Consumption of N2O by Flavobacterium azooxidireducens sp. nov. Isolated from Decomposing Leaf Litter of Phragmites australis (Cav.)

Microorganisms acting as sinks for the greenhouse gas nitrous oxide (N2O) are gaining increasing attention in the development of strategies to control N2O emissions. Non-denitrifying N2O reducers are of particular interest because they can provide a real sink without contributing to N2O release. The bacterial strain under investigation (IGB 4-14T), isolated in a mesocosm experiment to study the litter decomposition of Phragmites australis (Cav.), is such an organism. It carries only a nos gene cluster with the sec-dependent Clade II nosZ and is able to consume significant amounts of N2O under anoxic conditions. However, consumption activity is considerably affected by the O2 level. The reduction of N2O was not associated with cell growth, suggesting that no energy is conserved by anaerobic respiration. Therefore, the N2O consumption of strain IGB 4-14T rather serves as an electron sink for metabolism to sustain viability during transient anoxia and/or to detoxify high N2O concentrations. Phylogenetic analysis of 16S rRNA gene similarity revealed that the strain belongs to the genus Flavobacterium. It shares a high similarity in the nos gene cluster composition and the amino acid similarity of the nosZ gene with various type strains of the genus. However, phylogenomic analysis and comparison of overall genome relatedness indices clearly demonstrated a novel species status of strain IGB 4-14T, with Flavobacterium lacus being the most closely related species. Various phenotypic differences supported a demarcation from this species. Based on these results, we proposed a novel species Flavobacterium azooxidireducens sp. nov. (type strain IGB 4-14T = LMG 29709T = DSM 103580T).


Introduction
Mitigating global climate change requires a comprehensive understanding of the mechanisms that produce and consume greenhouse gases. Various biotic and abiotic processes are involved in the release of nitrous oxide (N 2 O), the potent greenhouse gas and the dominant source of stratospheric ozone depletion [1,2]. As part of efforts to develop strategies to control N 2 O emissions, increasing attention is being paid to the reduction of N 2 O by microorganisms. The most important biologically catalysed N 2 O reduction is conducted by the copper cluster-containing enzyme N 2 O reductase NosZ [3]. In the past, N 2 O consumption has been attributed primarily to canonical denitrifying bacteria. However, genomic-based studies identified "atypical" NosZ coding genes (Clade

Isolation and Primariy Classification
The isolates studied originate from a mesocosm experiment designed to investigate the litter decomposition of Phragmites australis (Cav.) under different redox conditions. Leaves were collected from Phragmites growing in a rewetted fen (Germany, 53 • 52 33.4 N, 12 • 53 20.7 E) placed in stainless steel litterbags (1 mm mesh size) and processed as described by Reuter, et al. [23]. The litterbags were transferred into 5 L Duran wide neck bottles with four port caps and incubated in artificial fen water medium (4.5 L). Therefore, organic-free, deionized water was supplemented with NaHCO 3 and fresh detritus mud from the plant-sampling site as an inoculum. Furthermore, 200 mgL −1 NO 3 − or SO 4 2− was added as an oxidant in comparison to a control without external oxidants. The mesocosms were incubated under dark conditions in a custom-built flow-through steadystate system which is described in detail by Rillig, et al. [24]. A temperature of 18 • C was maintained in the incubation vessels with the aid of a climate chamber. The headspace of the mesocosms (0.5 L) was continuously flushed with nitrogen (N 2, 6 Lh −1 ) using a nitrogen membrane-generator NGM-11-LC/MS (CMC Instruments GmbH, Eschborn, Germany). Gas flux measurements were performed continuously during the whole incubation period according to Rillig, et al. [24].
To investigate the microbial community, leaves of a litter bag (10 g) were immersed in 90 mL of 0.2 M Sörensen sodium phosphate buffer (pH 7) and treated for 2 min at high speed in the Stomacher 400 Circulator (Seward Ltd., Thetford, UK). Serially diluted samples were inoculated onto selective growth medium G3M12, which was optimized for the isolation of NO 3 − reducing bacteria [25]. The medium consisted of a standard mineral base according to Stanier, et al. [26] supplemented with Hutner's vitamin-free mineral base [27] and a thiamine and complex vitamin solution [25,28]. The C/N ratio was adjusted to 2.5, with ethanol added as a carbon source and KNO 3 as a nitrogen source. The pH of the medium was corrected to 7.5 after autoclaving and addition of sterile filtered supplements. The agar plates were incubated for 2 weeks at 21 • C in an anoxic chamber [gas composition: 10% carbon dioxide (CO 2 ), 5% hydrogen (H 2 ), and 85% N 2 ]. Forty isolates were randomly picked and purified on 1 2 strength nutrient agar II containing 15 gL −1 agar ( 1 2 NAII; composition according to Sifin, Berlin, Germany) to classify the isolates according their taxonomic affiliation and functional traits in the nitrogen cycle. Classification of isolates according to their taxonomic background was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) as described by Ulrich, et al. [29].

