Relative contributions of different substrates to soil N2O emission and their responses to N addition in a temperate forest

https://doi.org/10.1016/j.scitotenv.2020.144126Get rights and content

Highlights

  • Nitrate derived N2O through denitrification contributed most to the total N2O emission under ambient N deposition.

  • NH4NO3 addition of 50 kg N ha−1 yr−1 significantly increased organic N derived N2O on the 6th day after N addition.

  • Heterotrophic nitrification may be the dominating process under higher N deposition rate.

Abstract

With increasing nitrogen (N) deposition, soil nitrous oxide (N2O) emission is expected to increase, causing positive feedback to global warming. However, the substrates of soil N2O emission, especially their responses to N addition, are still unclear. Here, we conducted an in situ 15N tracing experiment to study the substrates of N2O (i.e., ammonium-derived, nitrate-derived and organic N-derived N2O emission) under N addition treatment in a temperate forest in northeast China. Nitrate derived N2O through denitrification contributed most to the total N2O emission, pointing to the importance of denitrification under ambient N deposition. NH4NO3 addition of 50 kg N ha−1 yr−1 significantly increased organic N derived N2O on the 6th day after N addition, which suggests that heterotrophic nitrification may be the dominating process with higher N deposition rate. However, because soil pH and the examined functional genes did not change after N addition, future studies should be carried out to understand if the increase of heterotrophic nitrification is transient. Our study emphasizes the role of organic N pool in soil N2O emissions, highlighting the importance of considering the heterotrophic nitrification process while studying soil N cycling or modeling soil N2O emission.

Graphical abstract

Soil N2O flux from the mixed Korean pine and broad leaved forest under ambient and N addition treatments from Jun. 14 to Sep. 23, 2014. Values are reported as mean ± standard deviation (n = 12). The insert shows the diurnal dynamics of soil N2O flux (mean ± standard deviation) during the first 24 h after N addition.

Unlabelled Image
  1. Download : Download high-res image (111KB)
  2. Download : Download full-size image

Introduction

Reactive nitrogen (N) deposition to the Earth's land surface has dramatically increased during the last few decades due to human activities (Galloway et al., 2004; Torres et al., 2009). Gaseous N losses to the atmosphere are important fates after anthropogenic N entering terrestrial ecosystems (Schlesinger, 2009), which include nitrous oxides (NOx), nitrous oxide (N2O), nitrogen (N2) and ammonia (NH3). Among these gases, N2O is a potent greenhouse gas and ozone-depleting agent (Ravishankara et al., 2009; Montzka et al., 2011), whose global warming potential is 298 times of that of CO2 over a 100-year timeframe. The current global rate of N2O emission is about 18 Tg N yr−1, and soil N2O substrates accounting for 37% (WMO, 2013; IPCC, 2014). With increasing N deposition, soil N2O emission is expected to increase, and causes a positive feedback to global warming (Lu et al., 2011).

Soil N2O emission can be produced from many processes, including nitrification, denitrification, nitrifier denitrification, co-denitrification, chemical denitrification and dissimilatory nitrate reduction to ammonium (DNRA), which can occur simultaneously (Levy-Booth et al., 2014). These processes are driven by different soil microbial groups and influenced by different soil and climate conditions (Booth et al., 2005; Gubry-Rangin et al., 2010; Qin et al., 2013; Xu et al., 2013; Levy-Booth et al., 2014). It is important to determine the substrates of N2O production and their contributions to better understand the responses of soil N2O emission to global and regional climate changes, like increased N deposition (De Boer and Kowalchuk, 2001). However, although numerous studies have investigated the response of soil N2O fluxes to increasing N deposition (Horvath et al., 2006; Pilegaard et al., 2006; Rojas-Garcia et al., 2009), we still know little about whether increasing N deposition would change the contributions of different substrates to N2O production and the processes involved in N2O production.

