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

Abstract

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 (N₂O, greenhouse gas), which contributes to climate change and ozone destruction (Beaulieu et al., 2011). Approximately 50% of the annual emissions of ocean N₂O 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 (NO₂^–), which is further reduced to gaseous N₂O and nitrogen (N₂) 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 N₂; some only produce N₂ in the complete absence of free oxygen. Nevertheless, they undergo partial denitrification under suboxic conditions and release N₂O (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 N₂O 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 (NO₃^–). 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 N₂O. 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.

Materials and Methods

Water sampling

Water samples were collected from the ASTS location using Niskin bottles on a CTD rosette from the surface (5 m), deep chlo­rophyll maxima (DCM) (~43–50 m), core OMZ depths (250 and 500 m), and 1,000‍ ‍m during three seasons i.e. the Spring intermonsoon (SIM), Fall intermonsoon (FIM), and Northeast monsoon (NEM). Water was processed through sterivex filters (0.22‍ ‍μm; Millipore), filled with 1.7‍ ‍mL of storage buffer (50 Mm, Tris pH 8.3, 40‍ ‍mM EDTA, and 0.75 M sucrose) and stored at –80°C until DNA extraction.

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, NO₂^– NO₃^–, and NH₄⁺) 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.

Results

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 (NH₄⁺ 1.01‍ ‍μmol L^–1) were noted at 250 and 500‍ ‍m during the NEM and SIM, respectively. The concentrations of DO, NO₂^– NO₃^–, and NH₄⁺ during all three seasons are shown in Fig. 1.

Table 1. 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.

Season		SIM			FIM			NEM
Depth (m)		250	500		250	500		250	500
Temperature (°C)		18.24±0.02	12.96±0.02		16.00±1.00	12.02±0.01		15.28±0.02	12.11±0.02
Salinity (ppt)		35.72±0.03	35.71±0.02		36.08±0.01	35.62±0.01		35.75±0.02	35.60±0.02
DO (μmol L–1)		11.79±0.03	4.58±0.02		3.47±0.01	0.77±0.02		1.41±0.07	1.51±0.02
pH		7.76±0.02	7.59±0.03		7.22±0.43	7.19±0.02		7.51±0.02	7.43±0.02
NO3– (μmol L–1)		22.41±0.02	15.33±0.03		25.00±0.43	32.79±0.02		19.05±0.02	25.98±0.02
NO2– (μmol L–1)		0.01±0.00	ND		ND	ND		2.57±0.02	0.06±0.00
PO43– (μmol L–1)		2.00±0.01	1.60±0.02		2.35±0.04	2.55±0.02		2.22±0.01	2.58±0.02
SiO32– (μmol L–1)		18.47±0.01	19.44±0.02		21.79±0.04	34.99±0.02		29.36±0.01	50.29±0.02
NH4+ (μmol L–1)		0.10±0.00	1.01±0.00		0.19±0.02	0.27±0.02		ND	ND
TOC (μmol L–1)		54.23±0.21	47.80±0.26		52.43±0.03	43.36±0.01		45.30±0.02	38.19±0.02

The Spring intermonsoon (SIM), Fall intermonsoon (FIM), and Northeast monsoon (NEM), Dissolved oxygen (DO), Nitrate (NO₃^–), Nitrite (NO₂^–), Phosphate (PO₄^3–), Silicate (SiO₃^2–), Ammonia (NH₄⁺), Total organic carbon (TOC), and ND=not detected

Fig. 1.

Vertical distribution of concentrations of dissolved oxygen (DO), nitrate (NO₃^–), nitrite (NO₂^–), and ammonia (NH₄⁺) during the spring intermonsoon (SIM), fall intermonsoon (FIM), and northeast monsoon (NEM) at the Arabian Sea Time Series (ASTS) location.

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 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.