Phylogenetic Analysis
The taxonomic classification was confirmed by sequencing the almost complete 16S rRNA gene [30]. The 16S rRNA gene was amplified using primers 8f and 1525r [31] according a protocol described by [32] and sequenced with the internal primers 1492r [31] and 782r [33]. Sequence similarity comparisons were carried out using the EzBiocloud database [34]. For the phylogenetic analysis, an alignment (1488 nt) of the 16S rRNA genes from closely related species was generated using the ClustalW algorithm with MEGA X [35]. A phylogenetic tree was constructed using the maximum-likelihood method based on evolutionary distances of the General Time Reversible model (+G+I) and the neighbourjoining algorithm applying the Kimura 2-parameter model (+G). To investigate a possible clonal origin Ribotyping was performed with restriction endonucleases EcoRI, PvuII as described by Behrendt, et al. [36]. Furthermore, ribosomal intergenic spacer regions of isolates were amplified with the primers 1492f and 115r and subsequently sequenced as described by Tokajian, et al. [37].

Genome Sequencing and Bioinformatic Analyses
Strain IGB 4-14 T was cultured in nutrient broth II (NBII; Sifin, Germany) for 2 days at 25 • C. The whole genomic DNA was extracted as described by Ulrich et al. [38]. DNA was sequenced using the Pacific Biosciences (PacBio) RS II sequencing platform at Eurofins Genomics (Konstanz, Germany). Sequence reads were de novo assembled using the PacBio hierarchical genome assembly process (HGAP4). The assembly resulted in one contig with an average genome coverage of 209x. The genome sequence was circularized with Circlator ver. 1.5.5 and deposited in the GenBank database under accession no. CP096205.
For the phylogenomic analysis, a tree based on core genome phylogeny [39] was constructed as described by Behrendt et al. [40]. The analysis of 120 bacterial core marker genes resulted in a concatenated amino acid sequence alignment, which was used to calculate a maximum-likelihood tree (LG substitution model with F+G+I) with MEGA X [35]. Digital DNA-DNA hybridization (dDDH) and genomic G+C content were determined on the Type Strain Genome Server (TYGS; [41]). Average nucleotide index (ANI) values were calculated by the OrthoANIu procedure [42]. To support the taxonomic classification, average amino acid identity (AAI) as a further genome relatedness index was calculated using the EzAAI tool [43].
The genome sequences of IGB 4-14 T and related species were annotated with Rapid Annotation using Subsystem Technology (RAST) version 2.0 [44,45]. Additional functional and pathway analyses were performed using the BlastKOALA web tool of the KEGG database [46] and the NCBI Prokaryotic Genome Annotation Pipeline [22]. To analyse the nosZ gene phylogeny amino acid sequences of type strains representing the genus Flavobacterium and reference strains for Clade II nosZ genes according to Sanford,et al. [4] were selected. A maximum-likelihood tree (LG substitution model with G+I) was calculated with MEGA X [35].

