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Soil Biology & Biochemistry

Estimation of Gross Nitrogen Transformations and Nitrogen Retention in Grassland Soils Using FLUAZ

^a European Commission, Joint Research Centre DG, Inst. for Reference Materials and Measurements, B-2440 Geel, Belgium
^b Lab. of Applied Physical Chemistry-ISOFYS, Faculty of Biosciences Engineering, Ghent Univ., Coupure Links 653, B-9000 Gent, Belgium
^c Chilean Nuclear Energy Commission, Amunátegui 95, P.O. Box 188-D, Santiago, Chile
^d Dep. of Soil Management and Soil Care, Faculty of Bioscience Engineering, Ghent Univ., Coupure Links 653, B-9000 Gent, Belgium

^* Corresponding author (pascal.boeckx{at}ugent.be)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
The flux of N through mineralization–immobilization turnover (MIT) in grassland soils is a major determinant for plant N uptake and for N loss processes. In this study we investigated the dynamics of gross N transformation rates and potential N retention on mineral fertilizer addition in three permanent grassland soils of varying texture (loamy sand, loam, and clay loam). Gross N transformation rates were calculated with the ¹⁵N-tracing model FLUAZ. Differentially ¹⁵N-labeled NH₄NO₃ (at a rate of 100 mg N kg^–1 soil) was added to the soils in paired laboratory incubation experiments. Size and ¹⁵N enrichment of the NH₄⁺, NO₃^–, and soil organic N pools were measured at 0, 1, 3, 7, 14, and 30 d after NH₄NO₃ addition. The accuracy of the simulations of the data using FLUAZ were robust, but tended to decrease (i) with increasing incubation times, (ii) with increasing duration of the time intervals considered, and (iii) with increasing experimental variability. The proportion of the initial N content mineralized on incubation was largest in the loamy sand soil (2.5%), followed by the clay loam soil (1.2%) and the loam soil (0.8%). The actual gross nitrification and N immobilization activity followed the same trend. The loam soil showed the lowest relative N retention (ratio N immobilization over [gross N mineralization + gross nitrification]), which was attributed to its low C availability. In general, the ¹⁵N retention after addition of ¹⁵NH₄¹⁴NO₃ was approximately five times larger than after addition of ¹⁴NH₄¹⁵NO₃.

Abbreviations: DNRA, dissimilatory nitrate reduction to ammonium • MIT, mineralization-immobilization turnover • MWE, mean weighted error • SOM, soil organic matter

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
THE FLUX OF N THROUGH MIT in grassland soils is a major determinant for plant N uptake and for N loss processes (Ledgard et al., 1998). Several field studies with ¹⁵N-labeled mineral fertilizer have shown that significant amounts of labeled fertilizer can be retained in the soil organic N pool, as a result of immobilization by the microbial biomass (Bristow et al., 1987; Hart et al., 1993; Whitehead, 1995). The extent to which inorganic N is immobilized is related to the supply of readily available C, because it regulates microbial activity (Okereke and Meints, 1985). Some of the immobilized fertilizer N in soil organic matter (SOM) is subsequently remineralized, though this is believed to occur relatively slowly (Whitehead, 1995).

The gross N transformation rates (mineralization, nitrification, and NH₄⁺– and NO₃^––immobilization) that occur in soils can only be estimated by using the ¹⁵N isotope pool dilution technique. The ¹⁵N isotope pool dilution methodology is based on the principle that after enrichment of an N pool with ¹⁵N, an influx of non-enriched N into this pool, via mineralization or nitrification, lowers the ¹⁵N-abundance (dilution) whereas an efflux, via NH₄⁺ immobilization and nitrification or via NO₃^– immobilization and denitrification, does not. Thus, the decrease in ¹⁵N abundance of the enriched pool is a measure for the gross production of the enriched N compound. In addition to this approach, the increase in ¹⁵N-abundance of other, non-enriched pools can be used to quantify the gross transformation rates of these pools (Wessel and Tietema, 1992). However, several N fluxes can simultaneously dilute or enrich the ¹⁵N abundance of a pool. As a result these fluxes can only be accurately estimated using numerical techniques (Mary et al., 1998). Moreover, long incubation times may hamper accurate estimates of the gross N transformation rates due to (i) a strong dilution of the initially enriched N pools, (ii) subsequent transformations of labeled N pools, such as remineralization of previously immobilized ¹⁵N or humification of the microbial biomass, and (iii) gaseous losses of ¹⁵N through volatilization or denitrification.

In this study we investigated the dynamics of gross N transformation rates and the potential N retention after mineral fertilizer application in three grassland soils with a varying texture, by means of long-term (30 d) ¹⁵N-labeling experiments. The principle objective was to evaluate whether the long-term dynamics of the size and ¹⁵N abundance of mineral and organic N pools in these ¹⁵N-labeling experiments could be accurately simulated with the numerical ¹⁵N tracing model FLUAZ (Mary et al., 1998), which enables to account for remineralization, humification, volatilization, and denitrification.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Site Description and Soil Sampling
Soil samples were collected in September 2002 from three permanent grassland soils of varying texture at three different locations in Belgium. At the time of sampling, these soils had been under permanent grassland (mainly Lolium perenne) for at least 35 yr. The first grassland soil was a wet, poorly drained Plagganthrept with a loamy sand texture, located at Wechelderzande (4°46' E, 51°15' N). The second grassland soil was a moderately drained Glossic Hapludalf with a loam texture, located at Melle (3°47' E, 50°59' N). The third grassland soil was a moderately drained Oxyaquic Udifluvent with a clay loam texture, located at Watervliet (3°35' E, 51°17' N). The grassland soils located at Wechelderzande, Melle, and Watervliet received an annual input of 150, 200, and 130 kg N ha^–1 as mineral fertilizer, respectively, and were grazed and cut one to two times per year.

