An evaluation of some manual colorimetric methods for the determination of inorganic nitrogen in soil extracts

Abstract

A number of manual colorimetric methods for the determination of inorganic nitrogen in 1 M KCl soil extracts were investigated to find techniques that were inexpensive, rapid, versatile and suitable for laboratories with limited analytical equipment. Three colorimetric methods for No^? ₃‐N determination were evaluated and only the copperised/cadmium reduction technique suffered no significant interference from the Cl^? present in the extracting solution. A phenol‐hypo‐chlorite (Berthelot) procedure for NH⁺ ₄‐N determination and the Griess‐Ilosvay method for NO^? ₂‐N determination were both found suitable for N determination in 1M KC1 soil extracts. The reliability and accuracy obtainable with the manual colorimetric methods described was shown to be comparable with that obtained from colorimetric analyses performed using an AutoAnalyser.     

Microscale determination of inorganic nitrogen in water and soil extracts

Abstract

Rapid, sensitive analysis of NH₄ ^‐ NO₃ ^‐, and NO₂ ^‐ in 1–150 μL of soil extract or water was achieved using a modified indophenol blue technique adapted to microtiter plate format. The microplate technique was similar to conventional steam distillation in accuracy and precision. By varying aliquot volume, a wide linear dynamic range (0.05 to 1000 mg of NH₄ ⁺‐ or NO₃ ^‐‐NL^‐1) was achieved without the need for sample dilution or concentration. High sample throughput (250–500 NH₄ ⁺ analyses d^‐1) was accomplished manually, but could be significantly increased by automation. Of considerable importance was the very low waste stream produced by the method. All equipment and supplies required are commercially available and need no modifications for this use. The microtiter plate format could be used for other soil colorimetric analyses with little or no down time for equipment setup, a major consideration for commercial soil‐testing laboratories. The method and equipment used are well suited to quality control and quality assurance programs, as required under FIFRA Good Laboratory Practices.     

Colorimetric interference and recovery of adsorbed ions from ion exchange resins

Abstract

Analytical interference in the colorimetric determinations of ammonium and nitrate was examined in various KCl extracts of several ion exchange resins. No analytical interference was found in the colorimetric NO₃ ^‐‐N determination in any extract of any resin. However, a mixed‐bed (cation + anion) exchange resin extract substantially affected the colorimetric determination of NH₄ ^‐ ‐N. Recovery of adsorbed ammonium and nitrate from ion exchange resins was also studied as a function of KCl extractant strength and number of extractions. The recovery of adsorbed NO₃ ^‐‐N in the first extraction increased with increasing KCl concentration, with a 2 M solution recovering about 80%. However, a 1 M KCl solution gave the greatest recovery of ammonium‐N, recovering about 75–80% of the adsorbed ammonium. The second extraction with the same concentration of KCl solution was greater with the 0.5 and 2.0 M than with the 1 M solution so that total NH₄ ⁺‐N recovery after two extractions was about the same for all three KCl concentrations. The recovery of resin‐adsorbed NH₄ ⁺‐N and NO₃ ^‐‐N appeared independent of their concentrations on the resins.     

Amino acid interference with ammonium determination in soil extracts using the automated indophenol method

Abstract

The formation of a colored indophenol complex is commonly used as a quantitative measure of the ammonium content of soil extracts. The potential interference with ammonium determination from co‐extracted amino acids was examined. The extent of color development was examined for 22 amino acids by subjecting pure solutions to ammonium determination by both the indophenol method and steam distillation. Apparent detection of amino acid as ammonium ranged from 0 to 94 % of total nitrogen for the indophenol procedure, whereas steam distillation resulted in little apparent ammonium recovery. With the exception of threonine, the extent of color development was inversely related to amino acid molecular weight. The range in recoveries for the indophenol procedure suggests both size and composition of the co‐extracted amino acid pool is important in determining the extent of interference.

Significantly (p=0.001) greater estimates, averaging 0.4 μg mL^‐1, were found in indophenol estimates of mineral‐N content of moist, fresh soil samples. Air drying, oven drying or chloroform fumigation significantly increased the difference (0.3 ‐ 0.7 μg mL^‐1) in estimates of ammonium content. At 10: 1 extract: soil ratios this could cause ? to be overestimated by 3–7 μg g^‐1soil. The increased interference was attributed to a release of amino acid as a result of pretreatment. The difference between distillation and indophenol estimates of ammonium content of 0.5 M K₂SO₄was found to be dependent upon ammonium content. The use of procedures employing a distillation step (manual or automated) is recommended to avoid amino acid interference when precise NH₄+‐N determinations are needed on dried or fumigated samples.     