Among the 87 clones sequenced from nosZ250m, 17 OTUs were obtained (Fig. S1), the majority of which were affiliated with the genera Rhodopseudomonas (18%, 3 OTUs) and Pseudomonas (18%, 3 OTUs). Azospirillum (2‍ ‍OTUs), Bradyrhizobium (2 OTUs), Achromobacter (2 OTUs), and Cupriavidus (2 OTUs) each contributed 12% of the total number of OTUs. Bacterial sequences belonging to the genera Nisaea (1 OTU), Thalassobaculum (1 OTU), and class Alphaproteobacteria (1 OTU) formed OTUs with a smaller number of clones.

Thirteen OTUs were generated from nosZ500m (84 sequences) (Fig. S2), the majority of which belonged to the genera Bradyrhizobium (15%, 2 OTUs), Pseudomonas (15%, 2 OTUs), and Alphaproteobacteria (15%, 2 OTUs). Thalassobaculum (1 OTU), Rhodopseudomonas (1 OTU), Sinorhizobium (1 OTU), Azospirillum (1 OTU), Herbaspirillum (1 OTU), Burkholderia (1 OTU), and Alcaligenes (1 OTU) were the other minor OTUs present.

Among the 171 clones sequenced from core OMZ depths, 27 OTUs were generated, which were affiliated with the classes Alpha-, Beta-, and Gammaproteobacteria (Fig. 2). The majority of OTUs were affiliated with the genera Bradyrhizobium (5 OTUs, 18.5%, 32 sequences), Azospirillum (5 OTUs, 18.5%, 32 sequences), and Pseudomonas (4 OTUs, 14.8%, 26 sequences). Three OTUs (11.1%) each were associated with Rhodopseudomonas (19 sequences), Sinorhizobium (19 sequences), and Achromobacter (19 sequences). One OTU (6 sequences) each was affiliated with the genera Thalassobaculum, Nisaea, and Burkholderia and the class Alphaproteobacteria (Fig. 3).

Fig. 2.

Phylogenetic tree constructed using the neighbor-joining method of nosZ gene OTU sequences obtained from CnosZ500m at the ASTS location. Sequences in bold are from the present study.

Fig. 3.

Percentages of the relative abundance and sequence numbers of nosZ gene OTU sequences obtained from CnosZ at the Arabian Sea Time Series (ASTS) location.

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).

Fig. 4.

Season-wise distribution of nosZ bacterial classes at 250 and 500‍ ‍m at the ASTS location. See Fig. 1 for abbreviations.

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 0.09×10⁶ copies L^–1 and between 0.07 and 0.09×10⁶ copies L^–1, respectively. At 250‍ ‍m, the abundance of nosZ was the highest during the SIM (1.32×10⁶ copies L^–1), followed by the NEM (0.63×10⁶ copies L^–1) and FIM (0.49×10⁶ copies L^–1). At 500 m, the abundance of the nosZ gene was the highest during the SIM (1.50×10⁶ copies L^–1), followed by the NEM (0.63×10⁶ copies L^–1) and FIM (0.41×10⁶ copies L^–1). Overall, the highest copy number of the nosZ gene was detected at 500‍ ‍m during the SIM (1.50×10⁶ copies L^–1). The abundance of the nosZ gene at 1,000‍ ‍m varied from 0.05 to 0.08×10⁶ copies L^–1. Fig. 5 shows the seasonal abundance and distribution of nosZ genes.

Fig. 5.

Season- and depth-wise distribution of nosZ gene copy numbers at the ASTS location. Depths are 5 m, deep chlo­rophyll maximum (DCM), 250 m, 500 m, and 1,000 m. See Fig. 1 for abbreviations.

