Soil Nitrogen Isotope Composition

83CHAPTER 4Soil nitrogen isotope compositionR. DAVE EVANSIntroductionThe isotope composition of a reaction product is determined by the isotope ratio of the substrate and fractionation during chemical transformations. Fractionation may not always occur so isotope ratios of some elements can be used as natural tracers, for example, the 2H and 18O of stem- and soil-water can be used to infer patterns of water use in plants (Dawson & Ehleringer 1991; Ehleringer et al. 1991). In other cases such as plant 13C, fractionation events predominate, but mechanistic models have been devel-oped to explain and predict product isotope ratios (Farquhar et al. 1982; Flanagan et al. 1991). Early interpretation of soil 15N was heavily infl uenced by enriched 15N labeling methodologies that were common in agriculture and ecology to understand soil nitrogen transformations, and it was hoped that 15N at natural abundance levels could be used as a natural tracer. Fraction-ation events can be ignored in labeling studies, and it was hoped a similar assumption could be made for 15N at natural abundance. For example, early natural abundance 15N studies attempted to trace fertilizer and soil nitrate into groundwater, or quantify the contribution of nitrogen fi xation to total plant nitrogen (reviewed in Shearer Robinson 2001). Scientists now realize that soil 15N is very complex, with multiple sources of nitrogen inputs, numerous internal transformations with associ-ated fractionation events, and multiple sources of nitrogen loss that all po-tentially discriminate against 15N (Figure 4.1). It is because of this complexity and the reliance on assumptions and methods for enrichment studies that our knowledge of the mechanisms controlling soil 15N has not progressed as rapidly as those for other elements. Nonetheless scientists have made sig-nifi cant progress in understanding soil 15N in the past decade and it is clear that it serves as a valuable integrator of soil processes (Robinson 2001). This chapter provides an overview of these recent advances.Sources of variation in soil d15NThe isotopic composition of nitrogen inputs and fractionation during nitrogen transformations and loss determine soil 15N. It is diffi cult to measure in situ 84 R.D. EVANSfractionation factors for most soil processes so the observed discrimination (15Nsubstrate 15Nproduct) is commonly used instead. The observed discrimina-tion exhibits considerable variation for individual transformations (Table 4.1). Shearer & Kohl (1993) and Hgberg (1997) identifi ed several reasons for this variation. First, a process may exhibit positive discrimination when substrates are plentiful, but discrimination will not be observed when a reac-tion is substrate limited because all substrate is converted to product. Second, there can be multiple substrates with different 15N for each product. For example, N2O and NO are produced by both nitrifi cation and denitrifi cation, which results in different isotope ratios depending upon the dominant pro-cess of formation. Third, some substrates such as NH4+and NO3are exposed to multiple, competing reactions with different fractionation factors. Fourth, related functional groups of organisms may have slightly different Soil OrganicMatter Pools Monomers Microbes NH4+NO3N2O, NO NO, N2O, N2NH3a dcffbgNitrogenInputseFigure 4.1 Soil nitrogen transformations based on the conceptual model of Schimel & Bennet (2004). Nitrogen inputs can enter into the organic and inorganic pools. The transformations are: a, depolymerization; b, gross mineralization; c, volatilization; d, nitrifi cation; e, denitrifi cation; f, microbial immobilization; g, death of soil microbes.Table 4.1 Observed discriminations for transformations in the nitrogen cycle. Process refers to the transformations indicated by letters in Figure 4.1. Values are taken from reviews by Shearer & Kohl (1986), Wada & Ueda (1996), Hgberg (1997) and Robinson (2001).Transformation Process Discrimination ()Gross mineralization b 05Nitrifi cation d 035NH4+ NH3equilibrium 2027Volatilization c 29N2O and NO production d 070during nitrifi cationN2O and N2production e 039during denitrifi cationNO3immobilization f 13NH4+immobilization f 1420SOIL NITROGEN ISOTOPE COMPOSITION 85discrimination values for the same process. Finally, it is diffi cult to predict the effects of interactions between abiotic and biotic factors, and they may vary between