The sand, silt, and clay content of the soil samples were determined by particle-size analysis following the pipette method of Robinson-Köhn (De Leenheer, 1966; Gee and Bauder, 1986). The soils were classified according to USDA (1999). The general soil characteristics of the 0- to 10-cm layer of the three soils are shown in Table 1. The three soils had comparable C/N ratios, ranging from 11.0 (Watervliet) to 11.3 (Wechelderzande). To obtain composite soil samples representative of each grassland soil, 20 replicate soil cores covering the whole area of the investigated grasslands (ranging from 0.1 to 0.25 ha) were taken from the 0- to 10-cm layer with a steel auger (3.5-cm diam.). The soil cores were bulked and stored in plastic bags at 4°C until the start of the ¹⁵N-isotope dilution experiments.

Before the start of the ¹⁵N-isotope dilution experiments, the fresh soil samples were homogenized and sieved through a 3.15-mm sieve to remove root material and shortly air-dried to obtain the gravimetric water content corresponding to a water filled pore space (WFPS) of 50% at the bulk density measured in the field (Table 1), minus the amount of ¹⁵N-labeling solution that would be added to the soils at the beginning of the experiment (corresponding with 6% gravimetric water content). The soils were pre-incubated during 7 d at 15°C. After the pre-incubation period, half the amount of the soil was labeled with a ¹⁵N-enriched (10.23 atom%) ¹⁵NH₄¹⁴NO₃–solution, equivalent to an addition of 50 mg NH₄⁺–N and 50 mg NO₃^––N kg^–1 soil. The other half of the soil was labeled with a ¹⁵N-enriched (10.40 atom%) ¹⁴NH₄¹⁵NO₃–solution at the same doses. The ¹⁵N-solutions were uniformly applied using a disposable syringe and thoroughly mixed into the soil. We added ¹⁵N-labeled NH₄NO₃ at this relatively high rate, because we intended to study the effect on soil N transformation processes of mineral fertilizer application, at a comparable dose as generally applied in agricultural practice. Taking into account the bulk densities measured in the field (Table 1), this ¹⁵N-labeling corresponded with a mineral fertilizer application of 119, 132, and 111 kg N ha^–1 in the grassland soils located at Wechelderzande, Melle, and Watervliet, respectively.

From both the ¹⁵NH₄¹⁴NO₃– and ¹⁴NH₄¹⁵NO₃–labeled bulk samples of each of the three grasslands soils, 15 disposable jars were filled with an amount of soil equivalent to 50 g oven-dry weight. The bulk densities of the soil samples were adjusted to the values measured in the field (resulting in a water filled pore space of 50%), covered with pin-holed parafilm to enable gas exchange and incubated at 15°C. After 1, 3, 7, 14, and 30 d of incubation, three replicate ¹⁵NH₄¹⁴NO₃– and ¹⁴NH₄¹⁵NO₃–labeled incubations were removed and extracted with 250 mL of 2 M KCl (60 min shaking). To remove any residual inorganic ¹⁵N from the soil samples, the extraction was repeated twice by shaking for 30 min with 150 mL of 2 M KCl. After shaking, the soil suspensions were centrifuged (Heraeus Sepatech, Labofuge GL) at 3000 rev min^–1 during 5 min and the clear supernatants was immediately frozen for later NH₄⁺, NO₃^–, and ¹⁵N analysis. The extracted soil samples were immediately dried at 50°C for 48 h and ground with a planetary ball mill (PM400, Retsch, Germany) for ¹⁵N analysis of the organic and fixed N, to study the ¹⁵N immobilization. This extraction procedure was also performed just before and 15 min after the ¹⁵NH₄¹⁴NO₃– and ¹⁴NH₄¹⁵NO₃–label additions (initial and Day 0 extraction).

Chemical Analysis
Analyses of the total C and N contents in the soil samples were performed using a CNS analyzer (Variomax, Elementar, Germany). The NH₄⁺ and NO₃^– concentrations in the KCl extracts were determined colorimetrically by means of a continuous flow analyzer (Skalar, The Netherlands). Isotope ratio analysis of the NH₄⁺ and NO₃^– pool was performed after chemical conversion to N₂O. The NH₄⁺ was converted to N₂O using NaOBr according to a protocol adapted from Hauck (1982) and Saghir et al. (1993). The samples with a NH₄⁺ concentration too low for conversion to N₂O were spiked by addition of 700 µL of an (NH₄)₂SO₄ solution at natural abundance, with a concentration of 10.7 mmol N L^–1, to 45 mL of the samples. NO₃^– was converted to N₂O according to Stevens and Laughlin (1994). Isotope ratio analysis of the produced N₂O was performed using an ANCA-TGII trace gas preparation unit (PDZ Europa, UK) coupled to a Continuous Flow Isotope Ratio Mass Spectrometer (20–20, PDZ Europa, UK). ¹⁵N analysis of the soil samples was performed using an ANCA-SL elemental analyzer coupled to a Continuous Flow Isotope Ratio Mass Spectrometer (20–20, PDZ Europa, UK).

