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Dep. of Natural Resources and Environmental Sciences, 1102 S. Goodwin Ave., Univ. of Illinois, Urbana, IL 61801
* Corresponding author (mulvaney{at}uiuc.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Several studies have been reported to compare the distribution of organic N in different soils, or among soils under different management practices (e.g., Stevenson, 1957; Keeney and Bremner, 1964; Porter et al., 1964; Moore and Russell, 1968; Sowden, 1968; Khan, 1971; Smith and Young, 1975; Meints and Peterson, 1977; Osborne, 1977). The results have generally indicated little variation in the distribution of N, regardless of soil type, cropping, or cultivation. The same sort of uniformity was observed during recent work in our laboratory to compare the distribution of organic N in soils with and without a history of heavy manuring. Subsequent studies revealed that steam-distillation methods of determining (NH4 + amino sugar)–N and amino acid–N are subject to serious error, so new methods were developed to fractionate the N in soil hydrolysates, utilizing Mason-jar diffusion methods described in previous publications for inorganic-N analysis of soil extracts and water (Khan et al., 1997; Mulvaney et al., 1997b) and total-N analysis of Kjeldahl digests (Stevens et al., 2000). The primary purpose of this article is to describe the diffusion methods that were developed to determine the following fractions for N-distribution analysis of soil hydrolysates: total hydrolyzable N, NH4–N, (NH4 + amino sugar)–N, amino acid–N, and (NH4 + amino sugar + amino acid)–N. To ensure a high level of reliability, extensive recovery tests were conducted to identify critical variables, optimize reaction conditions, and establish minimal diffusion periods. The resulting methods were evaluated by specificity tests using a wide variety of purified organic-N compounds, from the recovery of 15N added to soil hydrolysates as NH4, glucosamine, or glycine, and through comparison with N-distribution analyses by steam distillation.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Purified Compounds
Reagent-grade samples of the 53 organic-N compounds listed in Table 2 were obtained from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Before use, the samples were dried over anhydrous CaSO4 in a desiccator. Aqueous solutions containing 1 g N L-1 were prepared by dissolving each compound in 25 mL of deionized water. When necessary to facilitate dissolution, the pH was adjusted by adding one or two drops of 5 M H2SO4 or 10 M NaOH. The solutions were used within 24 h after preparation and were stored in a refrigerator (5°C) before use.
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1.5 atom % in 15N, which were stored in a refrigerator and used within 24 h after preparation. Prior to use, an exact enrichment was determined experimentally for each solution by total 15N analysis (Stevens et al., 2000).
Diffusion Methods
Apparati
Diffusion unit
The diffusion unit used consisted of a 473-mL (1-pint) wide-mouth Mason jar equipped with a lid that was modified to support the bottom of a 60-mm (diam.) Pyrex petri dish (Khan et al., 1997; Mulvaney et al., 1997b).
Aluminum block digester or micro-Kjeldahl digestion stand
Kjeldahl digestions were performed using a Tecator Model 1016 Digester equipped with a Model 1012 Autostep Controller (Foss Tecator AB, Höganäs, Sweden). To prevent bumping caused by droplets of H2SO4 condensate falling into the hot digest from the stem of a funnel in the mouth of the digestion tube, one side of the block digester was supported so that the digestion tubes would be inclined 20°.
Electric hot plate
A commercial griddle was used for carrying out diffusions and as a source of heat for oxidizing amino acids with ninhydrin. To perform diffusions, the heat control was adjusted such that a temperature of 48 to 50°C was obtained when a thermometer was immersed in 100 mL of deionized water in a Mason jar placed in the center of the griddle. A temperature of 95 to 100°C was employed to carry out the ninhydrin reaction, in which case temperature measurements were made using a Mason jar sealed with a lid having a central hole, through which a thermometer was inserted and immersed into 100 mL of deionized water.
Microburette or automatic titrator
Titrations were performed using a 5-mL microburette having a three-way stopcock, or a Metrohm Model 678 EP/KF Processor equipped with a Model 665 Dosimat (Metrohm, Herisau, Switzerland) and a combination electrode designed for flat-surface measurements (Fisher Scientific Model 13-620-289).
Reagents
Potassium sulfate–catalyst mixture
Twenty grams of CuSO4 · 5H2O was powdered by grinding in a mortar, and was then mixed intimately with 2 g of Se and 200 g of K2SO4 (powder).
