Plant and Soil 39, 71-80 (1973) Ms.

1899

UREASE ACTIVITY IN SOILS

b y A. B. L L O Y D and M. J A N E S H E A F F E
Department of Agricu!tural Biology, University of New England, ArmidaIe, New South
Wales, Australia

SUMMARY

Bacteria which can hydrolyse urea are common in soils. Of six soils exa-
mined, some 17--30per cent of the total bacterial populations, including aero-
bes, micro-aerophiles and anaerobes, could hydrolyse urea. One of the soils
had been enriched with urea for at least ten years, yet the proportion of
ureolytic bacteria (24 per cent) was similar to that of normal soils.
Addition of urea to a red-yellow podzolic soil low in available carbon and
under different moisture conditions did not increase the total urease activity,
the size of the bacterial population or the ratio of ureolytic to non-ureolytic
bacteria. However, when available carbon as glucose was added with urea to
this soil, nrease activity and size of the bacterial population both increased,
but the ratio of ureolytic to non-ureolytic bacteria in the population remained
unchanged.

INTRODUCTION

W h e n urea is a d d e d to soil it can be r a p i d l y h y d r o l y s e d to N H 4 -

ions and C02 b y urease enzymes in the soil, especially when the soil
is w a r m a n d moist 1 11 13 14. One of the effects of this hydrolysis is to
raise the p H of the soil locally to a level where a m m o n i a m a y be lost
to the atmosphere.
Urease enzymes can exist in two possible states in soil. T h e y
m a y be intracellular, t h a t is present within the cells of ureolytic
micro-organisms, 01- alternatively, extracellular, h a v i n g been releas-
ed from disrupted p l a n t a n d microbial cells a 11. E x t r a c e l l u l a r urease
is largely adsorbed on clay and organic colloids which h a v e a high
affinity for urease 5 6 7. This a d s o r p t i o n on soil colloids possibly
protects ureases from destruction b y soil micro-organisms, while the
a c t i v i t y of the e n z y m e s remains essentially u n i m p a i r e d 7.
A large n u m b e r of micro-organisms including bacteria, actino-
72 A. B. LLOYD AND M. JANE SHEAFFE

mycetes and fungi can hydrolyse urea intracellularly 9 10 12 The

urea molecules enter the bacterial cell where they are hydrolysed;
very little urease is released extracellularly be viable metabolizing
cells 10. In general, the ratio of ureolytic to non-ureolytic bacteria in
soil remains constant, even though the total bacterial population may
fluctuate considerably 9. Thus the amount of urease activity in soil
is often correlated with the size of the microbial population, although
it often seems that the relationship is more with microbial activity
than actual number of cells 9 11
In the work described here, we made no effort to identify the
location of urease enzymes in soil. Instead, we have defined the
'total urease activity of soil' as the sum of urease activity from all
sources, both intracellular and extracellular. This has allowed us to
avoid the addition to soil of such chemicals as toluene, which al-
though often used as a selective inhibitor of microbial activity, is
now known also to partly inhibit urease enzymes and induce lysis of
microbial cells s 11

MATERIALS AND METHODS

Six separate soils collected from within I00 k m of Armidale, N.S.W., were
used in this study: a garden soil which contained a considerable a m o u n t of
organic matter; a brown earth of heavy texture which was situated below
housed animals and so received intermittent enrichment with urine ; a coarse-
textured solodic; a reddish-chocolate soil which had a considerable clay con-
tent; a grey brown podzolic and a red-yellow podzolic which were both acid
sandy loam soils. The red-yellow podzolic and the garden soil were collected
from sites which were protected from grazing animals. All sites except the
red-yellow podzolic and the garden soil had some plant cover. Soil samples
were taken from the surface (0-I 0 cm), passed through a 2 m m sieve and then
kept in plastic bags at 8°C for up to 24 h before use.
The total aerobic bacterial population was estimated by the dilution-plate
method a using a soil extract agar m e d i u m 2 with cycloheximide (actidione)
added at 50~g/ml to inhibit fungal growth. The final soil dilution was spread
over the solidifiedm e d i u m and then incubated at 28°C for up to one week.
The percentage of aerobic bacteria that could hydrolyse urea (ureolytic
bacteria) was determined by transferring randomly selected colonies from the
soil e x t r a c t a g a r m e d i u m to sections of urea agar (Oxoid) w h i c h c o n t a i n e d t h e
i n d i c a t o r p h e n o l red. All t h o s e colonies w h i c h p r o d u c e d a d e f i n i t e colour
c h a n g e f r o m orange to b r i g h t p i n k w i t h i n 24 h a t 28°C were classed as aerobic
a n d ureolytic.
T h e p e r c e n t a g e s of micro-aerophilic a n d a n a e r o b i c b a c t e r i a w h i c h were
also u r e o l y t i c were e s t i m a t e d in t h e s a m e w a y e x c e p t for t h e c o n d i t i o n s of
 UREASE ACTIVITY IN SOILS 73

