
==== Front
Appl Environ Microbiol
Appl Environ Microbiol
aem
Applied and Environmental Microbiology
0099-2240
1098-5336
American Society for Microbiology 1752 N St., N.W., Washington, DC

39136491
aem02177-23
10.1128/aem.02177-23
aem.02177-23
Environmental Microbiology
applied-and-industrial-microbiologyApplied and Industrial MicrobiologyUnraveling the genetic potential of nitrous oxide reduction in wastewater treatment: insights from metagenome-assembled genomes
https://orcid.org/0000-0002-3850-4971
Schacksen Patrick Skov 1
https://orcid.org/0000-0002-8747-6938
Nielsen Jeppe Lund 1 jln@bio.aau.dk

1 Department of Chemistry and Bioscience, Aalborg University , Aalborg, Denmark
Editor Semrau Jeremy D. University of Michigan , Ann Arbor, Michigan, USA

Address correspondence to Jeppe Lund Nielsen, jln@bio.aau.dk
The authors declare no conflict of interest.

9 2024
13 8 2024
13 8 2024
90 9 e02177-2306 12 2023
22 7 2024
Copyright © 2024 Schacksen and Nielsen.
2024
Schacksen and Nielsen.
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

ABSTRACT

This study explores the genetic landscape of nitrous oxide (N2O) reduction in wastewater treatment plants (WWTPs) by profiling 1,083 high-quality metagenome-assembled genomes (HQ MAGs) from 23 Danish full-scale WWTPs. The focus is on the distribution and diversity of nitrous oxide reductase (nosZ) genes and their association with other nitrogen metabolism pathways. A custom pipeline for clade-specific nosZ gene identification with higher sensitivity revealed 503 nosZ sequences in 489 of these HQ MAGs, outperforming existing Kyoto Encyclopedia of Genes and Genomes (KEGG) module-based methods. Notably, 48.7% of the total 1,083 HQ MAGs harbored nosZ genes, with clade II being predominant, accounting for 93.7% of these genes. Taxonomic profiling highlighted the prevalence of nosZ-containing taxa within Bacteroidota and Pseudomonadota. Chloroflexota exhibited unexpected affiliations with both the sec and tat secretory pathways, and all were found to contain the accessory nosB gene, underscoring the importance of investigating the secretory pathway. The majority of non-denitrifying N2O reducers were found within Bacteroidota and Chloroflexota. Additionally, HQ MAGs with genes for dissimilatory nitrate reduction to ammonium and assimilatory nitrate reduction frequently co-occurred with the nosZ gene. Traditional primers targeting nosZ often focus on short-length amplicons. Therefore, we introduced custom-designed primer sets targeting near-full-length nosZ sequences. These new primers demonstrate efficacy in capturing diverse and well-characterized sequences, providing a valuable tool with higher resolution for future research. In conclusion, this comprehensive analysis enhances our understanding of N2O-reducing organisms in WWTPs, highlighting their potential as N2O sinks with the potential for optimizing wastewater treatment processes and mitigating greenhouse gas emissions.

IMPORTANCE

This study provides critical insights into the genetic diversity of nitrous oxide reductase (nosZ) genes and the microorganisms harboring them in wastewater treatment plants (WWTPs) by exploring 1,083 high-quality metagenome-assembled genomes (MAGs) from 23 Danish full-scale WWTPs. Despite the pivotal role of nosZ-containing organisms, their diversity remains largely unexplored in WWTPs. Our custom pipeline for detecting nosZ provides near-full-length genes with detailed information on secretory pathways and accessory nos genes. Using these genes as templates, we developed taxonomically diverse clade-specific primers that generate nosZ amplicons for phylogenetic annotation and gene-to-MAG linkage. This approach improves detection and expands the discovery of novel sequences, highlighting the prevalence of non-denitrifying N2O reducers and their potential as N2O sinks. These findings have the potential to optimize nitrogen removal processes and mitigate greenhouse gas emissions from WWTPs by fully harnessing the capabilities of the microbial communities.

KEYWORDS

nitrous oxide reductase
NosZ
nitrous oxide sink
wastewater treatment
nitrogen metabolism
Teknologi og Produktion, Det Frie Forskningsråd (FTP, DFF) 9041-00367B Nielsen Jeppe Lund cover-dateSeptember 2024
==== Body
pmcINTRODUCTION

Nitrous oxide (N2O) in the atmosphere has significantly contributed to stratospheric ozone depletion over the last 150 years, increasing by more than 20% since 1750 (1, 2). This increase is pronounced, given that N2O possesses a warming potential almost 300 times that of carbon dioxide (CO2) (3). This N2O emission is further exacerbated by the estimated annual increase of 2%, attributing to nearly 80% of the carbon footprint in modern wastewater treatment plants (WWTPs) globally (1, 4). In the context of modern WWTPs, nitrogen removal stands as a cornerstone, playing a pivotal role in reducing nitrogen compounds and thereby mitigating N2O emissions (5). However, paradoxically, the nitrogen removal process itself gives rise to a substantial portion of anthropogenic N2O emissions, constituting approximately 3% of the total N2O emissions (6).

Ammonia-oxidizing bacteria (AOB) and incomplete denitrifiers are identified as the primary N2O producers during the nitrification and denitrification process. Additionally, other nitrogen-related metabolisms influenced by environmental factors, such as fluctuating levels of ammonia (NH3), hydroxylamine (NH2OH), or nitrite (NO2−), have also been associated with N2O formation (6, 7). Notably, bacteria performing dissimilatory nitrate reduction to ammonium (DNRA), where nitrate (NO3−) and NO2− are reduced to ammonium (NH4+), also contribute to N2O formation. This process is primarily catalyzed by either the periplasmic cytochrome c nitrate reductase (nrfA) or the soluble siroheme-containing enzyme nitrite reductase (nirB). The amount of N2O released by DNRA-performing bacteria is uncertain but is considered to constitute only a marginal fraction of the overall N2O emissions compared to denitrification and AOB activities (8–11).

During the complete denitrification process, NO3− and NO2− are reduced to gaseous dinitrogen (N2) (10, 12). However, since the reduction of NO3− to NO2− is not exclusive to denitrification, the genes responsible for catalyzing this reaction (nap/nar) are not considered strictly denitrifying genes. The reduction of N2O to N2 is catalyzed by N2O reductase (nosZ) and is the best-known and most well-characterized biological sink for N2O in the biosphere (9). Recent studies have also identified another biological sink for N2O: N2O fixation (N2O to NH4+) in freshwater, linking a subcommunity of nitrogenase (nifH)-containing microorganisms to this process (13). The modular denitrification process can be carried out by a single organism reducing NO3−/NO2− to N2, referred to as “complete denitrifier,” or by a consortium of specialized cooperating microorganisms known as “partial denitrifiers.” These partial denitrifiers, defined as lacking one or more denitrifying genes, contribute to variations in the net N2O emission or consumption, depending on the microbial community’s genetic potential and environmental constraints (9). NosZ-containing non-denitrifying organisms are characterized by the absence of genes required for converting NO2− to gaseous nitric oxide (NO) (nirK/S) and subsequent NO to N2O (norB/C). They offer an alternative pathway for N2O reduction, making them essential for understanding and managing N2O emissions in WWTPs and other environments (6, 14). The recent evidence of microorganisms directly utilizing N2O as a nitrogen source through N2O fixation (N2O to NH4+) further highlights the complexity and significance of microbial interactions in N2O dynamics (13).

