Nitrous oxide (N2O) is a potent greenhouse gas and contributor to stratospheric ozone destruction. Anthropogenic N2O emissions are predominantly associated with microbial nitrification and denitrification in agricultural and industrial practices, including in wastewater treatment. In denitrification, N2O is an intermediate in a cascade of enzymatic reactions catalyzed by metalloenzymes. Denitrifying polyphosphate accumulating organisms (DPAOs) appear to be particularly prone to N2O accumulation under certain environmental conditions, including exposure to nitrite (NO2-). However, mechanisms underlying N2O generation by DPAOs are little understood. To address this knowledge gap, we’ve employed genome-resolved metagenomics, long-term kinetic analyses in a lab-scale bioreactor, and ex-situ batch assays to investigate nitrogen transformation potential in an enrichment culture of the as-yet-uncultivated DPAO Candidatus ‘Accumulibacter phosphatis’. Results demonstrate adaptation of this culture to NO2- as the terminal electron acceptor and a surprising capacity to reduce NO2- to N2O (70-80%) via incomplete denitrification. Metagenomic analyses yielded 41 near-complete draft genomes, including two co-occurring Accumulibacter strains affiliated with clades IA and IC and a diverse flanking denitrifying community. The dominant Accumulibacter clade IA encoded genes for complete denitrification, while the lower abundance Accumulibacter clade IC (the first published genome for clade IC) contained all denitrification genes except for nitric oxide reductase (NOR). Analysis of 39 flanking bacterial genomes revealed a high prevalence of taxa harboring incomplete denitrification pathways, suggesting that long-term NO2- feeding selects for denitrifying N2O producers that lack nitrous oxide reductase (NOS) and against N2O consumers. Our results suggest that the unusually high levels of N2O production observed in this microbial consortium is caused by a combination of different mechanisms, including selection of flanking microorganisms with truncated denitrification pathways. By resolving functional potential within a denitrifying N2O-producing culture, our results provide insight into genomic and environmental factors underlying N2O emissions by denitrifiers in complex environment systems.

Probing mechanisms of nitrous oxide generation in a denitrifying polyphosphate accumulating bacteria enrichment culture

255th National Meeting (2018)

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