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, NO₃^–, and copy numbers between 250 and 500‍ ‍m (Table S2) during all three seasons. PCA revealed three principal components with eigenvalues >1, which explained 92.39% of all variations in physicochemical parameters. The first component (PC1) explained a total variance of 42.20% and reflected a strong gradient caused by DO (0.943) and TOC (0.916). The second component (PC2) accounted for 27.06% of variance and was reflected by NH₄⁺ (0.958). The third component (PC3) explained 23.13% of variance and included NO₂^– (0.947) (Fig. 6). Multiple regression ana­lyses showed that OTUs and copy numbers were influenced by DO, TOC, NO₂^–, and NO₃^–, while copy numbers were influenced by DO, TOC, and NH₄⁺ (Table S3).

Table 2. 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 for abbreviations.

Location	Season	Genes	Variable	r	P value
ASTS	SIM	nosZ	Depth	0.352	>0.05
			DO	–0.701	0.01
			NO3–	0.223	>0.05
			NO2–	–0.985	0.001
			NH4+	0.682	0.01
			TOC	–0.593	0.05
	FIM	nosZ	Depth	–0.356	>0.05
			DO	–0.018	>0.05
			NO3–	–0.192	>0.05
			NO2–	–0.576	0.05
			NH4+	–0.059	>0.05
			TOC	0.083	>0.05
	NEM	nosZ	Depth	0.318	>0.05
			DO	–0.747	0.01
			NO3–	0.332	>0.05
			NO2–	0.827	0.001
			NH4+	–0.483	>0.05
			TOC	–0.605	>0.05

P<0.05; P<0.01; P<0.001—levels of significance

Fig. 6.

Principal component ana­lysis (PCA) plot of the rotated space with DO, TOC, NO₂^– NO₃^–, NH₄⁺, OTU, and copy numbers. See Fig. 1 for abbreviations.

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 non-parametric 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).

Table 3. Diversity of nosZ denitrifier libraries constructed from the ASTS location during the SIM, FIM, and NEM. See Table 1 for abbreviations.

Seasons	Depth (m)	Sequences	OTUs	Richness estimation
				Shannon	Simpsons	Chao	ACE
SIM	250	29	15	3.56	0.03	16.8	23.4
	500	28	13	3.49	0.01	28.3	26.28
FIM	250	30	11	3.27	0.02	85.2	107.31
	500	27	10	2.9	0.04	25	28.39
NEM	250	28	15	2.92	0.04	25.5	27.1
	500	29	11	2.27	0.09	11.33	11.69

Fig. 7.

Rarefaction curves of nosZ gene sequences at the ASTS location. See Fig. 1 for abbreviations.

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 N₂O 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 identified 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 suitable 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 group-specific 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 consistent 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), and Wang 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 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 (Wang et al., 2012). 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 (Wang et al., 2015), and marsh soils (Henry et al., 2006), respectively. The majority of sequences identified 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 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 N₂O-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 N₂O-consuming bacteria contain a complete denitrification gene sequence (Sanford et al., 2012). nosZ gene-associated 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).

Diversity and richness estimation

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 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 N₂O 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 nosZ-denitrifying bacteria at core OMZ depths of the AS.

Citation

Bandekar, M., Ramaiah, N., Seleyi, S. C.., Nazareth, D. R.., and Kekäläinen, J. (2023) Diversity and Quantitative Detection of Clade I Type nosZ Denitrifiers in the Arabian Sea Oxygen Minimum Zone. Microbes Environ 38: ME22056.

https://doi.org/10.1264/jsme2.ME22056

Acknowledgements

The authors thank the Director of CSIR-NIO for the facilities and encouragement. This work was supported by grants from the Ministry of Earth Sciences, the Government of India. M.B. and N.R. were supported under the Sustained Indian Ocean Biogeochemical and Ecological Research (SIBER) program (GAP 2658). S.S. was funded by the UGC grant (RF83600), D.N. was supported by the VNJCT fellowship, and J.K. was funded by the Academy of Finland (grant number 308485). This is CSIR-NIO contribution number 6983.