Sulfuric acid
Kjeldahl digestions to determine total hydrolyable N were performed using concentrated (18 M) H2SO4. A 5 M solution of H2SO4 was prepared by diluting 278 mL of the concentrated reagent to 1 L with deionized water in a volumetric flask. Titrations were done using 0.01 M H2SO4, which was prepared by adding 5.6 mL of concentrated H2SO4 to 10 L of deionized water in a 10-L Pyrex solution bottle. After thorough mixing with a motorized stirrer, the latter solution was standardized using primary standard-grade THAM obtained from Sigma.
Sodium hydroxide solution (10 M)
Reagent-grade NaOH pellets (400 g) were dissolved in 800 mL of deionized water in a 1-L volumetric flask. After cooling, the solution was diluted to 1 L and mixed thoroughly.
Boric acid-indicator solution
A reagent containing 40 g of H3BO3 L-1 was prepared as described by Khan et al. (1997) or Mulvaney et al. (1997b).
Magnesium oxide
The heavy powder available from Fisher Scientific was used.
Methanol
Anhydrous grade was used.
Ninhydrin solution
Twenty-five grams of certified ninhydrin (triketohydrindene hydrate) obtained from Fisher Scientific was dissolved in 250 mL of methanol. This solution was stored at room temperature in a tightly stoppered bottle.
Procedures
Total hydrolyzable nitrogen
Five milliliters of soil hydrolysate was pipetted into a 50-mL Pyrex digestion tube and treated with 0.5 g of K2SO4–catalyst mixture and 2 mL of 18 M H2SO4. A 25-mm Kimax filtering funnel was placed in the mouth of the tube to minimize loss of H2SO4 during digestion, and the tube was then transferred to an Al block digester and heated for 1.5 h at 150°C, then for 1 h at 250°C, and finally for 3 h at 350°C. After cooling, the digest was diluted with 2 mL of deionized water from a wash bottle, homogenized by vortex mixing, and decanted into a Mason jar. The latter operation was repeated three times to ensure complete transfer of N in the digest to the Mason jar, after which the wall of the jar was rinsed with 5 mL of deionized water from a wash bottle. Using a graduated cylinder, the digest was neutralized with 10 mL of 10 M NaOH, and within 30 s the jar was sealed by attaching a lid equipped with a petri dish containing 5 mL of H3BO3–indicator solution, swirled to mix the contents, and then transferred to a hot plate maintained at 48 to 50°C. After 4 h, the jar was removed from the hot plate, 5 mL of deionized water was added to the H3BO3 solution in the petri dish, and NH4–N was determined by titration with 0.01 M H2SO4.
Hydrolyzable ammonium-nitrogen
Ten milliliters of soil hydrolysate in a Mason jar was treated with 0.05 g of MgO using a calibrated spoon. The jar was swirled to mix the contents and then sealed by attaching a lid equipped with a petri dish containing 5 mL of H3BO3–indicator solution. Diffusion was performed for 28 h in an incubator maintained at 20°C (the preferred technique), for 26 h at room temperature (25°C), or for 2 h on a hot plate (48–50°C), followed by titrimetric determination of NH4–N.
(Ammonium + amino sugar)–nitrogen
Two milliliters of 10 M NaOH was added to 10 mL of soil hydrolysate in a Mason jar. After swirling the jar to mix the contents, a lid equipped with a petri dish containing 5 mL of H3BO3 solution was attached within 30 s, and the jar was heated on a hot plate (48–50°C) for 5 h. The amount of NH4–N collected was determined as described previously.
Amino acid–nitrogen
After completing diffusion of (NH4 + amino sugar)–N, 2.5 mL of 5 M H2SO4 was added to the jar, followed by 1 mL of ninhydrin solution. The jar was swirled to mix the contents, and after being covered (but not sealed) with an unmodified lid to minimize the loss of water, was placed within the central two-thirds of the surface area of a hot plate, and heated for 90 min at 95 to 100°C. A few minutes were allowed for the jar to cool, after which the contents were treated with 1 mL of 10 M NaOH and mixed by swirling. Within 30 s, the jar was sealed by attaching a lid with 5 mL of H3BO3 solution in a petri dish, and then heated at 48 to 50°C for 2 h on a hot plate. The H3BO3 solution was titrated as described previously.
