Nitrification is an integrative component in the nitrogen cycle, which includes oxidization of ammonia to nitrite by ammonia-oxidizing Bacteria (AOB) and ammonia-oxidizing Archaea (AOA), and then nitrite is further oxidized to nitrate by nitrite-oxidizing Bacteria (NOB). As the first and also the rate-limiting step, ammonia oxidation to nitrite is mediated by both AOB and AOA (Norton et al., 2002; Schleper and Nicol, 2010). The chemolithotrophic AOB are placed taxonomically in the beta- and the gamma-subdivision of Proteobacteria, and their natural population/distribution has been widely investigated by molecular tools, such as using the 16S rRNA gene sequences (Stephen et al., 1996, 1998; Kowalchuk et al., 2000) and the genes encoding ammonia monooxygenase (amoA; Bothe et al., 2000; Purkhold et al., 2000). In recent years, novel archaeal groups that function as ammonia oxidizers containing amoA genes have also been observed and they may contribute greatly to nitrification processes as well (Venter et al., 2004; Schleper et al., 2005). Environmental AOA sequences are mainly affiliated with several groups, such as marine group I (MGI), putatively marine group pSL12, thermophilic AOA (ThAOA) and soil group I.1b (Durbin and Teske, 2011; Pester et al., 2011; Hatzenpichler, 2012). In order to better understand their roles in global nitrogen cycling, it is critical to characterize the abundance, diversity, and distribution of AOA and AOB in natural environments.

Nitrogen is likely the limiting nutrient in marine ecosystems (Gruber and Sarmiento, 1997), and therefore the nitrogen cycling in the ocean is of particular interest (Deutsch et al., 2007). AOA and AOB have been commonly found in marine sediments all over the world, including the East China Sea (Dang et al., 2008), the South China Sea (Jin et al., 2011), hydrothermal vents of the Pacific Ocean (Wang et al., 2009; Nunoura et al., 2010), the western Pacific Ocean (Cao et al., 2011a), the tropical West Pacific Continental Margin (Dang et al., 2009), the Northeastern Japan Sea (Nakagawa et al., 2007), the Southern North Sea (Lipsewers et al., 2014), and also the deep-sea environments (Nunoura et al., 2013; Xu et al., 2014; Lagostina et al., 2015; Luo et al., 2015). Among these studies, ratios of AOA/AOB gene copy numbers varied from 0.0003 to 232.3, and no consistent patterns have been observed. Obviously the distribution and variations of AOA and AOB are relevant to their metabolic characteristics and their responses to environmental gradients as well. For instance, AOA has been reported to have a higher affinity to ammonium than AOB in oligotrophic environment (Martens-Habbena et al., 2015). The dominant group and distribution of AOB could be driven by salinity, pH, and nutrient availability (Bernhard et al., 2007; Cao et al., 2012; Li et al., 2015), while the distribution of AOA varied along water depth, redox variation, and concentration of ammonium in water column or marine sediments (Wuchter et al., 2006; Dang et al., 2009; Roussel et al., 2009; Flood et al., 2015). Although a global distribution, diverse composition, and high abundance of AOA and AOB have been reported (Rotthauwe et al., 1997; Nicol and Schleper, 2006; Francis et al., 2007; Cavicchioli et al., 2007), compared to other regions, we have very limited knowledge on these microorganisms in the Indian Ocean.

The Indian Ocean is the third largest ocean in the world, which is characterized by two semi-enclosed basins in the north, and greatly influenced by seasonal monsoon (Fine et al., 2008; Rixen et al., 2009). Strong stratification induced by monsoon suppresses up welling and mixing of the deep waters, making the Eastern Equatorial Indian Ocean a typical oligotrophic area (Kumar et al., 2009). The Arabian Sea (AS) and the Bay of Bengal (BOB) are the two basins in the north part of the Indian Ocean and are relatively less oligotrophic compared to the Equatorial region. The frequent dust input to the Arabian Sea, enhanced advection, and vertical eddy mixing bring small detritus and nutrients to the Central/Eastern AS (McCreary et al., 1993). In contrast, lack of detritus transport limits organic matter accumulation in the BOB (Kumar et al., 2004), meanwhile, river runoff, evaporation, seasonal advection, and mixing result in low surface salinity and high surface stratification (George et al., 1994; Kumar et al., 2002). The oceanic circulation and heat storage variation caused by low surface salinity strengthens the vertical stratification (Nyadjro et al., 2013). Finally, the BOB is also characterized by its low productivity (Madhupratap et al., 2003; Kumar et al., 2004) and high nitrification rate (Srinivas et al., 2011). Remineralization that consumes oxygen in the BOB is also weaker, making the BOB less hypoxic than AS, but still stronger than the Equator and other areas in the Eastern Indian Ocean (EIO; Kumar et al., 2002).