Determining nitrogen‐15 enrichment in soil mineral nitrogen fraction with optical emission spectrometry

Abstract

Optical emission spectroscopy provides a rapid and precise method for determining nitrogen (N) as the ¹⁵N/¹⁴N ratios of ¹⁵N‐enriched plant and/or soil samples. The objective of this study was to test whether ¹⁵N/¹⁴N ratios of ¹⁵N‐enriched soil samples could be determined using direct combustion of KCl extracts without distillation and titration procedures. Twenty soil samples with ranges of mineral N concentration (10 to 43 μg g^‐1 of dry soil) and ¹⁵N atom % enrichment (a.e.) (0.37 to 1.10%) were tested. Our data indicate that soluble organic N in the KCl extract contained lower and more variable ¹⁵N% a.e. levels than those of the mineral N (NO₃ ^‐and NH₄ ⁺) fractions. Thus, direct combustion of KCl extract is not a reliable method for determining ¹⁵N% a.e. in the soil mineral N pool.     

A phenol‐hypochlorite manual determination of ammonium‐nitrogen in Kjeldahl digests of plant tissue

Abstract

A phenol‐hypochlorite (Berthelot) procedure for determining NH₄‐N in acid digests of plant tissue is described. EDTA suppresses Cu interference and precipitation of hydroxides and phosphates. The oxidizing reagent includes an alkaline buffer to account for variations in acidity between digestion solutions, the main source of error in an unbuffered procedure. The reagent combinations and addition sequence employed allow for delays of up to at least 30 minutes between reagent additions without affecting the final colour intensity. The proposed procedure is as sensitive as similar methods used for determining NH₄‐N in natural waters and yields results which agree well with those obtained by steam distillation.     

Use of diffusion for automated nttrogen‐15 analysis of soil extracts

Abstract

Studies to evaluate the use of diffusion for automated ¹⁵N analysis of inorganic N in soil extracts showed that serious error can arise from use of the Devarda's alloy recommended for steam distillations and that the error can be avoided by using a commercial product of higher purity. These studies showed that serious error can also arise when NO₃ ^‐‐N is diffused following NH₄ ⁺‐N and that separate diffusions should be performed for NH₄ ⁺‐N and (NH₄ ⁺ + NO₃‐)‐N. Other work demonstrated that the plastic specimen containers employed for diffusion can be reused if acid‐washed, that diffusions can be performed using either light or heavy MgO without ignition to decompose carbonate, and that labeled NO₂‐is completely removed from soil extracts by treatment with sulfamic acid before diffusion. A comparison of ¹⁵N analyses by steam distillation and diffusion using extracts from two soils revealed better agreement for the soil having a lower content of organic matter. Substantial differences in analyses by the two techniques for the soil having a higher organic‐matter content were attributed to enzymatic conversions of inorganic N during the 6‐d diffusion period.     

Comparison of Conductimetric and Colorimetric Methods with Distillation–Titration Method of Analyzing Ammonium Nitrogen in Total Kjeldahl Digests