The NosZ protein phylogeny reveals two major clades (clades I and II), each generally containing a unique translocation pathway in the N-terminal of the gene. Most NosZ clade I employ the twin-arginine translocation (tat) pathway, transporting the folded protein across the membrane (15, 16). Clade I is further characterized by the presence of the accessory membrane-bound Fe-S flavoprotein gene (nosR) found in the nos gene cluster (9). The larger NosZ clade II utilizes the secretory pathway (sec) at the N-terminal, which facilitates the transport of the unfolded protein across the cytoplasmic membrane (17). Clade II is also distinguished by the presence of a membrane-spanning protein gene (nosB) found directly downstream of nosZ (9). This protein, in some cases, forms a NosB-NosC2 fusion protein anchored in the membrane by a N-terminal helix (18). Exceptions to these general rules of the secretory pathway and accessory genes do occur, and whether the translocated pathway is related to taxa or clade-specific functional significance is unclear (9). An example of this exemption is the nosB gene not being exclusive to clade II with certain exceptions within archaea and specific phyla, e.g., Chloroflexota (18). This clade II subgroup of Chloroflexota can also harbor the tat pathway instead of sec (16). The larger clade II nosZ group includes most non-denitrifying N2O reducers (16, 19). Organisms harboring nosZ clade I are most commonly found to also harbor both norB/C and nirK/S. While these also occur for clade II, it is more common for clade II to exclude other denitrification-affiliated genes (20). Genetic analysis has shown a distinct clustering of nosZ genes to phylogenetic groups and a relatively conserved gene structure, indicating low horizontal gene transfer, and it has, therefore, been hypothesized to hold potential as a reference gene for gene-based phylogenies (21). To date, the majority of primers targeting nosZ are based on short-read amplicons, aimed at achieving diverse phylogenetic coverage or targeting specific clades in unspecific environments, sometimes resulting in unspecific primers amplifying a multitude of other genes in the process (19, 22, 23). However, advancements in sequencing techniques, such as Oxford Nanopore, have made it feasible to design full-length, environmentally specific nosZ targeting primers. The primary limitation now lies in the specificity and quality of these designed primers (24). Despite the pivotal role of the nosZ-containing organisms, the diversity of the nosZ gene and the microorganisms harboring it remains largely unexplored in WWTPs. In this study, we investigate the phylogenetic and genetic diversity of nosZ in wastewater treatment plants, shedding light on the microorganisms harboring clade-specific nosZ genes. Our approach involves the identification of clade-specific nosZ genes in a non-targeted metagenomic approach and the design of environmentally specific long-fragment primers based on these genes to address microdiversity. This allows for the potential detection of novel N2O-reducing bacteria, as demonstrated in high-diversity full-scale WWTPs, based on high-quality near-full-length nosZ genes thereby advancing the detection and understanding of nosZ-harboring organisms.

MATERIALS AND METHODS

Pipeline for identification, alignment, and phylogeny of clade-specific nosZ gene sequences

To identify and annotate nosZ genes in activated sludge, 1,083 high-quality metagenome-assembled genomes (HQ MAGs) were obtained from 23 Danish full-scale WWTPs (25). The definition of an HQ MAG is based on the MIMAG draft requirement as >90% completeness; <5% contamination; the presence of 5S, 16S, and 23S rRNA genes; and ≥18 tRNA genes (26). These HQ MAGs were initially processed using Prodigal v2.6.2 to predict protein-coding genes (27). From there, the nucleotide sequences and translated protein sequences were identified and isolated. The identified nucleotide genes were mapped to National Center for Biotechnology Information (NCBI) GenBank v 234 (28) using the BLASTn algorithm, BLAST+ v2.12.0 (29) with a cutoff value of “1e−10,” and also annotated using KEGG elements (30) utilizing EnrichM v0.5.0 (31) to identify nosZ genes. Following the annotation of nucleotide sequences, the translated proteins were mapped using the BLASTp algorithm “1e−10” to high-quality full-length clade I (n = 20) and II (n = 46) NosZ protein sequences obtained from the Functional Gene Pipeline and Repository database version v9.9.11 (28). The translated proteins were subsequently mapped using the hmmsearch algorithm from HMMER v3.3.2 (32) to three full-length NosZ Hidden Markov Model files (HMMfiles) [one clade I (638aa) and two clade II (765 and 656aa)] (Text S1). The identified nosZ genes were all subset and manually screened for length (1,050–2,200 bp or 350–800aa). The respective taxonomy of the individual HQ MAGs was coupled to the identified nosZ genes from which they originated and used throughout.

To obtain additional clade-specific information on the identified NosZ proteins, the secretory pathway was identified through the presence of signal peptides associated with the sec/tat signal peptides, predicted using PRED-TAT (33). The presence of the clade-specific accessory genes, nosB and nosR, was verified using BLAST+ v2.12.0 (29). Reference genes for NosB-NosC (n = 5) and NosR (n = 1) (Text S1) were obtained from previous studies (18, 34), along with a manually curated NosR reference gene database from NCBI (n = 64) (Text S1) (35). The identified nosB were manually validated to be within seven genes up or downstream of nosZ and within three genes up or downstream for nosR (29). The nosZ genes identified were aligned using MUSCLE v5.0.1428 (36) and subsequently used to construct a maximum likelihood phylogenetic tree using IQ-TREE v2.0 (37), wherein the best-fit model was identified using the IQ-TREE model finder utilizing the minimized Bayesian information criterion score and 1,000 ultrafast bootstrap iterations (38). Consensus among different alignment strategies [MAFFT v7.490 (39) and MUSCLE v5.0.1428 (36)] and tree construction algorithms [maximum likelihood, unweighted pair group method with arithmetic mean (UPGMA), and neighbor joining] ensured systematic grouping of phylogenetic groups (data not shown), with MUSCLE and maximum likelihood trees chosen for their higher protein alignment accuracy (40). The tree was manually curated for misclassified genes.

The constructed trees were visualized using RStudio 2021.09.0+351 (41), R 4.1.2 (42), and the ggtree v3.2.0 package (43) and included taxonomy from the MiDAS 4 full-length v4.8.1 database (44). The HQ MAGs underwent genome-wide taxonomic classification, and their nosZ genes were then subset and assigned the taxonomic classification from the HQ MAG they originated from. This subset of taxonomically annotated nosZ genes served as a manually curated database throughout the study (30, 31).

Gene annotation

Using the predicted HQ MAGs translated protein-coding gene sequences from Prodigal v2.6.2 (27), the genes were annotated using EnrichM v0.5.0 (31) and KO annotations (30). A manually curated list of nitrogen metabolism-related KEGG modules was used to investigate the partial and complete KO modules found within the 1,083 HQ MAGs. Pathways were assumed present if 100% of the genes from the investigated list of KEGG modules were present (Table S1). MiDAS 4.8.1 taxonomy and nosZ clade-specific information were incorporated into HQ MAG annotations to allow insights into the physiology of relevant nosZ-related subgroups.

nosZ clade-specific primer design of taxonomic subgroups

Phylogenetic trees served as a reference to identify and subset distinct subclades based on nucleotide sequences. The subset nucleotide references were processed using CLC Genomic Workbench 20 (45) to generate primer combinations targeting multiple diverse taxonomic genera within the same phylum. Primers were generated based on the following criteria: primer melting temperature (tm) between 51°C and 58°C, GC content between 40% and 60%, allowed degeneracy of 3, allowed mismatch of 3, and target fragments longer than 1,000 bp. The nosZ primers (Table S2) were tested in silico against the HQ MAG gene sequences using EMBOSS v6.6.0 (46). The best primer combinations, targeting nosZ longer than 1,000 bp with <20% mismatch, and none-to-low ratio of other genes were tested by PCR (data not shown).

Sampling and DNA extraction

DNA from activated sludge, collected from Aalborg West WWTP (22-01-2020), were extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, USA). Aalborg West WWTP was chosen as a representative site for the nosZ primer test due to its long-term involvement in microbial community studies since 2006 and well-described, high-diversity, and relatively stable microbial community (47). DNA extraction followed the manufacturer’s instructions with minor alterations, using 478 µL sodium phosphate buffer and increased bead beating time to 4 × 40 s on FastPrep FP120 (MP Biomedicals, USA). DNA concentrations were measured with the Qubit dsDNA BR Assay Kit (Life Technologies, USA) on a Tecan Infinite F200 Pro (Tecan, Switzerland), and genomic DNA quality was assessed with a Genomic DNA ScreenTape and TapeStation 2200 (Agilent Technologies, USA).