Calculation of the Nitrogen Fluxes
The N fluxes during the incubation experiments were estimated numerically using the FLUAZ model developed by Mary et al. (1998). The eight N fluxes that can be taken into account in the FLUAZ model are (Fig. 1) : mineralization = ammonification (m), immobilization of NH₄⁺ (ia) and NO₃^– (in), remineralization or release of previously immobilized N (r), humification (h), (autotrophic) nitrification (n), volatilization (v), and denitrification (d). The calculations in this model are based on the isotopic dilution and isotopic enrichment principles (Monaghan and Barraclough, 1995). To use the model, measurements of the size and atom% ¹⁵N excess of the NH₄⁺, NO₃^–, and total soil organic N pool from either a single or a paired labeling experiment are needed as input data. In the case of a paired labeling experiment eight completely independent variables are measured (since the NH₄⁺ and NO₃^– concentrations are supposed to be similar in both experiments), which enables accurate calculation of up to six N fluxes per time interval considered (Mary et al., 1998). The biomass N pool that is simulated in the model is that part of the biomass that is actively growing and is responsible for the immobilization and remineralization of added mineral N (Mary et al., 1998). The FLUAZ model combines a numerical integration method (Runge-Kutta algorithm) for the differential equations, describing the N and ¹⁵N fluxes between the four N pools (NH₄⁺–N, NO₃^––N, humus and biomass N; Fig. 1), with a nonlinear fitting method (Haus-Marquardt algorithm) for the calculation (optimization) of the different N fluxes in the model. The model thus calculates the evolution of the size and atom% ¹⁵N excess of the N pools, using fixed or fitted N transformation rates, during each time interval considered. The simulated values at the end of a time interval are then used as initial values for the next interval, and the N transformation rates can vary from one interval to another. The optimal fit of the experimental data was calculated by minimizing the MWE (mean weighted error) criterion, which is a function of the difference between simulated and measured variables and the experimental variance of the measured variables. In this way, the measured variables with the largest experimental variability have the lowest weight in the optimization procedure (Mary et al., 1998). Further details on the FLUAZ model can be found in Mary et al. (1998).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Compartment model of the N pools and fluxes considered in FLUAZ; m = mineralization (= ammonification), n = nitrification, v = volatilization, d = denitrification, ia = immobilization of NH4+, in = immobilization of NO3–, r = remineralization, h = humification.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Size and Atom% ¹⁵N Excess of the NH₄⁺ and NO₃^– Pool
The initial NH₄⁺ concentration in the loamy sand (Wechelderzande), loam (Melle), and clay loam soil (Watervliet), just before addition of the labeled NH₄NO₃ solutions, was 42.4, 1.6, and 1.5 mg N kg^–1 soil, respectively. The initial NO₃^– concentration in the three soils was 28.6, 39.3, and 57.6 mg N kg^–1 soil, respectively. This indicates that, during the pre-incubation period, a considerable accumulation of NH₄⁺–N had occurred in the loamy sand soil, which was not the case in the loam and clay loam soils. The loam and clay loam soils, however, showed a relatively larger accumulation of NO₃^––N than the loamy sand soil during the pre-incubation period.

The dynamics of the NH₄⁺ and NO₃^– concentrations in the three soils during the incubation (between 15 min [Day 0] and 30 d after addition of the ¹⁵N-labeled NH₄ NO₃–solutions) are shown in Fig. 2, 3, and 4 (a and b). In the loamy sand soil (Fig. 2), the NH₄⁺ concentration increased in the intervals 0 to 3 d, followed by a linear decrease in the intervals 3 to 30 d. The NO₃^– concentration showed a steady increase between 0 and 30 d. The NH₄⁺ concentration in the loam soil (Fig. 3) decreased linearly between 0 and 14 d, and remained nearly constant afterward. The NO₃^– concentrations showed a steady increase between 0 and 14 d. In the clay loam soil (Fig. 4), the NH₄⁺ concentration showed a very fast decrease during the first 3 d after the NH₄NO₃ addition, which coincided with a very fast increase of the NO₃^– concentration, and remained nearly constant after 3 d.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Dynamics of the NH4+ and NO3– concentrations (a and b), and the atom% 15N excess of the NH4+ and NO3– pool (c and d) between 15 min (Day 0) and 30 d after addition of 15NH414NO3 and 14NH415NO3 in the loamy sand soil (average values of three replicates, error bars represent plus/minus one standard deviation).

View larger version (22K): [in this window] [in a new window]   Fig. 3. Dynamics of the NH4+ and NO3– concentrations (a and b), and the atom% 15N excess of the NH4+ and NO3– pool (c and d) between 15 min (Day 0) and 30 d after addition of 15NH414NO3 and 14NH415NO3 in the loam soil (average values of three replicates, error bars represent plus/minus one standard deviation).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Dynamics of the NH4+ and NO3– concentrations (a and b), and the atom% 15N excess of the NH4+ and NO3– pool (c and d) between 15 min (Day 0) and 30 d after addition of 15NH414NO3 and 14NH415NO3 in the clay loam soil (average values of three replicates, error bars represent plus/minus one standard deviation).