ecosystems or even within a site.Isotopic composition of nitrogen inputsNitrogen fi xationPrior to the industrial age the primary external source of nitrogen input for soils was biological nitrogen fi xation (Galloway et al. 1995). Scientists initially hoped that quantifying 15N could be used to trace the relative contribution of nitrogen fi xation to plants and soils. One assumption of this approach is that fractionation does not occur during nitrogen fi xation, therefore the 15N of nitrogen-fi xing organisms will refl ect that of the atmospheric source. This assumption is often not met because the discrimination observed during ni-trogen fi xation can vary from 0 to 3 (Shearer & Kohl 1986, 1993), so the 15N of organisms that derive the nitrogen from biological fi xation varies from 3 to 0 (Fry 1991). Soil 15N can, however, be used to infer the dominant sources of nitrogen input in very simple soil systems. In arid ecosystems the primary source of nitrogen input is the biological soil crust dominated by cyanobacteria and lichens that are capable of nitrogen fi xation. The crusts form a continuous cover in undisturbed plant communities, and spatial cov-erage is often higher than for vascular plants (see Evans & Johansen 1999). Surface disturbance in arid ecosystems is widespread and eliminates biologi-cal soil crusts and nitrogen fi xation. Therefore identifying whether nitrogen input is dominated by a physical (atmospheric deposition) or biological (N2-fi xation) process is important to determine the potential impacts of surface disturbance on ecosystem nitrogen cycles. Evans & Ehleringer (1993) used a Rayleigh relationship to assess the relative contribution of physical and bio-logical processes in a PinyonJuniper community on the Colorado Plateau. The predicted linear relationship between soil 15N and the log of soil nitrogen content was established using soil values (Figure 4.2). Values for the biologi-cal soil crust fell immediately along this relationship while values for atmo-spheric deposition fell well off the relationship. This indicates that the primary source of nitrogen input was biological nitrogen fi xation, and land-use change may alter the balance between nitrogen input and loss by eliminating this source. This result was confi rmed by Evans & Belnap (1999), who observed lower soil nitrogen contents and greater soil 15N values in disturbed versus undisturbed sites.Atmospheric depositionAn initial goal of early studies measuring soil nitrogen isotope composi-tion was to identify and trace sources of atmospheric input into ecosystems. 86 R.D. EVANSHeaton (1987) pointed out the early promise of this approach by measuring the 15N of oxidized and reduced nitrogen gases. The 15N ranged from an extreme of 150 for oxidized nitrogen gases from the stack of a nitric acid plant to 5.2 for emissions from a coal-burning power station. However, the 15N of atmospheric deposition can vary signifi cantly depending upon the nitrogen form and season. Heaton (1986) reviewed 15N of NO3and NH4+in wet and dry deposition. In general, values for wet deposition were 0. Weighted values for wet and dry deposi-tion estimated the 15N of total atmospheric deposition to be 3 for both NO3and NH4+. A drawback to point measurements of atmospheric deposition is that signifi cant temporal variation is common. The 15N of NO3and NH4+ranged from 7 to +4 and 9 to 2, respectively, in a single year, and could vary as much as 5 in a single storm.The observed temporal changes in the 15N of atmospheric deposition can be caused by several factors including changes in the source or intensity of precipitation. Pichlmayer et al. (1998) reconstructed sources of NO3in at-mospheric deposition in European alpine ecosystems by correlating 15N in snow and ice with known storm tracks. Storm tracks were calculated twice daily so that specifi c strata in the snow could be correlated with individual precipitation events and their region of origin. The observed seasonal varia-tion was ca. 5 and appeared to be the result of storms originating from northsouth tracks versus eastwest. All atmospheric values were 2.6.Bragazza et al. (2004, 2005) recently adopted a novel approach to inte-grate temporal and source changes at 16 sites across 11 European countries 10.05.00.05.04 2 0 2 4SoilSoil CrustNitrogenDepositionLn(mg N/g)15N()Figure 4.2 Relationship between soil 15N and ln(soil nitrogen content) in soils and in two potential