Phenotypic Characterisation
Phenotypic analyses were performed for strain IGB 4-14 T and the reference strain Flavobacterium lacus NBRC 109715 T . Standard methods for morphological and physiological characterization were conducted as described by Behrendt, et al. [47]. Unless otherwise stated, strains were cultivated on 1 2 NAII or in the respective broth ( 1 2 NBII) at 22 • C. Presence of oxidase was tested on Cytochrome Oxidase Test Strips (Merck). Production of flexirubintype pigments and Congo red absorption were assessed following the methods of Bernardet, et al. [48]. Oxidation of carbon compounds and resistance to inhibitory chemicals were determined using GEN III MicroPlates (Biolog) according to the manufacturer's instructions. Results were scored visually after 24 and 48 h. Additional physiological and enzymatic characteristics were determined using the API 20E and API 20NE test strip (bioMérieux) after 48 h.
Analysis of cellular fatty acids was performed at DSMZ GmbH (Braunschweig, Germany) after cultivation on tryptic soy agar (TSA) for 2 days at 20 • C. Cellular fatty acids were converted into fatty acid methyl esters (FAMEs) using minor modifications of the method of Miller [49] and Kuykendall, et al. [50]. The FAMEs were separated by gas chromatography and detected by a flame ionisation detector using Sherlock Microbial Identification System (MIDI, Microbial ID, Newark, DE, USA). Peaks were automatically integrated, and fatty acid identifications and percentages were calculated by standard software (Microbial ID, Library TSBA40, 4.10).

N 2 O Consumption
Strain IGB 4-14 T was cultured under oxic conditions in 1 2 NBII for 48 h at 20 • C. 100 µL of this culture was inoculated into sterile 12 mL flat bottom vials (Labco, Lampeter, UK) containing 5 mL 1 2 NBII broth. To create anoxic or micro-oxic conditions, the headspace was replaced three times with helium. For micro-oxic conditions, sterile air was added to achieve 4% (v/v headspace) oxygen (O 2 ). Furthermore, N 2 O and nitric oxide (NO) were added into vials of all experiments (oxic, micro-oxic and anoxic) at a final concentration of 0.8% (v/v headspace) and 6 ppm (v/v headspace), respectively. NO was included as an inducer molecule for transcription of the nos genes [51]. To avoid diffusion with the ambient air, an overpressure (0.05 MPa) was created with helium in the anoxic and micro-Microorganisms 2022, 10, 2304 5 of 17 oxic experiment and with sterile air in the oxic experiment. All tests were carried out in six replicates. For each experiment, a control was performed in triplicate without inoculation. The inoculated and control vials were incubated at 20 • C and sampled for gas analysis according to the time sequence shown in Figure 4. N 2 O, O 2 and CO 2 were measured via gas chromatography (7890B GC Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an autosampler (Combi Pal-xt system, CTC analytics, Zwingen, Switzerland). A thermal conductivity detector (TCD, G3440B, Agilent Technologies Inc., Santa Clara, CA, USA) was used for measurement of O 2. A pulsed discharge helium ionization detector (PDHID, V1D-3-I-HP220, Valco Instruments Company Inc. VICI AG International, Houston, TX, USA) detected CO 2 and N 2 O. Details of the method are described in Zaman, et al. [52].
Increase in optical density (OD 600 ) was used for estimating cell growth. It was measured at the start and the end of the experiment in a microplate with a Tecan plate reader (Infinite M Plex, Tecan Trading AG, Männedorf, Switzerland) and path length correction.
Loss of N 2 O, O 2 and CO 2 due to gas sampling was accounted for in the subsequent calculations. Equilibrated headspace concentrations of gases were used to allow comparative assessments of reduction or production of gas. Statistical significance was analyzed with a Kruskal-Wallis-Test and Dunn's post hoc test of multiple comparisons using rank sums adjusted with the Bonferroni method.

Results and Discussion
Based on a similarity analysis of the spectral data obtained by MALDI TOF-MS, the isolated strains were classified into taxonomic units at the species level. A group of isolates that could not be assigned to any taxa using the MALDI biotyper reference database originated from the 200 mgL −1 NO 3 − -supplemented mesocosm. The abundance of this group was 2 × 10 5 colony forming units per gram of fresh leaf litter. By 16S rRNA gene sequence comparison, the group could be assigned to the genus Flavobacterium of the phylum Bacteroidota, but a clear species affiliation was still not possible. Analysis of ribosomal intergenic spacer regions of five randomly selected isolates revealed identical sequences. Furthermore, the riboprint patterns showed no differences at the strain level. These results clearly indicated a clonal origin of the isolates. Therefore, only one isolate, strain IGB 4-14 T , was selected for detailed taxonomic and physiological studies.