In the loamy sand and loam soil, the atom% ¹⁵N excess of the NH₄⁺ pool after ¹⁵NH₄¹⁴NO₃ addition (Fig. 2c and 3c) showed a gradual decrease during the whole incubation period. This indicates that there was a continuous input of NH₄⁺ N at natural abundance into the ¹⁵N-labeled NH₄⁺ pool, resulting from mineralization (ammonification) of soil organic N. The atom% ¹⁵N excess of the NO₃^– pool after ¹⁵NH₄¹⁴NO₃ addition in the loamy sand and loam soil (Fig. 2d and 3d) showed a continuous increase as the incubation proceeded, which indicates that ¹⁵N-enriched NO₃^––N, resulting from nitrification, continuously entered the NO₃^– pool. In the clay loam soil, the rapid decrease of the NH₄⁺ concentration between 0 and 3 d after ¹⁵NH₄¹⁴NO₃ addition coincided with a very fast decrease of the atom% ¹⁵N excess of the NH₄⁺ pool between 0 and 3 d, followed by a small but continuous decrease between 3 and 30 d (Fig. 4c). During the first day after the ¹⁵NH₄¹⁴NO₃ addition, the atom% ¹⁵N excess of the NO₃^– pool (Fig. 4d) increased rapidly from 0.08 (Day 0) to 2.1 atom% excess (Day 1). The observation that the atom% ¹⁵N excess of the NO₃^– pool at Day 0 (15 min after addition of the ¹⁵NH₄¹⁴NO₃ solution) was already larger than natural abundance levels, and the very fast increase of the atom% ¹⁵N excess between 0 and 1 d indicate that the added ¹⁵N-labeled NH₄⁺–N was very rapidly converted to NO₃^––N through nitrification.

In the loamy sand and loam soil, the atom% ¹⁵N excess of the NO₃^– pool after ¹⁴NH₄¹⁵NO₃ addition (Fig. 2d and 3d) showed a gradual decrease during the whole incubation period. In the clay loam soil, however, the atom% ¹⁵N excess of the NO₃^– pool (Fig. 4d) decreased strongly between 0 and 3 d, followed by a very slow decrease between 3 and 30 d. The observed dilution of the ¹⁵N-labeled NO₃^– pools can be attributed to nitrification of NH₄⁺–N at natural abundance.

In the loam and the clay loam soil, an increase in the atom% ¹⁵N excess of the NH₄⁺ pool was observed between 14 (0.03 atom% excess) and 30 d (0.15 atom% excess), and between 7 (0.01 atom% excess) and 30 d (0.22 atom% excess) after ¹⁴NH₄¹⁵NO₃ addition, respectively (Fig. 3c and 4c). However, this increase was only significant (P < 0.05) in the clay loam soil. This ¹⁵N-enrichment in the NH₄⁺ pool when ¹⁴NH₄¹⁵NO₃ was applied could be explained by the remineralization of previously immobilized ¹⁵N, by a direct conversion of NO₃^– to NH₄⁺ (via dissimilatory reduction) or by a combination of both processes occurring simultaneously in these soils.

¹⁵N Recovery in the Soil Organic and Fixed N Pool
The atom% ¹⁵N excess of the non-labeled loamy sand, loam, and clay loam soil was 0.0019, 0.0020, and 0.0026 atom%, respectively. The atom% ¹⁵N excess of the soil organic and fixed N pool in the loamy sand and the loam soil showed a significant increase from 0 until 14 d after ¹⁵NH₄¹⁴NO₃ addition (data not shown), whereas in the clay loam soil a significant increase was only observed between 0 and 1 d (data not shown).

The amount of ¹⁵NH₄⁺–N recovered in the soil organic and fixed N pool immediately (15 min) after ¹⁵NH₄¹⁴NO₃ addition was smallest in the loamy sand soil (84 µg ¹⁵N kg^–1 soil) and approximately four times larger in the clay loam (353 µg ¹⁵N kg^–1 soil) and loam soil (357 µg ¹⁵N kg^–1 soil). This represented 1.7 and 7.2% of the total amount of ¹⁵NH₄⁺–N added in the loamy sand soil and the loam and clay loam soils, respectively. The amount of ¹⁵NH₄⁺–N recovered in the soil organic and fixed N pool at the end of the incubation was largest in the loam soil (1043 µg ¹⁵N kg^–1 soil), followed by the clay loam (664 µg ¹⁵N kg^–1 soil) and the loamy sand soil (590 µg ¹⁵N kg^–1 soil), which represented 21, 13, and 12%, respectively, of the total amount of ¹⁵NH₄⁺–N added.

The amounts of ¹⁵NO₃^––N recovered immediately after ¹⁴NH₄¹⁵NO₃–addition ranged from 27 µg ¹⁵N kg^–1 soil in the clay loam soil to 47 and 50 µg ¹⁵N kg^–1 soil in the loam and loamy sand soil, respectively. At the end of the incubation, the largest amount of ¹⁵NO₃^––N was recovered in the loam soil (206 µg ¹⁵N kg^–1 soil), followed by the clay loam (131 µg ¹⁵N kg^–1 soil), and the loamy sand soil (106 µg ¹⁵N kg^–1 soil). These amounts corresponded with 4.1, 2.6, and 2.1% of the total amount of ¹⁵NO₃^––N added, respectively.