(Ammonium + amino sugar + amino acid)–nitrogen
Ten milliliters of soil hydrolysate was treated with 4 mL of deionized water, 0.5 mL of 5 M H2SO4, and 1 mL of ninhydrin solution. The jar was swirled to mix the contents and then covered and heated on the central two-thirds of a hot plate at 95 to 100°C for 90 min. After cooling, the sample was treated and then mixed with 1 mL of 10 M NaOH, and within 30 s a lid with a petri dish containing 5 mL of H3BO3 solution was attached to the jar and sealed with a screw band. The NH3 liberated during a 5-h period of diffusion at 48 to 50°C was determined by titration with 0.01 M H2SO4.
Distillation Methods
Steam-distillation procedures to determine total hydrolyzable N, NH4–N, (NH4 + amino sugar)–N, and amino acid–N were performed as described by Bremner (1965) and Stevenson (1982)( 1996). The latter three procedures were evaluated for accuracy and specificity by performing distillations (4 replicates) on aliquots of soil hydrolysate that had been treated with 0 or 300 µg of labeled N as (NH4)2SO4 (1.455 atom % 15N), glucosamine · HCl (2.206 atom % 15N), or glycine (1.516 atom % 15N). Replicate distillations were performed using the same distillation unit, after distilling formic acid and ethanol to minimize cross-contamination error (Mulvaney, 1993). Percentage recovery of the labeled N (R) was calculated as
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Nitrogen-Isotope Analysis
In cases involving diffusion or distillation of 15N-treated soil hydrolysates, titrated samples were processed as described previously (Mulvaney, 1993; Khan et al., 1997; Mulvaney et al., 1997b) for N-isotope analysis with an automated Rittenberg system (Mulvaney et al., 1990; Mulvaney and Liu, 1991; Mulvaney et al., 1997a).
Development of Diffusion Methods
To check the effect of the Kjeldahl digestion period on recovery of total hydrolyzable N, digestions were performed (4 replicates) on three purified organic-N compounds (1 mg of N as alanine, 
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-diaminopimelic acid, or nicotinic acid) and on aliquots of the hydrolysate from one of the soils used (Xenia). In each case, the digest was heated for 1.5 h at 150°C, then for 1 h at 250°C, and finally for 0.5, 1, 3, or 5 h at 350°C.
The amount of MgO required to determine hydrolyzable NH4 was ascertained by studying the effects of different additions of this reagent (0.05, 0.1, 0.2, 0.5, or 1 g) on percentage recovery and solution pH (measured with a glass electrode), after carrying out diffusions (4 replicates) for 2 h on a hot plate (50°C) from 10 mL of 1.2 M NaCl containing 2 mg of N as (NH4)2SO4. The salt solution used provided the same concentration of NaCl that existed in the neutralized soil hydrolysates.
In a study to compare different alkaline treatments for recovery of (NH4 + amino sugar)–N, diffusions were performed for 2, 5, 6, 12, or 24 h at 50°C from 10 mL of 1.2 M NaCl containing 1 mg of N as glucosamine · HCl, after addition of 1.25 g of powdered phosphate–borate buffer (Bremner, 1965; Stevenson, 1982, 1996), 1.0 g of Na3PO4, 1 mL of 5 M NaOH, or 1 or 2 mL of 10 M NaOH. A subsequent study was done to optimize the temperature and diffusion period for recovery of glucosamine-N (1 mg) using 2 mL of 10 M NaOH, in which diffusions were done from 10 mL of 1.2 M NaCl for 4, 5, 6, 12, or 24 h, either at room temperature (25°C) or with heating on a hot plate at 45, 50, or 55°C. There were four replicates in both studies.
The optimal pH for ninhydrin oxidation of amino acid–N was established by comparing recovery of alanine–N when this reaction was performed (4 replicates) at a pH of 1.0 to 1.5, 1.6 to 1.8, 1.9 to 2.0, 2.5 to 3.0, or 3.5 to 5.0. In each case, the reaction pH was measured with a glass electrode following addition of 2 mL of 10 M NaOH to 10 mL of 1.2 M NaCl containing 1 mg of alanine–N, and was adjusted to fall within the desired range by adding 5 M H2SO4. One mL of ninhydrin solution was then added, and amino acid–N was determined as described previously.
To optimize heating conditions for ninhydrin oxidation of amino acid–N, recovery tests were performed using 1 mg of N as alanine in 10 mL of 1.2 M NaCl, in which heating was done at 90, 95, or 100°C for 15, 30, 45, 60, 75, 90, 120, or 180 min. In all other respects, the procedure for determining amino acid–N was performed as specified previously. There were four replicates.