The Indian Ocean and sediments are highly oligotrophic, and most of the areas including the BOB are covered by volcanogenic sediments that are characterized by high illite and chlorite in the clay mineral assemblages (Venkatarathnam and Biscaye, 1973; Madhupratap et al., 2003). River borne solids with terrestrial organic carbon (Goldberg and Griffin, 1970; Fontugne and Duplessy, 1986) also contribute to the surface sediments in the BOB, where bacterial oxidation and respiration occurred (Peterson and Prell, 1985; Middelburg, 1989; Walsh, 2014). Oxidation and reduction of organic matter in the deep-sea sediments are active for resource and energy flow, which provide ideal environments for microbial nitrogen transformations. However, no direct measurements were carried out on the nitrification/denitrification processes in sediment of the BOB. The diversity and distribution of bacterial communities in the water column of the BOB and Equatorial area of the EIO has been screened by a high throughput sequencing analysis (Wang et al., 2016), but the details of the composition, diversity and distribution of nitrogen transforming microbes in these areas still remain largely unknown.

In this study, deep-sea sediments (>3,300 m) were sampled to investigate the diversity and distribution of archaeal and bacterial amoA genes. Clone library and q-PCR analysis were applied to characterize the community composition and relative abundances of ammonia oxidizing genes (amoA). Based on the multivariate statistics and phylogenetic analysis, diversity and distribution patterns of AOA and AOB in the EIO were compared and discussed. Environmental factors driving the distributions were also analyzed by redundancy analysis (RDA).

Deep-sea floor sediment constitutes the largest land on earth. It is characterized by extreme conditions including high pressure, low temperature, lack of light etc., and it harbors taxonomically and metabolically diverse microorganisms (D'Hondt et al., 2004; Inagaki et al., 2006; Kallmeyer et al., 2012). Among those microbes, bacteria dominated over archaea in the oxic surficial sediments, and the dominant groups varied with environmental gradients (Orcutt et al., 2011). More recently, high throughput sequencing data showed similar observations in the largest oceanic oxygen minimum zone (OMZ)—the North Pacific Ocean (Beman et al., 2012b), Arctic deep-sea sediments (1,200–5,500 m water depth; Rapp et al., 2016), and the marginal region of the Indian Ocean (Wang et al., 2016). However, as Orcutt et al. (2011) pointed out, compared to other regions, our knowledge of deep ocean sediment is still scarce. Based on the authors' knowledge, detailed community composition and distribution patterns of nitrogen transforming microorganisms in the EIO have not been explored to date.

Our data showed a dominance of AOA over AOB within the ammonia-oxidizing community and the ratios of AOA/AOB amoA genes ranged from 1.42 to 8.49. This observation corroborated the previous results in most deep ocean sediments including the SCS, the Ogasawara Trench, the Northwest Pacific Ocean, and the Mariana Trench (Beman et al., 2012a; Dang et al., 2013; Nunoura et al., 2013; Luo et al., 2015). However, a few studies showed the opposite trend, where the dominance of AOB over AOA was found in the Mid-Atlantic Ridge of the South Atlantic Ocean sediments (Xu et al., 2014) and in surface sediments in the oligotrophic South Atlantic Gyre (Lagostina et al., 2015). The inconsistent results suggest that further investigations are required to explore the distribution and function of these ammonia oxidizers in deep ocean environments. We summarized and compared the diversity and abundance of archaeal and bacterial amoA genes in Table 3, including the results from this study and also other deep ocean environments. In general, OTU numbers of AOA were greater than AOB in the sediments from deep oceans such as the Pacific Ocean, the South Atlantic Ocean, the South China Sea, the Ogasawara Trench, and the EIO (Table 3). Among the eight studies, the abundance (gene copy number) of archaeal amoA genes was greater than bacterial amoA genes in five of them, with water depths ranging from 3,386 to 5,725 m. In contrast, the OTU numbers of AOB were higher than AOA in the shallower environment, like the Gulf of Mexico (Table 3). However, we are also aware that the number of sequences/reads (sequencing depth) varied in each study, which make the comparisons not straightforward. Another impacting factor is the potential PCR bias. Since identical primers were used to amplify the amoA genes among these studies, we assumed the PCR bias would be minimal and it is possible for us to make these direct comparisons.

