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

Printed in Great Britain

ASSAY OF UREASE 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 37C
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
UREA 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
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 37C for 2 h. Ammonium release is deter-
mined 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 appli-
cable 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 crush-
ed to pass a 2-mm screen. The analyses reported in Table 1 were performed as described by
Tabatabai and Bremner (1969).
TABLE 1. ANALYSES OF SOILS
Soil
Organic Total
carbon nitrogen Clay Silt Sand
No. Series
PH (%I (%) (%) (%) (%I
-
I Thurman 7.1 0.47 O-046 5 2 93
2 Hagener 6.4 0.92 0.093 13 23 64
3 Edina 6.1 1.46 0.147 20 76 4
4 Weller 5.7 1.72 0.161 18 80 2
5 Marshall 6.8 2.38 0.238 33 65 2
6 Ida 7.5 2.57 0.253 21 4
7 Nicollet 6.6 3.14 0.273 23 :: 47
8 Webster 7.4 4.35 0,356 32 49 19
9 Luton 6.8 4.35 0,388 42 3
10 Glencoe 7.3 5.24 0.460 36 :: 13
METHOD FOR ASSAY OF UREJ ASE 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 qf ammonium (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 37C. 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 am-
monium 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 des-
cribed 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 am-
monium 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 37C 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 investi-
gated 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 ammon-
ium N in waterlogged samples of a Crowley soil adjusted to different pH values by addition
482 M. A. TABATABAI AND J. M. BREMNER
2
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 67-7.2) phos-
phate 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 (23C).
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 concen-
tration 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 incu-
bation 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
ASSAY OF SOIL UREASE ACTIVITY 483
0 I DA SOI L
0 WEBSTER SUI L
A i DI HA SOI L
FIG. 2. Effect of substrate (urea) concentration on release of ammonium N in assay of soil
urease activity by method described.
1
0 I DA SOI L
^ 800. 0 MARSHALL SOI L
;
m
A EDI NA SOI L
t
"
, 630 -
2
0 1 2 3 4 5 6
I NCUBATI ON TI ME ( hj
7
FIG. 3. Effcxt of time of incubation on release of ammonium N in assay of soil urease activity by
method described.
484 M. A. TABATABAI 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 sur-
face 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 37C 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 37C was checked by experiments with soils that had been autoclaved at 120C for 1 h to
destroy urease activity. No hydrolysis of urea was detected when these autoclaved soils were
incubated with urea at 37C for 2 h under the conditions of the method described.
Amounts of soil and toluene
Figure 4 shows the effect of varying the amount of soil in the method described. The
observed linear relationship between amount of soil and amount of ammonium N released
is further evidence that the method proposed measures urease activity and that the substrate
concentration is not a limiting factor in this method.
600 - . I DA SOI L
o WEBSTER SOI L
~ 500- . EDI NA SOI L
N
I I I I I I I (
12 3 4 5 6 7 8 3 l o
AMOUNT OF SOI L ( 0)
FIG. 4. Effect of amount of soil on release of ammonium N in assay of soil urease activity by
method described.
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
ASSAY OF SOIL UREASE ACTIVITY 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 37C 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 37C 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 am-
monium 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 *O THAM
buffer, 1 ml of ammonium sulfate solution containing 1000 pg of ammonium N and O-2 ml
of toluene at 37C for 2 h. The vermiculite used in these experiments was obtained from
Wards 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 37C for 2 h). Am-
monium N was determined in these fixation tests with vermiculite by the procedure des-
cribed for determination of ammonium released when soil is incubated with pH 9 ~0 THAM
buffer, urea solution, and toluene at 37C 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. PRECISION OF METHOD
No. of
Urease activity
Soil No. analyses Range* Mean* S.D.t
1 6
2 6
3 6
4 7
5 6
6 8
7 6
8 6
9 8
10 9
27-28 27.5 0.4
51-53 51.8 0.8
5&55 53.3 1.6
103-106 104.9 1.1
148-153 149.8 1.6
324-329
142-144
221-226
306-314
352-360
326.6
143.2
224.3
310.3
356.5
1.8
0.8
1.8
2.0
2.1
* Activity expressed as pg of ammonium N released/g of soil/2 h.
t Standard deviation.
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.
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