The distillation–titration method (DTM) is a standard procedure used by most laboratories to measure ammonium-nitrogen (NH₄-N) in the total Kjeldahl N (TKN) digests of various kinds of agricultural and environmental samples. These samples may have TKN contents ranging from less than 100 ppb to as high as percentage levels. However, the DTM procedure generally leads to a very low throughput because it is labor intensive and time-consuming. At the current practical quantitation limit (PQL) of 300 ppb established at the Feed and Environmental (FEW) Laboratory, University of Georgia, the DTM procedure is less applicable to low TKN surface water samples. In this study, we therefore compared the performance of diffusion conductivity method (DCM) and colorimetric method (CM) with DTM in measuring NH₄-N in the TKN digests of 29 different samples representing surface waters, lagoons, manures, poultry litters, and environmental wastes. Acceptable accuracy and precision were achieved for various QC samples by all three methods. For widely different sample matrices and TKN contents, the NH₄-N in the TKN digests measured by DCM and CM both agreed well with that measured by DTM. However, the linear working range of CM is limited within 0.2 to 5.0 ppm, whereas DCM is linear at a wider range of 0.01 to 2000 ppm. With DCM, the PQL of TKN is at 13 ppb, much less than the 300 ppb in DTM and 520 ppb in CM. Both DCM and CM require increasing the pH of the working TKN digest to a highly alkaline range. To meet such pH requirement, the minimum dilution need for DCM is twofold, where as that CM is fourfold. Because of greater mandatory dilution requirement coupled with a greater PQL, CM may often fail to measure NH₄-N in the working TKN digest of some low TKN surface water samples. On the other hand, with some environmental waste samples containing TKN at percentage level, CM would require multistep dilution of the digests prior to measurement, thus allowing dilution-related error as well as requiring additional labor. In contrast, DCM can measure both low TKN surface waters and high TKN environmental wastes without any major limitations. Moreover, DCM may work well without any adjustment of sample background in the calibration standards. Thus DCM appears to be an attractive alternative to the labor-intensive and time-consuming DTM for measuring NH₄-N in the TKN digests of various kinds of agricultural and environmental samples in the analytical services laboratories.     

Evaluation of methods for soil inorganic nitrogen analysis

Abstract

This study determined the effects of soil preservation methods on inorganic nitrogen (N) analysis and evaluated methods of soil inorganic N analysis. Soils were preserved by oven‐drying at 55'C, air‐drying at 27°C, and freezing at ‐ 7°C. Inorganic N results were compared with initial N levels prior to imposing preservation treatments. Soil preservation effects on ammonium‐nitrogen (NH₄ ⁺‐N) were not consistent across soil types. Soil nitrate‐nitrogen (NO₃ ^‐‐N) levels after air‐drying and freezing compared most favorably with initial levels indicating that both are acceptable methods of soil inorganic‐N preservation. Levels of NH₄ ⁺‐N averaged across soils were 3.9 mg/kg for steam distillation, 4.2 mg/kg for sodium salicylate‐hypochlorite, and 3.7 mg/kg for indophenol blue. When compared with steam distillation averaged across soils, NO₃ ^‐‐N for cadmium‐copper (Cd‐Cu) reduction was 4 mg/kg greater, followed by nitrate electrode at 3 mg/kg, and salicylic acid at 2 mg/kg. Recovery of added N ranged from 83.3 to 94.8% for the NH4+‐N methods and from 74.8 to 112.4% for the NO₃ ^‐‐N methods with the nitrate electrode averaging 98.3%.     

Interference by amino acids during the determination of N ammonium in soil

In the study of terrestrial N cycling, NH₄⁺ concentration and ¹⁵N enrichment are routinely determined by colorimetric continuous flow analysis and microdiffusion methods. Amino acids can interfere in these determinations; consequently the aim of the present study was to evaluate the significance of the interference. Glycine and glutamine are key amino acids in soil and were therefore used as ‘models’. Both glycine and glutamine interfered during continuous flow analysis, whereas interference during microdiffusion was of little importance. The effects of interference can be significant, e.g. estimates of gross mineralisation rate were reduced up to 33%, where we allowed for amino acid interference during determination of NH₄⁺ concentration. The potential influence of amino acid interference emphasises that development of continuous flow analysis to increase NH₄⁺ specificity is needed.     

Nitrate‐nitrogen determination—a critical review

Abstract

This paper reviews the published methods of nitrate‐nitrogen (NO₃‐N) determination with the objective to assess their applicability to soil and plant tissue anarysis. The methods are separated into three categories on the basis of the analytical approach utilized for NO₃‐N determination. Strengths and weaknesses of the methods are discussed. The first analytical approach utilitizes direct measurement of NO₃‐N by the following methods: (a) colorimetric (after a color producing reaction with NO₃‐N), (b) potentiometric, (c) absorption of UV radiation by NO₃‐N in a complex matrix, (d) transnitration of salicylic acid, and (e) chromatographic (separation and measurement of NO₃‐N) methods. The second approach is based on the reduction of NO₃‐N to nitrite‐nitrogen (NO₂‐N), ammonium‐nitrogen (NH₄‐N), or nitric oxide and measurement of the reduction product. When NO₃‐N is reduced to NO₂‐N, the measurement may be achieved by (a) colorimetric, (b) fluorimetric, (c) coulometric, and (d) catalytic kinetic methods. When NO₃‐N is reduced to NH₄‐N, the measurement is done by (a) colorimetric (after a color producing reaction with NH₄), (b) potentiometric, (c) steam distillation, and (d) gas diffussion conductimetric methods. A chemiluminescence detection method is utilized when NO₃‐N is reduced to nitric oxide. The third approach determines NO₃‐N concentration by measuring the change in the concentration of the chemical species that react with NO₃‐N and form a complex.     

