assay of urease activity in soils - Science Direct

Soil Biol. Biochem.Vol. 4, pp. 479-487.Pergamon Press 1972.Printed in

ASSAY OF UREASE

Great

Britain

ACTIVITY

IN SOILS

M. A. TABATABAI and J. M. BREMNER Department

of Agronomy, Iowa State University, Ames, Iowa, U.S.A. (Accepted 13 March 1972)

Summary-A simple and precise method of assaying urease activity in soils is described. It involves determination of the ammonium released by unease activity when soil is incubated with tris(hydroxymethyl)aminomethane (THAM) buffer, urea solution, and toluene at 37°C for 2 h, ammonium release being determined by a rapid procedure involving treatment of the incubated soil sample with 2.5 M KC1 containing a unease inhibitor (Ag,SO4) and steam distillation of an aliquot of the resulting soil suspension with MgO for 3.3 min. Studies reported showed that the optimal buffer pH and substrate (urea) concentration for assay of soil unease activity using THAM buffer are 9.0 and 0.02 M, respectively, and that the method described is satisfactory for assay of urease activity in ammonium-fixing soils. INTRODUCTION

added to soils as fertilizer or as animal urine is hydrolyzed enzymatically by soil urease (NH,CONH2 + Hz0 > 2NH3 + COZ), and the resulting release of ammonia and rise in pH can lead to several problems, including damage to germinating seedlings and young plants, nitrite toxicity and volatilization of urea N as ammonia, which may cause air and water pollution problems (see Gasser, 1964; Hutchinson and Viets, 1969). The need for research to reduce these problems has been emphasized by the growing importance of urea as a nitrogen fertilizer in world agriculture (seecooke, 1969) and as a potentialpollutant where animals are confined to feedlots and such research requires reliable information concerning the distribution of urease activity in soil profiles and the factors affecting soil urease activity. A variety of methods have been used for assay of soil urease activity (e.g. Conrad, 1940; Hofmann and Schmidt, 1953; McLaren, Reshetko and Huber, 1957; Stojanovic, 1959; Hoffman and Teicher, 1961; Porter, 1965; McGarity and Myers, 1967; Simpson, 1968; Paulson and Kurtz, 1969; Skujins and McLaren, 1969). Most of these methods involve estimation of the ammonium released on incubation of toluene-treated soil with buffered urea solution, but assay has also beenperformed by estimation of the carbondioxide released or the urea decomposed on incubation (e.g. Conrad, 1940; Porter, 1965; Simpson, 1968; Skujins and McLaren, 1969). Several methods adopted have not involved use of a buffer to control pH (e.g. Conrad, 1940; Porter, 1965; Simpson, 1968) or addition of toluene to inhibit microbial activity (e.g. McLaren, Reshetko and Huber, 1957; Porter, 1965; Simpson, 1968 ; Paulson and Kurtz, 1969). The optimal conditions for assay of soilurease activity have not been investigated and no studies to evaluate methods proposed for this assay have been reported. It has not been demonstrated, for example, that methods involving estimation of the ammonium released by incubation of urea-treated soil are applicable to soils that fix ammonium or that those involving long incubation times (e.g. 24-80 h) are not vitiated through microbial activity during incubation. The purpose of this paper is to describe a simple and precise method of assaying soil urease activity developed from studies of factors affecting this assay. This method involves UREA

479

480

M. A. TABATABAI

AND J. M. BREMNER

determination of the ammonium released by urease activity when soil is incubated with buffered (pH 9.0) urea solution and toluene at 37°C for 2 h. Ammonium release is determined by a rapid procedure involving treatment of the incubated soil sample with 2.5 M KC1 containing a urease inhibitor (Ag,SO,J and steam distillation of an aliquot of the resulting suspension with MgO for 3-3 min. Studies reported show that the conditions of this method are satisfactory for assay of soil urease activity and that the method is applicable to soils that fix ammonium. MATERIALS

The soils used (Table 1) were surface (O-15 cm) samples selected to obtain a wide range in pH, texture and organic-matter content. Before use, each sample was air-dried and crushed to pass a 2-mm screen. The analyses reported in Table 1 were performed as described by Tabatabai and Bremner (1969). TABLE 1. ANALYSESOF SOILS

No.