Stable isotope tracing technique combined with stable isotope mixing model is an effective method to quantitatively differentiate substrates responsible for N2O production, as 15N-enriched substrate can be used to trace N2O (Beline et al., 2001; Wolf and Brumme, 2002; Mathieu et al., 2006; Menyailo and Hungate, 2006; Kato et al., 2013; Morse and Bernhardt, 2013; Lan et al., 2014; Müller et al., 2014). Originally, the two-source model assumes that NH4+ and NO3 were the only two substrates for N2O production (Stevens et al., 1997) and nitrifiers are thought to use NH4+ as their only substrate and their only energy source, hence are autotrophic microbes and unable to use organic matter to any extent (Ward, 2013). However, many previous researches have proposed that N2O can also be produced by heterotrophic bacteria and fungi using organic matter as their N substrate and energy source (Beleneva and Zhukova, 2009; Chen et al., 2015). These microbes are called heterotrophic nitrifiers and the process involved is called heterotrophic nitrification. Although some studies showed that heterotrophic nitrifiers were abundant in some ecosystems, particularly under conditions with low N input rate and high organic matter concentration (Aerts, 1995; Bending and Read, 1997; Pedersen et al., 1999; Zhang et al., 2011; Stange et al., 2013), the quantitative importance of heterotrophic nitrification is still uncertain. Recently, the three-source model using triple 15N tracer treatments (Rütting et al., 2010) or the inverse abundance approach (Stange et al., 2013) were able to quantify the relative contribution of the organic N pool to N2O production. The triple 15N tracer approach uses three 15N tracers (15NH4NO3, NH415NO3 and 15NH415NO3) to quantify the fractions of the three substrates (organic N pool, NH4+-N pool and NO3-N pool) by using three independent equations (Stange and Dohling, 2005; Laughlin et al., 2008). Heterotrophic nitrification was found to be important under conditions unfavorable for autotrophic nitrifiers such as in acidic soils or soils with naturally occurring nitrification inhibiting substances (Huygens et al., 2008). Heterotrophic nitrifiers may have superior degrading capabilities for recalcitrant soil organic matters (SOM) originated from evergreen leaves (Aerts, 1995; Bending and Read, 1997; Pedersen et al., 1999; Islam et al., 2007), thereby playing an important role in carbon and nitrogen cycling in forests with evergreen plants. However, most triple 15N tracer approach were applied in laboratory culture experiment and needed high tracer amount to achieve the required 15N atom% enrichment (i.e. NH4+ pool and NO3 pool) (Stange et al., 2009; Zhang et al., 2019), which may unable to represent the contribution of substrate N pools to soil N2O fluxes in situ and natural nitrogen deposition. Meanwhile, the both labeled treatment (15NH415NO3) may not necessary when we use the atom% of 15N, but not atom% excess of 15N. Therefore, an in situ experiment with a simpler method to model soil N2O fluxes and its sources is needed to demonstrate the sources of soil N2O fluxes under both ambient and increased nitrogen deposition.

Quantification and characterization of microbial functional genes in the nitrification and denitrification pathways provide further information on N transformation processes and the substrates of soil N2O emissions, as the process-specific functional genes are responsible for N transformations (Richardson et al., 2009; Morales et al., 2010). Autotrophic nitrification is the oxidation of NH4+ to NO3, and N2O is a byproduct during the process (Stevens et al., 1997). The rate-limiting step is the oxidation of NH3 to hydroxylamine, which is carried out mainly by autotrophic ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). AOA and AOB are differentiated by their diversity and the abundance of respective amoA genes, which encode the subunits of ammonia monooxygenase oxidizers (AMO) proteins (Yao et al., 2011). Denitrification is a full or partial dissimilative reduction of NO3 to dinitrogen gas (N2), and N2O as intermediate product. Denitrification is the primary pathway of N2O released from soil (Colliver and Stephenson, 2000; Shaw et al., 2006). Nitrate is first converted to nitrite and then nitrite is converted to NO by nitrite reductase (NIR), which is encoded by nirK (Cu-containing) or nirS (cytochrome cd1) genes (Levy-Booth et al., 2014), and then NO is converted to N2O. NirK and nirS genes are examples of convergent evolution and generally do not appear in the same organism. N2O can be further reduced to N2 by nitrous oxide-reducing bacteria (Billings, 2008), where nosZ gene encodes the catalytic subunit of N2O reductase (Chan et al., 1997). Therefore, N2O emission via denitrification is the net balance between the production and reduction of N2O, mainly regulated by nirK, nirS and nosZ genes (Henry et al., 2006). These genes can provide intrinsic view for understanding soil N2O emission, and its response to global climate change (Sun et al., 2017).

