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

Abstract

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.

Biogeochemical Implications of N2O-Reducing Thermophilic Campylobacteria in Deep-Sea Vent Fields, and the Description of Nitratiruptor labii sp. nov.

“…The sequences of clade II nosZ have been detected in similar abundance to those of clade I nosZ in various environments ( Sanford et al., 2012 ; Jones et al., 2013 ). Recent biochemical approaches for two types of NosZ revealed relatively lower whole-cell half-saturation constants for N 2 O with clade II bacteria compared with those with clade I bacteria ( Yoon et al., 2016 ; Suenaga et al., 2019 ), suggesting a high affinity for N 2 O in clade II nosZ organisms. In addition, ecological niche partitioning between two nosZ clade microorganisms has been demonstrated by culture-dependent and -independent analyses ( Dini-Andreote et al., 2016 ; Graf et al., 2016 ; Wittorf et al., 2016 ; Juhanson et al., 2017 ).…”

Temperature and oxygen level determine N2O respiration activities of heterotrophic N2O‐reducing bacteria: Biokinetic study

“…Harnessing N 2 ORB in engineered systems for reducing N 2 O emissions entails more knowledge of their ecophysiologies (Hallin et al, 2018; Suenaga et al, 2019). In addition to typical nosZ clade I type canonical denitrifying bacteria serving as a N 2 O sink (Table S1), the applicability of newly isolated nosZ clade II type denitrifying bacteria has demanded further investigation (Suenaga et al, 2019).…”

“…The two isolates affiliated to the genus Azospira are denitrifiers possessing clade II type nosZ , whereas those of genera Pseudomonas and Paracoccus are canonical clade I type nosZ denitrifying bacteria. These Azospira isolates, previously acquired from a bioreactor, were chosen as they have a proven high affinity for N 2 O (Suenaga et al, 2019). Contrarily, Ps.…”

“…These strains, stored in a deep freezer (−80°C), were aerobically pregrown in the nutrient medium (pH 7.0) consisting of 5 g L −1 NaCl, 5 g L −1 Bacto Peptone, and 3 g L −1 OxoidTM Lab‐Lemco meat extract, at 30°C with a shaking speed 150 rpm. This medium favors the resuscitation of stored bacteria for subsequent biokinetic experiments and avoids bacterial cell aggregation (Suenaga et al, 2018b; 2019). The incubation time was preliminarily optimized by the growth curves of the tested bacteria, as shown in Figure S1.…”

“…This pathway represents a pivotal role in wastewater treatment to mitigate N 2 O emissions (Kim et al, 2020), strongly dependent on the intrinsic N 2 O reduction capacity of N 2 ORB. Therefore, understanding the physiologies of N 2 ORB and developing N 2 O mitigation strategies has attracted more attention in recent years (Conthe et al, 2018c; Hallin et al, 2018; Suenaga et al, 2019; Yoon et al, 2016).…”