Nitrous oxide (N2O)-reducing bacteria, which reduce N2O to nitrogen in the absence of oxygen, are phylogenetically spread throughout various taxa and have a potential role as N2O sinks in the environment. However, research on their physiological traits has been limited. In particular, their activities under microaerophilic and aerobic conditions, which severely inhibit N2O reduction, remain poorly understood. We used an O2 and N2O micro-respirometric system to compare the N2O reduction kinetics of four strains, i.e., two strains of an Azospira sp., harboring clade II type nosZ, and Pseudomonas stutzeri and Paracoccus denitrificans, harboring clade I type nosZ, in the presence and absence of oxygen. In the absence of oxygen, the highest N2O-reducing activity, Vm,N2O, was 5.80 ± 1.78 × 10−3 pmol/h/cell of Azospira sp. I13, and the highest and lowest half-saturation constants were 34.8 ± 10.2 μM for Pa. denitirificans and 0.866 ± 0.29 μM for Azospira sp. I09. Only Azospira sp. I09 showed N2O-reducing activity under microaerophilic conditions at oxygen concentrations below 110 μM, although the activity was low (10% of Vm,N2O). This trait is represented by the higher O2 inhibition coefficient than those of the other strains. The activation rates of N2O reductase, which describe the resilience of the N2O reduction activity after O2 exposure, differ for the two strains of Azospira sp. (0.319 ± 0.028 h−1 for strain I09 and 0.397 ± 0.064 h−1 for strain I13) and Ps. stutzeri (0.200 ± 0.013 h−1), suggesting that Azospira sp. has a potential for rapid recovery of N2O reduction and tolerance against O2 inhibition. These physiological characteristics of Azospira sp. can be of promise for mitigation of N2O emission in industrial applications.

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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 towards 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 an 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 non-woven sheet substratum, was operated using the same inoculum. We hypothesized that supplying N 2 O vs. NO 3would enhance the dominance of distinct N 2 Oreducing 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, N2O : 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. 81 * Converted using the 0.28 pg dry-weight per bacterial cell (Madigan and Martinko, 2006) ** Attained dividing V max ×10 -9 by K m,N2O *** n.d. represents no growth.

The goal of this study was to investigate the effectiveness of a membrane-aerated biofilm reactor (MABR), a representative of counter-current substrate diffusion geometry, in mitigating nitrous oxide (NO) emission. Two laboratory-scale reactors with the same dimensions but distinct biofilm geometries, i.e., a MABR and a conventional biofilm reactor (CBR) employing co-current substrate diffusion geometry, were operated to determine depth profiles of dissolved oxygen (DO), nitrous oxide (NO), functional gene abundance and microbial community structure. Surficial nitrogen removal rate was slightly higher in the MABR (11.0 ± 0.80 g-N/(m day) than in the CBR (9.71 ± 0.94 g-N/(m day), while total organic carbon removal efficiencies were comparable (96.9 ± 1.0% for MABR and 98.0 ± 0.8% for CBR). In stark contrast, the dissolved NO concentration in the MABR was two orders of magnitude lower (0.011 ± 0.001 mg NO-N/L) than that in the CBR (1.38 ± 0.25 mg NO-N/L), resulting in distinct NO emission factors (0.0058 ± 0.0005% in the MABR vs. 0.72 ± 0.13% in the CBR). Analysis on local net NO production and consumption rates unveiled that zones for NO production and consumption were adjacent in the MABR biofilm. Real-time quantitative PCR indicated higher abundance of denitrifying genes, especially nitrous oxide reductase (nosZ) genes, in the MABR versus the CBR. Analyses of the microbial community composition via 16S rRNA gene amplicon sequencing revealed the abundant presence of the genera Thauera (31.2 ± 11%), Rhizobium (10.9 ± 6.6%), Stenotrophomonas (6.8 ± 2.7%), Sphingobacteria (3.2 ± 1.1%) and Brevundimonas (2.5 ± 1.0%) as potential NO-reducing bacteria in the MABR.

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