Nitrogen loss by nirS-type denitrifying bacterial communities in eutrophic coastal sediments
Graphical abstract
Introduction
Denitrification is a significant biological process that removes nitrogen fixed in aquatic and continental ecosystems into the atmosphere (Zheng et al., 2015). Under anaerobic, or anoxic, conditions, heterotrophic denitrifying bacteria produce N2 or N2O using organic matter as electron donors and NO3− or NO2− as electron acceptors (Abell et al., 2009). The denitrifying metabolic pathway involves several different functional genes, such as nar, nap, nir, nor and nos. Among them, the nir gene has become the most widely used in denitrifying bacterial community analysis (Braker et al., 1998; Mosier and Francis, 2010). nirS and nirK, two different types of nir genes, have been used to analyze the community composition of denitrifying bacteria, where nirS was found to be the gene with the widest distribution within ecosystems (Braker et al., 2000; Coyne et al., 1989; Lee and Francis, 2017; Nogales et al., 2002; Santoro et al., 2006; Wei et al., 2015; Zeng et al., 2016). Therefore, nirS has been increasingly used as a functional biomarker of denitrifying communities.
Many studies have investigated the presence of denitrifying bacteria in sediments (Zheng et al., 2015), soil (Mo et al., 2009), coastal wetlands (Gao et al., 2016), estuaries (Lisa et al., 2017; Zhang et al., 2014), and wastewater treatment plants (Zhang et al., 2019) based on the nirS-encoding gene. Francis et al. (2013) found that β-proteobacteria and α-proteobacteria dominated the community of denitrifiers in freshwater environments and seawater ecosystems, respectively. In addition, most of the nirS-encoding denitrifiers present in the coastal wetlands of China have been affiliated with uncultured bacteria due to the high diversity of the denitrifier phylotype (Gao et al., 2016). Dang et al. (2009) found that the sequences from these denitrifying bacteria, which were revealed by clone library in Jiaozhou Bay (JZB), matched those found in marine and estuarine sediments, especially in Chesapeake Bay. However, the data generated from the nirS gene clone library may not be sufficient to properly identify the diversity and distribution of denitrifiers.
Denitrification controls the amount of fixed nitrogen present in oceans (acting at a global scale), but it also acts in processes related to nitrogen loss at local and regional scales. (Seitzinger et al., 2006). Therefore, microbial denitrification is an essential component of the nitrogen cycle being extensively present throughout natural ecosystems. Isotope pairing technique (IPT) was first used to estimate the denitrification rate of marine, lake, and wetland sediments with the 15N labeling method (Nielsen, 1992; Trimmer et al., 2003). Based on this method, the contribution of denitrification to total nitrogen loss has been detected in marsh sediments of the Yangtze Estuary (87.1%–93%; Hou et al., 2013), in Arctic marine sediments (65%–99%; Rysgaard et al., 2004), surface flow constructed wetlands (76%; Erler et al., 2008), paddy soils (5.3%–17.4%; Yang et al., 2017), and coastal mangrove swamps of Daya Bay (90%; Xiao et al., 2018). Wu et al. (2019) reported that the activity and contribution to N2 production of anammox in JZB was approximately 0.01–0.24 nmol N g−1 h−1 and 0.07–18.55%, respectively. Nevertheless, the denitrifying activities in JZB sediments remained unknown.
JZB is a fan-shaped semi-enclosed natural bay located on the south coast of the Shandong Peninsula in China, with approximately 367 km2 and an average depth of 7 m (Zheng et al., 2019). Over the past decades, JZB has been seriously polluted due to the speedy development of agriculture, aquaculture, and industries in the area (Dang et al., 2008). At the same time, the scale of human disturbance affecting the nitrogen cycle is alarming, exacerbating the eutrophication of JZB. Microbial nitrogen removal has been carried out in eutrophic estuaries, thus, the excess nitrogen present in the bay could be removed by denitrification and anammox, reducing the level of eutrophication (Francis et al., 2013; Seitzinger et al., 2006). In this context, it is urgent to determine the nitrogen flux and analyze the nitrogen removal process to provide evidence that denitrification is an effective method to reduce the level of eutrophication in JZB.
In this study, nine representative surface sediments from JZB were obtained to (i) measure the denitrification rates and its contribution to nitrogen removal using the 15N tracing method; (ii) analyze the community composition and diversity of nirS-type denitrifying bacteria via high-throughput sequencing; (iii) quantify the abundance of nirS-type denitrifying bacteria; and (iv) investigate the relationship between environmental factors and characteristics of the denitrifying bacteria community at JZB. Our main aim was to provide a more comprehensive understanding of the denitrification process in eutrophic JZB.
Section snippets
Study area, sampling, and assay of physicochemical parameters
Site description, sampling details (Fig. 1), and analytical methods were previously reported by Wu et al. (2019). To analyze salinity, pH, and concentrations of ammonium, nitrite, and nitrate, porewater was extracted from sediments using the centrifugal method as described by Guan et al. (2017) and Wu et al. (2016).
Measurement of potential denitrification and anammox rates
In each sample, denitrification and anammox rates were calculated with isotope pairing. The slurry incubation experiments were carried out by adding (1) 15NO3−, (2) 15NH4 + 14NO3−,
Physicochemical properties
We used depth, pH, salinity and DIN (NO3−, NO2−, and NH4+) to characterize the environment of the nine sampled sites of JZB based on (Wu et al., 2019) (Table 1). Water depth in sampling sites ranged from 3.7 to 28 m, while pore-water pH varied from 7.54 to 8.42 and pore-water salinity varied from 28 to 35 psu. High levels of nitrate were found in all samples, which ranged from 233.85 ± 5.69 to 555.92 ± 10.08 μM. Additionally, nitrite and ammonium concentrations ranged from 0.31 ± 0.09 to
Discussion
The presence of nirS-encoding denitrifying bacteria in different habitats has been previously reported (Francis et al., 2013; Li et al., 2013, 2017; Lisa et al., 2017; Yu et al., 2018). Our findings revealed the activity, abundance, and community composition of nirS-type denitrifying bacteria in surface sediments of JZB using high-throughput sequencing and 15N isotope tracing method (Fig. 6). The potential denitrification rates in JZB sediments showed the same range values as determined in
Conclusions
In general, the contribution of denitrification to N2 production (81.45–99.93%) was considerably higher than that of anammox. The community of denitrifying bacteria varied geographically and was mainly assigned to Thiothrix, Woeseia, and uncultured denitrifiers in coastal estuaries and bays. Moreover, NH4+ was the limiting factor affecting the community composition of denitrifying bacteria. As the major process of nitrogen removal (5.66 × 104 t N y−1), microbial denitrification contributed
Ethical approval
This article does not contain human participants or experiments on animals.
Declaration of competing interest
All authors declare they have no conflicts of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China [Grant No. 91851111 and No.31870100], Natural Science Foundation of Guangdong Province [Grant No. 2019B1515120066], Research Foundation for Talented Scholars of Guangzhou University [Grant No.GU2017001], and Graduate Innovative Research Grant Program of Guangzhou University [Grant No. 019GDJC-M09].
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