i n c u b a t i o n . W i t h t h e m i c r o - a e r o p h i l i c b a c t e r i a , t h e p e t r i dishes were k e p t in
a n a n a e r o b i c j a r in w h i c h a c a n d l e h a d b e e n b u r n t t o r e d u c e t h e o x y g e n con-
t e n t in t h e enclosed air. T h e a n a e r o b i c p o p u l a t i o n was g r o w n i n i t i a l l y u n d e r
N~ gas w i t h 5 % CO2 or t-I2 gas w i t h 5 % CO~., b u t as t h e r e was e s s e n t i a l l y no
difference in t h e n u m b e r of a n a e r o b i c colonies recorded, N2 w i t h 5 % CO2 was
used t h r o u g h o u t . B e c a u s e of t h e slower g r o w t h of t h e m i c r o - a e r o p h i l i c a n d
a n a e r o b i c b a c t e r i a , t h e s e b a c t e r i a were c o n s i d e r e d u r e o l y t i c if t h e y p r o d u c e d
t h e colour c h a n g e w i t h i n 48 h. T h e m i c r o - a e r o p h i l i c e n v i r o n m e n t was includ-
ed t o e n c o u r a g e t h o s e slow g r o w i n g soil b a c t e r i a w h i c h m i g h t o t h e r w i s e be
selected a g a i n s t u n d e r a e r o b i c c o n d i t i o n s , b u t recognising t h a t t h e two g r o u p s
are n o t a b s o l u t e .
T h e a s s a y m e t h o d for d e t e c t i o n of u r e o l y t i c b a c t e r i a is b a s e d on t h e
a s s u m p t i o n t h a t if t h e b a c t e r i a c a n h y d r o l y s e urea, N H 4 - i o n s are f o r m e d
w h i c h cause a n increase in t h e p H of t h e m e d i u m , r e s u l t i n g in a colour c h a n g e
of t h e i n d i c a t o r , p h e n o l red. H o w e v e r , as t h e m e d i u m also c o n t a i n s p e p t o n e
t h e r e is t h e p o s s i b i l i t y t h a t c e r t a i n n o n - u r e o l y t i c b a c t e r i a m i g h t d e a m i n a t e
t h e p o l y p e p t i d e s , releasing N H 4 - i o n s i n t o t h e m e d i u m w h i c h w o u l d t t l e n in-
crease t h e p H s u f f i c i e n t l y to give a false p o s i t i v e result. To o v e r c o m e t h i s
defect, a w e a k p h o s p h a t e b u f f e r was i n c o r p o r a t e d into t h e m e d i u m . S u b -
s e q u e n t l y , in a n u m b e r of t r i a l s i t was s h o w n t h a t w h e n u r e a was o m i t t e d
f r o m t h e m e d i u m i t t o o k a t l e a s t 4 d a y s before a n y of a large n u m b e r of
r a n d o m l y selected b a c t e r i a could p r o d u c e a s l i g h t colour c h a n g e . T h u s i t
s e e m e d t h a t u p to 48 h a t least, t h e r e was little c h a n c e of r e c o r d i n g n o n - u r e o -
l y t i c b a c t e r i a as positive.