References

•  Amberkar,  U.,  Khandeparker,  R.,  Menezes,  L., and  Meena,  R. (2021) Phylogenetic diversity of culturable marine bacteria from sediments underlying the oxygen minimum zone of the Arabian Sea and their role in nitrate reduction. Mar Ecol 42: 1–12.
•  Arévalo-Martínez,  D.L.,  Kock,  A.,  Loscher,  C.R.,  Schmitz,  R.A., and  Bange,  H.W. (2015) Massive nitrous oxide emissions from the tropical South Pacific Ocean. Nat Geosci 8: 530–533.
•  Bandekar,  M.,  Ramaiah,  N.,  Jain,  A., and  Meena,  R. (2018a) Seasonal and depth-wise variations in bacterial and archaeal groups in the Arabian Sea oxygen minimum zone. Deep Sea Res Part II 156: 4–18.
•  Bandekar,  M.,  Ramaiah,  N., and  Meena,  R. (2018b) Diversity and abundance of denitrifying and anammox bacteria from the Arabian Sea oxygen minimum zone. Deep Sea Res Part II 156: 19–26.
•  Beaulieu,  J.J.,  Tank,  J.L.,  Hamilton,  S.K.,  Wollheim,  W.M.,  Hall Jr,  R.O.,  Bruce,  P.J., et al. (2011) Nitrous oxide emission from denitrification in stream and river networks. Proc Natl Acad Sci U S A 108: 214–219.
•  Braker,  G.,  Fesefeldt,  A., and  Witzel,  K.P. (1998) Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Appl Environ Microbiol 64: 3769–3775.
•  Bryant,  J.A.,  Stewart,  F.J.,  Eppley,  J.M., and  DeLong,  E.F. (2012) Microbial community phylogenetic and trait diversity declines with depth in a marine oxygen minimum zone. Ecology 93: 1659–1673.
•  Carpenter,  J.H. (1965) The Chesapeake Bay Institute technique for the Winkler dissolved oxygen method. Limnol Oceanogr 10: 141–143.
•  Castresana,  J. (2000) Selection of conserved blocks from multiple alignments for their use in phylogenetic ana­lysis. Mol Biol Evol 17: 540–552.
•  Castro-González,  M.,  Ulloa,  O., and  Farías,  L. (2015) Structure of denitrifying communities reducing N2O at suboxic waters off northern Chile and Perú. Rev Biol Mar Oceanogr 50: 95–110.
•  Castro-González,  M.,  Rodríguez-Rubio,  E., and  Castro,  A. (2018) Denitrifying community structure variability in the Colombian Pacific. Lat Am J Aquat Res 46: 392–410.
•  Codispoti,  L.A., and  Christensen,  J.C. (1985) Nitrification, denitrification and nitrous oxide cycling in the eastern tropical South Pacific Ocean. Mar Chem 16: 277–300.
•  Codispoti,  L.A.,  Brandes,  J.A.,  Christensen,  J.P.,  Devol,  A.H.,  Naqvi,  S.W.A.,  Paerl,  H.W., and  Yoshinari,  T. (2001) The oceanic fixed nitrogen and nitrous oxide budgets: moving targets as we enter the anthropocene? Sci Mar 65: 85–105.
• Codispoti, L.A., Yoshinari, T., and Devol, A. (2005) Suboxic respiration in the oceanic water column. In Respiration in the Aquatic Ecosystems. Del Giorgio, P., and Williams, P. (eds). Oxford, UK: Oxford University Press, pp. 225–247.
•  Codispoti,  L.A. (2007) An oceanic fixed nitrogen sink exceeding 400Tg N a-1 vs the concept of homeostasis in the fixed-nitrogen inventory. Biogeosciences 4: 233–253.
•  Conrad,  R. (1996) Soil microorganisms as controllers of atmospheric trace gases (H₂, CO, CH₄, OCS, N₂O, and NO). Microbiol Rev 60: 609–640.