Series

PH

Organic carbon (%I

I 2 3 4 5 6 7 8 9 10

Thurman Hagener Edina Weller Marshall Ida Nicollet Webster Luton Glencoe

7.1 6.4 6.1 5.7 6.8 7.5 6.6 7.4 6.8 7.3

0.47 0.92 1.46 1.72 2.38 2.57 3.14 4.35 4.35 5.24

Soil

Total nitrogen (%)

Clay (%)

Silt (%)

Sand (%I -

O-046 0.093 0.147 0.161 0.238 0.253 0.273 0,356 0,388 0.460

5 13 20 18 33 21 23 32 42 36

2 23 76 80 65

93 64 4 2 2 4 47 19 3 13

:: 49 ::

METHOD FOR ASSAY OF UREJASE ACTIVITY

Reagents ~o~ue~e. Fisher certified reagent {Fisher Scientific Co., Chicago, Illinois).

T&U4 buffer (pH 9-O), 0.05 M. Dissolve 6.1 g of tris(hydroxymethyl)aminomethane (THAM, Fisher certified reagent) in about 700 ml of water, bring the pH of the solution to 9.0 by addition of ca. 0.2 M sulfuric acid and dilute with water to 1 1. Urea solution, O-2 M. Dissolve 1 a2 g of urea (Fisher certified reagent) in about 80 ml of THAM buffer and dilute the solution to 100 ml with THAM buffer. Store the soiution in a refrigerator. Potassium chloride (2 *5 @-silver s@ate (100 parts/106) solution. Dissolve 100 mg of reagent-grade Ag,SO, in about 700 ml of water, dissolve 188 g of reagent-grade KC1 in this solution and dilute the solution to 1 1. Reagents for determination qfammonium (magnesium oxide, boric acid-indicator solution, 0.005 N su~ric acid). Prepare as described by Bremner and Keeney (1966).

Procedure

Place 5 g of soil ( < 2 mm) in a 50-ml volumetric flask, add O-2 ml of toluene and 9 ml of THAM buffer, swirl the flask for a few seconds to mix the contents, add 1 ml of 0.2 M urea solution and swirl the flask again for a few seconds. Then stopper the flask and place itinan

ASSAY OF SOIL UREASE ACTIVITY

481

incubator at 37°C. After 2 h, remove the stopper, add approximately 35 ml of KCl-Ag2S04 solution, swirl the flask for a few seconds and allow the flask to stand until the contents have cooled to room temperature (cu. 5 min). Then make the contents to volume (50 ml) by addition of KCl-Ag,SO, solution, and stopper the flask and invert it several times to mix the contents. To determine ammonium N in the resulting soil suspension, pipette a 20-ml aliquot of the suspension into a IOO-ml distillation flask designed for use with the steam distillation apparatus described by Bremner and Edwards (1965) and determine the ammonium N released by steam distillation of this aliquot with 0.2 g of MgO for 3.3 min as described by Bremner and Keeney (1966). Controls should be performed in each series of analyses to allow for ammonium N not derived from urea through urease activity. To perform controls, follow the procedure described for assay of urease activity, but make the addition of 1 ml of 0 -2 M urea solution after the addition of 35 ml of KCl-Ag2S04 solution. The KCl-Ag,SO, solution must be prepared by addition of KC1 to Ag,SO, solution as specified (Ag,S04 will not dissolve in KC1 solution) and the soil suspension analyzed for ammonium must be mixed thoroughly immediately before sampling for ammonium analysis. RESULTS AND DISCUSSION

The method described is based on systematic studies of factors affecting release of ammonium on incubation of soil with buffered urea solution in the presence of toluene (type of buffer, buffer pH, urea concentration. incubation time, etc.). Choice of bufer and bufer pH

Choice of buffer in the method described was based on studies showing that the amount of ammonium released by incubation of 5-g samples of different soils with 10 ml of buffered (pH 7 -0) 0 -02 M urea solution and 0 -2 ml of toluene at 37°C for 2 h was considerably higher with 0.05 M THAM buffer than with other buffers tested. The buffers employed included phosphate and citrate buffers similar to those previously used in assay of soil urease activity (e.g. Hofmann and Schmidt, 1953; Hoffman and Teicher, 1961) and several universal and modified universal buffers described by Britton (1955). Use of THAM buffer was investigated because Wall and Laidler (1953) found that, unlike other buffers used in studies of urease activity (e.g. sodium or potassium phosphate), THAM buffer has no activating or inhibiting effect on hydrolysis of urea by jack-bean urease. It should be noted that the expression ‘THAM buffer’ is used throughout this paper to describe buffer prepared by treatment of THAM solution with sulfuric acid. Wall and Laidler (1953) found that buffers prepared by treatment of THAM solutions with hydrochloric acid had an activating effect on hydrolysis of urea by jack-bean urease. Choice of buffer pH was based on studies showing that maximal release of ammonium in assay of soil urease activity using 0.05 M THAM buffer was obtained with pH 9-O buffer. Some of the results obtained in these studies are given in Fig. 1, which shows the effect of varying the pH of the 0.05 M THAM buffer used in the method described. Studies with soils 1-8 showed that, when 5-g samples of these soils were treated with pH 9 -0 THAM buffer as in the method proposed, the pH of the soil-buffer mixtures ranged from 8 *3 to 8 *7. The average pH after incubation for 2 h (8 ‘5) was higher than the pH 8 -0 optimum reported by Wall and Laidler (1953) for the activity of jack-bean urease in THAM buffer. Delaune and Patrick (1970) recently noted that the rate of conversion of urea N to ammonium N in waterlogged samples of a Crowley soil adjusted to different pH values by addition

