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An Unknown Non-denitrifier Bacterium Isolated from Soil Actively Reduces Nitrous Oxide under High pH Conditions

A nitrous oxide (N2O)-consuming bacterium isolated from farmland soil actively consumed N2O under high pH conditions. An acetylene inhibition assay did not show the denitrification of N2 to N2O by this bacterium. When N2O was injected as the only nitrogen source, this bacterium did not assimilate N2O. A polymerase chain reaction demonstrated that this bacterium did not have the typical nosZ gene. This bacterium belonged to Chitinophagaceae, but did not belong to known families that include bacteria with the atypical nosZ. This is the first study to show that a non-denitrifier actively reduces N2O, even under high pH conditions.

Although carbon dioxide is a well-known greenhouse gas (GHG), other GHG also influence climate change (Montzka et al., 2011). Among these GHG, nitrous oxide (N 2 O) has a major impact on global warming. N 2 O absorbs infrared radiation, and its potential to cause global warming is 298-fold that of carbon dioxide and, thus, is regarded as the most important ozone-depleting substance in this century (Ravishankara et al., 2009;Montzka et al., 2011). The emission of N 2 O from agricultural soil is accelerated by the addition of large amounts of nitrogen-containing fertilizers to farmlands, and accounts for 60% of the atmosphere (Mosier et al., 1998;Zhou et al., 2015). Various methods have been attempted to mitigate N 2 O emissions. Recent studies reported that N 2 O emissions may be suppressed by the addition of a substance used as an agrochemical (Obia et al., 2015;Abbruzzini et al., 2019;Takatsu et al., 2019). However, the use of agrochemicals is associated with a number of issues, such as the loss of soil biodiversity and the persistence of soil chemicals (Stolte et al., 2016;Silva et al., 2018), thereby necessitating other methods. Therefore, N 2 Oreducing microorganisms have been attracting increasing attention (Hallin et al., 2018). N 2 O is emitted from soil into the atmosphere through the processes of nitrification and denitrification by soil microorganisms, which are major sources of N 2 O in soil (Skiba and Rees, 2014 (Zumft, 1997;Wunsch and Zumft, 2005). Furthermore, some non-denitrifying N 2 Oreducing microorganisms lack the pathway for the conversion of NO 3 to N 2 O, but have the capacity to convert N 2 O to N 2 (Payne et al., 1982;Simon et al., 2004). Therefore, nondenitrifying N 2 O-reducing microorganisms have the potential to be true N 2 O sinks without contributing to N 2 O production (Hallin et al., 2018). NosZ protein phylogeny has two distinct groups, clade I and II nosZ (Hallin et al., 2018). These clades have been reported as typical and atypical nosZ (Sanford et al., 2012). Clade I nosZ comprises alpha-, beta-, or gamma-proteobacteria, while clade II nosZ consists of a large range of archaeal and bacterial phyla (Jones et al., 2013). Non-denitrifying N 2 O-reducing microorganisms belong to clade II and possess abundant diversity in all ecosystems (Hallin et al., 2018).
Complete denitrifiers utilize the N 2 O present in soil gas as the final electron acceptor in the nitrate respiratory system and emit N 2 as the final product into the atmosphere (Hutchins, 1991;Wunsch and Zumft, 2005). N 2 O is used to promote cell survival, even in the absence of oxygen (Park et al., 2017). Based on the assimilation of N as a nutrient, when N 2 O is abundant, from the perspective of activation energy, it is more efficient in the assimilation of N 2 O than N 2 fixation (Kryachko et al., 2001). Available N (NO 3 -and NH 4 + ) in soil is limited, even in relatively fertile soils because these nitrogen sources are competitively assimilated by plants and other microorganisms (Kaye and Hart, 1997). In terms of a survival strategy for bacteria, the assimilation of N 2 O is advantageous when N 2 O is abundant. Therefore, some bacteria that positively absorb N 2 O for assimilation may exist; however, this has not yet been demonstrated.
Therefore, the purpose of the present study was to search for a bacterium in soil that consumes N 2 O. We hypothesized that some bacteria among N 2 O-consuming microorganisms in farmland soil may assimilate N 2 O when it is abundant through the denitrification process. By detecting changes in N 2 O concentrations in gas chromatography vials injected with N 2 O before incubations, strains with the potential to consume N 2 O were screened among bacteria isolated from Article ME20100 farmland soil. Furthermore, an acetylene inhibition assay was conducted to establish whether the decrease in N 2 O concentrations was due to assimilation or reduction. We herein report the taxonomic affiliation and optimal pH conditions required for N 2 O reduction by this isolated N 2 Oreducing bacterium.
Andisol was collected on April 14, 2016 from a pasture farmland and the maize field at the Hokkaido University Shizunai Experimental Livestock Farm (Shinhidaka, Hokkaido, Japan [42°25'9"N, 142°29'1"E]) (Katayanagi et al., 2008). Soil samples were collected at a depth of 0-10 cm and used in the N 2 O reduction assay and the isolation of microorganisms. We used soil from the maize field. The soil suspension was prepared as described previously (Hashidoko et al., 2008).