•  Dalsgaard,  T.,  Thamdrup,  B.,  Farias,  L., and  Revsbech,  P.N. (2012) Anammox and denitrification in the oxygen minimum zone of the eastern South Pacific. Limnol Oceanogr 57: 1331–1346.
•  Dang,  H.,  Chen,  R.,  Wang,  L.,  Guo,  L.,  Chen,  P.,  Tang,  Z., et al. (2010) Environmental factors shape sediment anammox bacterial communities in hyper nitrified Jiao zhou Bay, China. Appl Environ Microbiol 76: 7036–7047.
•  Devol,  A.H. (1978) Bacterial oxygen uptake kinetics as related to biological processes in oxygen deficient zones of the oceans. Deep-Sea Res 25: 137–146.
•  Divya,  B.,  Annie,  F., and  Shanta,  N. (2017) Bacterial community profiling of the Arabian Sea oxygen minimum zone sediments using cultivation independent approach. Examines Mar Biol Oceanogr 1: 22–28.
•  Ferrari,  V.C., and  Hollibaugh,  J.T. (1999) Distribution of microbial assemblages in the Central Arctic Ocean Basin studied by PCR/DGGE: ana­lysis of a large data set. Hydrobiologia 401: 55–68.
•  Gomes,  J.,  Khandeparker,  R.,  Bandekar,  M., and  Meena,  R. (2018) Quantitative ana­lyses of denitrifying bacterial diversity from a seasonally hypoxic monsoon governed tropical coastal region. Deep Sea Res Part II 156: 34–43.
•  Graf,  D.R.,  Jones,  C.M., and  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.
• Grasshoff, K., Ehrhardt, E., and Kremling, K. (1983) Methods of Seawater Analysis, 2nd edn. New York, NY: Verlag Chemie Weinheim.
•  Hein,  S., and  Simon,  J. (2019) Bacterial nitrous oxide respiration: electron transport chains and copper transfer reactions. Adv Microb Physiol 75: 137–175.
•  Henry,  S.,  Bru,  D.,  Stres,  B.,  Hallet,  S., and  Philippot,  L. (2006) Quantitative detection of the nosZ Gene, encoding nitrous oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ Genes in Soils. Appl Environ Microbiol 72: 5181–5189.
•  Hou,  J.,  Li,  L.,  Zhang,  S.,  Wang,  P., and Wang, C., (2012) Diversity of NosZ gene in three municipal wastewater treatment plants located in different geographic regions. Afr J Microbiol Res 6: 3574–3581.
•  Hu,  Y.,  Du,  H.,  Jiao,  N., and  Zeng,  Y. (2006) Abundant presence of the γ-like Proteobacterial pufM gene in oxic seawater. FEMS Microbiol Lett 263: 200–206.
•  Jain,  A.,  Bandekar,  M.,  Gomes,  J.,  Shenoy,  D.M.,  Meena,  R.M.,  Naik,  H., et al. (2014) Temporally invariable bacterial community structure in the Arabian Sea oxygen minimum zone. Aquat Microb Ecol 73: 51–67.
•  Jayakumar,  D.A.,  Francis,  C.A.,  Naqvi,  S.W.A., and  Ward,  B.B. (2004) Diversity of nitrite reductase genes (nirS) in the denitrifying water column of the coastal Arabian Sea. Aquat Microb Ecol 34: 69–78.
•  Jayakumar,  D.A.,  Balachandran,  D.,  Rees,  A.P.,  Kearns,  P.J.,  Bowen,  J.L., and  Ward,  B.B. (2018) Community composition of nitrous oxide reducing bacteria investigated using a functional gene microarray. Deep Sea Res Part II 156: 44–50.
•  Jones,  C.M.,  Graf,  D.R.,  Bru,  D.,  Philippot,  L., and  Hallin,  S. (2013) The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink. ISME J 7: 417–426.
