Elsevier

Science of The Total Environment

Volume 668, 10 June 2019, Pages 224-233
Science of The Total Environment

Adaptive responses of comammox Nitrospira and canonical ammonia oxidizers to long-term fertilizations: Implications for the relative contributions of different ammonia oxidizers to soil nitrogen cycling

https://doi.org/10.1016/j.scitotenv.2019.02.427Get rights and content

Highlights

  • Long-term fertilizers inputs increased the abundance of comammox.

  • Specific comammox clades responded distinctly to fertilizers inputs.

  • Comammox played a less important role in soil nitrification than AOB.

  • The abundance of AOB increased with the gradients of fertilizers inputs.

Abstract

The new discovery of complete ammonia oxidizers (comammox), single organisms capable of oxidizing ammonia into nitrate, redefined the traditional view of nitrification. However, little is known about the relative contributions of comammox and other nitrifiers to nitrification, particularly in agricultural soils with long-term intensive input of nutrients. Herein, we investigated the communities of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and comammox Nitrospira in agricultural soils under nutrients input gradient of nitrogen (0–675 kg N ha−1 year−1), phosphorus (0–405 kg P2O5 ha−1 year−1), and potassium (0–675 kg K2O ha−1 year−1) fertilizers for 19 years. The results showed that N and K fertilizers input significantly (P < 0.05) increased the AOB-amoA gene abundance, while AOA were not as sensitive as AOB. The comammox-amoA gene copies were increased in all fertilizer treatments and was significantly correlated (P < 0.05) with the amount of N fertilizer added. Terminal restriction fragment length polymorphism (T-RFLP) combined with clone-library assays of comammox-amoA gene showed that increasing gradient of nutrients input increased the relative abundance of 73 bp T-RF (assigned to Clade A) but decreased the relative abundance of 198 bp T-RF (representing Clade B). Correlation analyses and stepwise linear regression analyses demonstrated that AOB were the dominate contributors to soil potential nitrification, while comammox Nitrospira did not play a significant role (P > 0.05). This study provided insights into the adaptive responses of comammox Nitrospira and canonical ammonia oxidizers to long-term fertilizations and their relative contributions to potential nitrification in arable soils.

Introduction

Nitrification, a biological oxidation of ammonia to nitrite and then to nitrate, is conventionally thought of a two-step process carried out by separate groups of nitrifiers: ammonia-oxidizing bacteria (AOB) and archaea (AOA), and nitrite oxidizing bacteria (NOB). However, researchers were puzzled by this two-step process because it is not the most kinetically optimal pathway for nitrification. Scientists predicted the presence of a single organism which could completely oxidize ammonia to nitrate with a lower energy consumption when compared with separate nitrification steps, and that this type of organism might have a competitive advantage over canonical ammonia oxidizers, i.e., AOB and AOA, in environments with low substrate concentrations (Costa et al., 2006). Researchers were able to successfully enrich microbes of the genus Nitrospira from a deep oil well (Daims et al., 2015) and an aquaculture system (van Kessel et al., 2015). These microbes were able to catalyze complete nitrification, and thus were called complete ammonia oxidizers (comammox). Since then, through molecular or culture methods, comammox have been detected in many engineered and natural ecosystems (Chao et al., 2016; Daims et al., 2015; Gao et al., 2016; Palomo et al., 2016). Recently, a kinetic analysis of Nitrospira inopinata revealed that it had a higher growth yield and affinity for ammonia than canonical nitrifiers, which indicated that comammox Nitrospira had an oligotrophic lifestyle and might be more competitive than canonical ammonia oxidizers under low ammonia substrate conditions (Kits et al., 2017). Indeed, comammox Nitrospira have often been reported to outnumber the canonical ammonia oxidizers in low ammonia environments such as biofilters (Fowler et al., 2018; Palomo et al., 2016), drinking water systems (Y. Wang et al., 2017) and forest soils (Hu and He, 2017). These findings have improved our understanding of nitrification and suggest that there is an urgent need to assess both the ecological distribution of comammox and their roles in nitrification and nitrogen (N)-cycling in various environments. This is of particular importance for soils with frequent additions of nutrients, such as those found in like agricultural ecosystems.