Winograsky's mineral solution containing 0.5% (w/v) sucrose and 5 mM KNO 3 (0.52 g L -1 ) was used as the medium in the culture-based N 2 O reduction assay (Hara et al., 2009;Nie et al., 2015). Since pH plays a key role in the emission of N 2 O (Nie et al., 2015), the pH of the solution was adjusted to various values (4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0) using 2 M H 2 SO 4 and 1 M KOH that was gelled with 0.5% (w/v) gellan gum and then autoclaved. The same medium was used in subsequent experiments. N 2 O levels were measured as described in a previous study (Nie et al., 2015). N 2 O was emitted in the culture at pH 4.5-7.5 ( Fig. 1). However, N 2 O emissions decreased in the culture at pH 8.5. This decrease in N 2 O emissions indicated the presence of N 2 O-consuming microorganisms. Therefore, we focused on this culture and isolated the bacterium from N 2 O-consuming microorganisms.
To screen for N 2 O-consuming microorganisms, colonies were isolated as described in a previous study (Nie et al., 2015). Fifteen distinguishable bacterial colonies, marked A to O, were identified. Standard N 2 O gas (GL Sciences) was injected using a gas-tight syringe into the headspace of gas chromatography vials to a final concentration of 2,000 ppmv. After incubations for 0, 1, and 4 days at pH 8.5, N 2 O concentrations in the headspace gas were measured. The results obtained showed that strain A (Sac-f1) exhibited the greatest consumption of N 2 O (Fig. 2).
To examine the potential of N 2 O reducers to reduce N 2 O to N 2 , 10% volume (2.25 mL) acetylene gas and N 2 O (12,000 ppmv) were injected into the headspace of the assay vials immediately after the inoculation of the isolated bacterium, and media were then incubated at 25°C for 0, 1, 2, 3, and 6 weeks. The concentration of N 2 O was measured after these incubation periods. N 2 O concentrations did not decrease with the acetylene gas treatment, which confirmed that the bacterium reduced N 2 O (Fig. 3). Based on the results of OD 660 measurements in the medium, when N 2 O was injected into gas chromatography vials as the only nitrogen source, this bacterium displayed no growth. This result indicated that this bacterium did not use N 2 O as a nutrient. Furthermore, N 2 O concentrations did not increase during the incubation with the acetylene gas treatment (Fig.  3). Therefore, the bacterium reduced, but did not assimilate, -N 2 O and did not denitrify NO 3 to N 2 O.
The DNA of this bacterium was extracted using an Isoplant II DNA Extraction kit (Nippon Gene), and the nosZ gene was subjected to a polymerase chain reaction (PCR) using nosZ gene-specific primers (nosZ-1111F and nosZ-1773R) (Scala and Kerkhof, 1998). The 16S rRNA region was amplified with PCR using the primers 27F and 1525R (Lane, 1991;Weisburg et al., 1991). PCR amplicons using the specific primers were purified by agarose gel electrophoresis. Pseudomonas denitrificans NBRC 12442 was used as the positive control. This bacterium did not have a nosZ gene (Fig. 4), and the region of 16S rRNA was successfully amplified from the DNA template.
The 16S rRNA sequence of the isolated bacterium was highly homologous to those of the species belonging to    Chitinophagaceae. The closest species to the isolated bacterium was Chitinophaga eiseniae (96.35% similarity). A phylogenetic analysis was performed based on the neighborjoining method using MEGA X (Kumar et al., 2018). The sequences of the species belonging to Chitinophagaceae were retrieved from the GenBank database. A similar phylogenetic analysis was performed using the 16S rRNA sequence data of previously characterized bacteria showing an atypical nosZ gene (Liu et al., 2008;Sanford et al., 2012;Jones et al., 2013;Park et al., 2017;Hallin et al., 2018) to identify the taxonomic group of the isolated bacterium. We reviewed these studies for species with an atypical nosZ gene in cases where 16S rRNA sequence data were not available. Consequently, this isolated bacterium belonged to the genus Chitinophaga (Fig. 5A). However, it was not reported whether the bacteria from this family belonged to clade II nosZ (Fig. 5B).
To assess the effects of pH on N 2 O reduction by the iso-   lated bacterium, the pH of the media was adjusted to various values (4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0), followed by incubations for 0, 1, 2, and 3 weeks. N 2 O was injected as described earlier. This isolated bacterium reduced N 2 O at pH in the range of 4.5 to 9.0, with the optimum pH being 8.5 (Fig. 6). Previous studies reported that soil microorganisms belonging to clade II reduce N 2 O at pH 7.0-7.5 (Liu et al., 2008;Sanford et al., 2012;Jones et al., 2013;Park et al., 2017;Hallin et al., 2018), whereas the isolated bacterium in the present study reduced N 2 O under alkaline rather than neutral conditions (Fig. 6). The present results clearly demonstrated that the isolated bacterium did not assimilate N 2 O, but reduced N 2 O to N 2 . The results of the phylogenetic tree analysis revealed that this bacterium was an unknown species belonging to Chitinophagaceae and reduced N 2 O at high pH (8.5). Since the application of nitrogen fertilizers, such as urea, to farmlands results in the largest increase in pH (Black et al., 1985) and accelerates N 2 O emissions (Zhou et al., 2015), a fertilizer inoculated with this isolated bacterium may be used to suppress the N 2 O flux from agricultural soil. Further investigations, draft genome analyses, and measurements of enzyme activity are needed to clarify the genetic background of this isolated bacterium.

Nucleotide sequence accession number
The 16S rRNA sequence obtained in the present study has been deposited under the following GenBank/ENE/ DDBJ accession number: LC554186.