Phylogenetic Analysis
Comparative 16S rRNA gene analysis of strain IGB 4-14 T revealed the highest sequence similarity of 97.3% to the species F. lacus (Table 1). Considering the species boundary of 98.2-99.0% for the 16S rRNA gene sequence similarity proposed by Meier-Kolthoff, et al. [53], a separate species position of the isolated strain is indicated. Furthermore, the high abundance of the isolates indicated an active growth under the tested redox conditions. The phylogenetic tree demonstrated that the isolated strain clearly clusters with F. lacus NP180 T in both treeing methods applied, which was additionally supported by a high bootstrap value of 82% ( Figure 1). Their position in relation to a cluster formed by further species of the genus Flavobacterium could also be found by both treeing methods. However, only the position related to the species Flavobacterium piscinae was confirmed by a supporting bootstrap value of 70%.   It is known that phylogenetic analyses based on the 16S rRNA gene do not always provide the desired taxonomic resolution depending on the genus studied [54]. Nevertheless, it gives a complete picture regarding the relationships within the genus because genomes are not available for all species. However, the study of evolutionary relationships using orthologous genes of the core genome can be used for a more solid and alternative It is known that phylogenetic analyses based on the 16S rRNA gene do not always provide the desired taxonomic resolution depending on the genus studied [54]. Nevertheless, it gives a complete picture regarding the relationships within the genus because genomes are not available for all species. However, the study of evolutionary relationships using orthologous genes of the core genome can be used for a more solid and alternative way for phylogenetic analysis [39]. Therefore, the genome of the strain was sequenced. The assembled genome comprises a single circular contig with a total length of 3,823,204 bp exhibiting 100% completeness. No plasmid is present. The genome encoded 3263 protein-coding sequences and the DNA G+C content calculated from the genome sequence is 33.8%, which is consistent with the description of the genus Flavobacterium [18].
The phylogenomic analysis revealed similar results like the 16S rRNA gene analysis. Strain IGB 4-14 T formed a branch with F. lacus within a joint cluster with F. piscinae, Flavobacterium orientale and Flavobacterium filum ( Figure 2). Overall genome relatedness indices substantiated the novel species position of strain IGB 4-14 T ( Table 1). The dDDH of closely related species resulted in values far below the species boundary of 70% [53]. Accordingly, the ANI and AAI values were below the recommended species cut-off level of 95% [55,56]. way for phylogenetic analysis [39]. Therefore, the genome of the strain was sequenced. The assembled genome comprises a single circular contig with a total length of 3,823,204 bp exhibiting 100% completeness. No plasmid is present. The genome encoded 3263 protein-coding sequences and the DNA G+C content calculated from the genome sequence is 33.8%, which is consistent with the description of the genus Flavobacterium [18]. The phylogenomic analysis revealed similar results like the 16S rRNA gene analysis. Strain IGB 4-14 T formed a branch with F. lacus within a joint cluster with F. piscinae, Flavobacterium orientale and Flavobacterium filum (Figure 2). Overall genome relatedness indices substantiated the novel species position of strain IGB 4-14 T ( Table 1). The dDDH of closely related species resulted in values far below the species boundary of 70% [53]. Accordingly, the ANI and AAI values were below the recommended species cut-off level of 95% [55,56].
In the summary of the results, it becomes clear that strain IGB 4-14 T deserves its own species position from a phylogenetic point of view.

Phenotypic Analysis
Strain IGB-14 T was comprehensively characterized by a morphological, physiological and chemotaxonomic analysis. The features characterizing the strain and thus the proposed novel species in detail are listed in the protologue (see Section 5). This section focuses on characteristics that allow the strain to be distinguished from the type strain F. lacus NBRC 109715 T , which possess the highest phylogenetic relationship.
The fatty acid profile of strain IGB 4-14 T is similar to the type strain of the related In the summary of the results, it becomes clear that strain IGB 4-14 T deserves its own species position from a phylogenetic point of view.

