Diversity and Quantitative Detection of Clade I Type nosZ Denitrifiers in the Arabian Sea Oxygen Minimum Zone

A significant amount of nitrous oxide (N2O) is effluxed into the atmosphere as a result of marine denitrification in the Arabian Sea (AS) oxygen minimum zone (OMZ). An assessment of temporal variations in the diversity and abundance of nosZ denitrifiers was performed to establish the relative importance of these bacteria in denitrification. Sampling was conducted at the Arabian Sea Time Series (ASTS) location and a quantitative PCR (qPCR) analysis was performed. We detected a high abundance of the nosZ gene at core OMZ depths (250 m and 500 m), indicating the occurrence of denitrification in the AS-OMZ. The maximum abundance of the nosZ gene was observed during the Spring Intermonsoon (SIM) at 250 m (1.32×106 copies L–1) and 500 m (1.50×106 copies L–1). Sequencing analysis showed that nosZ denitrifiers belonged to the classes Alpha-, Beta-, and Gammaproteobacteria. Taxonomic analysis revealed that most OTUs were affiliated with Pseudomonas, Rhodopseudomonas, and Bradyrhizobium. Diversity indices and richness estimators confirmed a higher diversity of nosZ denitrifiers at 250 m than at 500 m during all three seasons. The present results also indicated that dissolved oxygen (DO) and total organic carbon (TOC) are critical factors influencing the diversity and abundance of the nosZ-denitrifying bacterial community.

By contributing up to 40% of oceanic nitrogen loss (Codispoti, 2007), predominantly due to the biogeochemical processes occurring in the oxygen minimum zone (OMZ) (Stramma et al., 2008;Ulloa et al., 2008), oceanic denitrification markedly affects the global nitrogen cycle. Denitrification is a large source of nitrous oxide (N 2 O, greenhouse gas), which contributes to climate change and ozone destruction (Beaulieu et al., 2011). Approximately 50% of the annual emissions of ocean N 2 O are from OMZs located in the Arabian Sea (AS), Eastern Tropical South Pacific (ETSP), and Eastern Tropical North Pacific (ETNP) (Naqvi et al., 2005). In OMZs, the reduction of nitrate increases the concentration of nitrite (NO 2 -), which is further reduced to gaseous N 2 O and nitrogen (N 2 ) largely through heterotrophic microbe-mediated denitrification (Codispoti and Christensen, 1985).
The AS-OMZ is recognized as the largest suboxic region of extreme upwelling and high productivity (Wyrtki, 1973) with the low exchange of intermediary waters (150-1,000 m) and is responsible for the greatest marine nitrogen loss (20%) (Codispoti et al., 2001). Denitrification dominates the biogeochemistry of the oxygen-depleted water column of the AS and this process occurs through the diverse assemblage of heterotrophic microbes (Zumft, 1997). High abundance and disparate groups of denitrifying bacteria are present in the AS-OMZ (Jayakumar et al., 2004). Not all denitrifying bacteria convert nitrous oxide to N 2 ; some only produce N 2 in the complete absence of free oxygen. Nevertheless, they undergo partial denitrification under suboxic conditions and release N 2 O (Takaya et al., 2003). Therefore, denitrification is a potential source of nitrous oxide and a critical process in the AS-OMZ, which is continuously expanding Nitrous oxide reduction is the final step in the denitrification pathway, and the only known process for the utilization of N 2 O by microbial communities is by nitrous oxide reductase (nosZ; Hein and Simon, 2019), a copper-containing enzyme found in all denitrifiers capable of reducing nitrate (NO 3 -). The nosZ gene has two distinct clades, Clade I (typical) and Clade II (atypical). Nitrous oxide genes associated with conventional denitrifiers are grouped as Clade I nosZ genes.
However, the nosZ gene is not always affiliated with denitrifying microbes; the recent discovery of novel nosZ sequences in non-denitrifying bacterial groups (Sanford et al., 2012;Jones et al., 2013) showed that they possess Clade II (atypical) nosZ genes. Clade II nosZ genes lack one or more denitrification genes and are known as incomplete denitrifiers (Sanford et al., 2012).
The climatic impact of marine nitrous oxide emissions due to deoxygenation have led to appeals for a more detailed understanding of the biological sink and sources of this gas in the ocean. The key to assessing the budget of nitrous oxide in OMZs is clarifying the distribution and abundance of microorganisms involved in the production and consumption of N 2 O. In the present study, we investigated the diversity and abundance of denitrifying bacteria from suboxic waters of the Arabian Sea Time Series (ASTS, 17°0.126′ N, 67°59.772′ E) location. We targeted the nosZ gene from DNA extracts to assess community and temporal variations. Furthermore, season-and depth-wise differences in the distribution of these bacteria were examined using quantitative PCR (qPCR). This information is critical for recognizing the responses of denitrifiers to changing environmental factors. Additionally, the identification of these bacteria will provide insights into the influence of nosZ denitrifiers on nitrogen transformation and fluxes.

