Direct flux and 15N tracer methods for measuring denitrification in forest soils

Estimates of denitrification are one of the key uncertainties in the terrestrial nitrogen (N) cycle, primarily because reliable measurements of this highly variable process—especially the production of its terminal product (N₂)—are difficult to obtain. We evaluated the ability of gas-flow soil core and ¹⁵N tracer methods to provide reliable estimates of denitrification in forest soils. Our objectives were to: (1) describe and present typical results from new gas-flow soil core and in situ ¹⁵N tracer methods for measuring denitrification, (2) discuss factors that affect the relevance of these methods to actual in situ denitrification, and (3) compare denitrification estimates produced by the two methods for a series of sites in a northern hardwood forest ecosystem. Both methods were able to measure accumulations of N₂ over relatively short (2–5 h) incubations of either unamended or tracer-amended intact soils. Denitrification rates measured by the direct flux soil core method were very sensitive to incubation oxygen (O₂) concentration and decreased with increased O₂ levels. Denitrification rates measured by the in situ ¹⁵N tracer method were very sensitive to the ¹⁵N content of the nitrate (NO₃ ^?) pool undergoing denitrification, which limits the applicability of this method for quantifying denitrification in N-poor ecosystems. While its ability to provide accurate estimates of denitrification was limited, the ¹⁵N tracer method provided estimates of the short-term abiotic and biotic transformations of atmospheric N deposition to gas. Furthermore, results suggest that denitrification is higher and that N₂O:N₂ ratios are lower (<0.02) than previously thought in the northern hardwood forest and that short-term abiotic and biotic transformations of atmospheric N deposition to gas are significant in this ecosystem.     

A 15N tracer technique for assessing fine root production and mortality

We tested a ¹⁵N tracer technique to assess fine root production and mortality based on temporal measurements of the ¹⁵N mass in fine root structural tissues and the ¹⁵N concentration of the plant-available soil N pool. The results of a pilot study indicated that this technique is based on sound methods and reasonable assumptions. The ¹⁵N tracer technique avoids most of the major limitations which hinder existing methods and may provide valuable insight into the rates and controls of fine root production and mortality in terrestrial ecosystems. Received: 23 September 1996 / Accepted: 10 June 1997     

Towards an understanding of the dynamics of compost N in the soil-plant-atmosphere system using 15N tracer

Aims

The principal aim of the present review is to synthesize and evaluate published information on the N fertilizer value of composts, and their effect on the utilization of conventional N fertilizers by crops.

Methods

We have examined the literature where the dynamics of N in the soil-plant-atmosphere continuum are traced using composts that were either artificially enriched in the ¹⁵N stable isotope (in units of atom % ¹⁵N excess) or had a natural ¹⁵N abundance (δ¹⁵N in units of ‰ or per mil) due to isotope discrimination processes that occur during composting. The methods used to produce artificially-enriched composts and to test uniformity of labelling are reviewed.

Results

Limited data show that composts are generally inferior sources of N for crops compared with their raw materials due to a lower N mineralization capacity. Immobilization of fertilizer N increases in compost-amended soils and may reduce recovery by a crop, but fertilizer N losses are reduced overall. However, co-application of compost and urea should be avoided due to the risk of increased NH₃ volatilization due to the action of compost-derived urease. High annual rates of compost application can exacerbate environmental problems including nitrate contamination of groundwater.

Conclusions

Efforts are required to improve the N fertilizer value of composts by minimizing NH₃ volatilization losses during composting. More attention should also be given to the use of the natural ¹⁵N abundance of compost as a tracer.     

Growth and N2 fixation in mixed cropping of Medicago arborea and Atriplex halimus grown on a salt-affected soil using a 15N tracer technique

Abstract

The objective of this study was to evaluate dry matter (DM), nitrogen yield, N₂ fixation (Ndfa) and soil N uptake (Ndfs) in the shrubby medic (Medicago arborea) and saltbush (Atriplex halimus) grown in pots either solely or in a mixture on a salt-affected soil, using ¹⁵N dilution method. The combined DM of both species was considerably higher than that of solely grown shrubs. The inclusion of saltbush in the mixed cropping system decreased Ndfs by shrubby medic and enhanced % Ndfa without affecting amounts of N₂ fixed. It can be concluded that the use of mixed cropping system of shrubby medic and saltbush could be a promising bio-saline agricultural approach to utilize salt affected soils in terms of forage yield and N₂ fixation.     

