Fertilizer types shaped the microbial guilds driving the dissimilatory nitrate reduction to ammonia process in a Ferralic Cambisol

https://doi.org/10.1016/j.soilbio.2019.107677Get rights and content

Highlights

  • The potential DNRA rate ranged from 0.5 to 1.5 μg N g−1 dry soil h−1 in the Ferralic Cambisol.

  • Top DNRA OTUs belong to Proteobacteria, Verrucomicrobia and Acidobacteria.

  • The composition of DNRA microbes was associated with pH and soil CNP.

  • DNRA microbial composition × soil properties explained DNRA rates.

Abstract

Dissimilatory nitrate reduction to ammonia (DNRA) is one of the three processes of soil nitrate reduction. However, relationships between DNRA microbes and nutrient fertilization are poorly known. We studied the DNRA microbial community in a Ferralic Cambisol containing plots including control without fertilization, swine manure fertilization (M), chemical fertilization (NPK), and chemical/manure combined fertilization (MNPK) treatments. The abundance of DNRA microbes, represented by the nrfA gene abundance, ranged from 2 × 107 to 5.8 × 107 g−1 dry soil and was positively correlated with soil moisture and total phosphorus (TP) and negatively correlated with NH4+ and total potassium (TK). The potential DNRA rate ranged from 0.5 to 1.5 μg N g−1 dry soil h−1. The α-diversity of the DNRA bacteria increased in the M-treated plots, and the dominant DNRA bacterial OTUs were assigned to the phyla Proteobacteria, Verrucomicrobia and Acidobacteria. PCoA and redundancy analysis indicated that the composition of the DNRA bacteria was strongly impacted by the long-term fertilization regimes and was associated with pH, TN, TP and TC followed by moisture, NH4+ and C/NO3. Interestingly, the composition of the DNRA bacterial community, the properties of the soil (TP, AK and C/N) and the interactions of these factors (soil properties × DNRA composition) explained the DNRA rate. Collectively, these data suggested that the DNRA potential in the Ferralic Cambisol is possibly controlled by the stoichiometry of macronutrient and the composition of DNRA microbes but not their total abundance.

Introduction

Dissimilatory nitrate reduction to ammonia (DNRA) is one of the three major pathways of microbial mediated nitrate reduction, which also includes denitrification and anaerobic ammonia oxidation (Kraft et al., 2011). The microbes that drive DNRA and denitrification compete for nitrate/nitrite and electron donors. Among these microbes, the microbial communities that drive denitrification and anaerobic ammonium oxidation in soils have been extensively studied (Henry et al., 2004; Wei et al., 2015; Zhu et al., 2013, 2019). DNRA plays an important role in soil nitrogen retention (Huygens et al., 2007; Ma and Aelion, 2005; Page et al., 2003). For example, in paddy soils in China, this process contributes 0.4%–25.4% of the total nitrate reduction (Lu et al., 2012, 2013; Shan et al., 2016; Yin et al., 2002). DNRA is significantly associated with C/N, extractable organic carbon/nitrate and sulphate. The DNRA activities are generally enhanced in reductive environments with relatively higher organic matter content (Lu et al., 2012; Nizzoli et al., 2010; Rütting et al., 2011; Song et al., 2014). For example, in a subtropical pasture soil, which is rich in carbon, the redox potential plays a more important role in modulating the DNRA and denitrification partitioning than C/NO3 (Friedl et al., 2018). In a Molisol, DNRA is positively correlated with precipitation, clay, soil organic matter, C/NO3 and water filled pore space (Chen et al., 2015). The effects of nitrate and soil pH were contradictory in different studies (Bu et al., 2017). Nitrate could be positively, negatively or not correlated with DNRA in soils (Chen et al., 2015; Gao et al., 2016; Minick et al., 2016; Schmidt et al., 2011; Silver et al., 2005; Yang et al., 2017). Soil pH was also found to influence DNRA (Baldos et al., 2015; Schmidt et al., 2011), but this has not yet been confirmed (Baldos et al., 2015; Minick et al., 2016; Schmidt et al., 2011; Yang et al., 2017). These results suggested that the regulation of the DNRA process in soils is complex.