Dominance of AOA in the deep EIO sediment indicated that Archaea may adapt to low oxygen and low nutrient environments. Concentrations of [NO2− + NO3−] and NH4+ in our samples ranged from 0.002 to 0.0045 mg/L and 0.0019 to 0.0045 mg/L, respectively, which were lower than that in the Pacific Ocean and the shallow SCS (Cao et al., 2011a; Dang et al., 2013). Although AOB seems to have at least 10-fold higher cell activity than those of AOA, AOA appeared to be more sensitive to ammonia than AOB (Prosser and Nicol, 2012). Simulations based on experimental data from cultured AOA and AOB strains suggested that AOA grow faster than AOB at lower ammonia concentrations (Prosser and Nicol, 2012). Indeed, AOA have kinetic advantages over AOB under low substrate concentrations due to their up to 200-fold higher affinity for ammonium (Martens-Habbena et al., 2009, 2015). Furthermore, Archaea have developed low-permeability membranes in order to facilitate catabolic pathways, by which they are able to thrive under energy stress (Valentine, 2007). Moreover, deep-sea Archaea even might recycle membrane lipids between growing cells and the surrounding sediment in order to save energy (Takano et al., 2010). All the above evidences indicate the fact that Archaea could outcompete Bacteria or even phytoplankton in oxidizing ammonia under nutrient-limited conditions, such as the oligotrophic deep-sea sediments of the EIO.

All the archaeal amoA gene sequences from this study were clustered within the group of marine sediment, consisting of previously reported AOA sequences from the West Pacific Continental Margin, the SCS, and others (Figures 3, 4). No AOA clones were grouped into the clusters affiliated with estuarine or terrestrial environments. Meanwhile, the AOB sequences identified from the current study were all related with Nitrosospira-like species, which were commonly found in high salinity environments (see Section Discussion below). All these indicated the pure marine origin of the AOA and AOB clones revealed from this study. What's more, we did not observe distinct separation of the SCS sample from the EIO samples by forming unique clusters containing clones from the SCS only, with the exception of one small subcluster (Cluster II, Sub-cluster 2) containing two SCS clones in the AOA phylogenetic tree (Figure 3). We did observe distinct EIO clusters in both AOA and AOB phylogeny (Figures 3, 4). The Indian Ocean seemed to harbor large amount of local species of AOA and AOB in the sediments. In addition, due to the location specialization, novel or unique microorganisms were also expected to be present in deep EIO sediments. In our study, defined with 97% similarity, 5.8% of the archaeal and 3.6% of the bacterial amoA clones were previously un-revealed environmental sequences, suggesting the possible existence of unique ammonia-oxidizing microbes in the EIO sediments.

As expected, our data also indicated that the archaeal amoA genes contained higher diversity than bacterial amoA genes (Table 2). More variations as well as greater divergences of AOA genotypes than AOB sequences were observed from the constructed phylogeny (Figures 3, 4). High diversity of AOA has been observed from deep-sea sediments in the Pacific Ocean (5,017–7,068 m), the SCS (up to 2,370 m) and the northeastern Japan Sea (2,000–3,000 m; Nakagawa et al., 2007; Cao et al., 2011b; Luo et al., 2015; Table 3). However, in sediments from the Coastal North Sea, the SCS and the Mariana Trench, AOA was more abundant than AOB but gene variation of AOA was less significant than AOB (Wuchter et al., 2006; Cao et al., 2011b; Luo et al., 2015). Some previous studies also demonstrated that AOA and AOB were quite stable in terms of amoA gene copy numbers and both showed equal levels of variation (Mosier and Francis, 2008; Park et al., 2008; Jin et al., 2011; Zheng et al., 2013; Lagostina et al., 2015). Nevertheless, the disagreement of abundance and genetic variations between AOA and AOB distributions strongly suggested these two types of microbes are highly adapted to local environments and therefore their population dynamics are likely driven by ambient environmental gradients.

