Rates and microbial communities of denitrification and anammox across coastal tidal flat lands and inland paddy soils in East China
Graphical abstract
Introduction
China is one of the predominant rice producers in the world, with approximately 28% of arable land used for rice production (Baker, 2004). Nitrogen (N) is the primary limiting element for crop productivity in terrestrial ecosystems, and the application of N fertilizers intensively performs a key role in rice cultivation (Galloway et al., 2004; Long et al., 2013). However, the excessive input of N fertilizers is closely related with the low N-utilization efficiency and negative impacts on the environment in association with water pollution and the emission of nitrous oxide (N2O), a powerful greenhouse gas and atmospheric ozone destroyer (Galloway et al., 2003; Zhang et al., 2015). Particularly, nitrogen input via rice cultivation has strong influences on the biogeochemical cycling of nitrogen in coastal ecosystem nearby, as it provides favorable conditions for microbially mediated nitrate reduction processes including denitrification (dissimilatory denitrification) and anaerobic ammonium oxidation (anammox) (Ishii et al., 2011a; Kuypers et al., 2018). There is clearly therefore a need to understand, in marine-terrestrial ecotone, the microbial mechanisms of nitrate reduction process and to clarify the main influential factors driving the process and functional microbial diversity.
Dissimilatory denitrification has been intensively studied as it has long been considered the sole pathway for N2 loss until the discovery of anammox, while it is arguably the most poorly understood process in the terrestrial N cycle (Lennon and Houlton, 2017). The denitrification process comprises four reduction steps from NO3− to N2 via NO2−, NO, N2O, which were successively mediated by nitrate reductase (Nar or Nap), cytochrome cd1-type (NirS) or copper-containing type (NirK) nitrite reductase, nitric oxide reductase (Nor) and nitrous oxide reductase (Nos). The microbial mechanisms of denitrification also remain poorly understood to a large extent, as multiple functional genes and highly diverse microbes are involved in these processes (Philippot et al., 2007; Zumft, 1997). The last step, N2O reduction to N2 by the nitrous oxide reductase encoded by nosZ gene, is acting as the sole sink of atmospheric N2O. The nosZ gene, is thus widely used as an indicator of genetic marker for denitrifying community (Chapuis-Lardy et al., 2007; Ishii et al., 2011b). More recently, metagenomics-based studies have substantiated that a new clade of the nosZ gene container, named clade II, is widely present in soils (Domeignoz-Horta et al., 2017), salt marsh (Dini-Andreote et al., 2016), coastal sediment (Wittorf et al., 2016) and mine drainage water (Herbert Jr et al., 2014), and comprises a wide variety of ranges of bacterial and archaeal taxa (Jones et al., 2013; Sanford et al., 2012). Studies based on pure cultures and environment samples also demonstrated that the newly discovered nosZ clade II denitrifiers display distinct characteristics in terms of the gene expression, electron transport, and N2O-reducing activity in comparison with the typical nosZ clade (i.e. nosZ clade I) (Duan et al., 2018; Hein et al., 2017; Throbäck et al., 2004; Yoon et al., 2016), while their distribution pattern in soil and contribution to N2O reduction are greatly disputed (Assémien et al., 2019; Espenberg et al., 2018; Zhao et al., 2018). Further exploration on the distribution pattern of nosZ gene, particularly the newly discovered nosZ type II denitrifiers, and their correlation with N2O reduction in various wetland ecosystems are of paramount importance to understand the potential of different ecosystems as a N2O sink and the underlying mechanisms.
Since the first discovery in wastewater treatment plant in 1995, the anammox process, ammonium oxidized to dinitrogen by anammox bacteria using nitrite as an electron acceptor under severely anoxic conditions, has been widely reported in aquatic environments such as marine, coastal and estuarine sediment, freshwater and lake sediment (Dale et al., 2009; Dalsgaard et al., 2003; Kartal et al., 2006; Kuypers et al., 2003; Kuypers et al., 2005; Schubert et al., 2006). In some marine areas like Skagerrak, anammox even accounted for up to 79% of the N2 production (Engström et al., 2005). Compared to aquatic habitats, studies on the rate of anammox and their functional microbes in terrestrial habitats are still limited. The available estimations on the contribution of anammox to N2 loss and the key influential factors in paddy soils are variable. For example, it was reported that anammox accounted for 0.6–15% of N2 production and anammox activity was significantly correlated with soil nitrate concentration and C/N ratio in typical paddy soils across 11 provinces in southern China (Yang et al., 2015). Anammox accounted for 1.0–5.0% of N2 production in ravine paddy field soil from Japan (Sato et al., 2012), and 4.5–9.2% in 11 typical paddy soils from main rice production area in China and the rate of which was significantly correlated to multiple edaphic factors like soil total C, total N, soil organic carbon (SOC), dissolved organic carbon (DOC) etc. (Shan et al., 2016). However, anammox contributing up to 22.3–37% N2 loss was also reported in some sites with high organic matter and ammonium inputs (Zhang et al., 2017; Zhu et al., 2011). Meanwhile, few studies tried to link the rate of the process and anammox bacterial community, and the studies on which showed no consistent results. For instance, Candidatus Scalindua, the well-known dominant anammox genus in marine environments, was reported as the main anammox bacteria genus in paddy soils in northeast China (Wang and Gu, 2013), while Candidatus Brocadia and Candidatus Kuenenia were detected as the main anammox bacteria in ravine paddy soils from Japan (Sato et al., 2012), and in paddy soils from southeast and northwest China (Bai et al., 2015a; Yang et al., 2015). Our previous work on the vertical profiles of three paddy soils also suggested that anammox was more active in alkaline soil and neutral soil than in acidic soil, and the anammox bacterial community differentiated among three soil types (Bai et al., 2015b). Considering the large area of paddy soils (25% of arable land) across diverse soil types in China, further investigation on the activity and functional microbial distribution pattern of anammox is still necessary to estimate the N loss and develop possible mitigation regimes.