Amplification and sequencing of nosZ primers

PCR was made in 25-µL reactions using 10 ng template genomic DNA, 0.4 µM forward and reverse primer, and LongAmp Taq Master Mix (New England Biolabs, USA). The thermocycling program used was as follows: initial denaturation at 95°C (3 min), 35 cycles of 95°C denaturation (30 s) followed by gradient annealing depending on the primers used average Tm, Tm – 5, and Tm – 10 (60 s), 65°C extension for 100 s, and a final extension at 65°C for 10 min. PCR amplicons were visualized using gel electrophoreses on a ChemiDoc MP Imaging system (Bio-Rad, USA), with a 1% agarose gel (DNA Pure Grade) (Electran, Belgium) and 1× TAE Buffer (AppliChem PanReac, Germany) stained with GelRed (EMD Millipore, USA).

Samples resulting in clear bands with the theoretical target length and no unspecific bands were sequenced using Oxford Nanopore sequencing technologies due to the ability to retrieve full-length nosZ amplicons. Sequencing was performed using a MinION Nanopore R9.4.1 flow cell (FLO-MIN106D) with the Ligation Sequencing Kit (SQK-LSK109) and PCR Barcoding kit (EXP-PBC096) (Oxford Nanopore Technologies, UK). The samples were pooled in equimolar concentration according to the manufacturer’s instructions, and the flow cell was run for 14 hours.

Bioinformatic processing and clade-specific identification of nosZ genes

Raw sequenced fast5 Nanopore data were basecalled using guppy v4.2.2 and the dna_r9.4.1_450bps_hac.cfg model from Oxford Nanopore, yielding 0.57 Gbp. Barcodes and adapters were trimmed and demultiplexed using Porechop v0.2.3 (parameters --discard_middle --require_two_barcodes --barcode_threshold 75) (48) and filtered for low-quality reads <80% basecall accuracy (Q-score >7) using NanoFilt v2.6.0 (49), keeping only reads between 800 and 2,500 bp. Filtered reads were clustered into operational taxonomic units (OTUs) and denoised using VSEARCH v2.13.4 (parameters --cluster_unoise --minsize 1 --centroids) (50). The amplicons were mapped to a manually curated database with the nosZ gene sequences using QIIME2 v2020.6 (51) and VSEARCH v2.13.4 (QIIME feature-classifier classify-consensus-vsearch) consensus taxonomy classifier (50). Unassigned reads were extracted using seqtk v1.3 subseq function and mapped to the GenBank database release 248 (35) using the BLASTn algorithm and an e-value cutoff of “1e−10.”

RESULTS

Profiling of nitrogen metabolism genes in WWTP HQ MAGs

The 1,083 HQ MAGs analyzed in this study originate from a study linking the community structure in activated sludge to their complete genetic repertoires. The HQ MAGs were collected from 23 Danish full-scale WWTPs and were estimated to account for approximately 30% of the relative community abundance, based on 16S rRNA relative community abundance (Fig. S1) (25). Annotating the nitrogen-related metabolisms of the HQ MAGs using KO modules and the EnrichM software revealed the presence of 503 nosZ sequences across 489 HQ MAGs. The custom pipeline, designed for clade-specific nosZ gene identification, outperformed EnrichM and KO annotations in terms of full-length gene specificity and provided additional information regarding secretory pathways and accessory nos genes. It successfully identified 443 unique (cutoff 100% sequence identity) clade-specific nosZ genes with a minimum length of 1,050 bp in 428 HQ MAGs (doi.org/10.5061/dryad.p5hqbzkwq), including 38 HQ MAGs missed by EnrichM and the KO annotations (Fig. 1). Among the total 1,083 HQ MAGs, 527 (48.7%) harbored nosZ genes. However, only 34 and 70 possessed all the genes necessary for the complete reduction of NO3− and NO2− to N2, respectively. Almost half of all HQ MAGs (517) contained norB or norC, indicating the potential to reduce NO to N2O (Fig. 2). However, among these HQ MAGs containing norB/C, 216 lacked a nosZ gene for further reduction, highlighting incomplete or partial denitrification pathways in over 20% of the 1,083 HQ MAGs.

Fig 1 Circular maximum likelihood taxonomic tree of all clade annotated nosZ sequences on phylum level longer than 350aa (n = 443), identified using the custom pipeline (see Results). The tip of each branch is colored by phylum with the presence/absence of indication of the respective accessory nosB/R genes and sec/tat secretory pathway. Ultrafast bootstrap values = 1,000. The scale bar denotes the amino acid substitution rate. The taxonomic classification is made through the MiDAS 4.8.1 database (44).

Fig 2 Barplot showing the abundances of nitrogen-related metabolisms and partial pathways in 1,083 HQ MAGs from activated sludge, based on the EnrichM data utilizing the KEGG orthology gene list found in Table S1.

Phylogenetic analysis of clade-specific nosZ genes revealed 27 clade I and 401 clade II nosZ-containing HQ MAGs (Fig. 1). From the clade II-containing HQ MAGs, 15 contained two nosZ genes, showcasing the diversity of their denitrifying potential within the community. Within the HQ MAGs containing two nosZ genes, no two genes were identical nor positioned within 45 genes up or downstream of each other. A complete set of denitrifying genes allowing for the complete reduction of NO2− to N2 was found in 13 out of the 27 clade I annotated HQ MAGs, with only 9 also containing a gene to reduce NO3−. Furthermore, among the clade II annotated HQ MAGs, 46 out of 401 HQ MAGs contained the respective genes for fully reducing NO2− to N2, and 17 of these had the genetic potential to fully reduce NO3−. Remarkably, 146 HQ MAGs were classified as non-denitrifying N2O reducers, defined by the presence of nosZ but lacking norB/C and nirK/S. Among these, two contained nosZ clade I and 144 clade II (Fig. S3 and S4). From these non-denitrifying N2O reducers, 21 (13.4%) contained the nap/nar genes; however, as the reduction of NO3− to NO2− is not limited to denitrification, these HQ MAGs were also considered non-denitrifying N2O reducers.

Denitrifying and non-denitrifying N2O reducers’ connection to DNRA

Of the 1,083 annotated HQ MAGs, 237 contained genes for the complete DNRA pathway, and 9 for complete assimilatory nitrate reduction (ANR) pathway genes. Within these HQ MAGs, genes capable of reducing NO2− to NH4+ were found in 432 HQ MAGs, with 417 via the DNRA pathway and 36 via the ANR pathway. Among these, 21 contained genes from both pathways. Out of the 432 HQ MAGs, 167 also contained nosZ, where 91 of these nosZ-containing HQ MAGs lacked norB/C. From the 27 nosZ clade I-containing HQ MAGs, 20 (74.1%) contained genes to reduce NO2− to NH4+, with 7 (26.9%) of these lacking norB/C. From the 401 nosZ clade II-containing HQ MAGs, 126 (31.4%) contained genes to reduce NO2− to NH4+, 70 (17.5%) of which lacked norB/C.

Taxonomic profiling of nosZ clade I-containing HQ MAGs

Phylogenetic diversity among the HQ MAGs identified as belonging to nosZ clade I was investigated using the MiDAS v4.8.1 reference database (44). A total of 27 HQ MAGs contained nosZ clade I, primarily within Pseudomonadota and a single hit of Myxococcota (Fig. S5). Within the clade I-containing HQ MAGs, 9 families (2 being taxonomic placeholders) and 12 genera (3 being taxonomic placeholders) were annotated. A list of reports describing denitrification capabilities of genera identified to contain nosZ clade I or II from this study is collected in Table S3.

