Comammox Nitrospira play an active role in nitrification of agricultural soils amended with nitrogen fertilizers

https://doi.org/10.1016/j.soilbio.2019.107609Get rights and content

Highlights

  • Comammox Nitrospira clade A played a potential role in nitrification of fertilized agricultural soils.

  • The N fertilizer input significantly increased the abundances of comammox Nitrospira clade A, AOA and AOB.

  • Acetylene significantly impeded comammox clade A, AOA and AOB, but 1-octyne only inhibited AOB abundance.

  • Comammox Nitrospira clade A incorporated 13CO2 into their genomes during the incubation.

  • A new cluster of comammox Nitrospira may play an important role in nitrification of fertilized soils.

Abstract

The recent discovery of complete ammonia oxidizers (comammox Nitrospira) challenged the paradigm of the two-step nitrification mediated by two distinct groups of nitrifiers, and raised fundamental questions regarding their niche specialization and relative contribution to nitrification in agricultural soils. Previous studies suggest that comammox Nitrospira have an oligotrophic lifestyle and would outcompete canonical ammonia oxidizers (ammonia-oxidizing bacteria and ammonia-oxidizing archaea) under ammonia-limited conditions. Here, we demonstrated that comammox Nitrospira clade A were significantly more abundant than canonical ammonia oxidizers and 13CO2-DNA-stable isotope probing revealed that comammox Nitrospira clade A incorporated 13CO2 into their genomes in fertilized agricultural soils during the microcosm incubation. Phylogenetic analysis of the amoA gene revealed that 13CO2-labelled comammox Nitrospira clade A belonged to the Nitrospira inopinata-related cluster and a new cluster that was distinct from the known comammox isolates. These results demonstrated the potential important role of comammox Nitrospira in autotrophic ammonia oxidation in agricultural soils amended with nitrogen fertilizers and their lifestyle may be not strictly restricted to oligotrophic habitats. There is a potential contribution of comammox Nitrospira to soil nitrification, which calls re-evaluation of the microbial nitrogen cycling processes and the subsequent impacts on agriculture and the environment.

Introduction

Nitrification, the sequential microbial oxidation of ammonia (NH3) via nitrite (NO2) to nitrate (NO3), is a central process of the global nitrogen (N) cycling. The classic two-step nitrification process includes conversion from NH3 to NO2 by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), and conversion from NO2 to NO3 by nitrite-oxidizing bacteria (NOB) (Costa et al., 2006). Since the identification of archaea being capable of oxidizing ammonia to nitrite (Könneke et al., 2005), substantial efforts have been devoted to exploring the ecological significance and niche specialization of AOA and AOB in terrestrial ecosystems during the past decade (He et al., 2007; Hu et al., 2014; Prosser and Nicol, 2012). Although the relative contribution of AOA and AOB to nitrification is still debated, numerous physiological and genomic studies have supported their potential niche separation: AOA are more adapted to oligotrophic conditions characterized by acidic soils and nitrogen-depleted environments (He et al., 2007; Lehtovirta-Morley et al., 2011; Zhang et al., 2012); AOB have a eutrophic lifestyle and are more dominant in nitrogen-rich environments (Di et al., 2009).

In 2015, the discovery of complete ammonia oxidizers (termed “comammox”) within the Nitrospira genus that are capable of oxidizing NH3 to NO3 in a single organism radically challenged our long-held perspective of classic nitrification and the relative contribution of canonical ammonia oxidizers (Daims et al., 2015; van Kessel et al., 2015). Notably, in earlier theoretical studies, comammox organisms were predicted to play a crucial role under substrate-limiting conditions, such as in ammonia-depleted biofilms (Costa et al., 2006). Indeed, the enrichment of comammox organisms was successfully obtained from a deep oil exploration well (Daims et al., 2015) and an aquaculture system (van Kessel et al., 2015) containing low ammonium substrate influx. In 2017, a pure culture of comammox bacterium, Nitrospira inopinata was isolated from a microbial biofilm (Kits et al., 2017), and kinetic characterization revealed its high affinity for ammonia which facilitates the competitive advantage of comammox in oligotrophic habitats. Comparison of the ammonia oxidation kinetics for N. inopinata, AOA and AOB cultures suggested that, in contrast to earlier perceptions (He et al., 2012; Hu et al., 2014; Prosser and Nicol, 2012), comammox Nitrospira may out-compete AOA and AOB in ecological niches characterized by low ammonia availability (Kits et al., 2017).