482

M. A. TABATABAI

2

AND J. M. BREMNER

BUFFER?H

FIG. I. Effect of pH of buffer on release of ammonium N in assay of soil urease activity by method described.

of 2 N NaOH or 2 N HCI was highest with samples adjusted to pH 8. Most workers have assayed soil urease activity by methods involving use of almost neutral (pH 6’7-7.2) phosphate or citrate buffer to control pH (see Hofmann and Schmidt, 1953; McLaren, Reshetko and Huber, 1957; Stojanovic, 1959; Hoffman and Teicher, 1961; McGarity and Myers, 1967) but Skujins and McLaren (1969) used pH 5 *5 acetate buffer. It should be noted that the pH of THAM buffer is affected by temperature (see Good et al., 1%6), and that the pH values reported here for THAM buffer and soil-THAM buffer mixtures were determined at room temperature (23°C). Substrate concentration For valid assay of enzymatic activity, it is necessary to ensure that the enzyme substrate concentration is not a limiting factor in the assay procedure. A study of the effect of varying the substrate (urea) concentration in the method described (Fig. 2) showed that the concentration adopted (0.02 M) was satisfactory for assay of urease activity in the soils studied. This concentration is equivalent to 1120 pg of urea N/g of soil. The substrate concentrations in methods previously proposed for assay of soil urease activity have ranged from less than 400 to more than 40,000 pg of urea N/g of soil. Time and temperature of incubation Figure 3 shows results obtained in studies of the effect of varying the time of incubation in the method described. The observed linear relationship between time of incubation up to 4 h and amount of ammonium N released is evidence that the method proposed (2-h incubation time) measures enzymatic hydrolysis of urea and that assay of soil urease activity by this method is not complicated by microbial growth or assimilation of enzymatic reaction products by microorganisms. Release of ammonium N in assay of urease activity in the Thurman, Hagener, and Edina soils by the method described was a zero-order reaction for at least 10 h, but with soils having higher urease activities the substrate concentration became a limiting factor when the incubation time exceeded 4 h (see data for Ida soil in


FIG. 2.

0

IDA SOIL

0

WEBSTER SUIL

A

iDIHA SOIL

483

Effect of substrate (urea) concentration on release of ammonium urease activity by method described.

N in assay of soil

1

0 IDA SOIL ^

800.

; m

0 MARSHALL SOIL A EDINA SOIL

t " ‘, 630 2

0

1

2

3

4

5

INCUBATIONTIME (hj

FIG.

6

7

3. Effcxt of time of incubation on release of ammonium N in assay of soil urease activity by method described.

M. A. TABATABAI

484

AND J. M. BREMNER

Fig. 3). As Skujins (1967) has pointed out, it is important in assay of soil enzyme activity to use a procedure that does not require a long incubation time becausetherisk of error through microbial activity increases with increase in the time of incubation. The sensitivity of the assay procedure described here is such that precise results can be obtained with most surface soils even if the short incubation time recommended (2 h) is reduced to 1 h. Most of the methods previously proposed for assay of soil urease activity require incubation for times ranging from 6 to 80 h. Incubation was performed at 37°C because this temperature has been used extensively for assay of urease activity and preliminary work with soils 14 showed that it was not necessary to use a higher temperature to obtain precisely determinable hydrolysis of urea by soil urease under the conditions of the method described. The possibility that some chemical hydrolysis of urea might occur on incubation of soils with buffered (pH 9.0) urea solution at 37°C was checked by experiments with soils that had been autoclaved at 120°C for 1 h to destroy urease activity. No hydrolysis of urea was detected when these autoclaved soils were incubated with urea at 37°C for 2 h under the conditions of the method described.

Amounts of soil and toluene Figure 4 shows the effect of varying the observed linear relationship between amount is further evidence that the method proposed concentration is not a limiting factor in this

600 -

amount of soil in the method described. The of soil and amount of ammonium N released measures urease activity and that the substrate method.