Saline soils accounted for 5% of arable land in the world (Lambers, 2003), the occurrence of which greatly limit the crop yield in arid and semi-arid regions (Rengasamy, 2010). The total area of saline soil in China is about 3.6 × 107 ha, accounting for 4.9% of the total available land (Wang et al., 2011). Yancheng city in Jiangsu Province possesses the longest coastline and the largest tidal flat wetland in China, with a tidal flat area of 13 million hectares, accounting for 43.02% of the total land area (Zhou et al., 2007). A large area of rice has been cultivated in this area to expand the cultivated area and improve crop yield (Abrol and Bhumbla, 1979; Chi et al., 2012), and consequently increased large amount of N input in these marine-terrestrial ecotones, which would exert strong influences on the biogeochemical cycling of nitrogen and the transport of excessive nitrogen into coastal waters. A comprehensive understanding on the rates and influential factors of denitrification and anammox processes, and the functional microbial community involved in are therefore critical to enable better prediction on the mitigation of excessive nitrogen from these areas. Moreover, the marine-terrestrial ecotone represents drastic physical-chemical gradients like salinity, pH and organic matter, which might greatly influence the denitrification and anammox processes and their performance on mitigation of excessive nitrogen and N2O. We hypothesized that saline paddy soils developed from intertidal saline mud possessed the characteristics of both coastal wetland and paddy soil, and would exhibit different patterns from coastal sediment and inland paddy soil. We tested this by estimating the rates of anammox and denitrification using 15N labeling technique across coastal tidal flat, saline and inland paddy soils, combining with the molecular ecology survey on functional genes associated with N2 production (hzsB gene encoding hydrazine synthase beta subunit, and nosZ clade I and II genes). This study aimed to address the following questions: 1) How anammox and denitrification processes vary across coastal tidal flat to inland paddy soils, and what are the key influential factors; 2) What's the distribution pattern of anammox bacteria and nosZ gene-containing denitrifiers, and how are they shaped across coastal tidal flat to inland paddy soils.
Section snippets
Field sites and sample collection
Samples were collected from ten sites along the longitude from the Yellow sea coastal bay in Yancheng, Jiangsu province, to inland directions in August 2015, including a mud flat site daily flushed by tide, a tidal flat weed wetland site, and eight sites of rice fields (Table S1 and Fig. S1S1). Except site 8, all paddy soil sites were chosen at an interval of ~30 km between each other. Site 8 was 328 km far from site 7 and chosen as an inland paddy soil control without seawater intrusion or
Soil physiochemical properties
Soil physical and chemical properties were highly variable across the ten sampling sites (Table 1). Generally, soil pH and EC contents decreased along the coastal mud flat (CMF) to inland paddy soils except site S5. Among all samples, CMF and tidal flat wetland (TFW) had the highest pH values ranging between 8.97 and 9.41 in surface and subsurface layers (P < 0.05), while the pH ranged from 5.55 to 8.70 in surface layer and between 6.09 and 8.93 in subsurface layer among paddy soils (Table 1).
Discussion
Here we showed that the denitrification process contributed to 87.1–100% of the total N2 production while anammox accounted for the remaining 0% - 12.9% across the coastal mud flats, tidal flat wetlands and paddy soils with a gradient of salinity, pH and N inputs, indicating that denitrification played a dominant role in N2 formation in these ecosystems. This result was consistent with previous studies showing that the contribution to N2 production by anammox varied between 0 and 15% in coastal
Conclusions
In summary, our study based on 15N tracer showed that denitrification rather anammox was the main process driving N2 formation across coastal tidal flats, saline and inland paddy soils. The result was corroborated by the molecular ecology survey showing that both nosZ clade I and II gene abundance were 1–2 orders of magnitude higher than the hzsB gene in those samples. Our results further showed that salinity was the most influential factor driving the anammox bacterial community which was
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (41771288) and National Key R&D Program (2017YFD0800604). Limei Zhang was supported by the Youth Innovation Promotion Association (Y201615), Chinese Academy of Sciences. We would like to thank Dr. Lili Han and Yaqi Wang for assistance in soil sampling, and Ren Bai for helps in laboratory analysis.
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