Pseudomonadota, identified to contain both clade I and II annotated nosZ genes, accounted for 26 nosZ clade I HQ MAGs. These were divided into multiple subclades with diverse genetic sequences, with 24 out of 26 hits affiliated to the class Gammaproteobacteria. The largest family, Comamonadaceae (10 HQ MAGs), contained two genera previously affiliated with denitrification, namely, Ottowia (two HQ MAGs) and Rhodoferax (six HQ MAGs). Within the Rhodocyclaceae family (four HQ MAGs), two genera were affiliated with denitrification, Candidatus Accumulibacter (one HQ MAG) and Zoogloea (three HQ MAGs). From the Hahellaceae family (one HQ MAG), Hahella (one HQ MAG) was associated with denitrification and DNRA. The less abundant family Pseudomonadaceae (one HQ MAG) containing Pseudomonas (one HQ MAG) was also a known denitrifying organism. The single other annotated HQ MAGs affiliating within clade I was within Myxococcota, previously classified with the same phyla as Pseudomonadota, and was only annotated to the placeholder order UASB-TL25.

Taxonomic profiling of non-denitrifying N2O-reducing clade I-containing HQ MAGs

Examining the taxonomic diversity of the clade I non-denitrifying N2O reducers revealed two HQ MAGs (Fig. S3 and S4). Both HQ MAGs were affiliated with the phylum Pseudomonadota and the family Comamonadaceae and were annotated as belonging to the genera Limnohabitans and Rhodoferax. Additionally, both HQ MAGs contained nap/nar and nosR genes.

Taxonomic profiling of nosZ clade II-containing HQ MAGs

The nosZ clade II-containing HQ MAGs exhibited higher diversity at the phylum level compared to clade I, constituting a substantial portion of the data set (401 of 1,083 HQ MAGs) (Fig. S6). Within the clade II-containing HQ MAGs, 15 contained two nosZ genes, summing up to 416 nosZ clade II genes. These HQ MAGs were affiliated with Bacteroidota (10 HQ MAGs), Latescibacterota (2 HQ MAGs), Acidobacteriota (1 HQ MAG), and Chloroflexota (1 HQ MAG). For simplicity, HQ MAGs containing multiple nosZ genes are henceforth referred to as single HQ MAGs unless otherwise specified.

Bacteroidota dominated the nosZ clade II-containing HQ MAGs, comprising 70.6% of the total, followed by Pseudomonadota (6.5%), Chloroflexota (5.5%), Myxococcota (4.2%), Gemmatimonadota (4%), Latescibacterota (3.5%), Planctomycetota (1.7%), Acidobacteriota (1.2%), Spirochaetota (1.2%), and six other phyla, each with a single HQ MAG. In total, 66 families and 144 genera were annotated among clade II-containing HQ MAGs, with 37 of the families and 116 genera being placeholders (Table S3). Within the predominant phylum Bacteroidota (283 HQ MAGs), the order Chitinophagales (154 HQ MAGs) contained the known denitrifying genera Ferruginibacter (20 HQ MAGs), Terrimonas (18 HQ MAGs), and Phaeodactylibacter (2 HQ MAGs). In addition to the known denitrifiers, the genus Haliscomenobacter (4 HQ MAGs) has been associated with the presence of nosZ. The family Saprospiraceae (88 HQ MAGs) has also previously been associated with denitrification. Other orders, like Sphingobacteriales (46 HQ MAGs) and Flavobacteriales (45 HQ MAGs), also contained known denitrifiers. From the less abundant Ignavibacteriales order (10 HQ MAGs), Ignavibacteriaceae (4 HQ MAGs) was known to be affiliated with both nosZ and the ability to reduce NO3− via the DNRA pathway. The 26 clade II annotated Pseudomonadota HQ MAGs were predominantly in the class Gammaproteobacteria (23 HQ MAGs) and a few in Alphaproteobacteria (3 HQ MAGs). Noteworthy genera affiliated with denitrification were found in Rhodocyclaceae, with Dechloromonas (5 HQ MAGs), Denitratisoma (3 HQ MAGs), Ferribacterium (1 HQ MAG), and Sulfuritalea (5 HQ MAGs). Affiliated with the Comamonadaceae family were two other denitrifying genera, namely, Rhodoferax (1 HQ MAG) and Sphaerotilus (1 HQ MAG). The 22 clade II annotated Chloroflexota HQ MAGs were primarily in the Anaerolineae class (17 HQ MAGs). Among these, Candidatus Amarolinea (2 HQ MAGs) was associated with DNRA and non-denitrifying N2O reduction. In the class Chloroflexia (1 HQ MAG), Kouleothrix was previously associated with denitrifying capabilities. A total of four HQ MAGs were identified to contain the tat pathway generally affiliated with clade I (16). All four HQ MAGs were affiliated to the class OLB14, and only one family placeholder was identified, namely, midas_f_731 with two genera: midas_g_731 (3 HQ MAGs) and midas_g_1314 (1 HQ MAG). Myxococcota (17 HQ MAGs) featured a single genus annotation, Haliangium (4 HQ MAGs), associated with denitrification within the Haliangiaceae family. Gemmatimonadota (16 HQ MAGs) belonged to the Gemmatimonadaceae family, with one HQ MAG annotated as Gemmatimonas, known to be associated with nosZ. Acidobacteriota (5 HQ MAGs) showed potential as non-denitrifying N2O reducers, particularly within the orders Thermoanaerobaculales and Vicinamibacterales, known to be associated with nosZ and DNRA, but not complete denitrification. All five HQ MAGs affiliated with Spirochaetota were identified within the same genus Leptospira, which had previously been associated with non-denitrifying N2O reduction.

Taxonomic profiling of non-denitrifying N2O-reducing clade II-containing HQ MAGs

Among nosZ clade II-containing non-denitrifying N2O reducers, 144 HQ MAGs were identified (Fig. S3 and S4). These were predominantly affiliated with Bacteroidota (77.1%), followed by Chloroflexota (11.1%), Gemmatimonadota (2.8%), Planctomycetota (2.8%), Acidobacteriota (2.1%), Myxococcota (1.4%), and four less abundant phyla, each with a single hit. At the genus level, 64 taxonomic placeholders were found, and only 12 nomenclature genus-level annotations. HQ MAGs containing the nap/nar genes and the DNRA genes for the reduction of NO2− to NH4+ within the clade II non-denitrifying N2O reducers were not specifically allocated to a single phylum. Out of the 144 HQ MAGs, 16 contained the nap/nar genes to reduce NO3− to NO2−, distributed across various phyla.

Phylogenetic affiliation of amplified nosZ genes

For the investigation of Aalborg West WWTP sludge (selected for its well-characterized status, high microbial diversity, and stable community), 52 primer combinations were systematically assessed. The resulting amplicons yielded 39,592 reads using the custom-designed primers (Table S2). All tested primer combinations produced amplicons with the expected lengths, and 35 resulted in sequences mapped to the manually curated nosZ clade-specific database (Fig. 3). Utilizing the custom-designed clade-specific nosZ-targeting primers and the 443 identified nosZ genes as a reference database, 268 nosZ clade I reads and 24,136 clade II reads were taxonomically annotated. The effectiveness of these primers exhibited substantial variability, with a pronounced preference for nosZ clade II over clade I, with combinations targeting Bacteroidota yielding the highest number of OTUs. From the clade I-targeting primers, two primer combinations produced reads mapped to two distinct clade-specific genera: specifically Rhodoferax, a recognized denitrifying genus, and the placeholder BD1-7_clade, described at the family level of Spongiibacteraceae. Unfortunately, the development of universal primer combinations capable of amplifying a broader taxonomic diversity beyond one to two phyla and a maximum of five annotated genera proved unattainable. Notably, two primer combinations, F4-CII - R2-CII (designed for targeting Latescibacterota) and F7-CII - R5-CII (Pseudomonadota) exhibited cross-reactivity with Bacteroidota, targeting the genus Terrimonas. Additionally, the primer combination F6-CII - R4-CII (Pseudomonadota) unintentionally targeted the placeholder genus midas_g_753 within Latescibacterota. The nosZ clade II-targeting primers exhibited versatility, hitting 38 different genera to varying degrees, encompassing known denitrifiers such as Dechloromonas, Denitratisoma, Sulfuritalea, Terrimonas, and Phaeodactylibacter. Other genera, including Haliscomenobacter and Ferruginibacter, known for their affiliation with nosZ and varying denitrification abilities, were identified. Two genera, Lacihabitans and Candidatus Epiflobacter, previously unaffiliated with denitrification or nosZ, were also hit. The remaining 29 genera were categorized as MiDAS placeholders. The prominent amplicons, as indicated by the number of primer hits from clade II, were annotated as Haliscomenobacter, Lacihabitans, and Terrimonas, commonly found in Danish WWTPs (44). Among the clade II targeting primer combinations, seven resulted in a single genus-level annotation, while the remaining combinations hit between two and nine genera annotations per combination (Fig. 3).