The total ¹⁵N-recovery (in the soil organic and fixed N pool plus the mineral N pools) immediately after NH₄NO₃ addition was approximately 100% in the three soils. At the end of the incubations, the total ¹⁵N-recoveries ranged from 91% (loamy sand and clay loam soil) to 95% (loam soil) after ¹⁵NH₄¹⁴NO₃ addition, and from 92% (loam and clay loam soil) to 94% (loamy sand soil) after ¹⁴NH₄¹⁵NO₃ addition.

Simulation of the Data by FLUAZ
The simulated values of the NH₄⁺ and NO₃^– concentrations and the atom% ¹⁵N excess of NH₄⁺ and NO₃^– in the three soils are plotted versus time in Fig. 2, 3, and 4, respectively. The best overall fit of the data by the FLUAZ model, based on the MWE criterion, was obtained for the loam soil (average MWE of 1.6), followed by the loamy sand soil (average MWE of 2.8) and the clay loam soil (average MWE of 4.6). The simulated values of the NH₄⁺ and NO₃^– concentrations and the atom% ¹⁵N excess of the NH₄⁺ (Fig. 3) and NO₃^– pool were generally within the variation of the measured values. However, a discrepancy between the simulated and measured NO₃^– concentrations was observed at 30 d in the loamy sand and loam soil (19 mg N kg^–1 higher or 20 mg N kg^–1 lower, respectively) (Fig. 2 and 3) and the simulated NO₃^– concentrations at 3 d in the clay loam soil (56 mg N kg^–1 lower) (Fig. 4). A discrepancy between simulated and observed values was also observed for the atom% ¹⁵N excess values of the NH₄⁺ pool in the clay loam soil, after addition of ¹⁴NH₄¹⁵NO₃ (Fig. 4). The FLUAZ model simulated an increase in the ¹⁵N-enrichment of the NH₄⁺ pool between 7 and 14 d, but the simulated atom% ¹⁵N excess values were still 1.7 and 2.7 times smaller than the measured values on Days 14 and 30, respectively.

The atom% ¹⁵N excess of the soil organic and fixed N pool was very well fitted for the whole duration of the experiment in the loamy sand soil and for the first 14 d of the experiment in the loam soil (data not shown). In the loam soil, the simulated atom% ¹⁵N excess of the soil organic and fixed N pool at 30 d was slightly smaller than the measured value after addition of ¹⁵NH₄¹⁴NO₃. In the clay loam soil, the simulated atom% ¹⁵N excess of the soil organic and fixed N pool after addition of ¹⁵NH₄ ¹⁴NO₃ were systematically lower than the measured values from 3 d till the end of the incubation period.

Gross N Transformation Rates Calculated by FLUAZ
The gross N mineralization (m), nitrification (n), NH₄⁺ immobilization (ia), NO₃^– immobilization (in), remineralization (r), and denitrification (d) rates, which were calculated by FLUAZ for the five time intervals considered in the three soils are summarized in Table 2. In the loamy sand soil, the gross N mineralization rates during the intervals 0 to 1 and 1 to 3 d were considerably larger than the average gross N mineralization rates in the time intervals between 3 and 30 d. In the loam and clay loam soil the same trend was observed, but only for the gross mineralization rate in the interval 0 to 1 d. This observation indicates that a flush of gross N mineralization (priming effect) may have occurred at the beginning of the incubation experiments after the addition of the labeling solution. The cumulative gross N mineralization at the end of the incubation period was largest in the loamy sand soil (68 mg N kg^–1 soil or 81 kg N ha^–1), followed by the clay loam soil (58 mg N kg^–1 soil or 64 kg N ha^–1) and the loam soil (21 mg N kg^–1 soil or 28 kg N ha^–1). These values of cumulative gross N mineralization correspond to 2.5, 1.2, and 0.8% of the initial total N content in the loamy sand, clay loam, and loam soil, respectively.

The FLUAZ model indicated that in the three soils NH₄⁺ and NO₃^– immobilization occurred simultaneously in each interval considered, except during the interval 14 to 30 d in the clay loam soil (Table 2). In nearly all time intervals considered, the estimated NH₄⁺ immobilization rates were larger than the NO₃^– immobilization rates and in these cases, the proportion of the NH₄⁺ immobilization ranged from 60 to 95% of the total mineral N immobilization. In the three soils investigated, the gross NH₄⁺ and NO₃^– immobilization rates were considerably larger during the first day after addition of the NH₄NO₃–solutions in relation to the immobilization rates observed during the rest of the incubations.

In the loamy sand and the clay loam soils, the large NH₄⁺ and NO₃^– immobilization rates in the interval 0 to 1 d coincided with significant remineralization rates, which was not the case in the loam soil.