Measurements of physicochemical parameters
Physicochemical parameters (depth, temperature, salinity, and pH) in every water sample were measured using different sensors mounted onto the CTD rosette. The standard Winkler titration method (Carpenter, 1965) was used to measure dissolved oxygen (DO). Nutrient (ammonia, nitrate, nitrite, phosphate, and silicate) concentrations were assessed according to previously reported methods (Grasshoff et al., 1983).

DNA extraction and PCR amplification
Nucleic acid extraction from water samples was performed according to standard methods (Ferrari and Hollibaugh, 1999). The amplification of DNA samples was conducted using the primer set nosZ1F (5′-WCSYTGTTCMTCGACAGCCAG-3′) and nosZ1R (5′-ATGTCGATCARCTGVKCRTTYTC-3′) (Henry et al., 2006). The primers nosZ1F and nosZ1R targeted Clade I genes; Clade II genes were not amplified separately. PCR was performed using a Thermocycler machine (Applied Biosystem) following temperature conditions of 95°C for 5 min, for initial denaturation, 30 cycles at 95°C for 30 s, annealing at 60°C for 45 s, and a final extension step at 72°C for 90 s. A negative control (PCR mix and primers) was used in each PCR reaction and amplification was confirmed by agarose gel (1%) electrophoresis.

Cloning and sequencing
NosZ gene amplicons were purified, cloned into the PGEM-T Easy Vector (Promega), transformed inside high efficiency JM109 cells, and grown overnight at 37°C on LB/X-gal/IPTG plates. A minimum of 40 clones were selected per plate for colony PCR. Temperature conditions for colony PCR were as follows: an initial denaturation step at 94°C for 10 min, followed by 30 cycles at 94°C for 1 min, annealing at 55°C for 1 min with an elongation step at 72°C for 1 min, and a final extension at 72°C for 10 mins. PCR products were purified, measured, and sequenced using 15-50 ng of amplicons, adding 10 pmol each of the nosZ1F and nosZ1R primers in an ABI 3130 Genetic Analyzer (Applied Biosystems). Temperature conditions for sequencing were an initial denaturation at 96°C for 1 min, 30 cycles at 96°C for 10 s, annealing at 60°C for 45 s, elongation at 60°C for 4 mins, and a final extension at 60°C for 1 min.

Sequence analysis
NosZ gene sequences were assembled using DNA Baser sequence assembly software version 2. VecScreen was used to eliminate vector contamination. Non-chimeric consensus sequences without vector and primer residues were submitted to the National Center for Biotechnology Information (NCBI) database to obtain accession numbers and were used in additional analyses. Sequences were classified using 1,000 pseudo-bootstrap replications at a bootstrap value of 80% (standard error of only 1.3%). Alignments were trimmed using Gblocks (Castresana, 2000). Clone sequences were compared with the NCBI database and assigned to a phylum if their identity was more than 95%.