Fig 3 Maximum likelihood taxonomic tree displaying the amplified nosZ sequences mapped to the manually curated nosZ clade-specific database. The tree is constructed with ultrafast bootstrap values = 1,000 and a scale bar denoting the amino acid substitution rate. Each branch’s tip represents the secretory pathway type, tat (blue) and sec (brown), of reference genera color-coded according to their respective phyla. Additionally, a joined heatmap illustrates amplicon hits mapped to the reference. The axis names of the heatmap indicate the forward and reverse primers, the nosZ clade they target, and the theoretical length of the amplicon in base pairs (see Table S2 for additional information).

Following mapping to the manually curated clade-specific nosZ database, unassigned reads underwent further analysis using BLASTn (e-value 1e−10), mapping to the NT GenBank v248 database. The subsequent mapping revealed that 5,148 unassigned reads were mapped to various genes, with 2,566 mapped to partial or full-length nosZ genes. This mapping encompassed 37 of the evaluated primer combinations.

DISCUSSION

In this study, a comprehensive analysis of 1,083 HQ MAGs from 23 Danish WWTPs (25) was conducted to identify clade-specific full-length genes of nosZ and their taxonomic distribution, utilizing the MiDAS 4.8.1 database (44). The data have been reported to represent around 30% of the microbial community, with almost half (515) of the organisms observed in at least one sample with a relative read abundance above >0.1% of the total population (25). This data set represents the most complete HQ MAG database from activated sludge published to date and provides valuable insight into the most common organisms in modern WWTPs.

Phylogeny of nosZ

The observation that 48.7% of all investigated HQ MAGs contain at least one copy of the nosZ gene highlights the prevalence, distribution, and potentially essential role of nitrous oxide-reducing bacteria within WWTPs. Notably, the distribution of clade I (6.3%) and clade II (93.7%) nosZ resembles the tendencies reported in previous studies from soil (9, 52). Further investigation into HQ MAGs containing multiple nosZ genes revealed heterogeneous genetic sequences, indicating potential genetic diversity within nosZ, even within the same species. The purpose of multiple heterogenous nosZ genes remains unclear, as does their origin, which could result from horizontal gene transfer, mutations, gene duplication, diversification, or other mechanisms (21, 53). The phylogenetic relationship of nosZ clades, their respective translocation pathway, and the accessory nos gene cluster has been extensively studied (9, 54). Despite the typical association of the tat pathway and accessory nosR gene with clade I, and the sec pathway with the accessory nosB gene with clade II, exceptions have been reported, particularly within archaea or Chloroflexota (9). The structural alignment, shown in Fig. S2, indicates that some Chloroflexota contain the tat pathway, despite their relation to clade II. This suggests a complex interplay between nosZ clades, translocation pathways, and accessory gene clusters, highlighting the need for further research to elucidate the mechanisms driving nosZ diversity and function in microbial communities.

Taxonomic profiling of clade I and clade II

The taxonomic profiling of nosZ found in activated sludge revealed a diverse microbial community predominantly affiliated with Bacteroidota (66.1%), followed by Pseudomonadota (12.2%), Chloroflexota (5.1%), Myxococcota (4.2%), Gemmatimonadota (3.7%), Latescibacterota (3.3%), and less abundant phyla (5.4%). These findings align with the typical microbial composition observed in Danish WWTPs (9, 16). This implies a consistent preference of the nosZ-containing community across different environments, likely reflecting the natural distribution of the gene within the core microbial community of the specific environment (47, 55). Moreover, it could suggest a widespread energetic advantage associated with having an active nosZ enzyme. The largest nosZ-containing taxonomic group, Bacteroidota, was affiliated with clade II, consistent with previous observations (9). Despite their classification as nosZ clade II denitrifiers, many of these HQ MAGs also carried other denitrifying genes, such as norB/C and nirK/S. This diversity suggests the potential for partial denitrification or non-denitrifying N2O reduction, indicating the key role of these organisms in activated sludge as potential N2O sinks (16, 54). Phylogenetic analysis revealed genetic variation within Pseudomonadota, with affiliations to both nosZ clade I and clade II. The different clades displayed a clear association between the genetic structure, the tat/sec translocation pathway, and surrounding nosB/R genes, with both exhibiting genes typical of denitrification. This underscores their primary related role as complete denitrifiers, further supported by the numerous described genera within clade I and Pseudomonadota in general. In contrast, Chloroflexota did not adhere to the typical clade-specific trend with secretory pathway and accessory genes. Analysis of the phylogenetic relationship (Fig. S2) revealed that the nosZ genes within the phylum Chloroflexota exhibited a genetic structure similar to other clade II genes. However, four genes (highlighted in green in Fig. S2) containing the tat pathway, commonly associated with clade I, also contained the accessory nosB gene generally associated with clade II. Consequently, the phylum does not conform to the typical clade-associated trends for nosZ. Despite this, all identified Chloroflexota in this study were annotated as clade II based on structural alignment. The study emphasizes the importance of investigating the sec/tat secretory pathway, accessory nos genes, and structural alignment to other nosZ genes for accurate classification (9, 16, 56). Further exploration of denitrifying genes within the HQ MAGs annotated as Chloroflexota revealed that no HQ MAG contained norB/C genes (Fig. S2) and that approximately 25% contained nirK or DNRA/ANR genes, suggesting a denitrifying repertoire more akin to clade II than clade I, regardless of the sec/tat pathway. The HQ MAGs identified within the largely undescribed Latescibacterota phylum contained nosZ clade II, highlighting specialized nitrogen-related metabolic potentials for the reduction of NO and N2O, and the genes for NO2− reduction to NH4+ via DNRA. Notably, none of the HQ MAGs contained nirK/S, emphasizing a unique metabolic profile of this phylum. Overall, the high abundance of organisms harboring nosZ, particularly those lacking the preceding norB/C genes for the conventional denitrifying pathway, may indicate multiple functions. This potential function may encompass the utilization of excess or freely available N2O to access an additional energy source, thus serving as a beneficial gene for net energy conservation or acting as an electron sink. Additionally, nosZ might facilitate the conversion of residual N2O from bacteria performing reduction of NO2− to NH4+ via DNRA or ANR (8, 57, 58).

Distribution of the nosZ gene and other related metabolic pathways in the HQ MAGs

The distribution of clade-specific nosZ genes within the 1,083 HQ MAGs exhibits a similar distribution with nosZ clade II (93.7%) displaying greater diversity compared to clade I (6.3%) as previously described (9). Although this distribution is consistent, a more detailed taxonomic classification of the organisms harboring nosZ clade I was observed within this data set. This could result from cultivation biases associated with the species containing nosZ when first discovered (16, 59). Despite its larger and more diverse nature, nosZ clade II contains fewer described organisms at the genus level. This extensive novelty and number of unique nosZ clade II genes demonstrate substantial novelty within this group, suggesting the presence of numerous species still to be characterized.