Uptake of 15N fertilizer in compost-amended soils

Composts are considered low analysis fertilizers because their nitrogen and phosphorus content are around 1% and the organic nitrogen mineralization rate is near 10%. If compost is added to agricultural land at the N requirement of grain crops (40 – 100 kg N ha^–1), application rates approach 40–100 mg ha^–1. Much lower rates may be advisable to avoid rapid accumulation of growth limiting constituents such as heavy metals found in some composts. Combining low amendment rates of composts with sufficient fertilizer to meet crop requirements is an appealing alternative which (a) utilizes composts at lower rates than those needed to supply all the crop N requirement, (b) reduces the amount of inorganic fertilizer applied to soils, and (c) reduces the accumulation of non-nutrient compost constituents in soils. A study was conducted to compare the effects of blends of biosolids compost (C) with ¹⁵N urea(U) or ¹⁵NH4 ¹⁵NO3 (N) fertilizers to fertilizer alone on tall fescue (Festuca arundinacea L.) growth and N uptake. Blends which provided 0, 20, 40 or 60 mg N kg^–1 application rate as compost N and 120, 100, 80 or 60 mg N kg^–1 as fertilizer N, respectively, were added to Sassafras soil (Typic Hapludults). Fescue was grown on the blends in a growth chamber for 98 days. Fescue yields recorded by clippings taken at 23, 46 and 98 days and roots harvested after the 98-day clipping increased with increasing fertilizer level for both NH₄NO₃ and urea and with or without compost. Nitrogen uptake by fescue responded similarly to yield with increases recorded with increasing fertilizer levels with or without compost. Paired comparisons based on cumulative 98-day clippings data showed that yields from blends were equal to yields from fertilizer treatments containing the same percentage of fertilizer as the blends. These data indicated that compost did not provide sufficient plant-available N to increase yields or N uptake. None of the blends equaled 120 mg N kg^–1 fertilizer rate except for 100 mg NH₄NO₃-or urea-N kg^–1 –20 mg compost-N kg^–1blends. The data suggest that biosolids compost blended with fertilizer at a rate of 2–6 mg ha ^–1 did not supply sufficient additional available N to increase yields or N uptake over those of fertilizer alone.     

Nitrogen fixation and nutrient uptake by two chickpea genotypes cultivated in sandy soils of Egypt

A field experiment was conducted to compare local chickpea (Giza 2) and its recently developed cultivar L₃. The two genotypes were examined for biological dinitrogen fixation and P uptake as enhanced by inoculation withRhizobium (Rh) and/or arbuscular mycorrhiza (AM). Significant increase in dry mass was obtained by biological inoculation. Soil chemical and physical properties have a significant effect on the ability of the two genotypes to fix nitrogen on their P uptake and N content. Growth of the local genotype (G₂) was pronounced in sandy loam soil as compared with the sandy one.     

Crop uptake and leaching losses of15N labelled fertilizer nitrogen in relation to waterlogging of clay and sandy loam soils