Urease activity
U r e a s e a c t i v i t y of soil was a s s a y e d following a n l o d i f i c a t i o n of t h e colori-
m e t r i c t e c h n i q u e d e v e l o p e d b y H o f f m a n n a n d T e i c h e r 4. T e n - g r a m s a m p l e s
of fine e a r t h were p l a c e d in 5 0 - m l v o l u m e t r i c flasks a n d 10 m l of 0.1 M p h o s -
p h a t e b u f f e r (pH 6.7) a d d e d to e a c h a n d t h e n t h e flasks k e p t in a w a t e r b a t h
a t 30°C for a p p r o x i m a t e l y 1 h to allow t h e soil t e m p e r a t u r e to e q u i l i b r a t e .
T e n m l of 2 0 % u r e a s o l u t i o n was t h e n a d d e d a n d t h e flasks g e n t l y a g i t a t e d in
t h e w a t e r b a t h for 4 h a t 30°C. T h e c o n t r o l s c o n t a i n e d distilled w a t e r as a
s u b s t i t u t e for t h e urea. A f t e r i n c u b a t i o n , u r e a s e a n d m i c r o b i a l a c t i v i t y were
i n h i b i t e d b y t h e a d d i t i o n of m e r c u r i c chloride s o l u t i o n w i t h a final c o n c e n t r a -
t i o n of 1 : 1,000. To release all t h e N H 4 - i o n s p r e s e n t i n t o solution, p o t a s s i u m
was a d d e d as KC1 a t t h e r a t e of 0.8 g/kg soil. T h e c o n t e n t s of t h e flasks were
d i l u t e d to 50 m l e a c h w i t h distilled water, t h e flasks s h a k e n , s t o r e d o v e r n i g h t
a t 4°C a n d t h e n t h e c o n t e n t s filtered. T h e a m o u n t of a m m o n i a in t h e f i l t r a t e
was d e t e r m i n e d b y t h e i n d o p h e n o l blue m e t h o d 4. U r e a s e a c t i v i t y was ex-
p r e s s e d as m g N H 4 - N released p e r 100 g o v e n - d r i e d soil d u r i n g t h e 4-h in-
c u b a t i o n p e r i o d a t 30°C.
74 A. B. LLOYD AND M. J A N E S H E A F F E

RESULTS

Percentage o/ ureolytic bacteria in soil

Table 1 shows the percentages of bacteria, from six different soils,
which could hydrolyse urea. Each value is based on the examination
of between t00-200 randomly selected colonies.

TABLE 1

Percentage of bacteria from six different soils which could hydrolyse urea

Soil type Percentage ureolytie bacteria

Aerobic Micro- Anaerobic

aerophitic

Garden soil 35 14 13
Brown earth 29 15 27
Solodic 39 21 27
Reddish-chocolate 23 24 7
Grey-brown podzolic 23 11 18
Red-yellow podzolic 25 22 11

Ureolytic bacteria were abundant in all the soils examined. Be-

tween 20 to 40 per cent of the aerobes were ureolytic, while 10 to 25
per cent of the micro-aerophiles and 5-30 per cent of the anaerobes
were ureolytic. Taken collectively, some 25 per cent of all soil bac-
teria sampled could readily hydrolyse urea with the subsequent
production of NH4-ions. The brown earth was of special interest as
this soil was situated below an animal house where urine from the
housed animals had passed into the soil for at least ten years. Yet
in spite of this sustained enrichment with urea, the proportion of
ureolytic bacteria to the total bacterial population was similar to that
of other soils.

Supplementation o/ soil with urea

Urea was applied in solution at the rate of 500 ppm urea-N to a
red-yellow podzolic collected from three different sites: a bare soil
with a low moisture content (t7 per cent) ; a bare soil with a medium
moisture content (25 per cent) ; a soil under Phalaris pasture with a
moisture content of 22 per cent. Each soil was enclosed in plastic to
prevent drying, incubated at 28°C, and sampled for up to 9 days for
 UREASE ACTIVITY IN SOILS 75

u r e a s e a c t i v i t y , size of t h e b a c t e r i a l p o p u l a t i o n ( b o t h t o t a l a n d ureo-
lyric) a n d m o i s t u r e c o n t e n t . F o r controls, distilled w a t e r w a s sub-
s t i t u t e d for t h e urea.
T h e results for t h e r e d - y e l l o w p o d z o l i c u n d e r t h r e e different m o i s t -
ure c o n d i t i o n s were similar a n d c a n t h e r e f o r e be c o n s i d e r e d collec-
tively.

3O

=0 It
25

=_ ;/\'\
"~ 20
"/ ~', / ~
I
~x, ,,'/ ~, /.,o~ \
g S I O
*-~ lc
lp

I i I I I I I I I I
6 12 24 36 48 60 72 84 101 125 1~0 1~0 1~3 197
Time (h)
Fig. I Changes in urease activity with addition ot urea or water to a
red-yellow podzolic soil.