. IDA SOIL o WEBSTER SOIL

~

500-

. EDINA SOIL

N

12

I 3

I I I I 5 6 7 4 AMOUNT OF SOIL (0)

FIG. 4. Effect of amount of soil on release of ammonium method described.

I 8

I 3

( lo

N in assay of soil urease activity by

Experiments with soils l-8 showed that the results obtained by the method described were not affected when the amount of toluene used was increased from 0 -2 to 0 - 5, 1 .O or 2-O ml. Several workers have found that use of toluene to inhibit microbial activity in assay of soil enzyme activity can increase the activity of some enzymes in soils (see Skujins, 1967), and we found that it increased the urease activity of all soils studied in our work. Other


485

workers (e.g. Conrad, 1940, 1942) have noted that toluene increases soil urease activity, and Skujins (1967) has suggested that toluene (a plasmolytic agent) may release urease from soil microorganisms. In contrast, McGarity and Myers (1967) found that the urease activity values obtained for some Australian soils by a method involving incubation with citrate buffer (pH 6.7) and urea solution for 6 h at 37°C were substantially reduced by addition of toluene. Determination of ammonium

Preliminary work showed that, when 1 ml of urea solution containing 1000 pg of urea N was treated with 9 ml of pH 9.0 THAM buffer and 0.2 ml of toluene, and the mixture was incubated at 37°C for 2 h and subsequently treated with 40 ml of 2 ~5 M KCI-Ag,S04 solution as in the method described, no ammonium was released by distillation of a 20-ml aliquot of the resulting solution with MgO for 3 -3 min. As noted previously, the possibility that some chemical hydrolysis of urea to ammonium might occur under the conditions of the method proposed was also checked by experiments with autoclaved soils. The effectiveness of the silver sulfate treatment used to terminate urease activity in the method proposed was confirmed by experiments showing that the ammonium release values obtained in analysis of soils l-8 by this method did not increase when the soil suspensions analyzed for ammonium were allowed to stand for 2 h before ammonium analysis. As noted in the Introduction, assay of soil urease activity by determination of the ammonium released through hydrolysis of urea by soil urease is complicated by the ability of many soils to fix ammonium. The soils used in our work were selected to include samples that tied ammonium against extraction with 2 M KC1 (when 5-g samples were treated with 2 ml of ammonium sulfate solution containing 1000 pg of ammonium N for 5 min, the amount of ammonium N fixed against extraction with 2 M KC1 ranged from 10, with the Hagener soil, to 170 pg with the Edina soil). But no fixation of ammonium N by these soils could be detected under the conditions of the method proposed (quantitative recovery of added ammonium N was obtained when 1 ml of ammonium sulfate solution containing 1000 pg of ammonium N was substituted for 1 ml of 0 -2 M urea solution in analysis of soils l-8 by the method described). The deduction from this finding that the pH 9.0 THAM buffer used in the method proposed prevents ammonium fixation by soil minerals was supported by experiments showing that no tiation of ammonium occurred when 1 g of finely ground (< lOO-mesh) Montana vermiculite was treated with 9 ml of pH 9 *OTHAM buffer, 1 ml of ammonium sulfate solution containing 1000 pg of ammonium N and O-2 ml of toluene at 37°C for 2 h. The vermiculite used in these experiments was obtained from Ward’s Natural Science Establishment, Inc., Rochester, New York, and it had a high capacity for ammonium tiation (1 g fixed 670 pg of ammonium N when treated with 10 ml of ammonium sulfate solution containing 1000 pg of ammonium N at 37°C for 2 h). Ammonium N was determined in these fixation tests with vermiculite by the procedure described for determination of ammonium released when soil is incubated with pH 9 ~0THAM buffer, urea solution, and toluene at 37°C for 2 h. Precision

The high precision of the method described is illustrated by Table 2, which gives the results of replicate analyses of 8 soils differing markedly in pH (5 *7-7.5), texture (542 per cent clay, 2-93 per cent sand) and organic-matter content (0.47-5 -24 per cent organic carbon). The urease activities of these soils, expressed as pg of ammonium N released/g of soil/2 h, ranged from 27 -5 to 356 *5 (average, 174.8) and the standard deviation of the activity determinations ranged from 0.4 to 2.1 (average, 1.4).

486

M. A. TABATABAI

AND J. M. BREMNER

TABLE 2. PRECISIONOF METHOD

Soil No. 1

2 3 4 5 6 7 8 9 10

No. of analyses

Range*

6 6 6 7 6 8 6 6 8 9

27-28 51-53 5&55 103-106 148-153 324-329 142-144 221-226 306-314 352-360

Urease activity Mean*

* Activity expressed as pg of ammonium t Standard deviation.