Previous studies using the stable isotope tracing technique were all applied in lab incubation experiments. Here we studied N2O emission and the relative contributions of its substrates in a temperate broad-leaved and Korean pine mixed forest using the in situ isotope labeling and gene detection techniques. Our objectives were to understand: 1) soil N2O fluxes under ambient N deposition and the relative contributions of different substrate N pools (NH4+, NO3 and organic N); and 2) the effects of N addition on soil N2O fluxes and relative contributions of different substrate N pools after N addition.

Section snippets

Study site

The study site was located in the Changbai Mountain National Nature Reserve, which is in Jilin province, northeast China (41°42′N, 127°38′E). The study area is characterized by a typical temperate climate, with a long and cold winter and a warm summer. The mean annual temperature in the study area is 3.6 °C, with the highest mean monthly temperature in July, and the lowest in January. Mean annual precipitation is 745 mm, mainly falling between May and September. The growing season is from June

N-addition effects on soil properties

Soil pH was not significantly different between ambient N plots (5.45 ± 0.32) and N-addition treatment plots (5.33 ± 0.29, Table 1). Soil clay content was similar between ambient N and N-addition plots (51.2 ± 6.06% and 47.3 ± 5.77%, respectively). Soil WFPS was also similar between ambient N (59.2 ± 13.8 kg H2O kg−1 dry soil) and N-addition plots (59.9 ± 15.6 kg H2O kg−1 dry soil). Mean soil NO3 concentration was significantly higher in N-addition plots (8.72 ± 12.9 mg N/ kg soil) than in

The contributions of the three N pools to N2O

Our results indicated that the NO3-N pool was the predominant N source of soil N2O production under ambient N deposition in our studied forest (Table 2). Denitrification, DNRA, chemo-denitrification, and coupled nitrification-denitrification were possible processes responsible for NO3-derived N2O (Wrage et al., 2001; Levy-Booth et al., 2014). The acidic (pH = 5.39) and aerobic (43%–99% WFPS) soil in this study were suitable for denitrification. Strict anoxic conditions with high soil moisture

Declaration of competing interest

All authors declare that no conflict of interest exists.

Acknowledgments

This work was supported by grants from the Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-DQC006), National Natural Science Foundation of China (41701605, 41807523).

References (91)

  • T. Lan

    Sources of nitrous and nitric oxides in paddy soils: nitrification and denitrification

    Journal of Environmental Sciences-China

    (2014)
  • D.J. Levy-Booth

    Microbial functional genes involved in nitrogen fixation, nitrification and denitrification in forest ecosystems

    Soil Biol. Biochem.

    (2014)
  • O. Mathieu

    Quantifying the contribution of nitrification and denitrification to the nitrous oxide flux using N-15 tracers

    Environ. Pollut.

    (2006)
  • J.L. Morse et al.

    Using 15N tracers to estimate N2O and N2 emissions from nitrification and denitrification in coastal plain wetlands under contrasting land-uses

    Soil Biol. Biochem.

    (2013)
  • C. Müller

    Quantification of N2O emission pathways via a 15N tracing model

    Soil Biol. Biochem.

    (2014)
  • F.C. Nichols

    Unique lipids from a common human bacterium represent a new class of toll-like receptor 2 ligands capable of enhancing autoimmunity

    Am. J. Pathol.

    (2009)
  • M. Nie

    Effects of salt marsh invasion by Spartina alterniflora on sulfate-reducing bacteria in the Yangtze River estuary, China

    Ecol. Eng.

    (2009)
  • S.S. Parker et al.

    Soil nitrogen availability and transformations differ between the summer and the growing season in a California grassland

    Appl. Soil Ecol.

    (2011)
  • D. Richardson

    Mitigating release of the potent greenhouse gas N2O from the nitrogen cycle - could enzymic regulation hold the key?

    Trends Biotechnol.

