Canonical ammonia oxidizers, rather than comammox Nitrospira, dominated autotrophic nitrification during the mineralization of organic substances in two paddy soils

https://doi.org/10.1016/j.soilbio.2021.108192Get rights and content

Highlights

  • Gln and low C/N rice straw stimulated the autotrophic nitrification in paddy soils.

  • AOB rather than AOA and comammox dominated nitrification in acidic paddy soil.

  • AOA dominated nitrification with mineralization of SOM or straw in neutral soil.

  • Nitrosospira cluster 3 was the active AOB in acidic and neutral paddy soils.

Abstract

Chemoautotrophic canonical ammonia-oxidizers and complete ammonia oxidizers (comammox) are responsible for ammonia oxidation, which is the rate-limiting step of nitrification in terrestrial ecosystems. However, the relative importance of autotrophic nitrification and the related active nitrifiers during mineralization of organic substances with different C/N ratios have not been fully elucidated to date. In this study, 15N tracing and 13CO2-DNA-based stable isotope probing (DNA-SIP) techniques were employed to investigate the responses of soil nitrification processes and the active nitrifying phylotypes in acidic and neutral paddy soils with amendment of glutamine (Gln) or rice straw with low C/N (LS) or high C/N (HS). Acetylene (C2H2) was utilized to differentiate autotrophic and heterotrophic nitrification. The results indicated that C2H2 completely blocked the production of 15NO3 in Gln and LS treatments, implying the predominance of autotrophic nitrification with the N derived from the mineralization of Gln and LS. Neither 15NH4+ nor 15NO3 was detected in the HS-amended soils, suggesting that NH4+ derived from organic N mineralization was immobilized immediately by microorganisms. In the DNA-SIP microcosms, the abundance of ammonia-oxidizing bacteria (AOB) in the unamended control (CK), Gln and LS treatments of acidic paddy soil increased by 22.1-, 71.9- and 27.6-fold, respectively. A significantly larger proportion of Nitrosospira-like AOB, rather than ammonia-oxidizing archaea (AOA) and comammox Nitrospira, was labelled by 13CO2, indicating that AOB made a stronger contribution to autotrophic nitrification in acidic paddy soil. The active nitrification of neutral paddy soil was predominated by AOA affiliated with the Nitrososphaera viennensis lineage in the CK and LS microcosms but by Nitrosospira-like AOB in the Gln microcosm. These results suggest the significance of autotrophic nitrification catalysed by canonical ammonia oxidizers, rather than comammox, during the mineralization of organic substances with low C/N in paddy soils.

Introduction

Nitrification, a critical component of the terrestrial nitrogen (N) cycle, can lead to significant N losses through nitrate (NO3–N) leaching and nitrous oxide (N2O) emissions (Beeckman et al., 2018; Hu et al., 2015). Autotrophic nitrification involves two steps: the first and rate-limiting step is ammonia oxidation (NH3 → NH2OH/HNO → NO2), which is traditionally thought to be driven by ammonia-oxidizing archaea (AOA) and bacteria (AOB); the second step, nitrite oxidation (NO2 → NO3), is executed by nitrite-oxidizing bacteria (NOB) (Gruber and Galloway, 2008). However, this two-step nitrification was challenged by the recent discovery of complete ammonia oxidizers (comammox) within the genus of Nitrospira, which are capable of oxidizing NH3 to NO3 in a single organism (Daims et al., 2015; van Kessel et al., 2015). The genomes of comammox Nitrospira harbour the full set of genes encoding ammonia monooxygenase and hydroxylamine dehydrogenase for ammonia oxidation and genes encoding nitrite oxidoreductase for nitrite oxidation (Daims et al., 2015; van Kessel et al., 2015). The discovery of comammox Nitrospira necessitates the re-evaluation of the niche differentiation of nitrifiers and their relative contributions to nitrification.

Soil pH is a vital factor influencing the niche differentiation of autotrophic nitrifiers. AOA have been reported to play a predominant role in autotrophic nitrification of acidic soils, benefiting from their high NH3 affinity (Lehtovirta-Morley et al., 2014; Lu and Jia, 2013; Yu et al., 2018; Zhang et al., 2012, 2016), though autotrophic nitrification is potentially impeded by low soil pH due to the ionization of NH3 (Li et al., 2018a; Zhang et al., 2018). However, recently, an acidic-tolerant AOB was isolated from acidic tea plantation soil (Hayatsu et al., 2017), and DNA-stable isotope probing (SIP) analysis indicated that Nitrosospira-like AOB dominated ammonia oxidation in acidic forest soil and paddy soil (Huang et al., 2018; Zhang et al., 2019). In neutral or alkaline soils, the relative importance of AOA or AOB in autotrophic nitrification may depend on soil fertility (Di et al., 2009; Jia and Conrad, 2009; Zhang et al., 2010). Furthermore, the newly discovered comammox Nitrospira were suggested to play an important role in oligotrophic environments, e.g., aquatic systems and biofilters characterized by low ammonium concentrations (Bartelme et al., 2017; Fowler et al., 2018; Kits et al., 2017; Liu et al., 2020). Thus, it is reasonable to presume that comammox Nitrospira can be a strong competitor to AOA for nitrification in acidic soil. However, compelling evidence is lacking for the contribution of comammox Nitrospira to nitrification across pH gradients. Therefore, the niches of canonical ammonia oxidizers and newly discovered comammox Nitrospira across pH gradients are still in debate.