The metabolic capabilities related to the clade-specific nosZ genes align with previous reports, indicating that clade I is more commonly associated with denitrification (39.4% clade I and 11.6% clade II complete denitrifiers) and other nitrogen-related pathways, such as DNRA and ANR (54.5% DNRA, 27.3% ANR clade I), compared to clade II (31.6% DNR, 5.8% ANR) (60). Results from this study show co-occurrence of DNRA genes found among non-denitrifying N2O reducers, with 49 of the 146 reducers harboring DNRA genes and only 8 possessing ANR genes. DNRA and ANR pathways, in contrast to denitrification, are undesired in WWTPs as they convert NO2− back to NH4+ rather than removing it from the system as gaseous N2 (61). The partial denitrifiers containing nirK/S but lacking norB/C constituted 8 of the 27 HQ MAGs affiliating with nosZ clade I and only 40 of the 401 with nosZ clade II, further strengthening the observation that nosZ clade I is more commonly associated with denitrification than clade II, thus true for partial denitrifiers as previously postulated (19).

Custom-designed nosZ targeting primer

The custom-designed primer sets, strategically crafted to target specific near-full-length genetically or taxonomically similar nosZ subclades, allow for the enhanced detection and characterization of known environmentally specific nosZ genes in WWTPs. While multiple studies have focused on nosZ targeting primers and their subclades, the vast majority have concentrated on short-read targeting, primarily due to previous restrictions with sequencing technologies (19, 22, 23). While developing universal primers for near-full-length amplification of nosZ was not possible using this strategy, the focus was placed on targeting environmentally diverse groups spanning multiple taxa to address the microdiversity of the different nosZ clades. The variable regions of the nosZ gene across these clades allow for distinct taxonomic identification, which makes the designed primers valuable candidates for supplementing previously published primers for more comprehensive investigations into a wider selection of nosZ genes (19, 21). For clade I-targeting primers, HQ MAGs identified as Pseudomonadota were selected as reference clades due to their taxonomic and genetic sequence similarity, containing semi-conserved regions. Concerning clade II-targeting primers, Bacteroidota emerged as the primary target, suggesting that the primer designed to target Acidobacteriota and Gemmatimonadota did not effectively target the intended phyla. A closer examination of HQ MAGs originating from Aalborg West WWTP revealed the expected presence of both phyla in the sludge. It, therefore, appears that the primers targeting phyla other than Bacteroidota had lower specificity to the targeted microorganisms or were present in low concentrations in the sample. Notably, from the nosZ clade II-targeting primers, seven combinations hit more than five different genera, demonstrating promising potential as Bacteroidota-targeting primers. The near-full-length amplicons produced with the newly designed primers revealed a large number of uncharacterized microdiversity within denitrification nosZ. These designed primers were also all demonstrated to exhibit high specificity to nosZ, providing superior sequence information compared to previous primers with shorter amplicon lengths (62). An optimization strategy for the primers entails considering multiplexing, as various primer combinations have shown the ability to amplify beyond the target genes across different genera and even phyla. Multiplexing primers known to amplify multiple genera could enhance the detection of organisms in a single run, thereby broadening the scope of the analysis. Further validation of primer specificity could involve incorporating additional samples from other WWTPs. However, the primary focus of designing and applying the new primers was to validate taxonomically specific full-length nosZ amplification, as evidenced by both in silico and in vitro tests. This validation encompassed some of the primary taxonomic targets, known to be prevalent genera in Danish WWTPs; hence, no additional plants were included in the in vitro test (44).

The N2O sink potential of the investigated HQ MAGs in activated sludge

The investigated HQ MAGs in this study highlight the potential of both described and undescribed organisms as N2O sinks, encompassing both clade I and II-containing organisms (56). Theoretically, these organisms could be harnessed by favoring their presence and/or activity. However, the majority of these relatively abundant organisms in WWTPs remain largely undescribed, with the majority of their genetic repositories unknown (44). From this study, the population of the non-denitrifying N2O reducers constitutes around 13.5% of the investigated community, suggesting that a significant portion of the WWTP community has the potential to act as a N2O sink and limit emissions, with especially the clade II-containing Chloroflexota and Bacteroidota emerge as abundant populations. It is important to note that the total 1,083 HQ MAGs analyzed were previously estimated to represent 30% of the activated sludge population (25). Due to the diverse representatives and frequent occurrence of these organisms in Danish WWTPs, a comprehensive exploration of their complete genetic repositories is warranted. Such investigations hold the potential to enhance our understanding of how these organisms may help reduce the carbon footprint by lowering greenhouse gas emissions from WWTPs. Notably, the developed primers offer the opportunity to target taxonomically specific organisms of interest and, thus, assist in specialized analysis of nosZ presence or distribution. This knowledge could be invaluable in designing pilot studies aimed at promoting the presence and/or activity of these organisms. The utilization of these organisms as potential N2O sinks aligns with the broader goal of optimizing wastewater treatment processes and mitigating greenhouse gas emissions. Further research into the genetic makeup and influencing factors associated with these HQ MAGs is crucial for advancing our strategies in wastewater management. This includes a broader examination of other metabolic pathways and general functions found within them. Such comprehensive analysis could serve as the foundation for targeted studies aimed at investigating various organisms or metabolic functions of interest.

Conclusion

In this study, we developed a robust pipeline utilizing 1,083 previously published HQ MAGs obtained from 23 full-scale WWTPs in Denmark to identify near-full-length nosZ sequences. This approach provided an in-depth analysis of the genetic repertoire of nosZ-containing bacteria within wastewater treatment systems.

Our results showed that nearly 48% of the HQ MAGs contained a nosZ gene, demonstrating its widespread distribution across various taxa. Additionally, we observed a high co-occurrence of bacteria containing genes associated with DNRA and nosZ. HQ MAGs featuring clade I nosZ gene exhibited a notable association with other denitrifying genes and complete denitrification, whereas clade II contained a majority of partial denitrifiers and a much lower abundance of complete denitrifiers. There was also a strong association between the secretory pathway and accessory nos genes to clade, with tat and nosR linked to clade I, and sec and nosB to clade II, except for a subgroup of Chloroflexota containing tat and nosB. Non-denitrifying N2O reducers were primarily found among clade II, with only two clade I-containing HQ MAGs identified as non-denitrifying N2O reducers. These findings indicate that the role of nosZ, especially among clade II, could serve as an additional energy-producing pathway for a substantial portion of the organisms inhabiting activated sludge.

ACKNOWLEDGMENTS

The authors would like to express their gratitude to Dr. Caitlin Margaret Singleton for help with data processing and access to the MGP1000 data and Prof. Per Halkjær Nielsen for access to the MGP1000 data and academic discussions and guidance.

Financial support for this research was provided by the Independent Research Fund Denmark Technology and Production Sciences (9041-00367B).

DATA AVAILABILITY

Genomic data used in this study have been published previously (25) and were accessed from the National Center for Biotechnology Information (NCBI) BioProject database with the accession number PRJNA629478. The sequence data presented in this study were deposited in the European Nucleotide Archive (ENA) database under accession number PRJEB58728.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/aem.02177-23.

10.1128/aem.02177-23.SuF1 Text S1 aem.02177-23-s0001.txt

nosR and nosB supplemental fasta files.

10.1128/aem.02177-23.SuF2 Supplemental figures aem.02177-23-s0002.pdf

Figures S1 to S6.

10.1128/aem.02177-23.SuF3 Table S1 aem.02177-23-s0003.xlsx

Taxonomy of all 1,083 HQ MAGs analyzed.

10.1128/aem.02177-23.SuF4 Table S2 aem.02177-23-s0004.docx

List of custom-designed, clade-specific nosZ primers.

10.1128/aem.02177-23.SuF5 Table S3 aem.02177-23-s0005.docx

List of clade-specific nosZ-containing genera and their nitrogen-related metabolism.