OTU assessment and phylogenetic tree
Sequences (nosZ gene) were assigned to operational taxonomic units (OTUs) by the average neighbor rule (Schloss and Westcott, 2011) using MOTHUR. Sequences obtained from all seasons from depths of 250 and 500 m were grouped as nosZ250m and nosZ500m, respectively, and all nosZ sequences obtained from 250 and 500 m were combined as CnosZ. OTUs were obtained at a sequence similarity of 95%, and representative sequences for each OTU of nosZ250m, nosZ500m, CnosZ, and top-hit nucleotide sequences from cultivated known strains from the NCBI database were used to build the phylogenetic tree (MEGA 6.0) with 1,000 replicate bootstrap analyses.

qPCR assay
Quantification of the nosZ gene was performed using the ABI 7500 Real-Time PCR system (Applied Biosystems). A plasmid carrying the nosZ gene fragment was cloned using the pCR4-TOPO vector and confirmed by sequencing. Ten-fold serial dilutions of a known copy number of plasmid DNA were subjected to a qPCR assay in triplicate to generate a standard curve and calculate the qPCR efficiency of the nosZ gene. A standard curve was generated by plotting threshold cycle values versus log10 of gene copy numbers. The slope, y-intercept, and coefficient of determination (r2) were assessed. The efficiency of amplification (E) was calculated using the following equation: E=-1+10 (-1/slope) .
The abundance of the nosZ gene was quantified in triplicate. Each reaction contained a mixture of DNA (4 μL), the primer pair nosZ1F/nosZ1R (0.5 μL), and 5× qARTA Green qPCR Mix (12.5 μL). PCR cycles were performed according to the standard protocol (Levy-Booth and Winder, 2010). The copy number of the target gene was calculated directly against the standard curve. The negative control had higher Ct values (~9 cycles) than the most diluted plasmid containing the target gene. Additionally, qPCR products were cloned and sequenced to confirm the identity of the gene. A post-amplification melting curve analysis showed that there was no target gene contamination in the reagents.

Statistical analysis
Spearman's correlation coefficient was used to evaluate the relationships between physicochemical parameters (DO, TOC, NO 2 -NO 3 -, and NH 4 + ) and gene copy numbers for every individual season. One-way analysis of variance (ANOVA) was performed for each physicochemical parameter, OTU, and copy number. Paired differences between depths within each season were tested using Tukey's post hoc tests. Principal component analysis (PCA) with varimax rotations for the above-described physicochemical parameters was performed to reduce the number of inter-correlated variables. Multiple regression analyses were conducted to investigate the relationships between OTUs, copy numbers (dependent variables), and principal component scores (predictor variables) obtained from PCA. Statistical analyses were conducted using IBM © SPSS 23.0. Each result was shown as the mean±standard deviation (SD).
Rarefaction curve, diversity indices (Shannon's and Simpson's), and richness estimators (Chao 1 and ACE) were evaluated using MOTHUR. Non-parametric richness estimators were used to extrapolate the total richness of clone libraries from the observed number of OTUs. Diversity and richness estimators were calculated for individual clone libraries.

NCBI Accession numbers
NCBI accession numbers for the 171 nosZ gene sequences obtained in the present study are KX784867 to KX784885, KX911214 to KX911243, KY065372 to KY065445, and KY100043 to KY100090.

Hydrography
Temperature, salinity, pH, and total organic carbon (TOC) were consistent, while the concentrations of DO and nutrients varied at core OMZ depths (Table 1). The average DO concentration decreased from 15.73 μmol L -1 at 250 m to 5.85 μmol L -1 at 500 m. The concentration of nitrate was higher at core OMZ depths during all three seasons and ranged between 15.33 to 32.79 μmol L -1 . The highest concentrations of nitrite (2.57 μmol L -1 ) and ammonia (NH 4 + 1.01 μmol L -1 ) were noted at 250 and 500 m during the NEM and SIM, respectively. The concentrations of DO, NO 2 -NO 3 -, and NH 4 + during all three seasons are shown in Fig. 1.