    (2009)
  • C.R. Rojas-Garcia

    Phenoloxidase and trypsin in germ-free larvae of Artemia fed with cooked unicellular diets: examining the alimentary and protective effects of putative beneficial bacterium, yeast and microalgae against vibriosis

    J. Exp. Mar. Biol. Ecol.

    (2009)
  • C.F. Stange

    Use of the inverse abundance approach to identify the sources of NO and N2O release from Spanish forest soils under oxic and hypoxic conditions

    Soil Biol. Biochem.

    (2013)
  • R.J. Stevens

    Measuring the contributions of nitrification and denitrification to the flux of nitrous oxide from soil

    Soil Biol. Biochem.

    (1997)
  • I.N. Throback

    Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE

    FEMS Microbiol. Ecol.

    (2004)
  • I. Wolf et al.

    Contribution of nitrification and denitrification sources for seasonal N2O emissions in an acid German forest soil

    Soil Biol. Biochem.

    (2002)
  • L.J. Yang

    Dielectrophoresis assisted immuno-capture and detection of foodborne pathogenic bacteria in biochips

    Talanta

    (2009)
  • S.J. You et al.

    Performance and dye-degrading bacteria isolation of a hybrid membrane process

    J. Hazard. Mater.

    (2009)
  • J.B. Zhang

    N2O production pathways in the subtropical acid forest soils in China

    Environ. Res.

    (2011)
  • J.B. Zhang

    Heterotrophic nitrification of organic N and its contribution to nitrous oxide emissions in soils

    Soil Biol. Biochem.

    (2015)
  • Y. Zhang

    Temperature effects on N2O production pathways in temperate forest soils

    Sci. Total Environ.

    (2019)
  • E. Bai

    Pulse increase of soil N2O emission in response to N addition in a temperate forest on Mt Changbai, Northeast China

    PLoS One

    (2014)
  • I.A. Beleneva et al.

    Seasonal dynamics of cell numbers and biodiversity of marine heterotrophic bacteria inhabiting invertebrates and water ecosystems of the Peter the Great Bay, Sea of Japan

    Microbiology

    (2009)
  • S.A. Billings

    Biogeochemistry: nitrous oxide in flux

    Nature

    (2008)
  • M.S. Booth

    Controls on nitrogen cycling in terrestrial ecosystems: a synthetic analysis of literature data

    Ecol. Monogr.

    (2005)
  • G. Braker

    Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples

    Appl. Environ. Microbiol.

    (1998)
  • G. Braker

    Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in pacific northwest marine sediment communities

    Appl. Environ. Microbiol.

    (2000)
  • J.M. Bremner

    Sources of nitrous oxide in soils

    Nutr. Cycl. Agroecosyst.

    (1997)
  • D. Castignetti

    Substrate diversity of an active heterotrophic nitrifier, an alcaligenes Sp

    Can. J. Microbiol.

    (1985)
  • C.B. Caston

    The relative importance of heterotrophic bacteria to pelagic ecosystem dynamics varies with reservoir trophic state

    Limnol. Oceanogr.

    (2009)
  • Y.K. Chan

    A new nos gene downstream from nosDFY is essential for dissimilatory reduction of nitrous oxide by Rhizobium (Sinorhizobium) meliloti

    Microbiology-Sgm

    (1997)
  • H. Chen

    Soil moisture and pH control relative contributions of fungi and bacteria to N2O production

    Microb. Ecol.

    (2015)
  • Z.J. Du

    Gilvimarinus chinensis gen. nov., sp nov., an agar-digesting marine bacterium within the class Gammaproteobacteria isolated from coastal seawater in Qingdao, China

    Int. J. Syst. Evol. Microbiol.

    (2009)
  • V. Figueiredo

    Soil organic matter content controls gross nitrogen dynamics and N2O production in riparian and upland boreal soil

    Eur. J. Soil Sci.

    (2016)
  • C.A. Francis

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

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

    (2005)
  • J.N. Galloway

    Nitrogen cycles: past, present, and future

    Biogeochemistry

    (2004)
  • C. Gubry-Rangin

    Archaea rather than bacteria control nitrification in two agricultural acidic soils

    FEMS Microbiol. Ecol.

    (2010)
  • Cited by (4)

    View full text