Evaluation of methods for nitrogen‐15 analysis of inorganic nitrogen in soil extracts: I. Steam‐distillation methods

Abstract

A study was conducted to evaluate conventional steam‐distillation techniques for N‐isotope analysis of inorganic forms of N in soil extracts. Extracts obtained with 2 M KCl from 10 diverse soils were treated with: (i) (¹⁵NH₄)₂SO₄ and KNO₃, (ii) (NH₄)₂SO₄ and K¹⁵NO₃, or (iii) KNO₃and Na¹⁵NO₂. Steam distillations were performed sequentially to determine NH₄ ⁺‐N and NO₃ ^‐‐N, and were also carried out to determine (NO₃ ^‐ + NO₂ ^‐)‐N or (NH₄ ⁺ + NO₃ ^‐ + NO₂ ^‐)‐N; a pretreatment with sulfamic acid was used to determine NO₃ ^‐‐N in the presence of NO₂ ^‐‐N. Recovery of added N ranged from 95 to 102%. Significant isotopic contamination was observed in sequential distillation of unlabeled NO₃ ^‐‐N following labeled NH₄ ⁺‐N; otherwise, analyses for ¹⁵N were usually within 1% of the values calculated by isotope‐dilution equations.     

Terrestrial N cycling associated with climate and plant‐specific N preferences: a review

The dramatic increase in anthropogenic reactive nitrogen (Nr) from agricultural activities negatively affects the environment. An additional challenge is to ensure food security while at the same time keeping the environmental impact to a minimum to prevent negative feedback effects on climate. To date, however, few studies have addressed the direct connection between soil N transformations, forms of N, species‐specific N preferences and climate, despite the fact that the fate of N and soil N biochemical cycling are known to be intimately linked. In this paper we review the connections between soil N transformation, species‐specific N preferences and climate, and explore how N‐use efficiency may be enhanced while minimizing the environmental effect. Gross rates of N mineralization and immobilization govern the amount of available N in soil, especially in natural ecosystems, while nitrification plays a central role in regulating the NO₃^? to NH₄⁺ ratio. Plant species prefer either NH₄⁺‐N or NO₃^?‐N, depending on the NO₃^?‐N to NH₄⁺‐N ratio in their habitat. Thus, plant N uptake could be optimized (i.e. Nr losses reduced) if species‐specific N preferences are maintained by matching N sources applied with prevailing soil‐specific N transformations. Therefore, whether N management practices can optimize N‐use efficiencies hinges on the coupling of soil N transformation with climate and species‐specific N preferences.

Highlights

• We review the inherent connections between the soil N cycle, plant N preference and climate.
• Nitrification plays a central role in regulating the NO₃^? to NH₄⁺ ratio in soil and soil solution.
• Soil N transformations regulate the composition of hydrological N export.
• Plant N uptake can be optimized if soil N cycle is well matched with plant N preference.

     

Added nitrogen interaction as affected by soil nitrogen pool size and fertilization – significance of displacement of fixed ammonium