27.5 51.8 53.3 104.9 149.8 326.6 143.2 224.3 310.3 356.5

S.D.t 0.4 0.8 1.6 1.1 1.6 1.8 0.8 1.8 2.0 2.1

N released/g of soil/2 h.

Acknowledgements-Journal Paper No. J-7126 of the Iowa Agriculture-and Home Economic Experiment Station, Ames, Iowa. Projects 1835 and 1845. This work was supported in part by the Tennessee Valley Authority. REFERENCES BREMNERJ. M. and EDWARDSA. P. (1965) Determination

and isotope-ratio analysis of different forms of nitrogen in soils-I. Apparatus and procedure for distillation and determination of ammonium. Proc. Soil Sci. Sot. Am. 29, 504-507. BREMNERJ. M. and KEENEY D. R. (1966) Determination and isotope-ratio analysis of different forms of nitrogen in soils-3. Exchangeable ammonium, nitrate, and nitrite by extraction-distillation methods. Proc. Soil Sci, Sot. Am. 30, 577-582. BRITTON H. T. S. (1955) Hydrogen Zons. Chapman & Hall, London. CONRADJ. P. (1940) The nature of the catalyst causing the hydrolysis of urea in soils. Soil Sci. 50,119-134. CONRADJ. P. (1942) The occurrence and origin of urease like activities in soils. Soil Sci. 54, 367-380. COOKE G. W. (1969) Fertilizers in 2000 A.D. Phosphorus in Agriculture, Bull. Doe. No. 53, pp. 1-13. International Superphosphate and Compound Manufacturers’ Association, Paris. DELAUNER. D. and PATRICKW. H. (1970) Urea conversion to ammonia in waterlogged soils. Proc. Soil Sci. Sot. Am. 34,603-607. GA~SERJ. K. R. (1964) Urea as a fertilizer. Soils Fertil. 27, 175-180. GOOD N. E., WINGET G. D., WINTER W., CONNOLLYT. N., IZAWA S. and SINGHR. M. M. (1966) Hydrogen ion buffers for biological research. Biochemistry 5,467-477. HOFFMANG. and TEICHER K. (1961) Ein kolorimetrisches Verfabren zur Bestimmung der Ureaseaktivitat in Boden. Z. PjZErmihr. Dung. Bodenk. 95, 55-63. HOFMANNE. and SCHMIDTW. (1953) Uber das Enzymsystem unserer Kulturboden-II. Urease. Biochem. 2.

324, 125-127. HUTCHIN~~NG. L. and VIETS F. G. (1969) Nitrogen enrichment of surface water by absorption of ammonia volatilized from cattle feedlots. Science N. Y. 166, 514-515. MCGARITY J. W. and MYERS M. G. (1967) A survey of urease activity in soils of northern New South Wales.

PI. Soil 27,217-238. MCLAREN A. D., RESHETKOL. HUBER W. (1957) Sterilization of soil by irradiation with an electron beam, and some observations on soil enzyme activity. Soil Sci. 83,497-502. PAULSONK. N. and KURTZ L. T. (1969) Locus of urease activity in soil. Proc. Soil Sci. Sot. Am. 33,897-901. PORTERL. K. (1965) Enzymes. In Methods of Soil Analysis, Part 2 (C. A. Black, Ed.) pp. 1536-1549, American

Society of Agronomy,

Madison, Wisconsin.

SIMPSONJ. R. (1968) Losses of urea nitrogen from the surface of pasture soils. Transactionsof

the Ninth

International Congress of Soil Science, Adelaide, Vol. II, pp. 459466. SKUJINS J. J. (1967) Enzymes in soil. In Soil Biochemistry (A. D. McLaren and G. H. Peterson, 371-414, Marcel Dekker, New York.

Eds) pp.

SKUJINS J. J. and MCLAREN A. D. (1969) Assay of unease activity using %-urea preserved, and in irradiated soils. Soil Biol. Biochem. 1, 89-99.

in stored, geologically


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STOJANOVICB. J. (1959) Hydrolysis of urea in soil as affected by season and added urease. Soil Sci. 88, 251-255. TABATABAIM. A. and BREMNERJ. M. (1969) Use of p-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol. Biochem. 1,301-307. WALL M. C. and LADLER K. J. (1953) The molecular kinetics of the urea-urease system-IV. The reaction in an inert buffer. Arch. Biochem. Biophys. 43,299-306.