Phylogenetic analyses
Evolutionary differences between OTUs and representative sequences were assessed by phylogenetic analyses. PCR amplification of the nosZ gene (259-bp amplification product) was only positive for samples collected at 250 and 500 m in all seasons. Among the 210 clones sequenced, 171 non-chimeric nosZ gene sequences were obtained and the following clone libraries were built: SIM-250, SIM-500, FIM-250, FIM-500, NEM-250, and NEM-500.

Phylogeny of nosZ denitrifiers
The sequences of nosZ clones showed 80-93% identity with one another and 77-88% similarity to sequences in GeneBank. All OTUs obtained in the present study matched the sequences of the phylum Proteobacteria in the NCBI Spatial (depth) variations at 250 and 500 m in different physicochemical parameters at the Arabian Sea Time Series (ASTS) location during SIM, FIM, and NEM seasons.  nosZ Bacteria from Core OMZ Depths of AS database. Taxonomic analyses reveals that most nosZ OTUs were associated with Pseudomonas, Rhodopseudomonas, Bradyrhizobium, and Alphaproteobacteria. A small percentage of bacteria was affiliated with Azospirillum, Achromobacter, Cupriavidus, Nisaea, Thalassobaculum, Sinorhizobium, Herbaspirillum, Burkholderia, and Alcaligenes. The phylogenetic analysis showed that OTUs in the present study closely matched the environmental sequences obtained from the suboxic zone of the AS, deep-sea waters of the Mediterranean Sea, the crop soil and wetland sediment of Mexico, terrestrial subsurface sediments, a marine aquaculture biofilter, and paddy soil.

Season-and depth-wise distribution of nosZ denitrifiers
The seasonal distribution patterns of nosZ gene sequences at the class level from 250 and 500 m showed that the maximum number of sequences was affiliated with the class Alphaproteobacteria. At 250 m, Alphaproteobacteria, Oscillatoriophycideae, Bacilli, and Actinobacteria were detected during all three seasons. Acidobacteria was found during the SIM and FIM, Chloroflexia only during the SIM, and Clostridia and Cytophagia only during the NEM. The maximum number of classes was detected in the SIM (7), followed by the FIM (5) and NEM (5) (Fig. 4). At 500 m, Alphaproteobacteria was predominant, followed by Bacilli, Actinobacteria, and Oscillatoriophycideae. Clostridia and Mollicutes were the other classes present at small percentages. No significant variations among classes were observed between seasons, except for Clostridia and Mollicutes, which were only found during the NEM (Fig. 4).
The number of classes detected at 250 m (8) was higher than that detected at 500 m (6).

Abundance of the nosZ gene
Standard curves for the nosZ gene were plotted and qPCR efficiency (81.93%) was calculated. The efficiency reading was used as a reference to calculate the concentrations of the gene in environmental DNA samples. At surface and DCM depths, the copy numbers of the nosZ gene were negligible, ranging between 0.05 and 0.09×10 6 copies L -1 and between 0.07 and 0.09×10 6 copies L -1 , respectively. At 250 m, the abundance of nosZ was the highest during the SIM (1.32×10 6 copies L -1 ), followed by the NEM (0.63×10 6 copies L -1 ) and FIM (0.49×10 6 copies L -1 ). At 500 m, the abundance of the nosZ gene was the highest during the SIM (1.50×10 6 copies L -1 ), followed by the NEM (0.63×10 6 copies L -1 ) and FIM (0.41×10 6 copies L -1 ). Overall, the highest copy number of the nosZ gene was detected at 500 m during the SIM (1.50×10 6 copies L -1 ). The abundance of the nosZ gene at 1,000 m varied from 0.05 to 0.08×10 6 copies L -1 . Fig. 5 shows the seasonal abundance and distribution of nosZ genes.