Displacement of NH₄⁺ fixed in clay minerals by fertilizer ¹⁵NH₄⁺ is seen as one mechanism of apparent added nitrogen interactions (ANI), which may cause errors in ¹⁵N tracer studies. Pot and incubation experiments were carried out for a study of displacement of fixed NH₄⁺ by ¹⁵N‐labeled fertilizer (ammonium sulfate and urea). A typical ANI was observed when ¹⁵N‐labeled urea was applied to wheat grown on soils with different N reserves that resulted from their long‐term fertilization history: Plants took up more soil N when receiving fertilizer. Furthermore, an increased uptake of ¹⁵N‐labeled fertilizer, induced by increasing unlabeled soil nitrogen supply, was found. This ANI‐like effect was in the same order of magnitude as the observed ANI. All causes of apparent or real ANI can be excluded as explanation for this effect. Plant N uptake‐related processes beyond current concepts of ANI may be responsible. NH₄⁺ fixation of fertilizer ¹⁵NH₄⁺ in sterilized or non‐sterile, moist soil was immediate and strongly dependent on the rate of fertilizer added. But for the tested range of 20 to 160 mg ¹⁵NH₄⁺‐N kg^–1, the NH₄⁺ fixation rate was low, accounting for only up to 1.3 % of fertilizer N added. For sterilized soil, no re‐mobilization of fixed ¹⁵NH₄⁺ was observed, while in non‐sterile, biologically active soil, 50 % of the initially fixed ¹⁵NH₄⁺ was released up to day 35. Re‐mobilization of ¹⁵NH₄⁺ from the pool of fixed NH₄⁺ started after complete nitrification of all extractable NH₄⁺. Our results indicate that in most cases, experimental error from apparent ANI caused by displacement of fixed NH₄⁺ in clay is unlikely. In addition to the low percentage of only 1.3 % of applied ¹⁵N, present in the pool of fixed NH₄⁺ after 35 days, there were no indications for a real exchange (displacement) of fixed NH₄⁺ by ¹⁵N.     

Fate of nitrogen and sulphur as affected by the rhizosphere of oilseed rape and barley

Abstract

Within plants, sulphur (S), and nitrogen (N) equilibrium is a requisite for their normal development. Pot experiments with oilseed rape and barley fertilized at different N to S ratios were carried out under glasshouse conditions by using the “rhizobag”; technique. The objective was to compare the induced‐influence of rhizosphere and non‐rhizosphere soil on N and S nutrition of the studied plants. Thus, SO₄ ²‐S, NC₃ ^‐‐N and NH₄ ⁺‐N concentrations, and total N and S taken up by the plants were examined. Barley increased the pH of rhizosphere soil whereas no real change of pH was observed with oilseed rape. Both plants took up all the NO₃ ^‐ present in the soil solution, but rapeseed took up greater quantities of NH₄ ⁺‐N and SO₄ ^2‐ ‐S than barley. Moreover, the ratio values of N to S of the aerial parts of the rapeseed were significantly and positively correlated to those of soil available‐N to ‐S ratios while this correlation was significant but negative with barley. This indicated a clear‐cut different influence between the two rhizospheres which oppositely induce the N and S nutrition of the two plant species.     

Method for the quantification of acid production by plants in gel

Abstract

Hydrogen (H⁺) and hydroxyl ion (OH^‐) production by the tropical grass, Brachiaria humidicola, is quantified using a method in which the plants are grown in soil then transferred to agar gel for 24 h. The amount of H⁺ and OH^‐produced was calculated from the pH of the melted gel and the gels’ buffer curve. Values were obtained for plants of different ages and with nitrogen (N) supplied in the gel as nitrate (NC₃ ^‐), ammonium (NH₄ ⁺), or ammonium nitrate (NH₄NO₃) and compared with data calculated using the sum of H⁺ changes in differently colored zones of the gel. Daily H⁺ and OH^‐ production increased with plant age and total dry matter for the NH₄ ⁺‐ and NO₃ ^‐‐fed plants, respectively. By integrating the data over time, a value of 0.33 mmol H⁺ plant^‐1 was obtained for the total H⁺ production over 62 d. The proposed method was sufficiently rapid and versatile to allow the comparison between plant species or genotypes, which were grown using a variety of nutrient supplies. This procedure may indicate how acid production affects plant nutrient acquisition and aid the prediction of soil acidification by different plant species or cultivars.     

Effect of nitrate/ammonium ratio on biomass production,nitrogen accumulation,and use efficiency in sorghums of different origin

