Comammox Nitrospira play an active role in nitrification of agricultural soils amended with nitrogen fertilizers
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.
References (33)
- et al.
Why is metabolic labour divided in nitrification?
Trends in Microbiology
(2006) - et al.
Current insights into the autotrophic thaumarchaeal ammonia oxidation in acidic soils
Soil Biology and Biochemistry
(2012) - et al.
Ammonia-oxidizing bacteria are more responsive than archaea to nitrogen source in an agricultural soil
Soil Biology and Biochemistry
(2016) - et al.
Archaeal and bacterial ammonia-oxidisers in soil: the quest for niche specialisation and differentiation
Trends in Microbiology
(2012) - et al.
Comammox Nitrospira clade B contributes to nitrification in soil
Soil Biology and Biochemistry
(2019) - et al.
Comammox in drinking water systems
Water Research
(2017) - et al.
Freshwater recirculating aquaculture system operations drive biofilter bacterial community shifts around a stable nitrifying consortium of ammonia-oxidizing archaea and comammox Nitrospira
Frontiers in Microbiology
(2017) - et al.
Design and assessment of species-level qPCR primers targeting comammox
Frontiers in Microbiology
(2019) - et al.
Complete nitrification by Nitrospira bacteria
Nature
(2015) - et al.
Nitrification driven by bacteria and not archaea in nitrogen-rich grassland soils
Nature Geoscience
(2009)
Ammonia-oxidizing bacteria and archaea grow under contrasting soil nitrogen conditions
FEMS Microbiology Ecology
Comammox Nitrospira are abundant ammonia oxidizers in diverse groundwater‐fed rapid sand filter communities
Environmental Microbiology
Archaea rather than bacteria control nitrification in two agricultural acidic soils
FEMS Microbiology Ecology
Quantitative analyses of the abundance and composition of ammonia‐oxidizing bacteria and ammonia‐oxidizing archaea of a Chinese upland red soil under long‐term fertilization practices
Environmental Microbiology
Archaea produce lower yields of N2O than bacteria during aerobic ammonia oxidation in soil
Environmental Microbiology
Comammox—a newly discovered nitrification process in the terrestrial nitrogen cycle
Journal of Soils and Sediments
Cited by (146)
Higher stochasticity in comammox Nitrospira community assembly in upland soils than the adjacent paddy soils at a regional scale
2024, Science of the Total EnvironmentAOB Nitrosospira cluster 3a.2 (D11) dominates N<inf>2</inf>O emissions in fertilised agricultural soils
2024, Journal of Environmental ManagementAbundance and community structure of comammox bacteria in the rhizosphere of aquatic plants in an urban eutrophic lake
2024, Ecohydrology and Hydrobiology