The cumulative net N immobilization was also calculated by FLUAZ (total mineral N immobilization minus remineralization). At 14 d after the NH₄NO₃ addition, the cumulative net immobilization was comparable in the three soils, and ranged from 29 mg N kg^–1 soil or 34 kg N ha^–1 in the loamy sand soil, 32 mg N kg^–1 soil or 42 kg N ha^–1 in the loam soil, to 35 mg N kg^–1 soil or 39 kg N ha^–1 in the clay loam soil. Between 14 and 30 d, however, a much lower gross immobilization rate was calculated for the clay loam soil in comparison with the loamy sand and loam soils, and a much higher remineralization rate was calculated for the clay loam and loam soils in relation to the loamy sand soil (Table 2). This resulted in a relatively larger cumulative net N immobilization in the loamy sand soil (52 mg N kg^–1 soil or 62 kg N ha^–1) at 30 d after the NH₄NO₃ addition, in relation to the loam (42 mg N kg^–1 soil or 56 kg N ha^–1) and clay loam soils (38 mg N kg^–1 soil or 42 kg N ha^–1). The FLUAZ model calculated that denitrification at a rate of 1.49 mg N kg^–1 soil d^–1 occurred in the clay loam soil during the last time interval.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
¹⁵N Recovery in the Soil Organic and Fixed N Pool
Shortly (15 min) after addition of the ¹⁵NH₄¹⁴NO₃ and ¹⁴NH₄¹⁵NO₃ solutions, significant amounts of ¹⁵N were already recovered in the soil organic and fixed N pool of the three soils investigated. The rapid ¹⁵N recovery after ¹⁵NH₄¹⁴NO₃ addition could be attributed to a rapid biological immobilization, a rapid abiotic immobilization or a combination of the two processes occurring simultaneously immediately after ¹⁵NH₄¹⁴NO₃ addition (Davidson et al., 1991). Processes of abiotic immobilization of ¹⁵NH₄⁺ which have been described in literature include fixation or adsorption on clay minerals, adsorption to SOM, and condensation reactions with humic compounds (Foster et al., 1985; Davidson et al., 1991; Strickland et al., 1992; Compton and Boone, 2002), and have been shown to occur within 15 min after addition of NH₄⁺–N (Newman and Oliver, 1966). In the loam and clay loam soil, a small amount of the NH₄⁺–N added was not KCl-extractable after 15 min (4.9 ± 3.9 and 4.3 ± 0.6 mg NH₄⁺–N kg^–1 soil, respectively), which was not the case in the loamy sand soil. This observation, together with the higher clay content in the loam and clay loam soils in relation to the loamy sand soil (Table 1), may explain why the ¹⁵N recovery in the soil organic and fixed N pool 15 min after the ¹⁵NH₄¹⁴NO₃–addition in the loam and clay loam soil was about four times larger than in the loamy sand soil. A fast recovery of ¹⁵N in the soil organic and fixed N pool after addition of ¹⁵NH₄⁺ or ¹⁵NO₃^– to mineral and organic soils has been reported in several other studies (Recous et al., 1990; Davidson et al., 1991; Mary et al., 1993; Berntson and Aber, 2000; Andersen and Jensen, 2001; Compton and Boone, 2002; Müller et al., 2004b). Davidson et al. (1991), who compared the ¹⁵N recovery in a sterilized and non-sterilized silt loam grassland soil after addition of ¹⁵NH₄⁺, concluded that the observed rapid immobilization was completely abiological.

Simulation of the Data by FLUAZ
When the MWE values corresponding with the data fits in the different time intervals were considered, the quality of the fit of the data generally tended to decrease (increasing MWE values) from the first toward the last time interval. This might be partially attributed to the increasing duration of the time intervals between the sampling dates, as the incubation experiments proceeded. All N transformation rates, except the nitrification rate, were estimated assuming zero-order kinetics, which implies that these rates are assumed to remain constant during the whole time interval considered. However, when longer time intervals were considered (especially the intervals 7–14 d and 14–30 d) this assumption of constant rates may not have been fully met, explaining the larger MWE values in the longer time intervals.

The simulated values of the size and atom% ¹⁵N excess of the NH₄⁺ and NO₃^– pools were generally within the variation of the measured values (Fig. 2, 3, and 4). The largest discrepancies between simulated and measured values were observed for the NO₃^– concentrations after 3 d in the clay loam soil (Fig. 4) and after 30 d in the loamy sand and loam soil (Fig. 2 and 3). This might be attributed to the relatively large standard deviations associated with these measured values, as measured variables with the largest experimental variability have the lowest weight in the optimization procedure of the FLUAZ model, due to the MWE criterion. The considerable underestimation of the NO₃^– concentrations after 3 d in the clay loam soil might imply that the gross nitrification rate in the interval 1 to 3 d has been underestimated by FLUAZ.

Gross N Immobilization and Remineralization Rates
In the three soils investigated, the gross NH₄⁺ and NO₃^– immobilization rates observed during the first day after addition of the NH₄NO₃ solutions were considerably larger than the immobilization rates observed during the rest of the incubations. This could possibly indicate a stimulation of both the NH₄⁺ and NO₃^– immobilization rates, due to the large amounts of NH₄⁺–N and NO₃^––N, which have been added to the soils. The smaller gross NH₄⁺ immobilization rate in the loamy sand soil during the first day in relation to the rates observed in the loam and clay loam soils, might then be partially explained by a smaller stimulation of the NH₄⁺ immobilization, due to the larger NH₄⁺ concentration already present in the loamy sand soil before the NH₄NO₃–addition (see above). In nearly all time intervals considered, NO₃^– immobilization occurred simultaneously with NH₄⁺ immobilization, but at a smaller proportion of the total immobilization than the NH₄⁺ immobilization. This is consistent with the findings of several other studies and reflects the generally observed preferential microbial uptake of NH₄⁺–N in relation to NO₃^––N, when both forms are present in soil (Jansson et al., 1955; Rice and Tiedje, 1989; Recous et al., 1990).