Summary Ammonium nitrate fertilizer, labelled with¹⁵N, was applied in spring to winter wheat growing in undisturbed monoliths of clay and sandy loam soil in lysimeters; the rates of application were respectively 95 and 102 kg N ha⁻¹ in the spring of 1976 and 1975. Crops of winter wheat, oilseed rape, peas and barley grown in the following 5 or 6 years were treated with unlabelled nitrogen fertilizer at rates recommended for maximum yields. During each year of the experiments the lysimeters were divided into treatments which were either freelydrained or subjected to periods of waterlogging. Another labelled nitrogen application was made in 1980 to a separate group of lysimeters with a clay soil and a winter wheat crop to study further the uptake of nitrogen fertilizer in relation to waterlogging. In the first growing season, shoots of the winter wheat at harvest contained 46 and 58% of the fertilizer nitrogen applied to the clay and sandy loam soils respectively. In the following year the crops contained a further 1–2% of the labelled fertilizer, and after 5 and 6 years the total recoveries of labelled fertilizer in the crops were 49 and 62% on the clay and sandy loam soils respectively. In the first winter after the labelled fertilizer was applied, less than 1% of the fertilizer was lost in the drainage water, and only about 2% of the total nitrogen (mainly nitrate) in the drainage water from both soils was derived from the fertilizer. Maximum annual loss occurred the following year but the proportion of tracer nitrogen in drainage was nevertheless smaller. Leaching losses over the 5 and 6 years from the clay and sandy loam soil were respectively 1.3 and 3.9% of the original application. On both soils the percentage of labelled nitrogen to the total crop nitrogen content was greater after a period of winter waterlogging than for freely-drained treatments. This was most marked on the clay soil; evidence points to winter waterlogging promoting denitrification and the consequent loss of soil nitrogen making the crop more dependent on spring fertilizer applications.     

Fate of nitrogen applied as Azolla and blue-green algae (Cyanobacteria) in waterlogged rice soils—a15N tracer study

Summary ¹⁵N tracer was used to detect the extent to which nitrogen of appliedAzolla caroliniana, Anabaena variabilis andNostoc muscorum was available for assimilation by the growing rice plants in pots under 4 cm flood water for 60 days. The rate of release of nitrogen from the above biofertilizers, the amount of nitrogen remaining in the soils and the amount that was lost from the soils during this period were also examined. Previously¹⁵N-labelled biomass of Azolla, Anabaena and Nostoc to provide 40 mg N was mixed thoroughly with 0.5 kg silt loam Bangladesh soil (Sonatola series) in each of three pots used for a single treatment. Each pot received four 16 days old IR8 rice seedlings. A parallet set of experiments was conducted without rice plants.It was found that nitrogen uptake in the rice plants was increased by 91, 176 and 215% on using Azolla, Anabaena and Nostoc which resulted in increased total dry matter yields (shoot plus root) of 74, 105 and 125%, respectively. Of the total¹⁵N applied at the start, 26, 49 and 53% was released from Azolla, Anabaena and Nostoc; about 7, 14 and 13% was lost by denitrification and 74, 51 and 47% remained in the soils as the undecomposed part of the biofertilizers, respeciively, after 60 days. Of 15.76, 22.72 and 25.92 mg N assimilated by the rice plants, 48, 61 and 62% was supplied by Azolla, Anabaena and Nostoc, respectively. The rest was obtained from the soil used.In the absence of the rice plants 30, 43 and 45% of applied¹⁵N of Azolla, Anabaena and Nostoc was released, respectively, in 60 days of which 93–96% was lost as N₂ through denitrification.     

Fate of airborne nitrogen in heathland ecosystems: a 15N tracer study

In the present study, we analyze the fate of airborne nitrogen in heathland ecosystems (NW Germany) by means of a ¹⁵N tracer experiment. Our objective was to quantify N sequestration and N allocation patterns in an ecosystem that is naturally limited by N, but that has been exposed to airborne N inputs exceeding critical loads for more than 3 decades. We hypothesized that the system has a tendency towards N saturation, which should be indicated by low N sequestration and high N leaching. We analyzed ¹⁵N partitioning (aboveground biomass and soil horizons) and investigated ¹⁵N leaching over 2 years following a ¹⁵N tracer pulse addition. ¹⁵N tracer recovery was 90% and 76% in the first and second year, respectively. Contrary to our expectations, more than 99% of the tracer recovered was sequestered in the biomass and soil, while leaching losses were <0.05% after 2 years. Mosses were the most important short‐term sink for ¹⁵N (64% recovery in the first year), followed by the organic layer. In the second year, the moss layer developed from a sink to a source (23% losses), and soil compartments were the most important sink (gains of 11.2% in the second year). Low ¹⁵N recovery in the current year's shoots of Calluna vulgaris (<2%) indicated minor availability of ¹⁵N tracer sequestered in the organic layer. N partitioning patterns showed that the investigated heaths still have conservative N cycling, even after several decades of high N loads. This finding is mainly attributable to the high immobilization capacities for N of podzols in soil compartments. In the long term, the podzol A‐ and B‐horizons in particular may immobilize considerable amounts of incoming N. Since N compounds of these horizons are not readily bio‐available, podzols have a high potential to withdraw airborne N from the system's N cycle.     