The N content of organic substances also plays an important role in controlling soil NO3- production. For instance, some studies assumed that the continuous flux of NH3 from the mineralization of organic N (e.g., L-glutamic acid, yeast extract, and rice callus) would supply a substrate for autotrophic nitrification and stimulate the growth of AOA (Levičnik-Höfferle et al., 2012; Liu et al., 2019a). However, other studies found that heterotrophic nitrification dominated soil nitrification under amino acid and maize straw amendments (Zhang et al., 2014, 2015a), and was positively correlated with soil carbon (C) to N ratio (Zhang et al., 2015b; Zhu et al., 2013), indicating that higher availability of C and N might favour heterotrophic nitrification and autotrophic nitrification, respectively. Moreover, heterotrophic nitrification was the dominant nitrification process in some acidic forest soils and dairy pasture with high organic C content (Liu et al., 2015; Zhang et al., 2015b, 2020), suggesting that soil pH may interact with C and N availability to regulate the relative contribution of heterotrophic and autotrophic nitrification. To date, few studies have investigated the relative importance of the C and N availability of organic substances with different C/N ratios for heterotrophic and autotrophic nitrification in soils across pH gradients.

The objectives of this study were to assess the relative importance of heterotrophic and autotrophic nitrification and to identify the active nitrifiers in acidic and neutral paddy soils with L-glutamine, low C:N ratio (21) rice straw and high C:N ratio (90) rice straw amendments. 15N-labelled organic substances and C2H2 inhibition were utilized to differentiate heterotrophic and autotrophic nitrification. 13CO2-DNA-SIP and MiSeq sequencing were employed to identify the active nitrifying communities. Based on previously published results, we hypothesized that (i) autotrophic and heterotrophic nitrification dominate in soils with low and high C/N organic substances, respectively, and (ii) AOA and comammox Nitrospira play active roles in acidic paddy soil with rice straw amendment, while AOB are active in neutral soil with glutamine amendment.

Section snippets

Soil sampling and physicochemical analysis

Neutral paddy soil (pHH2O 6.2) was collected from Changxing, Zhejiang Province (31°00′ N, 119°55′ E), and acidic paddy soil (pHH2O 4.9) was collected from Taishan, Guangdong Province (22°03′ N, 112°57′ E), China. Both sampling sites have a subtropical monsoon climate. The neutral paddy soil site has a mean annual rainfall of 1300 mm and a mean annual temperature of 15.6 °C. The acidic paddy soil site has a mean annual rainfall of 2443 mm and a mean annual temperature of 22 °C. Soil samples were

N mineralization and nitrification of organic amendments

In the 15N-labelled microcosms, the mineralization of organic substances and the subsequent nitrification were assessed by determining the changes in soil 15NH4+ and 15NO3 contents during the 56-day incubation with or without C2H2 (Fig. 1). In acidic paddy soil without C2H2, the amendment of Gln and LS significantly increased 15NH4+-N concentrations from 0.38 at day 0–3.48 and 0.63 mg kg−1 dry soil at day 56, respectively (P < 0.05) (Fig. 1a). 15NO3-N concentrations increased significantly

Autotrophic nitrification dominated soil nitrification with organic amendments

Acetylene, a specific inhibitor of autotrophic nitrification, is usually used to differentiate autotrophic and heterotrophic nitrification (Liu et al., 2015). C2H2 application impeded the production of 15NO3-N while 15NH4+-N accumulated with Gln and LS amendments (Fig. 1), indicating that NH3 mineralized from Gln and LS fuelled autotrophic nitrification in paddy soils. These results agreed with previous findings that the NH3 released from the mineralization of rice callus or soil native

Conclusions

Our results demonstrated that the NH3 generated from mineralization of soil native organic N, glutamine or low C/N rice straw stimulated autotrophic nitrification in acidic and neutral paddy soils, while no nitrification activity was observed in response to high C/N rice straw amendment. DNA-SIP indicated that canonical ammonia oxidizers exhibited greater growth than comammox Nitrospira in response to mineralization of soil native organic N, glutamine or low C/N rice straw. More specifically,

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 supported by National Key R&D Program of China (2016YFD0200302), National Natural Science Foundation of China (41671249, 42007033) and Fundamental Research Funds for the Central Universities (2019QNA6011). The authors sincerely appreciate two anonymous reviewers acknowledging their insightful and helpful suggestions for improving the manuscript.

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