•  Law,  C.S., and  Owens,  N.J.P. (1990) Significant flux of atmospheric nitrous oxide from the northwest Indian Ocean. Nature 346: 826–828.
•  Levy-Booth,  D.J., and  Winder,  R.S. (2010) Quantification of nitrogen reductase and nitrite reductase genes in soil of thinned and clear-cut Douglas-fir stands by using real-time PCR. Appl Environ Microbiol 76: 7116–7125.
•  Madhupratap,  M.,  Kumar,  S.P.,  Bhattathiri,  P.M.A.,  Kumar,  M.D.,  Raghukumar,  S.,  Nair,  K.K.C., and  Ramaiah,  N. (1996) Mechanism of the biological response to winter cooling in the northeastern Arabian Sea. Nature 384: 549–552.
•  Nagdev,  J.K.,  Kashyap,  S.R.,  Bhullar,  S.,  Purohit,  H.,  Taori,  G., and Daginawala. (2015) Comparison of real-time PCR and conventional PCR assay using IS110 region of Mycobacterium tuberculosis for efficient diagnosis of tuberculous meningitis and pulmonary tuberculosis. IJBT 14: 94–100.
•  Naqvi,  S.W.A.,  Noronha,  R.J.,  Somasundar,  K., and  Gupta,  S.R. (1990) Seasonal changes in the denitrification regime of the Arabian Sea. Deep Sea Res Part I 37: 593–611.
•  Naqvi,  S.W.A.,  Bange,  H.W.,  Gibb,  S.W.,  Goyet,  C.,  Hatton,  A.D., and  Upstill-Goddard,  R.C. (2005) Biogeochemical ocean-atmosphere transfers in the Arabian Sea. Prog Oceanogr 65: 116–144.
•  Nie,  Y.,  Li,  L.,  Wang,  M.,  Tahvanainen,  T., and  Hashidoko,  Y. (2015) Nitrous oxide emission potentials of Burkholderia species isolated from the leaves of a boreal peat moss Sphagnum fuscum. Biosci Biotechnol Biochem 79: 2086–2095.
•  Ramaiah,  N.,  Sarma,  V.V.S.S.,  Gauns,  M.,  Kumar,  M.D., and  Madhupratap,  M. (2000) Abundance and relationship of bacteria with transparent exopolymer particles during the 1996 summer monsoon in the Arabian Sea. J Earth Syst Sci 109: 443–451.
•  Sanford,  R.A.,  Wagner,  D.D.,  Wu,  Q.,  Chee-Sanford,  J.C.,  Thomas,  S.H.,  Cruz-García,  C., et al. (2012) Unexpected non denitrifier nitrous oxide reductase gene diversity and abundance in soils. Proc Natl Acad Sci U S A 109: 19709–19714.
•  Scala,  D.J., and  Kerkhof,  L.J. (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.
•  Scala,  D.J., and  Kerkhof,  L.J. (1999) Diversity of nitrous oxide reductase (nosZ) genes in continental shelf sediments. Appl Environ Microbiol 65: 11681–11687.
•  Schloss,  P.D., and  Westcott,  S.L. (2011) Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence ana­lysis. Appl Environ Microbiol 77: 3219–3226.
•  Staggemeier,  R.,  Bortoluzzi,  M.,  Heck,  S.M.T.,  Spilki,  F.R., and  Almeida,  S.E.M. (2015) Quantitative vs. conventional PCR for detection of human adenoviruses in water and sediment samples. Rev Inst Med Trop Sao Paulo 57: 299–303.
•  Stevens,  H., and  Ulloa,  O. (2008) Bacterial diversity in the oxygen minimum zone of the eastern tropical South Pacific. Environ Microbiol 10: 1244–1259.
•  Stramma,  L.,  Johnson,  G.C.,  Sprintall,  J., and  Mohrholz,  V. (2008) Expanding oxygen-minimum zones in the tropical oceans. Science 320: 655–658.