sources of N in a cold desert ecosystem in southern Utah.SOIL NITROGEN ISOTOPE COMPOSITION 87and identifying sources of atmospheric deposition by measuring the 15N of mosses. Rates of atmospheric deposition ranged from 1 to 20 kg N ha1yr1, and moss 15N varied from 8 to 3 across the gradient. The 15N values were not correlated with total deposition, annual temperature, or annual precipitation. Instead, the isotope compositions were signifi cantly correlated with the ratio of reduced to oxidized nitrogen (NHx/NOx) in the deposition. Mosses located in areas with greater emissions of NH3from agricultural ac-tivities had lower 15N values than those more heavily infl uenced by NOxemissions from industrial activity. This study is among the fi rst to provide integrated 15N values and to also identify potential agricultural and industrial sources and their impact on the 15N of nitrogen inputs.FertilizersConsideration of the rates of input and 15N of fertilizer N is essential to un-derstand current and future patterns of soil 15N in agricultural soils. Pre-industrial global rates of nitrogen input, primarily from biological nitrogen fi xation, are estimated at 90 to 130 Tg N yr1(Galloway et al. 1995). In con-trast, fertilizer application was about 80 Tg N yr1in 1990 and is predicted to exceed rates of biological nitrogen fi xation by 2020 (Galloway et al. 1995; Vitousek et al. 1997; Steffen et al. 2004). Vitoria et al. (2004) recently re-viewed published values for fertilizer isotope composition. The isotope com-position of fertilizers refl ects their origins from atmospheric nitrogen (0) and oxygen (22.5). The 15N of total nitrogen ranged from 3 to +4 with the highest frequency from 1 to +1. The 15N in NO3and NH4+exhibited much greater variation; 15N of NO3varied from 8 to +7 and values were primarily enriched in 15N. In contrast, 15N of NH4+were primarily all negative and ranged from 7 to +3. The difference between NO3and NH4+is thought to be due to fractionation during oxidation of NH3(Freyer & Aly 1974). Chilean nitrates can be a signifi cant source of fertilizer nitrogen in some regions. The 15N of these fertilizers was similar to the pattern observed for synthetic fertilizers, but the 18O was enriched from +40 to +50.Soil transformationsMineralization and organism-available nitrogenFractionation during mineralization has been estimated by comparing bulk soil and NH4+15N. The differences are often small and it is assumed that the observed discrimination is negligible (Table 4.1). Further research is needed on the processes and fractionation events that produce organism-available nitrogen in light of our changing understanding of soil nitrogen cycling and new technological advances. A view held for many decades was that miner-alization was the process that limited overall rates of nitrogen cycling in soils 88 R.D. EVANSand that plants primarily assimilated NH4+and NO3, but it is now believed that overall soil nitrogen cycling is limited by the rate of depolymerization of amino acids from soil organic matter that occurs at localized microsites within the soil (Schimel & Bennett 2004). In addition to uptake of inorganic nitrogen, organic nitrogen uptake may occur either directly by plants or through mycorrhizae. For these reasons, and because soil nitrogen is domi-nated by a large, non-reactive recalcitrant pool, bulk soil values may provide little information on the 15N of nitrogen assimilated by organisms. Research in this area is being facilitated by advances in compound-specifi c isotope analysis that now allows measurement of the 15N of individual amino acids, because it is these individual amino acids in litter and soil that serve as the substrate for subsequent soil reactions. The 15N of individual compounds can vary greatly within plants; proteins are generally enriched in 15N compared with secondary products such as chlorophyll, lipids, and amino sugars and differences among compounds can be as great as 20 (Werner & Schmidt 2002), and similar variation has been observed for amino acids in soils (Ostle et al. 1999; Bol et al. 2004).Many early studies assumed that plant 15N could be used as an indicator of plant nitrogen source because it was presumed that the progressive prod-ucts in the organic matter NH4+ NO3sequence would become increas-ingly depleted in 15N. This assumption is often not correct because competing reactions may enrich the soil inorganic nitrogen pool (Figure 