Our RDA analyses did not show significant correlations between total carbon and AOA/AOB distributions, but ammonia, nitrate and total nitrogen influenced the distribution of nitrogen transforming microbes (Figure 7). Distribution patterns inferred by NMDS showed a good separation of communities from the BOB and those close to the Equator; and the SCS was also separated from sites in the EIO (Figure 6). The distribution patterns revealed in this study were in certain accordance with the observations that presumably the environmental gradients including organic matter, nutrients and salinity impact the distribution and abundance of ammonia oxidizers (Urakawa et al., 2006; Dang et al., 2009; Bernhard and Bollmann, 2010; Wang et al., 2014; Xie et al., 2014). At the same time, AOA or AOB also responded differently to the environments. For example, Archaea tended to be less sensitive to the changes of nutrient availability (Leininger et al., 2006; Zheng et al., 2013), while the abundance of Bacteria responded to total carbon or ammonium concentrations (Zheng et al., 2013; Luo et al., 2015; Lagostina et al., 2015). Taken the above two points into consideration, likely the nutrient gradients (e.g., nitrate and ammonium in this study) between the BOB and the Equatorial area influenced the distribution patterns of nitrogen transforming microorganisms in the EIO sediments. Moreover, the total carbon (TC) ranged from 5.56% in the Equator (average of sites I412, I704, and I501A) to 10.87% in the BOB (average of sties I103, I105, 1202, and I207), which might also have contributed to the separation of these two groups of samples.

A close correlation of archaeal and bacterial amoA community distribution with water depth was also observed in our study (Figure 7). As reported, geographic location, light, oxygen concentration, and water depth were believed to influence vertical structure and richness of marine microbes in general (Walsh et al., 2014). Based on the nomenclature of AOB, all of our clones fell into deep-sea marine sediments cluster (Avrahami and Conrad, 2003), and were associated with high salinity and low ammonium environments (Dang et al., 2010). Ammonia oxidizers have been found to be associated with salinity gradients (Bernhard et al., 2005, 2007): Nitrosospira was more abundant in high-salinity environment, while Nitrosomonas preferred low-salinity environments (Dang et al., 2010; Jin et al., 2011). In most cases Nitrosospira-like species were dominant in marine environments (Bernhard and Bollmann, 2010), such as in deep-sea environment of Japan Sea, Atlantic Ocean, and Pacific Ocean (Nakagawa et al., 2007; Xu et al., 2014). Results from this study further strengthen this view by showing a preliminary dominance of Nitrosospira over Nitrosomonas in the deep-sea sediment of EIO (Figure 4).

Results from this study provided insights of diversity and distributions of AOA and AOB in the oligotrophic EIO sediments. Diversity and abundance of AOA outnumbered AOB in each sample, and water depth and nutrient gradients (e.g., [NO_2⁻ + NO_3⁻], NH_4⁺ concentration) were the main driving factors in shaping the distribution patterns from the Equator to the BOB. However, in natural settings, it is difficult to identify single environmental factors that explain the relative abundance of AOA and AOB. As Prosser and Nicol (2012) pointed out, further investigations on niche specialization and differentiation of AOA and AOB are needed, including in the BOB where the oxygen concentration is lower than other areas of the EIO. Future studies of these aspects will shed light on links between microbial distribution and nitrogen cycling in global oceans.

This work was designed by JS, JW, and JK. Samples were collected by JW. Experiments were carried by JW, XDZ, XCZ, ZX, and XL. Data were analyzed by JW, YM, and GQ. The manuscript was drafted by JW and revised by JK.

This work was supported by the Natural Science Foundation of China under contract Nos. 41276124 and 41506182, and Endowment from Stroud Water Research Center; Science Fund for University Creative Research Groups in Tianjin under contract No. TD12-5003; the Program for Changjiang Scholars to JS; the Science Fund from the Tianjin University of Science & Technology to JW under No. 0001001.