The use of mean pool abundances to interpret 15N tracer experiments

Details are presented of a simple mathematical framework that allows ¹⁵N tracer experiments to be interpreted in terms of the main processes of the soil/plant nitrogen cycle. The calculations, all of which can be performed on a scientific calculator, yield the rates of gross mineralization and nitrification and the crop nitrogen uptake occurring as ammonium and nitrate. Two procedures are presented. One requires paired experiments with labelled ammonium and unlabelled nitrate as one treatment, and unlabelled ammonium and labelled nitrate as the other. The second procedure requires only the labelled ammonium, unlabelled nitrate treatment. Example calculations are presented using actual experimental data. The interpretative procedure uses the fact that the rate of isotopic dilution in an ammonium pool labelled with ¹⁵N is a function of the rate at which unlabelled ammonium is introduced into the pool via mineralization. Similarly, the rate of isotope dilution in an ¹⁵N labelled nitrate pool is a function of the rate at which unlabelled nitrate is introduced into the pool via nitrification.     

The use of mean pool abundances to interpret 15N tracer experiments

A pulse dilution ¹⁵N technique was used in the field to determine the effect of the ammonium to nitrate ratio in a fertilizer application on the uptake of ammonium and nitrate by ryegrass and on gross rates of mineralization and nitrification. Two experiments were performed, corresponding approximately to the first and second cuts of grass. Where no substantial recent immobilization of inorganic nitrogen had occurred, mineralization was insensitive to the form of nitrogen applied, ranging from 2.1–2.6 kg N ha^-1 d^-1. The immobilization of ammonium increased as the proportion of ammonium in the application increased. In the second experiment there was evidence that high rates of immobilization in the first experiment were associated with high rates of mineralization in the second. The implication was that some nitrogen immobilized in the first experiment was re-mineralized during the second. Whether this was nitrogen taken up, stored in roots and released following defoliation was not clear. Nitrification rates in this soil were low (0.1–0.63 kg N ha^-1 d^-1), and as a result, varying the ratio of ammonium to nitrate applied markedly altered the relative uptake of ammonium and nitrate. In the first experiment, where temperatures were low, preferential uptake of ammonium occurred, but where >90% of the uptake was as ammonium, a reduction in yield and nitrogen uptake was observed. In the second experiment, where temperatures and growth rates were higher, the proportion of ammonium to nitrate taken up had no effect on yield or nitrogen uptake.     

Nitrogen uptake in barley after spring incorporation of 15N-labelled Italian ryegrass into sandy soils

A 5-month laboratory incubation experiment was conducted to study the immobilization-mineralization of N in soil to which dried or composted ¹⁵N labelled ryegrass (Lolium italicum L.) had been added. Cellulose was added to dried ryegrass to give a C/N ratio similar to that of composted ryegrass. Exchangeable NH₄ ⁺ and NO₃ ^–, HCl-hydrolyzable N forms, microbial biomass N, NaOH-soluble and insoluble N were monitored during incubation. Dried ryegrass brought about a significant increase in total and labelled exchangeable NH₄ ⁺, while a rapid immobilization and a subsequent slow release of exchangeable NH₄ ⁺ was observed in soil with composted ryegrass, together with a resistance to degradation of the labelled humic substances. Compounds synthesized during the composting process and resistant to microbial decomposition probably caused an increase in the amino-acid fraction of soil. These findings suggest that composting can reduce the risk of N losses.     