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.
==== Refs
REFERENCES

1 Tian H, Xu R, Canadell JG, Thompson RL, Winiwarter W, Suntharalingam P, Davidson EA, Ciais P, Jackson RB, Janssens-Maenhout G, et al. . 2020. A comprehensive quantification of global nitrous oxide sources and sinks. Nature 586 :248–256. doi:10.1038/s41586-020-2780-0 33028999
2 Lycus P, Lovise Bøthun K, Bergaust L, Peele Shapleigh J, Reier Bakken L, Frostegård Å. 2017. Phenotypic and genotypic richness of denitrifiers revealed by a novel isolation strategy. ISME J 11 :2219–2232. doi:10.1038/ismej.2017.82 28696424
3 Clarke L, Jiang K, Akimoto K, Babiker M, Blanford G, Fisher-Vanden K, Hourcade J-C, Krey V, Kriegler E, Löschel A, McCollum D, Paltsev S, Rose S, Shukla PR, Tavoni M, van der Zwaan BCC, van Vuuren DP. 2015. Climate change 2014 mitigation of climate change. Contribution of working group III to the fifth assessment report of the intergovernmental panel on climate change. change, intergovernmental panel on climate, p 418–490
4 Daelman MRJ, van Voorthuizen EM, van Dongen LGJM, Volcke EIP, van Loosdrecht MCM. 2013. Methane and nitrous oxide emissions from municipal wastewater treatment - results from a long-term study. Water Sci Technol 67 :2350–2355. doi:10.2166/wst.2013.109 23676409
5 Schmidt I, Sliekers O, Schmid M, Bock E, Fuerst J, Kuenen JG, Jetten MSM, Strous M. 2003. New concepts of microbial treatment processes for the nitrogen removal in wastewater. FEMS Microbiol Rev 27 :481–492. doi:10.1016/S0168-6445(03)00039-1 14550941
6 Ren Y, Ngo HH, Guo W, Ni BJ, Liu Y. 2019. Linking the nitrous oxide production and mitigation with the microbial community in wastewater treatment: a review. Bioresour Technol Rep 7 :100191. doi:10.1016/j.biteb.2019.100191
7 Todt D, Dörsch P. 2016. Mechanism leading to N2O production in wastewater treating biofilm systems. Rev Environ Sci Biotechnol 15 :355–378. doi:10.1007/s11157-016-9401-2
8 Stremińska MA, Felgate H, Rowley G, Richardson DJ, Baggs EM. 2012. Nitrous oxide production in soil isolates of nitrate-ammonifying bacteria. Environ Microbiol Rep 4 :66–71. doi:10.1111/j.1758-2229.2011.00302.x 23757231
9 Hallin S, Philippot L, Löffler FE, Sanford RA, Jones CM. 2018. Genomics and ecology of novel N2O-reducing microorganisms. Trends Microbiol 26 :43–55. doi:10.1016/j.tim.2017.07.003 28803698
10 Schreiber F, Wunderlin P, Udert KM, Wells GF. 2012. Nitric oxide and nitrous oxide turnover in natural and engineered microbial communities: biological pathways, chemical reactions, and novel technologies. Front Microbiol 3 :372. doi:10.3389/fmicb.2012.00372 23109930
11 Wang H, Gunsalus RP. 2000. The nrfA and nirB nitrite reductase operons in Escherichia coli are expressed differently in response to nitrate than to nitrite. J Bacteriol 182 :5813–5822. doi:10.1128/JB.182.20.5813-5822.2000 11004182
12 Simon J, Klotz MG. 2013. Diversity and evolution of bioenergetic systems involved in microbial nitrogen compound transformations. Biochim Biophys Acta Bioenerg 1827 :114–135. doi:10.1016/j.bbabio.2012.07.005
13 Si Y, Zhu Y, Sanders I, Kinkel DB, Purdy KJ, Trimmer M. 2023. Direct biological fixation provides a freshwater sink for N2O. Nat Commun 14 :6775. doi:10.1038/s41467-023-42481-2 37880204
14 Conthe M, Lycus P, Arntzen MØ, Ramos da Silva A, Frostegård Å, Bakken LR, Kleerebezem R, van Loosdrecht MCM. 2019. Denitrification as an N2O sink. Water Res 151 :381–387. doi:10.1016/j.watres.2018.11.087 30616050
15 Pohlschröder M, Giménez MI, Jarrell KF. 2005. Protein transport in Archaea: Sec and twin arginine translocation pathways. Curr Opin Microbiol 8 :713–719. doi:10.1016/j.mib.2005.10.006 16257258
16 Jones CM, Graf DRH, Bru D, Philippot L, Hallin S. 2013. The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink. ISME J 7 :417–426. doi:10.1038/ismej.2012.125 23151640
17 Berks BC, Sargent F, Palmer T. 2000. The Tat protein export pathway. Mol Microbiol 35 :260–274. doi:10.1046/j.1365-2958.2000.01719.x 10652088
18 Hein S, Witt S, Simon J. 2017. Clade II nitrous oxide respiration of Wolinella succinogenes depends on the NosG, -C1, -C2, -H electron transport module, NosB and a Rieske/cytochrome bc complex. Environ Microbiol 19 :4913–4925. doi:10.1111/1462-2920.13935 28925551
19 Chee-Sanford JC, Connor L, Krichels A, Yang WH, Sanford RA. 2020. Hierarchical detection of diverse Clade II (atypical) nosZ genes using new primer sets for classical- and multiplex PCR array applications. J Microbiol Methods 172 :105908. doi:10.1016/j.mimet.2020.105908 32234512
20 Graf DRH, Jones CM, Hallin S. 2014. Intergenomic comparisons highlight modularity of the denitrification pathway and underpin the importance of community structure for N2O emissions. PLoS One 9 :e114118. doi:10.1371/journal.pone.0114118 25436772
21 Palmer K, Drake HL, Horn MA. 2009. Genome-derived criteria for assigning environmental narG and nosZ sequences to operational taxonomic units of nitrate reducers. Appl Environ Microbiol 75 :5170–5174. doi:10.1128/AEM.00254-09 19502444
22 Maheshwari A, Jones CM, Tiemann M, Hallin S. 2023. Carbon substrate selects for different lineages of N2O reducing communities in soils under anoxic conditions. Soil Biol Biochem 177 :108909. doi:10.1016/j.soilbio.2022.108909
23 Scala DJ, Kerkhof LJ. 1998. Nitrous oxide reductase (nosZ) gene-specific PCR primers for detection of denitrifiers and three nosZ genes from marine sediments. FEMS Microbiol Lett 162 :61–68. doi:10.1111/j.1574-6968.1998.tb12979.x 9595664
24 MacKenzie M, Argyropoulos C. 2023. An introduction to nanopore sequencing: past, present, and future considerations. Micromachines (Basel) 14 :459. doi:10.3390/mi14020459 36838159
25 Singleton CM, Petriglieri F, Kristensen JM, Kirkegaard RH, Michaelsen TY, Andersen MH, Kondrotaite Z, Karst SM, Dueholm MS, Nielsen PH, Albertsen M. 2021. Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing. Nat Commun 12 :2009. doi:10.1038/s41467-021-22203-2 33790294
26 Bowers RM, Kyrpides NC, Stepanauskas R, Harmon-Smith M, Doud D, Reddy TBK, Schulz F, Jarett J, Rivers AR, Eloe-Fadrosh EA, et al. . 2017. Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea. Nat Biotechnol 35 :725–731. doi:10.1038/nbt.3893 28787424
27 Hyatt D, Chen G-L, Locascio PF, Land ML, Larimer FW, Hauser LJ. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11 :119. doi:10.1186/1471-2105-11-119 20211023
28 Fish JA, Chai B, Wang Q, Sun Y, Brown CT, Tiedje JM, Cole JR. 2013. FunGene: the functional gene pipeline and repository. Front Microbiol 4 :291. doi:10.3389/fmicb.2013.00291 24101916
29 Altschul SF, Gish W. 1996. Local alignment statistics. Meth Enzymol 266 :460–480. doi:10.1016/S0076-6879(96)66029-7
