Urea treatment decouples intrinsic pH control over N2O emissions in soils
Introduction
Nitrous oxide (N2O) is a potent greenhouse gas, with a global warming potential of 298 over a 100-year time period, and is involved in stratospheric ozone depletion (Ravishankara et al., 2009; IPCC, 2013). The atmospheric concentration of N2O has increased by 20%, from 271 ppb to 324 ppb, over the last 260 years due to increasing anthropogenic activities (IPCC, 2013). The major anthropogenic source of N2O is agricultural soils (Cole et al., 1997; Mosier et al., 1998), with grazed pasture soils being a key source (Oenema et al., 2005; van Groenigen et al., 2008).
Grazed pasture soils receive nitrogen (N) as a result of ruminant excreta, with urine being the dominant source (de Klein and Ledgard, 2005). A single dairy cow ruminant urination event can deliver on average 613 kg N ha−1 within the wetted urine patch area (Selbie et al., 2015a) making these areas key sources of N2O emissions (de Klein and Ledgard, 2005; Chadwick et al., 2018). Despite this, our understanding of factors controlling N2O emissions within ruminant urine patches is lacking.
In pastoral systems, urine-N is deposited onto soils predominately as urea (Selbie et al., 2015a) which is then hydrolysed to ammonium (NH4+). From here, an entire repertoire of biotic (e.g. nitrification, nitrifier-denitrification and denitrification) and/or abiotic processes (e.g. chemodenitrification) are initiated, resulting in the formation of N2O (Firestone et al., 1980; Delgado and Mosier, 1996). However, the dominant N2O generating processes in soils are nitrification and denitrification (Signor and Cerri, 2013; Soares et al., 2016), which proceed under aerobic and anaerobic conditions, respectively (Barnard et al., 2005; Saggar et al., 2013). Nitrification is the stepwise oxidation of ammonia to nitrate (NO3−) via nitrite (NO2−) catalysed by specialised microbial groups (Bothe et al., 2000; Hu et al., 2015). The conversion of ammonia to NO2− is performed by ammonia oxidising archaea and bacteria (AOA and AOB, respectively) (Bothe et al., 2000; Könneke et al., 2005; Prosser, 2007; Prosser and Nicol, 2012) while the second step is performed by NO2− oxidising bacteria (NOB) (Bothe et al., 2000; Prosser, 2007). However, it was recently discovered that this multistep process can be performed by a single group within the genus Nitrospira, through a process termed commamox (Daims et al., 2015). Generally, AOA tend to dominate in soils but external N inputs from fertilizers or urine favours AOB over AOA (Di et al., 2010; Sterngren et al., 2015). These findings indicate that these groups occupy varying niches and have different affinities towards ammonia (Di et al., 2010; Prosser and Nicol, 2012; Hink et al., 2018). If soil conditions become anaerobic, NO3− initially formed via nitrification, may undergo denitrification. Denitrification is a stepwise process catalysed by mainly heterotrophic bacteria in which NO3− is sequentially reduced to NO2−, nitric oxide (NO), N2O, and finally dinitrogen (N2), provided denitrification is complete (Wallenstein et al., 2006; Saggar et al., 2013; Hu et al., 2015). A series of genes and enzymes are involved in the subsequent conversions: nitrate reductase (narG/napA) [NO3− → NO2−], nitrite reductase (nirS/nirK) [NO2− → NO], nitric oxide reductase (norB/qnor) [NO → N2O], and nitrous oxide reductase (nosZ) [N2O → N2] (Wallenstein et al., 2006; Saggar et al., 2013).
The complex array of inorganic-N transformation pathways potentially able to generate N2O means that a variety of factors can govern the microbial production of N2O, but soil pH has emerged as one of the most cited and influential (Bergaust et al., 2010; Liu et al., 2010, 2014; Samad et al., 2016a, 2016b). Soil pH has been coined as a master regulator because it affects not only the microbiomes (functional + composition) (Lauber et al., 2009; Bergaust et al., 2010; Rousk et al., 2010; Samad et al., 2016b) but also resulting emissions, by regulating the gaseous end products (N2O or N2) and the denitrification product ratio/N2O ratio (Čuhel et al., 2010; Samad et al., 2016a, 2016b). In the past, denitrification studies have been conducted under ‘ideal conditions’ using NO3− as substrate under anaerobic conditions known to promote denitrification (Firestone et al., 1979; Gillam et al., 2008; Senbayram et al., 2012). Such studies have identified pH as a master regulator of denitrification and the N2O emission ratio (Čuhel et al., 2010; Saggar et al., 2013; Samad et al., 2016b). For example, under true (anaerobic) denitrification in soils the emission ratio of N2O (tendency of soil to emit N2O instead of N2) and soil pH are negatively correlated (Šimek and Cooper, 2002; Bakken et al., 2012; Raut et al., 2012; Qu et al., 2014; Samad et al., 2016a). The current hypothesis is that low pH hinders the post-translational assembly of a functional N2O-reductase enzyme (Bergaust et al., 2010; Bakken et al., 2012; Liu et al., 2014). However, it is unclear if the pH effect on emissions observed in ideal denitrification conditions (-O2, +NO3−) is conserved in urine amended soil exposed to aerobic conditions.