The large NH₄⁺ and NO₃^– immobilization rates during the first day after NH₄NO₃ addition were accompanied by significant remineralization rates during the first day in the loamy sand and clay loam soils, and between 1 and 3 d in the loam soil (Table 3). This indicates that a quick recycling through the microbial biomass of the recently immobilized mineral N already occurred during the first days after NH₄NO₃ addition in the three soils investigated, and that this fast recycling was most pronounced in the loamy sand and clay loam soils. This fast recycling was also observed by Mary et al. (1998).

As discussed before, the significant ¹⁵N recovery in the soil organic and fixed N pool shortly after both ¹⁵NH₄ ¹⁴NO₃ and ¹⁴NH₄¹⁵NO₃ addition to these soils could also be attributed to a rapid abiotic fixation of the added NH₄NO₃. Therefore, it is not unrealistic that this fixed ¹⁵N-enriched mineral N was entirely or partially released again into the available mineral N pool, shortly after fixation or later on during the incubation experiments. Fixation and subsequent release of this ¹⁵N-enriched mineral N, two processes that are not considered in FLUAZ, may thus also have affected the dynamics of the ¹⁵N excess of the NH₄⁺ and NO₃^– pool. If this (abiotic) exchange process of fixation and release would also be accounted for in FLUAZ, different optimization results might probably be obtained for the (biotic) immobilization and remineralization dynamics. As a consequence, we have to be careful with our conclusion that, based on the optimization results from FLUAZ, the fast (re)mineralization–immobilization turnover shortly after fertilizer addition could be entirely attributed to microbial recycling, as it might also be (partially) attributed to an abiotic exchange process. Executing identical ¹⁵N-tracing experiments on sterilized soil samples might clarify whether this abiotic exchange process is actually occurring in the soils we investigated and the relative importance of this abiotic process in relation to the biotic (re)mineralization–immobilization turnover.

An increase in the atom% ¹⁵N excess of the NH₄⁺ pool after addition of ¹⁴NH₄¹⁵NO₃ was observed after 14 d in the loam soil (Fig. 3c), after 7 d in the clay loam soil (Fig. 4c) and not in the loamy sand soil (Fig. 2c). This ¹⁵N enrichment in the NH₄⁺ pool when labeled ¹⁵NO₃ was applied could again be explained by the remineralization of previously immobilized ¹⁵N. Other processes that could explain this, but which are also not considered in FLUAZ, are the direct conversion of NO₃^– to NH₄⁺ via dissimilatory reduction (DNRA), or a combination of both DNRA and remineralization occurring simultaneously in these soils. The robust FLUAZ simulations (Fig. 2c, 3c, and 4c) indicated that this ¹⁵N enrichment of the NH₄⁺ pool in the loam soil could be largely explained by the remineralization after 14 d (Table 2) of recently immobilized ¹⁵NO₃^–. For the clay loam soil, however, FLUAZ simulated a smaller increase in the ¹⁵N enrichment of the NH₄⁺ pool than the observed enrichment between 7 and 30 d (Fig. 4c). This underestimation of the observed ¹⁵N enrichment in the NH₄⁺ pool by the FLUAZ model, which doesn't consider DNRA, might suggest that this enrichment should be partially attributed to DNRA occurring in the clay loam soil after 7 d. However, considering the strict anaerobic nature of the DNRA process (Paul and Clark, 1996) and the assumption that the soils were incubated under aerobic conditions (water filled pore space of 50%), it would be very unlikely that DNRA, nor denitrification would have occurred in the clay loam soil. Nevertheless, a decrease of the NO₃^– concentration was observed after 14 d (Fig. 4b) and FLUAZ estimated a significant denitrification rate between 14 and 30 d (Table 2). This might indicate that some anaerobic microsites could have been produced throughout the incubation period, enabling denitrification or DNRA to occur in the clay loam soil. Fazzolari et al. (1998) demonstrated that DNRA activity may be less sensitive than denitrification to an inhibitory effect by O₂ and therefore may also occur in aerobic soils. Recently, Müller et al. (2004a) suggested that DNRA, rather than remineralization, was responsible for the significant ¹⁵N enrichment of the NH₄⁺ pool which they observed during aerobic incubation of an old grassland soil after addition of equal amounts of ¹⁴NH₄¹⁵NO₃.