Metagenomic profiling in published databases and PCR-based molecular investigations revealed the broad distribution of comammox Nitrospira in various aquatic and terrestrial habitats and engineered environments, including forest soils, freshwater sediments, wastewater treatment plants, and drinking water treatment systems (Bartelme et al., 2017; Beach and Noguera, 2019; Daims et al., 2015; Fowler et al., 2018; Hu and He, 2017; Palomo et al., 2016; Pinto et al., 2015; Pjevac et al., 2017; van Kessel et al., 2015; Wang et al., 2017; Yu et al., 2018). The majority of these habitats are characterized by low ammonium concentrations. These findings raised fundamental questions regarding the functional importance of comammox Nitrospira and the niche separation between comammox Nitrospira and canonical nitrifiers in terrestrial ecosystems. However, we have a very incomplete understanding of the relative contribution of comammox Nitrospira and canonical nitrifiers to the global nitrogen cycle, especially under copiotrophic conditions. As a novel group of ammonia oxidizers, it is imperative to bridge our knowledge gap regarding the potential contribution of comammox Nitrospira in nitrification of agricultural soils, which usually receive a large amount of N fertilizers to sustain agricultural production and might be unfavourable for the growth of comammox Nitrospira.

The aim of this study was to examine the potential contribution of comammox Nitrospira to nitrification and the niche differentiation between comammox Nitrospira and canonical nitrifiers in fertilized agricultural soils. We established a laboratory microcosm incubation to: (1) determine the responses of net nitrification rates and comammox Nitrospira, AOA and AOB populations to addition of N fertilizer and two nitrification inhibitors (NIs) (C2H2 and C8H14) in the pasture and arable soils; (2) assess the incorporation of 13CO2 into the genomes of comammox Nitrospira, AOA and AOB using 13CO2-DNA-stable isotope probing (SIP) technique; and (3) characterize the community composition of 13CO2-incorporated comammox Nitrospira through cloning library, sequencing and phylogenetic analysis.

Section snippets

Site description and soil sampling

Soil samples were collected in May 2018 from two long-term research farms: a dairy farm site (pasture soil) at Dookie (36°33′ S, 145°69′ E) and a vegetable farm site (arable soil) at Clyde (38°13′ S, 145°33’ E), Victoria, Australia. The Dookie site has been used as a dairy farm for more than 30 years, including around 43 ha of irrigated pastures with a feeding capacity for 180 cows. The pastures at the Dookie site are a mixture of perennial ryegrass (Lolium perenne) and white clover (Trifolium

Changes in soil properties during the microcosm incubation

The changes of NH4+-N and NO3-N concentrations were measured after the application of (NH4)2SO4 fertilizer and two NIs, C2H2 and C8H14 in the pasture and arable soils in a short-term microcosm incubation. In the control treatments, the NH4+-N concentrations remained largely unchanged in both soils (Fig. 2A and B), while the NO3-N concentrations continuously increased over time, indicating a sustaining nitrification activity in both soils (Fig. 2C and D). In the (NH4)2SO4 treatment, the NH4+-N

Discussion

Previous studies have recognized ammonia availability as a major factor shaping the niche differentiation between AOA and AOB (Hu et al., 2014; Prosser and Nicol, 2012). Agricultural soils usually receive a large amount of N-based fertilizers to promote plant growth, and pasture soils receive a majority of N from animal excreta return (Di et al., 2009, 2010). The theoretical NH3 concentrations in soil aqueous solution calculated based on the ionization equilibrium (NH4+ ↔ NH3 + H+; pKa

Conclusions

Our understanding of the niche specification and metabolic versatility of comammox Nitrospira was restricted to investigations in engineered systems and aquatic and terrestrial ecosystems characterized by low ammonia concentrations. This study, by using 13CO2-DNA-SIP and molecular approaches, demonstrated that, contrary to previous perceptions of the oligotrophic lifestyle of comammox Nitrospira, they are abundant and might play an active role in the two tested agricultural soils amended with N

Acknowledgements

This work was finally supported by Australian Research Council (DP160101028, LP160101134). The authors would like to thank Ms Zhenzhen Yan and Dr Bing Han for their assistance with soil sampling and Mrs Qing Xie for her help during the incubation trial.

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