~ t 40
I,=
50f
=30 ~ ^~ /X
--
~= 20
IlL
t / '/,,,
• ft %I ~ /
\
~
water.._~. "
urea
~"
t,"

=~o= "~~/ v "...... x._'--~ ~/

10

t,4
I I I I
0L'III0
6 12 214 I 36l 4~ 610 712 814 101 125 173 197
Time (h)
Fig. 2. Changes in the percentage of ureolytic bacteria w i t h i n the total soil
population with addition of urea or water to a red-yellow podzolic soil.
76 A. B. L L O Y D AND M. J A N E S H E A F F E

For each treatment, the variability during the length of incubation

was often disappointingly high (Fig. 1, 2, and 3). This reflects to
some extent the difficulty in sampling soil and the crudeness of the
dilution-plate and enzyme-assay techniques. For these reasons, a
5 °/o level of significance was chosen.
There was no significant difference (5 per cent level) in urease
activity, or size of the bacterial population, or in the percentage of
ureolytic bacteria in the total bacterial population, when the same
soil was supplemented with either urea or water. This is illustrated
in Figures 1, 2, and 3. Thus the addition of urea to this red-yellow
podzolic even under conditions optimal for microbial activity did not
increase urease activity, the size of the bacterial population, or
selectivity towards increase in the number of ureolytic bacteria,
relative to the addition of water only.
4O
fa 1
I I
! !
I 1
l ', A i

3~
/\ /",, / ,.
t|l " Iv \: I ~; ",/ _)x". / u r e a /
.= ivl I \', I _II I'-'- ~ ,"
i,t
II li
I
/
V
~ /"
i\/I nr
-,. q"% •
,,
i\l '--" V -, /
"'~f_water

6 12 36 0 72 84 101 12!5 173 197

Time (h)
Fig. 3. Changes in tile size of the total bacterial population with addition of
urea or water to a red-yellow podzolic soil.

Supplementation o~ soil with both urea and glucose

The red-yellow podzolic was treated with urea (500 ppm urea-N)
as well as 0.2 per cent glucose, while the control soil received the
urea treatment only. As previous experiments had showed that most
activity was completed within 3 days, and because of tile great
number of samples required, sampling was confined to this period.
Approximately 12 h after the soil had been supplemented with
 UREASE ACTIVITY IN SOILS 77

25

=
20

+1,
=
Z

and glucose
~ " ~"---urea only

I , I 1 I I I
6 12 24 36 48 60
Time (h)
Fig. 4. C h a n g e s in u r e a s e a c t i v i t y w i t h a d d i t i o n of u r e a a n d glucose or
u r e a only, to a red-yellow podzolic soil.

both urea and glucose, the urease activity (Fig. 4) and the total
bacterial population (Fig. 6) increased significantly relative to the
same soil supplemented with urea only. These increases were main-
50

..~g 40

_m
30

20 I
e " " " " -... f ghlcose
.~ nrea on
¢J
¢¢

0 I I | I I I
6 12 24 36 48 60
Time (h)
Fig. 5. C h a n g e s in t h e p e r c e n t a g e of u r e o l y t i c b a c t e r i a w i t h i n t h e t o t a l soil
p o p u l a t i o n w i t h a d d i t i o n of u r e a a n d glucose or u r e a only, t o a red-yellow
podzolic soil.
78 A. B. LLOYD AND M. JANE SHEAFFE

tained throughout the remainder of the time. However, the per-

centage of ureolytic bacteria in the total bacterial population re-
mained unchanged (Fig. 5/.
In the example shown in Figure 4, urease activity in soil supple-
mented with urea only tended to increase almost linearly up to 48
hours. This was not a consistent feature, for in other experiments
(Fig. 1 for example) there was great variability during this period,
due perhaps to the inevitable disturbance of the soil when initially
supplemented with urea.
Thus in soil supplemented with urea, the addition of glucose as a
carbon source increased the urease activity and the size of the total
bacterial population, but did not selectively increase the proportion
of bacteria in the population which were ureolytic.

"2

~urea
jS and glucose

3o
tl~. iII "~ ~
I/I ~ II

. u

10 ",.f urea only

CJ

I I I I I t
10 20 30 40 50 60
Time (h)
Fig. 5. C h a n g e s in t h e size of t h e t o t a l b a c t e r i a l p o p u l a t i o n w i t h a d d i t i o n
of u r e a a n d glucose or u r e a only, to a red-yellow podzolic soil.