Phenotypic Analysis
Strain IGB-14 T was comprehensively characterized by a morphological, physiological and chemotaxonomic analysis. The features characterizing the strain and thus the proposed novel species in detail are listed in the protologue (see Section 5). This section focuses on characteristics that allow the strain to be distinguished from the type strain F. lacus NBRC 109715 T , which possess the highest phylogenetic relationship.
The fatty acid profile of strain IGB 4-14 T is similar to the type strain of the related species F. lacus (Table 2). Both strains display fatty acids iso-C 15:0 and iso-C 15:1 G as the major components. Minor amounts were detected for iso-C 15:0 3-OH, iso-C 17:0 3-OH and iso-C 17:1 w9c, with the proportions differing significantly. Qualitative differences were only found for fatty acids, which occurred in traces, so that a distinction between both strains was mainly based on quantitative differences. For type strains of species clustering in the phylogenomic analysis with the investigated strains, similar fatty acid compositions were described, especially with regard to the dominant acids [57,58]. The profiles match the general description of the genus Flavobacterium [18]. Furthermore, the strain IGB 4-14 T can be clearly distinguished from the related species F. lacus due to several differentiating physiological characteristics (Table 3). These results provide phenotypic support for the separate species status already demonstrated in the phylogenetic analysis. Therefore, the assignment of the investigated isolate to a novel species, Flavobacterium azooxidireducens sp. nov., is proposed.

Genome Inventory with Regard to the N-Oxide Reduction
The genome of strain IGB 4-14 T does not contain genes coding for NO 3 − , NO 2 − and NO reductases. Only the gene of the sec-dependent Clade II NosZ (locus_tag M0M57_RS11105), followed by genes of various accessory proteins of the NosZ, could be detected. Thus, this bacterium possesses only genes for N 2 O reduction, and therefore has the potential to be an important sink for N 2 O without the capabilities to act as an N 2 O source.
The nosZ phylogeny followed the phylogenetic relationship ( Figure 3). Strains of the genus Flavobacterium constitute a monophyletic branch separate from the reference strains for Clade II NosZ included in the analysis according to Sanford,et al. [4]. Type strains of the species F. orientale, F. filum and F. piscinae formed a cluster with the strain IGB 4-14 T similar to the phylogenomic analysis ( Figure 2). However, the type strain of F. lacus, the species with the closest relationship, does not possess any gene associated with N-transformation processes. These results are consistent with the general view that diversification of nosZ occurred predominantly through vertical inheritance, and on a finer scale the distribution of nosZ can be uneven, as nosZ may be present or absent in the genomes of closely related organisms [2]. alignment of NosB homologues by Spanning [59]. In strain IGB 4-14 T , they were located at positon 76 (LNHYIGM) and 144 (YGHN) of the fusion protein (M0M57_RS11110). These results imply that the hypothetical protein found in some strains is a NosB and the multidomain fusion protein of the other flavobacteria including IGB 4-14 T represents a NosB-L that combines the functions of NosB and NosL.

Consumption of N2O
Strain IGB 4-14 T consumed significant amounts of N2O under anoxic conditions (Figure 4a). Following a high consumption rate within the first 14 days, N2O consumption plateaued out until almost all N2O has been consumed by the end of the experiment. The N2O consumption activities were significantly affected by O2 levels (Figure 4c). Under microoxic conditions, N2O was consumed within the first 6 days. At the same rate, the O2 concentration in the test tubes decreased and the CO2 production (Figure 4b) increased. With this, the maximum of N2O consumption was reached and in the following course of the experiment there were no further changes in the investigated parameters. The average N2O consumption rate up to day 6 was 0.090 ± 0.066 µmol d −1 and was marginal higher than under anoxic conditions with 0.062 ± 0.022 µmol d −1 . In contrast, consumption of N2O under oxic conditions started around day 6, after most of the O2 amount had been consumed. Until then, only low consumption rates of 0.006 ± 0.016 µmol d −1 on average were achieved, which increased to 0.054 ± 0.019 µmol d −1 in the further course of the experiment. The increase in CO2 production correlated negatively with the O2 amount in the test tube. When CO2 reached its maximum at day 20, N2O consumption ceased.
N2O reduction by NosZ usually requires anoxic conditions, as NosZ copper centers are susceptible to O2 [6]. Nevertheless, N2O reduction under oxic and microoxic conditions has been described, for example, for strains of the species Pseudomonas stutzeri (Clade I The nosZ gene of strain IGB 4-14 T is followed by genes encoding NosZ asseccory proteins annotated as nosL (M0M57_RS11110), nosD (M0M57_RS11115), nosF (M0M57_RS11120) and nosY (M0M57_RS11125). An identical nos gene cluster was found for the closely related species, F. denitrificans and F. crocinum (Figure 3). In contrast, the other flavobacteria involved in the study exhibit two genes at this position between nosZ and nosD. The first gene is annotated as a hypothetical protein, whereas the second gene is clearly annotated as nosL. However, a nosB gene typical for a Clade II nos gene cluster was not identified. The nosB gene encoding a yet uncharacterized polytopic membrane protein [59] is often surrounded by nosZ and nosD [4,7]. In Wollinella succinogenes, Hein, et al. [60] demonstrated that NosB is essential for N 2 O respiration by characterising a non-polar nosB deletion mutant. It is assumed that the protein is involved in electron transport to NosZ, but possibly also in copper management [59]. The latter is derived from the fact that in certain bacteria like Dyadobacter fermentans NosB is fused to the copper binding protein NosL [59,60].
It is likely that a fusion protein is also produced by strain IGB 4-14 T and flavobacteria, which have an identical nos gene cluster (Figure 3). Sequence comparisons of the nosL gene of these bacteria with genes encoding the hypothetical protein and NosL of the other flavobacterial group revealed high similarity to the first gene segment and the following segment, respectively. A nosB amino-acid sequence comparison of Gemmatimonas aurantiaca T-27 T with the flavobacterial sequences of the hypothetical protein or the corresponding stretch of the fusion protein revealed identity values between 40 and 46%. Considering conservative substitutions in addition to identical amino-acids, similarity values of around 63% were achieved. In addition, two highly conserved amino-acid stretches were found in the investigated flavobacteria, which were described on the basis of an alignment of NosB homologues by Spanning [59]. In strain IGB 4-14 T , they were located at positon 76 (LNHYIGM) and 144 (YGHN) of the fusion protein (M0M57_RS11110). These results imply that the hypothetical protein found in some strains is a NosB and the multidomain fusion protein of the other flavobacteria including IGB 4-14 T represents a NosB-L that combines the functions of NosB and NosL.

