Enrichment, Isolation, and Characterization of High-Affinity N 2 O-Reducing Bacteria in a Gas-Permeable Membrane Reactor

The recent discovery of nitrous oxide (N 2 O)-reducing bacteria suggests a potential biological sink for the potent greenhouse gas N 2 O. For an application toward N 2 O mitigation, characterization of more isolates will be required. Here, we describe the successful enrichment and isolation of high-affinity N 2 O-reducing bacteria using a N 2 O-fed reactor (N 2 OFR). Two N 2 OFRs, where N 2 O 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 NO 3 – -fed reactor (NO 3 FR), filled with a nonwoven sheet substratum, was operated using the same inoculum. We hypothesized that supplying N 2 O vs NO 3 – would enhance the dominance of distinct N 2 O-reducing bacteria. Clade II type nosZ bacteria became rapidly enriched over clade I type nosZ bacteria in the N 2 OFRs, whereas the opposite held in the NO 3 FR. High-throughput sequencing of 16S rRNA gene amplicons revealed the dominance of Rhodocyclaceae in the N 2 OFRs. Strains of the Azospira and Dechloromonas genera, canonical denitrifiers harboring clade II type nosZ, were isolated with high frequency from the N 2 OFRs (132 out of 152 isolates). The isolates from the N 2 OFR demonstrated higher N 2 O uptake rates ( V max: 4.23 × 10 –3 –1.80 × 10 –2 pmol/h/cell) and lower N 2 O half-saturation coefficients ( K m,N 2 O: 1.55–2.10 μM) than a clade I type nosZ isolate from the NO 3 FR. Furthermore, the clade II type nosZ isolates had higher specific growth rates on N 2 O than nitrite as an electron acceptor. Hence, continuously and exclusively supplying N 2 O in an N 2 OFR allows the enrichment and isolation of high-affinity N 2 O-reducing strains, which may be used as N 2 O sinks in bioaugmentation efforts.