Effects of environmental parameters on OTUs and copy numbers
During the SIM, DO (r=-0.701; P<0.01), nitrite (r=-0.985; P<0.001), and TOC (r=-0.593; P<0.05) negatively correlated with nosZ gene copy numbers, whereas ammonia (r=0.682; P<0.01) showed a positive correlation. Environmental parameters did not correlate with the abundance of the nosZ gene during the FIM, except for nitrite (r=-0.576; P<0.05), which showed a negative correlation. DO (r=-0.747; P<0.01) negatively correlated with the distribution of the nosZ gene during the NEM, while this correlation was positive for nitrite (r=0.827; P<0.001) ( Table 2). A one-way ANOVA confirmed that physicochemical parameters, OTUs, and copy numbers significantly varied between depths (Table S1). Tukey's post-hoc tests showed significant differences in DO, TOC, NO 3 -, and copy numbers between 250 and 500 m (  (Fig. 6). Multiple regression analyses showed that OTUs and copy numbers were influenced by DO, TOC, NO 2 -, and NO 3 -, while copy numbers were influenced by DO, TOC, and NH 4 + (Table S3).

Richness, OTUs, and rarefaction curves
Shannon's and Simpson's diversity indices revealed that the diversity of nosZ denitrifiers was the highest during the SIM, followed by the FIM and NEM. Diversity was the highest at 250 m during all sampling seasons. The nonparametric estimators, Chao 1 and ACE showed a higher number of OTUs at 250 m during all seasons. The SIM (250 m) and NEM (250 m) had the highest number of OTUs (Table 3). A rarefaction curve was plotted based on the number of clones and OTUs to investigate the relationship between sampling efforts and diversity. The saturation of the rarefaction curve indicated that the sampling effort sufficiently covered nosZ-denitrifying bacteria (Fig. 7).

Discussion
In the highly productive surface waters (0-150 m) of the AS-OMZ, the in situ oxidation and rapid decomposition of organic matter leads to the near-exhaustion of DO at intermediate depths (~200-1,200 m column) triggering intense denitrification (Naqvi et al., 1990), which, in turn, causes the copious efflux of N 2 O into the atmosphere (Law and Owens, 1990;Arévalo-Martínez et al., 2015). Although present in small numbers, nosZ denitrifiers are diverse (Jones et al., 2013) and play a critical role in nitrous oxide production. However, most studies conducted on nosZ bacteria associated with denitrification are performed on cultures and do not represent the total microbial community. Therefore, detection and characterization using a metagenomic approach are important for recognizing the nosZ bacterial community, which is critical in denitrification.
Although mostly detected in the soil ecosystem (Sanford et al., 2012), recent metagenomic sequencing studies identi- nosZ Bacteria from Core OMZ Depths of AS fied Clades I and II nosZ genes in regions associated with the OMZ of the Eastern Tropical Pacific, the AS, and in oxygenated surface waters of the Arctic and Southern Oceans (Jayakumar et al., 2018). The majority of studies that attempted to characterize nosZ gene diversity using DNA-based PCR approaches mainly focused on Clade I nosZ because the high diversity of Clade II nosZ makes it challenging to design a universal primer set that effectively amplifies all nosZ genes in this clade. The primer pair (nosZ1F/nosZ1R; Henry et al., 2006) used in the present study was suitable for both marine and terrestrial nosZ sequences. This primer amplifies a shorter region of 259 bps than those developed by others (1,100 bp; Scala and Kerkhof, 1999) for marine targets, and, thus, was more suit-able for the present study. Although these primers may not efficiently amplify and cover the sequence divergence of Clade II nosZ sequences (Jayakumar et al., 2018), previous studies using microarrays showed that they amplified some of these sequences (Jayakumar et al., 2018). Rhodobacteraceaea affiliated with Clade II nosZ genes detected in the present study was identified in the anoxic section of a wastewater treatment plant using Clade II nosZ gene-specific primers (DaeHyun, D.K., et al., 2019 Development of groupspecific nosZ quantification method targeting active nitrous oxide reducing population in complex environmental samples. bioRxiv https://doi.org/10.1101/710483). Nevertheless, we acknowledge that the PCR primers used in the present study may have been biased towards the detection of Clade I  nosZ genes and, thus, we may have underestimated the real abundance of nosZ genes in our samples. Nevertheless, the present results indicate that marine nosZ denitrifiers (Clade I) inhabit core AS-OMZ depths and play an equal and significant role at the ASTS, leading to a high percentage of fixed nitrogen loss.