30 Kanehisa M, Sato Y, Furumichi M, Morishima K, Tanabe M. 2019. New approach for understanding genome variations in KEGG. Nucleic Acids Res 47 :D590–D595. doi:10.1093/nar/gky962 30321428
31 Boyd JA, Woodcroft BJ, Tyson GW. 2019. Comparative genomics using EnrichM. https://github.com/geronimp/enrichM.
32 Eddy SR. 1995. Multiple alignment using hidden Markov models. Proc Int Conf Intell Syst Mol Biol 3 :114–120.7584426
33 Bagos PG, Nikolaou EP, Liakopoulos TD, Tsirigos KD. 2010. Combined prediction of Tat and Sec signal peptides with hidden Markov models. Bioinformatics 26 :2811–2817. doi:10.1093/bioinformatics/btq530 20847219
34 Velasco L, Mesa S, Xu C-A, Delgado MJ, Bedmar EJ. 2004. Molecular characterization of nosRZDFYLX genes coding for denitrifying nitrous oxide reductase of Bradyrhizobium japonicum. Antonie Van Leeuwenhoek 85 :229–235. doi:10.1023/B:ANTO.0000020156.42470.db 15028871
35 Sayers EW, Bolton EE, Brister JR, Canese K, Chan J, Comeau DC, Connor R, Funk K, Kelly C, Kim S, Madej T, Marchler-Bauer A, Lanczycki C, Lathrop S, Lu Z, Thibaud-Nissen F, Murphy T, Phan L, Skripchenko Y, Tse T, Wang J, Williams R, Trawick BW, Pruitt KD, Sherry ST. 2022. Database resources of the national center for biotechnology information. Nucleic Acids Res 50 :D20–D26. doi:10.1093/nar/gkab1112 34850941
36 Edgar RC. 2004. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5 :113. doi:10.1186/1471-2105-5-113 15318951
37 Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, Lanfear R. 2020. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol 37 :1530–1534. doi:10.1093/molbev/msaa015 32011700
38 Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. 2018. UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol 35 :518–522. doi:10.1093/molbev/msx281 29077904
39 Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30 :3059–3066. doi:10.1093/nar/gkf436 12136088
40 Edgar RC. 2022. Muscle5: high-accuracy alignment ensembles enable unbiased assessments of sequence homology and phylogeny. Nat Commun 13 :6968. doi:10.1038/s41467-022-34630-w 36379955
41 RStudio Team. 2015. RStudio: integrated development environment for R. RStudio, Inc., Boston, MA.
42 R Core Team. 2022. R: a language and environment for statistical computing. 4.1.2. R Foundation for Statistical Computing, Vienna, Austria
43 Yu G. 2020. Using ggtree to visualize data on tree-like structures. Curr Protoc Bioinformatics 69 :e96. doi:10.1002/cpbi.96 32162851
44 Dueholm MKD, Nierychlo M, Andersen KS, Rudkjøbing V, Knutsson S, Albertsen M, Nielsen PH, MiDAS Global Consortium. 2022. MiDAS 4: a global catalogue of full-length 16S rRNA gene sequences and taxonomy for studies of bacterial communities in wastewater treatment plants. Nat Commun 13 :1908. doi:10.1038/s41467-022-29438-7 35393411
45 QIAGEN. 2022. CLC genomics workbench. 20. Aarhus, Denmark QIAGEN
46 Rice P, Longden I, Bleasby A. 2000. EMBOSS: the European molecular biology open software suite. Trends Genet 16 :276–277. doi:10.1016/s0168-9525(00)02024-2 10827456
47 Saunders AM, Albertsen M, Vollertsen J, Nielsen PH. 2016. The activated sludge ecosystem contains a core community of abundant organisms. ISME J 10 :11–20. doi:10.1038/ismej.2015.117 26262816
48 Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Completing bacterial genome assemblies with multiplex MinION sequencing. Microb Genom 3 :e000132. doi:10.1099/mgen.0.000132 29177090
49 De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. 2018. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics 34 :2666–2669. doi:10.1093/bioinformatics/bty149 29547981
50 Rognes T, Flouri T, Nichols B, Quince C, Mahé F. 2016. VSEARCH: a versatile open source tool for metagenomics. PeerJ 4 :e2584. doi:10.7717/peerj.2584 27781170
51 Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F, et al. . 2019. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37 :852–857. doi:10.1038/s41587-019-0209-9 31341288
52 Orellana LH, Rodriguez-R LM, Higgins S, Chee-Sanford JC, Sanford RA, Ritalahti KM, Löffler FE, Konstantinidis KT. 2014. Detecting nitrous oxide reductase (NosZ) genes in soil metagenomes: method development and implications for the nitrogen cycle. mBio 5 :e01193-14. doi:10.1128/mBio.01193-14 24895307
53 Birchler JA, Yang H. 2022. The multiple fates of gene duplications: deletion, hypofunctionalization, subfunctionalization, neofunctionalization, dosage balance constraints, and neutral variation. Plant Cell 34 :2466–2474. doi:10.1093/plcell/koac076 35253876
54 Sanford RA, Wagner DD, Wu Q, Chee-Sanford JC, Thomas SH, Cruz-García C, Rodríguez G, Massol-Deyá A, Krishnani KK, Ritalahti KM, Nissen S, Konstantinidis KT, Löffler FE. 2012. Unexpected nondenitrifier nitrous oxide reductase gene diversity and abundance in soils. Proc Natl Acad Sci U S A 109 :19709–19714. doi:10.1073/pnas.1211238109 23150571
55 Dueholm MKD, Andersen KS, Petersen A-K, Rudkjøbing V, Alves M, Bajón-Fernández Y, Batstone D, Butler C, Cruz MC, Davidsson Å, et al. . 2023. MiDAS 5: global diversity of bacteria and archaea in anaerobic digesters. Microbiology. doi:10.1101/2023.08.24.554448
56 Jones CM, Spor A, Brennan FP, Breuil MC, Bru D, Lemanceau P, Griffiths B, Hallin S, Philippot L. 2014. Recently identified microbial guild mediates soil N2O sink capacity. Nature Clim Change 4 :801–805. doi:10.1038/nclimate2301
57 Sullivan MJ, Gates AJ, Appia-Ayme C, Rowley G, Richardson DJ. 2013. Copper control of bacterial nitrous oxide emission and its impact on vitamin B12-dependent metabolism. Proc Natl Acad Sci U S A 110 :19926–19931. doi:10.1073/pnas.1314529110 24248380
58 Spiro S. 2012. Nitrous oxide production and consumption: regulation of gene expression by gas-sensitive transcription factors. Philos Trans R Soc Lond B Biol Sci 367 :1213–1225. doi:10.1098/rstb.2011.0309 22451107
59 Firestone MK, Firestone RB, Tiedje JM. 1980. Nitrous oxide from soil denitrification: factors controlling its biological production. Science 208 :749–751. doi:10.1126/science.208.4445.749 17771133
60 Graf DRH, Saghaï A, Zhao M, Carlsson G, Jones CM, Hallin S. 2019. Lucerne (Medicago sativa) alters N2O-reducing communities associated with cocksfoot (Dactylis glomerata) roots and promotes N2O production in intercropping in a greenhouse experiment. Soil Biol Biochem 137 :107547. doi:10.1016/j.soilbio.2019.107547
61 Chutivisut P, Isobe K, Powtongsook S, Pungrasmi W, Kurisu F. 2018. Distinct microbial community performing dissimilatory nitrate reduction to ammonium (DNRA) in a high C/NO3– reactor. Microbes Environ 33 :264–271. doi:10.1264/jsme2.ME17193 30089740
62 Ma Y, Zilles JL, Kent AD. 2019. An evaluation of primers for detecting denitrifiers via their functional genes. Environ Microbiol 21 :1196–1210. doi:10.1111/1462-2920.14555 30724437