Consumption of N 2 O
Strain IGB 4-14 T consumed significant amounts of N 2 O under anoxic conditions (Figure 4a). Following a high consumption rate within the first 14 days, N 2 O consumption plateaued out until almost all N 2 O has been consumed by the end of the experiment. The N 2 O consumption activities were significantly affected by O 2 levels (Figure 4c). Under microoxic conditions, N 2 O was consumed within the first 6 days. At the same rate, the O 2 concentration in the test tubes decreased and the CO 2 production (Figure 4b) increased. With this, the maximum of N 2 O consumption was reached and in the following course of the experiment there were no further changes in the investigated parameters. The average N 2 O consumption rate up to day 6 was 0.090 ± 0.066 µmol d −1 and was marginal higher than under anoxic conditions with 0.062 ± 0.022 µmol d −1 . In contrast, consumption of N 2 O under oxic conditions started around day 6, after most of the O 2 amount had been consumed. Until then, only low consumption rates of 0.006 ± 0.016 µmol d −1 on average were achieved, which increased to 0.054 ± 0.019 µmol d −1 in the further course of the experiment. The increase in CO 2 production correlated negatively with the O 2 amount in the test tube. When CO 2 reached its maximum at day 20, N 2 O consumption ceased. N 2 O reduction by NosZ usually requires anoxic conditions, as NosZ copper centers are susceptible to O 2 [6]. Nevertheless, N 2 O reduction under oxic and microoxic conditions has been described, for example, for strains of the species Pseudomonas stutzeri (Clade I nosZ) and Azospira sp. (Clade II nosZ) [12,[61][62][63]. N 2 O reducing bacteria are often facultatively aerobic bacteria isolated from anoxic to microoxic habitats that possess a respiratory chain involved in micro-aerobic respiration. It is assumed that this property gives the cells a certain aerotolerance and protects the N 2 O reductase from inactivation by O 2 [6]. This could be an explanation for the moderate consumption of N 2 O under microoxic conditions with decreasing O 2 content in this experiment. Nevertheless, the question arises why N 2 O consumption does not increase after the O 2 content has been completely reduced. The cessation of CO 2 formation could indicate exhausted growth. On the other hand, Nos recovery after exposure to O 2 might be restricted. The latter is considered an important parameter for the selection of strains for N 2 O mitigation in engineered systems [6,12].
Another important result is the lack of CO 2 formation during anaerobic N 2 O consumption. This indicates that the cells do not grow, which is also reflected in the optical density. During the incubation period, no significant increase in OD was recorded (data not shown). This result implies that N 2 O consumption by strain IGB 4-14 T is not coupled to energy conservation for growth, which is in contrast to the common assumption that the reduction of N 2 O is associated with energy conservation but in line with recent studies demonstrating that this is not the case for all microorganisms [10]. G. aurantiaca T-27 T is one of these organisms that has been extensively studied in terms of physiological properties [64,65]. Although it has been characterized as an obligate aerobic bacterium [66], it is able to reduce N 2 O not only under oxic, but also under microoxic and anoxic conditions when O 2 was regularly refed [64,65]. The reduction of N 2 O under anoxic conditions apparently acts as an electron sink for metabolism to sustain viability during transient anoxia and is not associated with growth. However, it is not known how widespread this NosZ function is, as it has not yet been tested on a broader diversity of isolates [10]. A further alternative NosZ function is the detoxification of high N 2 O