Plant nitrogen (N) uptake, growth, and N use efficiency may be affected by N form (NO₃ ^‐or NH₄ ⁺) available to the root. The objectives of this study were to determine the effect of mixed N form on dry matter production and partitioning, N uptake, and biomass N use efficiency defined as total dry matter produced per unit plant N (NUE₁) in U.S. and tropical grain sorghums [Sorghum bicolor (L.) Moench]. The U.S. derived genotype CK 60 and three tropical genotypes, Malisor‐7, M 35–1, and S 34, were evaluated in a greenhouse trial using three nutrient solutions differing in their NO₃ ^‐/NH₄ ⁺ ratio (100/0, 75/25, 50/50). Shoot and root biomass, N accumulation, and NUE, were determined at 10‐leaf and boot stages. Averaged over all genotypes, shoot and root biomass decreased when NH₄ ⁺ concentration was increased in the solution. Shoot biomass was reduced by 11% for 75/25 and 26% for 50/50 ratios, as compared to 100/0 NO₃ ^‐/NH₄ ⁺. Similarly, root biomass reduction was about 34% and 45% for the same ratios, respectively. Increasing NH₄ ⁺ concentration also altered biomass partitioning between shoot and root as indicated by decreasing root/shoot ratio. Total plant N content and NUE₁ were also reduced by mixed N source. Marked genotypic variability was found for tolerance to higher rates of NH₄ ⁺. The tropical line M 35–1 was well adapted to either NO₃ ^‐ as a sole source, or to an N source containing high amounts of NH₄ ⁺. Such a characteristic may exist in some exotic lines and may be used to improve genotypes which do not do well in excessively wet soil conditions where N uptake can be reduced.     

Correction of iron chlorosis in peanut (arachis hypogea shulamit) by ammonium sulfate and nitrification inhibitor

Peanut (Arachis hypogea cv. Shulamit) grown on very high calcium carbonate (CaCO₃) content soils is showing iron (Fe) chlorosis symptoms. Supplying the plant with ammonium sulphate ((NH₄)₂SO₄) in the presence of nitrapyrin (N‐Serv) for preventing nitrification reduced Fe chlorosis. Nitrate (NO^‐ ₃) developed in the soil with time, even with nitrapyrin present. When ammonium (NH⁺ ₄) was even less than 20% of the total mineral N in the soil, no Fe‐stress could be observed, suggesting that the NH⁺ ₄ uptake by the plant and the consequence of hydrogen (H⁺) efflux occurs from the root to the rhizosphere, resulting in a decrease of redox potential near the root, and solubilizing enough Fe near the root to overcome the chlorosis.     

Technique of soil sample pretreatment for analysis of 15N:14N isotope ratio

Abstract

The technique of simultaneous quantitative determination of mineral N soil forms (nitrates, exchangeable and non‐exchangeable ammonium, and total amount of these compounds) and sample pretreatment for the analysis of ¹⁵N:¹⁴N ratio is suggested. The technique is based on the selective association of NH₄ ⁺‐ions into indophenol complex and subsequent ethyl‐acetate extraction of this complex from solution. The mineralization of indophenol is carried out in alkaline medium with simultaneous NH₃ distillation into H₂SO₄ titrant. The application of given technique allows us to shorten significantly the time required for analysis and to increase the accuracy of analytical determination.     

Evidence that fungi can oxidize NH4+ to NO3− in a grassland soil

The contribution of bacteria and fungi to NH₄⁺ and organic N (N_org) oxidation was determined in a grassland soil (pH 6.3) by using the general bacterial inhibitor streptomycin or the fungal inhibitor cycloheximide in a laboratory incubation study at 20°C. Each inhibitor was applied at a rate of 3 mg g^?1 oven‐dry soil. The size and enrichment of the mineral N pools from differentially (NH₄¹⁵NO₃ and ¹⁵NH₄NO₃) and doubly labelled (¹⁵NH₄¹⁵NO₃) NH₄NO₃ were measured at 3, 6, 12, 24, 48, 72, 96 and 120 hours after N addition. Labelled N was applied to each treatment, to supply NH₄⁺‐N and NO₃^?‐N at 3.15 μmol N g^?1 oven‐dry soil. The N treatments were enriched to 60 atom % excess in ¹⁵N and acetate was added at 100 μmol C g^?1 oven‐dry soil, to provide a readily available carbon source. The oxidation rates of NH₄⁺ and N_org were analysed separately for each inhibitor treatment with a ¹⁵N tracing model. In the absence of inhibitors, the rates of NH₄⁺ oxidation and organic N oxidation were 0.0045 μmol N g^?1 hour^?1 and 0.0023 μmol N g^?1 hour^?1, respectively. Streptomycin had no effect on nitrification but cycloheximide inhibited the oxidation of NH₄⁺ by 89% and the oxidation of organic N by more than 30%. The current study provides evidence to suggest that nitrification in grassland soil is carried out by fungi and that they can simultaneously oxidize NH₄⁺ and organic N.