15N abundance of surface soils,roots and mycorrhizas in profiles of European forest soils

¹⁵N natural abundances of soil total N, roots and mycorrhizas were studied in surface soil profiles in coniferous and broadleaved forests along a transect from central to northern Europe. Under conditions of N limitation in Sweden, there was an increase in ¹⁵N of soil total N of up to 9% from the uppermost horizon of the organic mor layer down to the upper 0–5 cm of the mineral soil. The ¹⁵N of roots was only slightly lower than that of soil total N in the upper organic horizon, but further down roots were up to 5% depleted under such conditions. In experimentally N-enriched forest in Sweden, i.e. in plots which have received an average of c. 100 kg N ha^–1 year^–1 for 20 years and which retain less than 50% of this added N in the stand and the soil down to 20 cm depth, and in some forests in central Europe, the increase in ¹⁵N with depth in soil total N was smaller. An increase in ¹⁵N of the surface soil was even observed on experimentally N-enriched plots, although other data suggest that the N fertilizer added was depleted in¹⁵N. In such cases roots could be enriched in¹⁵N relative to soil total N, suggesting that labelling of the surface soil is via the pathway: — available pools of N-plant N-litter N. Under N-limiting conditions roots of different species sampled from the same soil horizon showed similar ¹⁵N. By contrast, in experimentally N-enriched forest ¹⁵N of roots increased in the sequence: ericaceous dwarf shrubs15N enriched compounds in fungal material, which could contribute to explain the observed ¹⁵N profiles if fungal material is enriched, because it is a precursor of stable organic matter and recalcitrant N. This could act in addition to the previous explanation of the isotopically lighter soil surface in forests: plant uptake of ¹⁵N-depleted N and its redeposition onto the soil surface by litter-fall.     

Protein degradation in different feedstuffs labelled with 15N by using the nylon bag technique

The nylon bag technique was used to determine the Nitrogen (N) and ¹⁵N degradation of ¹⁵N labelled feedstuffs in the rumen. The N and ¹⁵N degradation values were calculated according to ?rskov and McDonald (1979) and ranged from 46.8 to 92.0 and from 61.8 to 93.6%, respectively. The differences between N and ¹⁵N degradation values of high fibre content feedstuffs are the highest, thus the measuring errors were greatest here. But differences also existed in concentrates. This study indicated that especially barley had a higher proportion of microbial N in the bag residues after the washing than the other concentrates. Therefore it is necessary to correct the N degradation values not only in cases of high fibre content but also in cases of low nitrogen content of feedstuffs. The calculation of the N degradation values could be possible on the basis of crude fibre and crude protein contents of feedstuffs. But experiments with a much larger number of ¹⁵N labelled feedstuffs have to be realized to give an accurate prediction of N degradation.     

The use of optical emission spectroscopy for human 15N tracer studies.

Optical emission spectroscopy is a convenient method for determing ¹⁵N specific activity in a variety of metabolites following the administration of an ¹⁵N-labeled amino acid to humans. The only disadvantage, compared to the more conventional isotope ratio mass spectrometer analytical system is that more tracer is needed to give accurate results. When an ¹⁵N-labeled amino acid is used as the ¹⁵N carrier, the amount of ¹⁵N is not above a tracer dose. A suitable procedure is described for performing such studies using [¹⁵N]glycine as a tracer. As an experimental example, [¹⁵N]glycine was infused into a subject at a rate of 35 mg of N/hr for 8 hr. After 3 hr a meal was consumed by the subject. The urinary urea-¹⁵N, ammonia-¹⁵N and amino acid-¹⁵N profiles were determined.     