Indeed, the activities of DNRA in soils should be determined by both the environmental factors and DNRA microbes. DNRA microbes usually possess a pentaheme cytochrome C nitrite reductase (NrfA) to catalyse the reduction of nitrite (NO2) to NH4+ (Einsle et al., 1999). Therefore, nrfA genes were used as biomarkers to study DNRA microbial ecology (Mohan et al., 2004). For example, DNRA activity and nrfA gene abundance co-decreased along an environmental gradient in the river estuary. The DNRA microbial community in a shallow lagoonal estuarine system was regulated by nitrate, ammonium, organic matter content, temperature and salinity (Li et al., 2019; Song et al., 2014). In the freshwater sediment, the DNRA microbial community was closely linked to the variability of organic matter type and nitrate (Tomaszek and Rokosz, 2007). In a Eutric Cambisol, the DNRA rate was positively related to C/NO3, nrfA/nirS and crop rotation (Putz et al., 2018). In a paddy soil, DNRA was positively related to SOC/NO3 and nrfA, which was increased in situations of low N treatment (Pandey et al., 2019).

Ferralic Cambisol is one of the typical soil types in tropic and subtropic regions, and its characteristics includes high Fe, high exchangeable Al3+ and low levels of inorganic nutrients (ion exchange capacity, P, K, Mg and Ca) (Zhang et al., 2009; Wen et al., 2018). Cheng et al. (2018), found that long-term P addition may stimulate soil gross N dynamics and hence increase overall N availability for crops in the P-deficient Ferralic Cambisol. However, in the Ferralic Cambisol, how different fertilization regimes influence the DNRA microbes and their activities remains unclear. Enhancing DNRA activity in agriculture soils may improve N use efficiency (Putz et al., 2018). In the Ferralic Cambisol used for this study, chemical fertilization was found to acidify soil but the manure utilization counteracted the acidification effects. Both the inorganic and organic fertilizers increased organic C, soil N, and P (Han et al., 2018). Considering the present knowledge about the effectors on DNRA and DNRA microbes, we hypothesized that (i) fertilization would increase the nutrient availability including phosphorus for DNRA microbes and thus changed their abundances, structure and potential activity; (ii) the acidifying effects from the chemical fertilizers could also impact the DNRA microbial community and their activity; (iii) manure utilization would be a positive factor to regulate DNRA bacterial community as it increases organic carbon supply. To verify this, we determined the abundances and composition of the DNRA communities in a Ferralic Cambisol using nrfA based molecular techniques, including quantitative PCR (qPCR) and high throughput sequencing. We also measured the DNRA potential rate using a15N tracing experiment.

Section snippets

Study sites, field sampling and soil physicochemical properties

The soils were collected from the Qiyang Red Soil Experimental Station (26°45′N, 111°52′E), established in 1990, which is located in Hunan Province, China. The soil at this site was originated from the Quaternary red clay soil and is classified as Ferralic Cambisol. The long-term field was under a wheat-maize rotation, including the following four treatments: (1) Control, no fertilizer added; (2) M, swine manure; (3) NPK, chemical fertilizers including nitrogen, phosphorus, and potassium; (4)

DNRA rate and nrfA gene abundance

The DNRA rates ranged from 0.5 to 1.5 (μg N g−1 dry soil h−1) with MNPK plots possessing the highest rate, followed by those for the M plots (Fig. 1A). The lowest DNRA rate was detected in the control. We analysed the correlation between DNRA rate and physicochemical properties and found that the contents of TP (r = 0.69, P < 0.05), AK (r = 0.59, P < 0.05) and C/N (r = 0.72, P < 0.01) had significantly positive correlations with DNRA rates (Table S3).

The abundances of DNRA bacteria were

Discussion

Our results supported our hypothesis that nutrient fertilization influenced DNRA microbial community and their activities in the Ferralic Cambisol. First, activity-based data showed that the DNRA activity was greatest in the M plots. The activity was statistically related to the soil nutrients, including TP, AK and C/N, but not nrfA gene copies or C/NO3. Many studies showed that high input of organic C such as forest soils (Huygens et al., 2007), peatlands (Davis et al., 2008) and mangrove

Conclusion

In this study, we investigated the effects of different fertilization regimes on the DNRA microbes and their activities in a Ferralic Cambisol. We found that long term fertilizations influenced DNRA community along the pH, TN, TP and TC gradients. The potential DNRA activities were explained by the soil properties and DNRA bacterial composition. Higher DNRA activities would be closely linked to a higher C/N, P level, K availability and a subset of the DNRA bacterial members when the soil 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.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2018YFE0105600), and the National Natural Science Foundation of China (grant No. 41701284).

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