4.1). For example, Binkley et al. (1985) observed that soil NO3may not differ or may even be enriched compared with NH4+. The 15N of soil inorganic nitro-gen can also change rapidly over time because these pools are very labile (Figure 4.3); variation of 1020 can occur over days or months (Feigin et al. 1974; Herman Frank et al. 2004).It is important to emphasize that isotopic measurements of soil inorganic nitrogen should be interpreted with caution and that values may be the product of artifacts during collection and purifi cation rather than a soil bio-logical or physical process. Collection of soil samples disturbs the system and this can alter 15N values (Hgberg 1997), and many of the methods currently used to study soil inorganic nitrogen were developed for labeling studies and it is unclear whether they are appropriate at natural abundance levels. Rob-inson (2001) summarized the limitations of using methods that were devel-oped for other applications. First, inorganic nitrogen is often isolated using diffusion methods developed for labeling studies. The pH of the solution containing NH4+is raised causing NH3to volatilize where it is collected on an acidifi ed disk. Robinson (2001) calculates that recovery must be 99 percent for maximum accuracy, and recovery of only 95 percent would result in an error of 3. Second, many of the isolation methods currently in use are not NH4+-specifi c and contamination by organic nitrogen is common. This could introduce substantial errors considering the large variation in soil organic compounds (Ostle et al. 1999). Robinson (2001) states that methods should SOIL NITROGEN ISOTOPE COMPOSITION 89be used that have been specifi cally designed for natural abundance measure-ments to overcome the problems associated with diffusion and isolation. For NO3these included NO3-specifi c dye-coupling (Johnston et al. 1999), ion exchange (Silva et al. 2000; Stickrod & Marshall 2000), and denitrifying bacteria (Sigman et al. 2001). Suitable methods are still being developed for NH4+.Nitrogen lossVolatilizationRelatively few studies have measured the 15N of inorganic reduced nitrogen compounds compared with oxidized forms. This is likely to change in the future because 70 percent of atmospheric NH3originates from volatilization of NH4+deposited in the soil from domestic animals, fertilizers, and excrement (Schlesinger & Hartley 1992) leading to increased aerosol formation that can alter Earths energy budget (Chapin et al. 2002). The observed discrimination with volatilization can be large because the formation of gaseous NH3from NH4+involves several steps and each can discriminate against 15N (Table 4.1). The fi rst is the formation of NH3from NH4+. The isotope effect for this equi-librium reaction is ca. 20. NH3must then diffuse to the site volatilization, but the observed discrimination associated with this may be negligible (Shearer & Kohl 1986). Finally, there is volatilization of NH3into the atmo-sphere and the observed discrimination can be as great as 30 depending on the concentration gradient between the soil and atmosphere and the rate of removal as determined by atmospheric turbulence (Hgberg 1997).40.020.00.020.040.0TimeSoil NH4+NH315N()Figure 4.3 Predicted soil NH4+and atmospheric NH3based on a Rayleigh distillation model using data from Frank et al. (2004). The model assumes a closed system over a 10-day period.90 R.D. EVANSThe 15N of NH3emitted from the soil can increase over time and often follows Rayleigh distillation kinetics (Figure 4.3). The increase is caused by the large observed discrimination with volatilization that enriches the re-maining soil NH4+. The change over time can be signifi cant over short periods of time; Frank et al. (2004) observed a 25 increase for NH3over a 10-day period following application of an artifi cial urine patch. The 15N of soil NH4+was estimated over the same time period using the Rayleigh model and the predicted increase was from 0 on day 1 to almost 30 by day 10. The large observed discrimination with volatilization can have signifi cant effects on total soil 15N. Frank & Evans (1997) observed that grazed sites had soil 15N ca. 1 greater than sites where grazing had been excluded for 32 to 36 years, presumably due to microbial immobilization that retained NH4+enriched by volatilization from urine patches in the ecosystem.Nitrifi cation and denitrifi cationMeasuring the isotopic composition of oxidized nitroge