•  Takaya,  N.,  Catalan-Sakairi,  M.A.B.,  Sakaguchi,  Y.,  Kato,  I.,  Zhou,  Z., and  Shoun,  H. (2003) Aerobic denitrifying bacteria that produce low levels of nitrous oxide. Appl Environ Microbiol 69: 3152–3157.
•  Ulloa,  O.,  Belmar,  L.,  Farías,  L.,  Castro-González,  M.,  Galán,  A., et al. (2008) Microbial communities and their biogeochemical role in the water column of the oxygen minimum zone in the eastern South Pacific. Gayana (Concepción) 70: 83–86.
•  Urios,  L.,  Michotey,  V.,  Intertaglia,  L.,  Lesongeur,  F., and  Lebaron,  P. (2008) Nisaea denitrificans gen. nov., sp. nov. and Nisaea nitritireducens sp. nov., two novel members of the class Alphaproteobacteria from the Mediterranean Sea. Int J Syst Evol Microbiol 58: 2336–2341.
•  Wallenstein,  M.D.,  Myrold,  D.D.,  Firestone,  M., and  Voytek,  M. (2006) Environmental controls on denitrifying communities and denitrification rates: Insights from mole­cular methods. Ecol Appl 16: 2143–2152.
•  Wang,  C.,  Zhu,  G.,  Wang,  Y.,  Wang,  S., and  Yin,  C. (2012) Nitrous oxide reductase gene (nosZ) and N2O reduction along the littoral gradient of a eutrophic freshwater lake. J Environ Sci (Beijing, China) 25: 44–52.
•  Wang,  Q.,  Liu,  Y.R.,  Zhang,  C.J.,  Zhang,  L.M.,  Han,  L.L.,  Shen,  J.P., and  He,  J.Z. (2017) Responses of soil nitrous oxide production and abundances and composition of associated microbial communities to nitrogen and water amendment. Biol Fert Soils 53: 601–611.
•  Wang,  X.,  Yu,  P.,  Zeng,  C.,  Ding,  H.,  Li,  Y.,  Wang,  C., and  Lu,  A. (2015) Enhanced Alcaligenes faecalis denitrification rate with electrodes as the electron donor. Appl Environ Microbiol 81: 5387–5394.
•  Ward,  B.B.,  Devol,  A.H.,  Rich,  A.A.,  Chang,  B.X.,  Bulow,  E.S.,  Naik,  H., et al. (2009) Denitrification as the dominant nitrogen loss process in the Arabian Sea. Nature 461: 78–81.
•  Wyman,  M.,  Hodgson,  S., and  Bird,  C. (2013) Denitrifying Alphaproteobacteria from the Arabian Sea that express nosZ, the gene encoding nitrous oxide reductase, in oxic and suboxic waters. Appl Environ Microbiol 79: 2670–2681.
• Wyrtki, K. (1973) Physical oceanography of the Indian Ocean. In The Biology of the Indian Ocean. Zeitzschel, B. (ed.) Berlin, Heidelberg: Springer, pp. 18–36.
•  Zemtsova,  G.E.,  Montgomery,  M., and  Levin,  M.L. (2015) Relative sensitivity of conventional and real-time PCR assays for detection of SFG Rickettsia in blood and tissue samples from laboratory animals. PLoS One 10: e0116658.
•  Zhang,  G.I.,  Hwang,  C.Y., and  Cho,  B.C. (2008) Thalassobaculum litoreum gen. nov., sp. nov., a member of the family Rhodospirillaceae isolated from coastal seawater. Int J Syst Evol Microbiol 58: 479–485.
•  Zheng,  Y.,  Jun,  Y., and  Lemian,  L. (2014) Denitrifier community in the oxygen minimum zone of a subtropical deep reservoir. PLoS One 9: e92055.
•  Zumft,  W.G. (1997) Cell biology and mole­cular basis of denitrification. Microbiol Mol Biol Rev 61: 533–616.