Recovery tests were also conducted to check whether analyses for amino acid–N are affected by the amount of ninhydrin used for oxidation. Once again, determinations were done (4 replicates) on 1 mg of alanine–N in 10 mL of 1.2 M NaCl by the procedure specified previously, except for the use of ninhydrin solutions that differed in concentration (50, 75, 100, 125, 150, 200, 300, or 500 g L-1).
Evaluation of Diffusion Methods
The specificity of the five diffusion methods described was evaluated from recovery tests using the 53 organic-N compounds listed in Table 2. With each compound tested, analyses were performed in quadruplicate on 1-mL aliquots of an aqueous solution that contained 1 g N L-1, following addition of 4 (total hydrolyzable N) or 9 (all other analyses) mL of 1.2 M NaCl. All diffusions were carried out at 48 to 50°C on a hot plate.
Recovery tests using 15N were performed to evaluate the accuracy and specificity of the diffusion methods described for determining NH4–N, (NH4 + amino sugar)–N, and amino acid–N. In these tests, analyses were performed (3 or 4 replicates) on 10 mL of soil hydrolysate that had been treated with 1 mL of deionized water containing 0 or 300 µg of N as labeled (NH4)2SO4 (1.455 atom % 15N), glucosamine · HCl (1.621 atom % 15N), or glycine (1.516 atom % 15N). Percentage recovery of the labeled N was calculated by Eq. [1].
In a study to compare N-distribution analyses by distillation and diffusion, analyses were performed in quadruplicate on 5 or 10 mL of soil hydrolysate for quantitative determination of total hydrolyzable N, NH4–N, (NH4 + amino sugar)–N, or amino acid–N. All diffusions were done at 48 to 50°C on a hot plate. Amino sugar–N was obtained as the difference between determinations of (NH4 + amino sugar)–N and NH4–N.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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,-diaminopimelic acid and nicotinic acid is increased by prolonging digestion, whereas this is unnecessary with an amino acid such as alanine. Table 4 also suggests that, if desired, the digestion period after clearing can be reduced because values obtained for the soil hydrolysate were unaffected when this period was increased from 0.5 to 5 h.
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10, which is sufficient to liberate any NH4 present as gaseous NH3, but not high enough to effect hydrolysis of amino sugars and other alkali-labile organic-N compounds (Bremner and Shaw, 1955). The addition of MgO specified herein (0.05 g) is smaller than that recommended previously (0.2 g) for determining NH4–N in soil extracts or water (Khan et al., 1997; Mulvaney et al., 1997b) because there is no need to overcome excess acidity in a neutralized soil hydrolysate. Recovery tests with (NH4)2SO4 (Table 5) showed that larger additions of this reagent have the same effect on pH, but the increased quantity of solid phase may impede liberation of NH3 and require a longer diffusion period. Unlike distillation, the diffusion method described does not require the use of MgO that has been ignited to remove CO3, since any CO3 present would remain in the alkalized hydrolysate, given the mild temperatures employed for diffusion.
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The use of ninhydrin allows direct determination of amino acid–N following removal of (NH4 + amino sugar)–N by diffusion with NaOH, or determination of (NH4 + amino sugar + amino acid)–N if the prior addition of alkali has been omitted. This reagent liberates CO2 and NH3 from -amino acids and reacts with a variety of other organic-N compounds, including amino sugars, to release NH3 but not CO2. In all cases, heating is required to promote the reaction, and pH must be carefully controlled if a quantitative determination is to be made. According to MacFadyen (1944), recovery of NH3 will be quantitative if the pH does not exceed 2.5, and this value has been adopted in the steam-distillation methods described by Bremner (1965) and Stevenson (1982)(1996). Studies to optimize the pH for ninhydrin oxidation by the diffusion method of determining amino acid–N (Table 8) showed recovery to be incomplete at pH 2.5, and to become quantitative only when the pH did not exceed 1.8. Adequate acidity is provided by addition of H2SO4 but cannot be achieved using citric acid as specified by Bremner (1965) and Stevenson (1982)(1996).
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To check the specificity of the five diffusion methods described, analyses were performed on a wide variety of purified organic-N compounds, including 27 -amino acids, four amino acids lacking an -NH2 group, four amino sugars, four purines, four pyrimidines, three amides, and seven other biochemicals that occur widely in plants, animals, or microorganisms (Table 2). The results are summarized by Table 10, which categorizes the compounds into seven groups according to percentage recovery by each method, representing negligible (<1%), slight (1–10%), moderate (11–45%), intermediate (46–55%), substantial (56–90%), nearly quantitative (91–97%), and quantitative (>97%) recovery.
