Enrichment, Isolation, and Characterization of High-Affinity N₂O-Reducing Bacteria in a Gas-Permeable Membrane Reactor

Abstract

The recent discovery of nitrous oxide (N₂O)-reducing bacteria suggests a potential biological sink for the potent greenhouse gas N₂O. For an application toward N₂O mitigation, characterization of more isolates will be required. Here, we describe the successful enrichment and isolation of high-affinity N₂O-reducing bacteria using a N₂O-fed reactor (N₂OFR). Two N₂OFRs, where N₂O was continuously and directly supplied as the sole electron acceptor to a biofilm grown on a gas-permeable membrane, were operated with acetate or a mixture of peptone-based organic substrates as an electron donor. In parallel, a NO₃^- -fed reactor (NO₃FR), filled with a nonwoven sheet substratum, was operated using the same inoculum. We hypothesized that supplying N₂O vs NO₃^- would enhance the dominance of distinct N₂O-reducing bacteria. Clade II type <i>nosZ</i> bacteria became rapidly enriched over clade I type <i>nosZ</i> bacteria in the N₂OFRs, whereas the opposite held in the NO₃FR. High-throughput sequencing of 16S rRNA gene amplicons revealed the dominance of <i>Rhodocyclaceae</i> in the N₂OFRs. Strains of the <i>Azospira</i> and <i>Dechloromonas</i> genera, canonical denitrifiers harboring clade II type <i>nosZ</i>, were isolated with high frequency from the N₂OFRs (132 out of 152 isolates). The isolates from the N₂OFR demonstrated higher N₂O uptake rates (<i>V</i>_max: 4.23 × 10^-3-1.80 × 10^-2 pmol/h/cell) and lower N₂O half-saturation coefficients (<i>K</i>_m,N₂O: 1.55-2.10 μM) than a clade I type <i>nosZ</i> isolate from the NO₃FR. Furthermore, the clade II type <i>nosZ</i> isolates had higher specific growth rates on N₂O than nitrite as an electron acceptor. Hence, continuously and exclusively supplying N₂O in an N₂OFR allows the enrichment and isolation of high-affinity N₂O-reducing strains, which may be used as N₂O sinks in bioaugmentation efforts.