Gross Nitrification Rates
As NH₄⁺ is the substrate for the (autotrophic) nitrification process, the relatively large NH₄⁺ addition at the start of the incubation experiments has most probably stimulated the nitrification. Therefore, the calculated gross nitrification rates are initially stimulated by the NH₄⁺ addition and closer to the potential nitrification rates in these soils at the start of the incubation experiments, but decrease with the availability of NH₄⁺ as the incubations proceeded. The nitrification rate observed during the first day after NH₄NO₃ addition in the clay loam soil was 10 to 17 times larger than the rates observed in the loam and loamy sand soils, respectively (Table 2). This indicates that the clay loam soil had a much higher potential nitrifying activity, resulting in a much faster depletion of the NH₄⁺ concentration (after 3 d) than in the loam soil (after 14 d) and the loamy sand soils (after 30 d). These differences in potential nitrifying activity might be partially explained by differences in soil pH (Table 1). The conditions for nitrification might be more favorable in the clay loam soil due to its higher pH (7.2) in relation to the loam soil (6.3) and loamy sand soil (5.8). Paul and Clark (1996) reported that nitrification is curtailed when the soil pH is below 6.0. Another possible explanation is the difference in the naturally occurring, nitrifying microbial population in the three soils. The difference in the initial NH₄⁺ and NO₃^– concentration (after pre-incubation) which we observed in the three soils, indicates that the naturally occurring nitrification rate also tended to be much smaller in the loamy sand soil in relation to the loam and especially clay loam soils.

The ratio of net cumulative N immobilization/(gross cumulative mineralization + gross cumulative nitrification) were for the loamy sand, clay loam, and loam soil 0.29, 0.27, and 0.65, respectively. This suggests that the loam soil has the lowest capacity to immobilize N, which is attributed to the low C content and the relatively high silt plus clay content, resulting in the lowest C mineralization rates of the three soils (data not shown). A relationship between gross N immobilization rates and C mineralization rates has been observed in several studies (Schimel, 1986; Hart et al., 1994; Barrett and Burke, 2000) and also in this study (i = 0.50m_C – 0.54, R² = 0.85, P < 0.01). In an earlier study (Accoe et al., 2004) we could conclude that N retention through microbial N immobilization in grassland soils was limited by C availability.

Another process which may have occurred in these soils, but which is not considered in FLUAZ, is heterotrophic nitrification or the direct conversion of organic N to NO₃^– without passing through the exchangeable NH₄⁺ pool (Barraclough and Puri, 1995). Heterotrophic nitrification is well known in acidic forest soils (Barraclough and Puri, 1995), but Müller et al. (2004b) showed that heterotrophic nitrification can also be an important N transformation process in old grassland soils. Heterotrophic nitrification results in a dilution of the enriched ¹⁵NO₃^– pool, which is additional to the dilution resulting from autotrophic nitrification. As heterotrophic nitrification was not accounted for in FLUAZ, the gross autotrophic nitrification rates in the investigated soils may thus have been overestimated by FLUAZ. Additional ¹⁵N isotope dilution experiments using nitrification inhibitors (Barraclough and Puri, 1995) could be executed to separate autotrophic and heterotrophic nitrification in these soils, and to assess the need to account for heterotrophic nitrification in the FLUAZ model to optimize the gross N transformation rates.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
This study indicates that the dynamics of the size and ¹⁵N abundance of mineral and organic N pools from long-term (30 d) ¹⁵N-labeling experiments can be accurately simulated with the numerical ¹⁵N-tracing model FLUAZ. The accuracy of the simulations tended to decrease (i) with increasing incubation times, (ii) with increasing duration of the time intervals considered and (iii) with increasing experimental variability. This allowed to robustly estimate six gross N transformation rates (mineralization, immobilization of NH₄⁺, and NO₃^–, remineralization, autotrophic nitrification and denitrification). However, some processes that may also occur in grassland soils (abiotic fixation and release of mineral N, DNRA, and heterotrophic nitrification) are not accounted for by FLUAZ. As a consequence, the gross N transformation rates (especially NH₄⁺ and NO₃^– immobilization, remineralization, and nitrification) that were optimized by FLUAZ might be affected by not considering one or more of these processes. Additional ¹⁵N tracing experiments might clarify the actual occurrence and relative importance of these processes in the soils investigated and the need to consider them as well in ¹⁵N-tracing models like FLUAZ.

In the three soils investigated, significant amounts of ¹⁵N were recovered in the soil organic and fixed N pool shortly after addition of both ¹⁵NH₄¹⁴NO₃ and ¹⁴NH₄ ¹⁵NO₃, indicating a fast biotic or abiotic immobilization capacity. The total amounts of ¹⁵N recovered in the soil organic and fixed N pool 30 d after addition of ¹⁵NH₄¹⁴NO₃ were approximately five times larger than the amounts recovered after addition of ¹⁴NH₄¹⁵NO₃, which reflects the generally observed preferential microbial uptake of NH₄⁺–N in relation to NO₃^––N. The large NH₄⁺ and NO₃^– immobilization rates, which were observed during the first days after NH₄NO₃–addition, were accompanied by significant remineralization rates. This probably indicates that a quick recycling through the microbial biomass of the recently immobilized mineral N occurred shortly after NH₄NO₃ addition. The observed cumulative gross mineralization and nitrification were in the following order: loamy sand > clay loam > loam soil. The cumulative net N immobilization was in the order: loamy sand > loam > clay loam soil. The ratios of cumulative net N immobilization over cumulative gross mineralization plus gross nitrification were as follows: loamy sand clay loam > loam soil. Finally, N immobilization seemed to be limited by C availability.

Received for publication August 24, 2004.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 

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