Ammonia oxidation and nitrification are important processes in nutrient transformation after fertilizer input into soils. Since the discovery of ammonia oxidation function in AOA (Francis et al., 2005) and its predominance in different soil types when compared to AOB (Leininger et al., 2006), many studies have focused on the distribution of AOA and AOB in arable soils and their relative contributions to nitrification. Although most researchers agree that the AOB amoA gene abundance has a greater response to N additions than their archaeal counterparts (Carey et al., 2016; Prosser and Nicol, 2012), scientists have not reached an agreement about their relative contributions to nitrification. Researchers have speculated that AOA might contribute more to the process of nitrification in soils with low ammonia contents, high acidity and low oxygen (Zhang et al., 2012; Zhang et al., 2010), while AOB might play key roles in neutral and alkaline soils, or in soils with high ammonia content (Ke et al., 2013; Shen et al., 2012; Giguere et al., 2015; Taylor et al., 2010). The recent discovery of comammox, and their surprisingly high abundance, makes evaluations of the relative contribution of these three organisms to the process of nitrification exceedingly complex.

For decades, excessive use of fertilizers was the norm in limited areas of the world, notably in China, and agricultural ecosystems are now facing serious environmental and ecological problems (Yang et al., 2015). Low fertilizer use efficiency not only waste limited resources, it also causes environmental pollution such as the leaching of nitrate, NH3 volatilization and soil acidification (Guo et al., 2010). Nitrification through AOA and AOB produces the intermediate metabolite NO2 in the presence of sufficiently high available ammonia such as in arable soils; soil NO2 accumulation would lead to an increase in NO2-driven N2O production (Duan et al., 2018; Venterea et al., 2015). Comammox, which do not accumulate NO2 during ammonia oxidation (Daims et al., 2015), might reduce the production of NO2 and N2O, which would lessen environmental pollution and improve the use efficiency of fertilizers.

Recently, the newly-released PCR primer sets for the comammox amoA gene were successfully used in investigating the distribution of comammox Nitrospira in various environments (Fowler et al., 2018; Pjevac et al., 2017). A study using quantitative PCR revealed that increased rates of N deposition could increase the amoA gene abundance of comammox Nitrospira clade B in forest soils, which suggests that N input could influence comammox Nitrospira (Shi et al., 2018). However, the adaptive response pattern of comammox to long-term fertilizations in arable soils is still unknown, and elucidation of this pattern is vital for a holistic understanding of nitrification as it relates to the foundations of agricultural production.

In our present study, we collected soil samples from an experimental field that has undergone 19 years of fertilization using varying amounts of N, phosphorus (P) and potassium (K) fertilizers. The purposes of this study were as follows: 1) to investigate the impacts of different amounts of fertilizers on canonical ammonia oxidizers and comammox Nitrospira, and 2) to explore the relative contributions of canonical ammonia oxidizers and comammox Nitrospira to nitrification. We hypothesized that (i) AOB would be stimulated by increased fertilizer input, while AOA and comammox would not be as sensitive to fertilizer input as AOB, due to their different affinities for substrate, and (ii) that comammox Nitrospira play a less important role in nitrification in arable soils with high nutrient availability, when compared to the canonical ammonia oxidizers, due to the oligotrophic lifestyle of comammox Nitrospira.

Section snippets

Site description

The experimental site is situated at Qianyanzhou Ecological Station, Jiangxi province, China (26°44′ N, 115°03′ E) and is a double-season rice system. The site has a subtropical monsoon climate in a hilly region and the soil type is waterloggogenic paddy soil (soils under good conditions of irrigation and drainage). The average annual temperature is 16.7 °C. Annual precipitation varies from 1200 to 1900 mm.