The nosZ phylogeny
The absence of nosZ-denitrifying bacteria from the surface, DCM, and 1,000 m, and its invariable presence in samples from 250 and 500 m during all three seasons is con-Spearman's correlation coefficients (r-values) between physicochemical parameters and nosZ gene copy numbers at the ASTS location during the SIM, FIM, and NEM. See Table 1   sistent with previous findings from the AS-OMZ (Bandekar et al., 2018b). As the level of oxygen falls below the detection limit (Devol, 1978), conditions become favorable for denitrification (Codispoti et al., 2005). The persistence of low oxygen levels in the core of the OMZ may be a factor limiting nosZ-denitrifying bacteria to depths of 250 and 500 m (Castro-González et al., 2015). The AS-OMZ with intense upwelling and the low exchange of intermediate waters provides a high redox environment for the growth and multiplication of denitrifying bacteria. The present results demonstrated that nosZ denitrifiers inhabited the core of the AS-OMZ with DO levels fluctuating between 0.76-11 μmol L -1 , similar to findings from the Colombian Pacific Bay (CPB; Castro-González et al., 2018). All nosZ sequences obtained from the ASTS were affiliated with the phylum Proteobacteria, as was also reported by Gomes et al. (2018), Divya et al. (2017), andWang et al. (2017). Proteobacteria have been identified as the dominant phylum and play a significant role in denitrification in the AS-OMZ (Bandekar et al., 2018a;Gomes et al., 2018). OTUs obtained from the ASTS aligned into three classes: Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria, which is consistent with findings from the OMZ of the Subtropical Deep Reservoir (Zheng et al., 2014) and sediments of the AS (Amberkar et al., 2021). nosZ denitrifiers from the present study were predominant within the class Alphaproteobacteria, which is in accordance with the findings of Wyman et al. (2013) from the AS.
The nosZ denitrifiers identified in the present study were dominated by phylotypes affiliated to Pseudomonas, Rhodopseudomonas, Bradyrhizobium, and Alphaproteobacteria. This is similar to the findings of Gomes et al. (2018) and Castro-González et al. (2018). It is important to note that Bradyrhizobium, an aerobic anoxygenic phototrophic bacterium typically reported in oxic waters (Hu et al., 2006), was nosZ Bacteria from Core OMZ Depths of AS found at core OMZ depths in the present study. Few OTUs from the ASTS were homologous with the novel sequence of Nisaea denitrificans (class Alphaproteobacteria) isolated from the Mediterranean Sea, which is potentially involved in denitrification (Urios et al., 2008).
Sequences from the ASTS showed similarity to the nosZ gene isolates of Achromobacter detected from the OMZ of the CPB (Castro-González et al., 2018), Herbaspirillum identified in anaerobic wastewater treatment plants (Hou et al., 2012), Sinorhizobium reported in the sediments of the Atlantic (Scala and Kerkhof, 1998), and Azospirillum found in eutrophic freshwater lakes . We also identified NosZ denitrifiers affiliated with Burkholderia, Alcaligenes, and Cupriavidus, which were previously detected in boreal peat moss (Nie et al., 2015), wastewater treatment plants , and marsh soils (Henry et al., 2006), respectively. The majority of sequences identi-fied in the present study showed homogeneity to those reported by Gomes et al. (2018) from the seasonal OMZ in the AS. All of the nosZ denitrifiers identified in the present study were actively involved in nitrous oxide production. Some OTU sequences from this study aligned with the cultured, facultative anaerobe Thalassobaculum (family Rhodospirillaceae), the role of which remains unknown (Zhang et al., 2008).

Abundance and distribution of the nosZ gene
NosZ denitrifiers were uncommon at 5 m, DCM, and 1,000 m. Quantitative analyses of the nosZ gene from these depths showed lower Ct values than those at core OMZ depths (250 and 500 m). PCR with a higher concentration of DNA did not yield positive amplification for any of the samples taken from depths of 5 m, DCM, or 1,000 m. Therefore, nosZ genes at these depths were limited and hard  Table 1 for abbreviations.