Estimating crop N uptake from organic residues using a new approach to the 15N isotope dilution technique

Experiments were conducted to test a new approach to the ¹⁵N isotope dilution technique for estimating crop N uptake from organic inputs. Soils were pre-labelled with ¹⁵N fertiliser and a carbon source. These were then incubated until there was stabilisation of the ¹⁵N abundance of the inorganic N pool and resumption of inorganic N concentrations. Residues were then applied to the soils and planted with ryegrass (Lolium perenneL.) to determine the nitrogen derived from the residue (Ndfr) using the isotope dilution equations. This method was compared with the direct method, i.e. where ¹⁵N-labelled residues were added to the soil and Ndfr in the ryegrass calculated directly. Estimates of percentage nitrogen derived from the residue (%Ndfr) alfalfa (Medicago sativaL.) in the ryegrass, were similar, 22 and 23% for the direct and soil pre-labelling methods, respectively, in the Wechsel sandy loam. Also, estimates of the %Ndfr from soybean (Glycine max (L.) Merr) residues in the Krumbach sandy loam were similar 34% (direct) and 36% (soil pre-labelling approach). However, in the Seibersdorf clay loam, the %Ndfr from soybean was 49% using the direct method and 61% using the soil pre-labelling method; yet Ndfr from common bean residue was 46% using the direct approach and 40% using the pre-labelling, not significantly different (P > 0.05). The soil pre-labelling approach appears to give realistic values for Ndfr. It was not possible to obtain an estimate of Ndfr using the soil pre-labelling method from the maize residues (Zea mays L.) in two of the soils, as there was no increase in the total N of the ryegrass over the growing period. This was probably due to microbial immobilisation of inorganic N, as a result of the wide C:N ratio of the residue added. The results suggest that the new soil pre-labelling method is feasible and that it is a potentially useful technique for measuring N release from a wide range or organic residues, but it requires further field-testing. This revised version was published online in June 2006 with corrections to the Cover Date.     

Assimilation of 15N2 and 15NO3- by Partially Nitrate-Tolerant Nodulation Mutants of Soybean

Growth-chamber studies were conducted to evaluate nitrogen assimilationby three hypernodulated soybean [Glycine max (L.) Merr.] mutants(NOD1–3, NOD2–4, NOD3–7) and the Williamsparent. Seeds were inoculated at planting and transplanted atday 7 to nutrient solution with 1 mol m^–3 urea (optimizesnodule formation) or 5 mol m^–3 NO₃^– (inhibits noduleformation). At 25 d after planting, separate plants were exposedto ¹⁵NO₂ or ¹⁵NO₃^– for 3 to 48 h to evaluate N₂ fixationand NO₃^– assimilation. Plant growth was less for hypernodulatedmutants than for Williams with both NO₃^– and urea nutrition.The major portion of symbiotically fixed ¹⁵N was rapidly assimilated(30 min) into an ethanol-soluble fraction, but by 24 h aftertreatment the ethanolinsoluble fraction in each plant part wasmost strongly labelled. Distribution patterns of ¹⁵N among organswere very similar among lines for both N growth treatments aftera 24 h ¹⁵N₂ fixation period; approximate distributions were40% in nodules, 12% in roots, 14% in stems, and 34% in leaves.With urea-grown plants the totalmg ¹⁵N fixed plant^–1 24h^–1 was 1·18 (Williams), 1·40 (N0D1-3),107 (NOD2-4), and 0·80 (NOD3-7). The 5 mol m^-3 NO₃^- treatmentresulted in a 95 to 97% decrease in nodule mass and ¹⁵N₂ fixationby Williams, while the three mutants retained 30 to 40% of thenodule mass and 17 to 19% of the ¹⁵N₂ fixation of respectiveurea-grown controls. The hypernodulated mutants, which had restrictedroot growth, absorbed less ¹⁵NO₃^- than Williams, irrespectiveof prior N growthcondition. The ¹⁵N from ¹⁵NO₃^- was primarilyretained in the soluble fraction of all plant parts through24 h. The ¹⁵N incorporation studies confirmed that nodule developmentis less sensitive to external NO₃^- in mutant lines than in theWilliams parent, and provide evidence that subsequent metabolismand distribution within the plant was not different among lines.These results further confirm that the hypernodulated mutantsof Williams are similar in many respects to the hyper- or supernodulatedmutants in the Bragg background, and suggest that a common mutationalevent affectingautoregulatory control of nodulation has beentargeted. Key words: Glycine max (L.) Merr., soybean, N₂fixation, nitrate assimilation, nodulation mutants, ¹⁵N isotope