Experimental design and soil sampling

The long-term fertilization experiment was established in 1998 with randomized block

Soil properties, PAO and PNO

As shown in Table 2, all of the measured soil properties were significantly (P < 0.05) influenced by the input of fertilizers. In terms of the 7 inorganic fertilizer treatments, the soil pH, SOM, TN, TP, AN, AP, and AK tended to increase as the amount of N, P and K in the fertilizer increased, which peaked in the N4P4K4 treatment receiving the highest amounts of inorganic fertilizers. When the OM treatment was included in the comparison, the OM treatment had the highest SOM, TN, AN, and AP

Impacts of nutrients addition on canonical ammonia oxidizers

In the present study, the abundances of AOB and AOA were significantly (P < 0.05) influenced by the gradient of nutrients input over the course of 19 years (Fig. 2). Many previous studies have reported the impacts of fertilization regimes on canonical ammonia oxidizers, and it is not surprising that the addition of N fertilizers increased the abundance of AOB. This observation was in line with previous studies based on field experiments (Guo et al., 2017; J. Wang et al., 2017) and meta-analysis

Conclusion

Our present study revealed that the abundance of AOB increased with the increasing gradient of nutrients input, while the abundance of AOA was not as sensitive as AOB to fertilizers input. The abundance of comammox Nitrospira was lower than both AOB and AOA, the input of nutrients, especially N, could enhance the abundance of comammox Nitrospira. Specific clades of comammox Nitrospira had different responses to the fertilization. For instance, the relative abundance of the 73 bp T-RF (Nitrospira

Acknowledgements

This work was supported by the National Natural Science Foundation of China (41671254), the Chinese Academy of Sciences (XDB15020200, and Hundred Talents Program to Y. Ge), and the State Key Laboratory of Urban and Regional Ecology (SKLURE2017-1-7).

References (51)

  • J.I. Prosser et al.

    Archaeal and bacterial ammonia-oxidisers in soil: the quest for niche specialisation and differentiation

    Trends Microbiol.

    (2012)
  • X. Shi et al.

    Niche separation of comammox Nitrospira and canonical ammonia oxidizers in an acidic subtropical forest soil under long-term nitrogen deposition

    Soil Biol. Biochem.

    (2018)
  • Y. Wang et al.

    Comammox in drinking water systems

    Water Res.

    (2017)
  • J. Bengtsson et al.

    The effects of organic agriculture on biodiversity and abundance: a meta-analysis

    J. Appl. Ecol.

    (2005)
  • J.R. Brown

    Recommended Chemical Soil Test Procedures for the North Central Region

    Missouri Agricultural Experiment Station, University of Missouri-Columbia.

    (1998)
  • D. Bru et al.

    Determinants of the distribution of nitrogen-cycling microbial communities at the landscape scale

    ISME J

    (2011)
  • Y. Chao et al.

    Novel nitrifiers and comammox in a full-scale hybrid biofilm and activated sludge reactor revealed by metagenomic approach

    Appl. Microbiol. Biotechnol.

    (2016)
  • H. Daims et al.

    Complete nitrification by Nitrospira bacteria

    Nature

    (2015)
  • W. Dong et al.

    Effect of different fertilizer application on the soil fertility of paddy soils in red soil region of southern China

    PLoS One

    (2012)
  • M.C.P. e Silva et al.

    Microbe-mediated processes as indicators to establish the normal operating range of soil functioning

    Soil Biol. Biochem.

    (2013)
  • S.J. Fowler et al.

    Comammox Nitrospira are abundant ammonia oxidizers in diverse groundwater-fed rapid sand filter communities

    Environ. Microbiol.

    (2018)
  • C.A. Francis et al.

    Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean

    Proc. Natl. Acad. Sci. U. S. A.

    (2005)
  • J.-F. Gao et al.

    Diversity, abundance and activity of ammonia-oxidizing microorganisms in fine particulate matter

    Sci. Rep.

    (2016)
  • A.T. Giguere et al.

    Nitrification responses of soil ammonia-oxidizing archaea and bacteria to ammonium concentrations

    Soil Sci. Soc. Am. J.

    (2015)
  • J. Guo et al.

    Significant acidification in major Chinese croplands

    Science

    (2010)
  • Cited by (76)

    View all citing articles on Scopus
    View full text