Seasons
Depth ( to detect via conventional PCR. Additionally, Staggemeier et al. (2015); Nagdev et al. (2015), and Zemtsova et al. (2015) reported that the sensitivity of qPCR was higher than that of conventional PCR. Melt curves, the melting temperature, and all other protocols confirmed that qPCR amplification at 5 m, DCM, and 1,000 m was not an artefact. Although differences were observed in abundance, the highest copy numbers during all three seasons were detected at core OMZ depths. Therefore, the oxygen concentration at these depths was the most suitable for denitrifying bacteria, indicating the perennial survival of nosZ denitrifiers in the AS-OMZ. The higher abundance of nosZ denitrifiers during the SIM may be attributed to organic carbon in the OMZ being a significant substrate that supports the existence of denitrifying and anammox bacteria (Dang et al., 2010). During the SIM, bacterial communities in the AS-OMZ are sustained by slow-to-degrade dissolved organic carbon (DOC) (Ramaiah et al., 2000) i.e., the SIM is a transitional phase with low primary productivity (Madhupratap et al., 1996) due to the persistence of oligotrophic conditions and stratification.
We herein reported a higher abundance of N 2 O-reducing bacteria from the ASTS than nirS denitrifiers (Bandekar et al., 2018b). The nirS and nosZ genes are both assumed to be present in the genome as single-copy genes; however, there are exceptions for nosZ genes (Sanford et al., 2012). One possible explanation for differences in abundance is that not all N 2 O-consuming bacteria contain a complete denitrification gene sequence (Sanford et al., 2012). nosZ geneassociated bacteria lack the other steps required for conventional denitrification. In comparisons with other ecosystems, bacteria with only nosZ genes are over-represented in the genomes of marine bacteria (Graf et al., 2014). However, the nirS gene, is associated with bacteria that contain a complete denitrification pathway (Graf et al., 2014). Another contributing factor that may explain this difference is the specificity of PCR primers. The primers used in the present study represent a more extensive database of nosZ sequences (terrestrial and marine sequences), whereas the nirS primers used in previous studies (Bandekar et al., 2018b) are potentially biased towards marine sequences (Braker et al., 1998). Stevens and Ulloa (2008) reported that DO and organic matter were important factors affecting the microbial community composition in the OMZ. The present results suggest that DO and TOC play a critical role in influencing the diversity and abundance of nosZ denitrifiers during different seasons. The presence of denitrifiers at core OMZ depths (as is implicit in derived diversity indices and the richness estimators ACE and Chao 1) indicated that low concentrations of oxygen, nitrite, and ammonia provided an ideal environment for the presence of nosZ denitrifiers (Bandekar et al., 2018b). In contrast to the findings of Castro-González et al. (2018), the present results showed a higher diversity of nosZ denitrifiers at 250 m than at 500 m. Although the sampling size in the present study was not very large, the saturation of rarefaction curves indicates that the diversity of nosZ denitrifiers was adequately covered.

Conclusion
In the OMZ layers of the Arabian Sea, denitrification is a crucial pathway (Ward et al., 2009;Dalsgaard et al., 2012;Jain et al., 2014;Bandekar et al., 2018a) that has enabled the occurrence and sizable abundance of a diverse group of microbial communities, including those not taking part in nosZ Bacteria from Core OMZ Depths of AS the process, per se. The present results showed that the diversity nosZ denitrifiers was low and limited to core OMZ depths, suggesting that low concentrations of organic matter in OMZs not only reduce the number of available niches for microbes (Bryant et al., 2012), but also unfavorably influence the denitrifying microbial community structure. While nirS-possessing denitrifiers control N 2 O emissions (Conrad, 1996;Wallenstein et al., 2006), the higher abundance of the nosZ gene than nirS from the ASTS in the present study suggests otherwise. Furthermore, our results on hydrographic parameters indicate that the concentrations of DO and TOC influence the abundance and distribution of nosZdenitrifying bacteria at core OMZ depths of the AS.