In urine amended soil, pH is altered through the combination of alkaline urine deposition and biological process (e.g. nitrification) which occur within the soil matrix. Urine deposition triggers an N-cascade commencing with urea hydrolysis, generating NH4+ and carbonate ions, which later dissociate, raising the soil pH (pH ≥ 8) (Avnimelech and Laher, 1977). Over time, nitrification is initiated contributing to a decrease in pH, which happens gradually over several weeks (Sherlock and Goh, 1985; Rex et al., 2018; van der Weerden et al., 2021). These responses are common among soils receiving urea inputs but the extent of change in pH (increase or decrease) is very much soil dependent (Doak, 1952; Khan et al., 2011; Rex et al., 2018; van der Weerden et al., 2021). Although urine deposition alters intrinsic pH, the extent of intrinsic pH control on emissions especially under aerobic conditions have not been clearly elucidated. Here, we predict that pH, a master regulator characteristic of true denitrification (-O2, +NO3−) would still emerge as the dominant regulator of emissions under aerobic conditions in urine amended soil where urea is the dominant N compound (+O2, +urea).
To test this we used a fully automated high-resolution gas chromatography system for measuring gas kinetics immediately after applying artificial urine to simulate a urine patch using 13 different soils representing Northern (Ireland) and Southern Hemispheres (New Zealand) soils. Our objectives were: (1) to determine gas kinetics of urine amended soils under aerobic conditions, (2) to investigate the relationship between soil pH and the emission ratio of N2O (e.g. N2O/(NO + N2O + N2)) under aerobic conditions, (3) to compare the N2O emission ratios (e.g. N2O/(NO + N2O + N2)) and conditions within urine amended soil vs idealized/true anaerobic denitrifying conditions and (4) to identify the genetic potential for N cycling in soils prior to urine deposition (to provide background on whether response to urea is controlled by intrinsic factors or by urea deposition changes itself).
Section snippets
Study sites, and sample collection
Soil samples were collected from 13 different grassland sites in both Northern (Ireland [Moorepark, Johnstown, Solohead]) and Southern (New Zealand [Warepa, Otokia, Wingatui, Tokomairiro, Mayfield, Lismore, Templeton, Manawatu, Horotiu, Te Kowhai]) hemispheres as described previously (Samad et al., 2016a). From each site soil cores (n > 3) were collected randomly using a soil corer (25 mm diameter by 100 mm long) and excluding the grass layer. Replicate cores were sieved to <4 mm, composited
N kinetics in urine amended soils
Soil samples were incubated under oxic conditions for 180 h with urine (Fig. 1) or without urine (Supplementary Fig. S1). While a significant (p < 0.001) increase in all measured gases was observed in response to urine addition (Fig. 2, Table 1), kinetic profiles for all soils varied significantly within a treatment (Fig. 1 and Supplementary Fig. S1). Site to site variance across urea treated samples for all measured gases are shown in Table S3. Profiles demonstrate active respiration, with the
Discussion
In this study, we focused on the impact of pH on N2O emissions using soil exposed to urine simulating a urine patch. These soils were previously used to analyze denitrification kinetics under ideal denitrifying conditions (-O2, +NO3−) which identified pH as a master regulator of emissions (Samad et al., 2016a). Denitrification occurs optimally at pH 7–8 (Wijler and Delwiche, 1954; Peterjohn, 1991) but can also occur at low pH (Wijler and Delwiche, 1954; Parkin et al., 1985; Weier and Gilliam,
Conclusion
In this study, we simulated a urine patch under aerobic conditions to assess if the same set of regulators identified under anaerobic systems would dictate N2O formation in the current set-up. We observed that regulators of N2O emissions and N gas kinetics were not conserved under urine treated soils and ideal denitrification conditions. Instead, a new set of regulators was identified indicating a shift in conditions that creates a challenge for transferring mechanisms for emissions from
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
We thank all members of the Nitrogen group at the Norwegian University of Life Sciences (http://www.nmbu.no/en/research/groups/nitrogen) for technical advice in gas kinetics work and Surinder Saggar for providing the Manawatu soil samples used in this study. This work was funded by the New Zealand Government through the New Zealand Fund for Global Partnerships in Livestock Emissions Research to support the objectives of the Livestock Research Group of the Global Research Alliance on
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These authors contributed equally, authorship order was determined randomly.