concentrations [2,10,67]. Sullivan,et al. [68] demonstrated the cytotoxic effect of elevated N 2 O concentrations by inactivation of vitamin B 12 , thereby preventing enzyme reactions essential for microbial metabolisms. Thus, in Paracoccus denitrificans the B 12 -requiring methionine synthase MetH was inhibited, and the bacterium switched to the vitamin B 12 -independent methionine synthase MetE pathway, through the transcriptional modulation of genes controlled by vitamin B 12 riboswitches. The two synthases occur together in a large number of bacteria, but in some species only one is present but not the other. In the Bacteroidota phylum in particular, MetH is the predominant single enzyme [69]. Correspondingly, the strain IGB 4-14 T also possesses only the gene encoding the B 12 -depent synthase MetH (locus tag M0M57_09420), similar to G. aurantiaca T-27 T , which belong to the same clade. Therefore, NosZ can play an important role in the detoxification of high N 2 O concentrations in these organisms. However, the almost complete consumption of N 2 O indicates that strain IGB 4-14 T is capable of consuming N 2 O even in the range of natural concentrations in the environment, and along with the demonstrated absence of other denitrification associated enzymes (see above), could be a sink without contributing to N 2 O formation.
Cells are Gram-stain-negative, nonmotile rods, which are approximately 0.3-0.6 µm in diameter and 1.0-1.9 µm long. A capsule is present. Colonies are deep yellow, convex, circular, 1-4 mm in diameter with entire margins. Growth occurs at 4 to 30 • C (optimum 20-25 • C). No growth is observed at 37 • C. It is able to grow on TSA, Reasoner's 2A agar and nutrient agar. Flexirubin-type pigments are produced, and Congo red is absorbed by colonies. Catalase and oxidase activities are present. Hydrolyses gelatin but not tyrosine, casein, aesculin, starch and DNA. Nitrate is not reduced to nitrite. Arginine dihydrolase, lysine and ornithin decarboxylase, tryptophan deaminase and β-galactosidase is not present. Indole and acetoin production is negative.
The type strain IGB 4-14 T (LMG 29709 T = DSM 103580 T ) was isolated from a mesocosm experiment with decomposing leaf litter of Phragmites australis (Cav.) in artificial fen water enriched with fresh detritus originating from a rewetted fen (Germany, 53 • 52 33.4 N, 12 • 53 20.7 E). The DNA G+C content of the type strain is 33.8%. The genome sequence accession number is CP096205 and the 16S rRNA gene accession number is LT598610.

Conclusions
In this study, a bacterium associated with decomposing leaf litter of Phragmites australis (Cav.) was clearly differentiated from related Flavobacterium species in a polyphasic approach applying phylogenomics and phenotypic analysis. Based on these data, a novel species Flavobacterium azooxidireducens sp. nov. with the type strain IGB 4-14 T was proposed. The type strain is a non-denitrifying bacterium that was able to consume significant amounts of N 2 O under anoxic conditions by a N 2 O reductase of the Clade II. Due to the absence of other enzymes associated with denitrification, it has the potential to be a sink without contributing to N 2 O formation. The N 2 O consumption served as an electron sink for metabolism and/or to detoxify high N 2 O concentrations. It shows a high similarity in nos gene cluster composition and amino acid similarity of the nosZ gene with different type strains of the genus, so it can be assumed that the N 2 O reductase in these strains might also underlie this function and is typical for the genus Flavobacterium. However, in contrast to the NosZ function for energy conservation through anaerobic respiration, the importance and distribution of these alternative NosZ functions in the environment is not known and needs further investigation.