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,-diaminopimelic acid and nicotinic acid (Table 10). As noted previously (Table 4), a longer period of digestion would have been more effective in cases where recovery was not quantitative, but this is probably of no concern with soil hydrolysates. Recovery tests indicated that determinations of hydrolyzable NH4–N are highly specific. Of the compounds tested, slight recovery was detected only with galactosamine (Table 10).
As expected, analyses for (NH4 + amino sugar)–N were higher with the four amino sugars used than with the other compounds tested. Table 10 shows that recovery was quantitative for glucosamine and nearly quantitative for galactosamine or mannosamine, but was lower for N–acetylglucosamine, which is much more stable than glucosamine (Horton, 1969). About 50% of the N in asparagine or glutamine was recovered, and this can no doubt be attributed to hydrolysis of amide–N under alkaline conditions. With most of the remaining compounds, recoveries were very limited or undetectable.
As indicated by Table 10, the method described for determining amino acid–N was quantitative, or nearly quantitative, with about one-half of the -amino acids tested. Partial recovery was obtained with the remainder of these compounds and with a variety of others, including all three amides, two amino sugars, two imino acids (proline and hydroxyproline), a purine, and a pyrimidine. In some of the latter cases, the magnitude of recovery depended on the fact that analyses for amino acid–N were performed following a diffusion with NaOH to remove (NH4 + amino sugar)–N. For example, recovery as amino acid–N was higher with N–acetylglucosamine than with the other amino sugars tested because reduced alkaline decomposition increased carryover. As expected, recovery with asparagine and glutamine was about 50%, and represents -NH2–N that remained following removal of amide–N.
If desired, amino acid–N may be estimated as the difference between determinations of (NH4 + amino sugar + amino acid)–N and (NH4 + amino sugar)–N. This approach has the advantage that both analyses may be completed in a somewhat shorter period than is required to determine amino acid–N following a 5-h diffusion to estimate and remove (NH4 + amino sugar)–N, and Table 10 shows comparable specificity with either approach. When performed according to the method described for (NH4 + amino sugar + amino acid)–N, analyses were quantitative, or nearly quantitative, with three of the four amino sugars and 17 of the 27 -amino acids tested. With several of the latter compounds, higher recoveries were achieved by this method than by the method of determining amino acid–N, the only difference being whether ninhydrin oxidation is preceded by a treatment with NaOH. Presumably, this treatment had a chemical effect that reduced recovery, but deamination is unlikely since there was very little, if any, recovery of amino acid–N during diffusions to recover (NH4 + amino sugar)–N (Table 10).
In a further evaluation of analytical accuracy and specificity, the diffusion methods described for determination of NH4–N, (NH4 + amino sugar)–N, and amino acid–N were applied to soil hydrolysates that had been treated with 15N-labeled (NH4)2SO4, glucosamine, or glycine. The results (Table 11) differ considerably from those obtained in a similar evaluation of steam distillation (Table 3), and leave little doubt that diffusion is far superior for estimating amino sugar–N and amino acid–N. When 15N was added as NH4, quantitative recovery was achieved by analyses for NH4–N or (NH4 + amino sugar)–N, and carryover of 15NH4 was negligible during determination of amino acid–N. In cases involving the use of labeled glycine, complete recovery of 15N was usually obtained by performing diffusions to estimate amino acid–N, with very little recovery as NH4–N or (NH4 + amino sugar)–N. With labeled glucosamine, 15N was recovered quantitatively by the method to determine (NH4 + amino sugar)–N, and very little enrichment was observed for amino acid–N. There was, however, substantial recovery of glucosamine–15N when diffusions to estimate NH4–N were performed on a hot plate, particularly with the Bloomfield soil. This finding was unexpected, since no recovery had been observed in performing diffusions under the same conditions from a purified solution of glucosamine (Table 10), and can only be attributed to a matrix effect that promoted chemical decomposition. Subsequent work showed a marked reduction in interference by glucosamine when diffusions to recover NH4–N from 15N-treated soil hydrolysates were done at room temperature (25°C), and still lower interference at 20°C (Table 11). The latter temperature is thus recommended for determining NH4–N and will be of value in avoiding underestimation when amino sugar–N is calculated by difference.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication July 27, 2000.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-amino acids by ninhydrin. J. Biol. Chem. 153:507–513.This article has been cited by other articles:
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K. K. Moran, R. L. Mulvaney, and S. A. |