DISCUSSION

Ureolytic bacteria are present in soil in large numbers. Of six

soils examined, some 17-30 per cent of the total bacterial populations
including aerobes, micro-aerophiles and anaerobes could hydrolyse
urea rapidly. One of the soils, a brown earth, was enriched for at
least ten years with urine from housed animals, yet the proportion of
ureolytic bacteria (24 per cent) in the total soil population remained
similar to that of other soils not enriched with urine.
 U R E A S E A C T I V I T Y I N SOILS 79

Addition of urea to a red-yellow podzolic soil known to be low in

available carbon did not increase the total urease activity, or the
number of ureolytic bacteria, or the size of the bactexial population
This remained so even when the soil was collected from a number of
different sites, the moisture content adjusted, and the soil incubated
under conditions which appeared optimal for microbial activity.
This seems to mean that in soils like the red-yellow podzolic, where
available carbon is a limiting factor for microbial growth, added
urea is hydrolysed by a constant number of ureolytic bacteria al-
ready present in the soil, as well as by extracellular urease enzymes
largely adsorbed on soil colloids. Also, as the total urease activity
remains constant, there must be little, if any, further synthesis of
urease enzymes shortly after the soil is supplemented with urea.
When the same soil was supplemented with glucose as a readily
available carbon source, then the addition of urea did increase the
urease activity as well as the number of ureolytic bacteria and the
size of the total bacterial population. An interesting aspect is that
the ratio of ureolytic to non-ureolytic bacteria in the soil population
remained constant, due to the general increase in the total bacterial
population. Apparently, there is not a selection towards increasing
those bacteria which could hydrolyse urea.
Presumably the ureolytic bacteria obtain their urea-N mainly
from hydrolysis of urea by intracellular urease enzymes. Providing
there is adequate available carbon in the soil, the ureolytic popula-
tion increases in size, and as cells die, urease enzymes are released
into the soil and become adsorbed on soil colloids. It must be this
extracellular urease which hydrolyses urea to NH4-ions which then
become available as a nitrogen source to non-ureolytic bacteria. The
final result is that both the ureolytic and non-ureolytic groups of
bacteria increase in size, apparently in the same proportion.
Finally, it should be noted that we purposely added a large amount
of urea to soil so that urea-N would not be a limiting factor for micro-
bial activity. If, as shown by P a u l s o n and K u r t z 5, a small a-
mount of urea (13 ppm) is added to a soil low in available carbon,
then the ureolyLic bacteria are induced to produce more urease while
the number and proportion of ureolytic bacteria remains constant.
But the increase in urease activity is small, and considering the cru-
deness of the assay method, it is difficult to measure.
80 U R E A S E A C T I V I T Y IN SOILS

ACKNOWLEDGMENT

We wish to thank D r . V. B o f i n g e r for help with interpretations of t h e

statistical analysis.

Received February 15, 1972. Revised October 1972

REFERENCES

1 B r o a d b e n t , F. E., H i l l , G. N., and T y l e r , K. B., Transformations and m o v e m e n t

of urea in soils. Soil Sci. Soc. Am. Proc. 22, 303-307 (1958).
2 B u n t , J. S., and R o v i r a , A. D., Microbiological studies of some subantarctic soils.
J. Soil Sei. 6, 119-128 (1955).
3 C 1ar k, F. E., Agar-plate method for total microbial count. In Method of soil analysis,
ed. C. A. B l a c k . Agronomy 9, 1460-1466 (1965).
4 H o f f m a n n , G. a n d T e i c h e r , K., Colorimetric determination of urease activity in
soils. Z. Pflanzenern~hr. Dfing. Bodenk. 95, 55-63 (1961).
5 P a u l s o n . K. N. and K u r tz, L. T., Locus of urease activity in soil. Soil Sci. Soe. Am.
Proc. 33, 897-901 (1969).
6 P i c k , L. A. and A l l i s o n , F. E., Adsorption and release of nrease by and from clay
minerals. Soil Sci. 91, 183-188 (1969).
7 P i n e k , L. A., D y a l , R. S., and A l l i s o n , F. E., Proteinmontmorillonite complexes,
their preparations and the effects of soil micro-organisms on their decomposition.
Soil Sci. 78, 109-118 (1954).
8 R o b e r g e , M. R., Effects of toluene on microftora and hydrolysis of urea in a black
spruce h u m u s . Canad. J. Microbiol. 14, 999-1003 (1968).
9 R o b e r g e , M. R. and K n o w l e s , R., The ureolytie mieroftora in a black spruce
(Picea mariana Mill.) h u m u s . Soil Sci. Soc. Am. Proe. 31, 76-79 (1967).
10 S e n e c a , H., P e e r , P., and N a l l y , R., Microbial urease. Nature 193, 1106-1107
(1962).
11 S k u j i n s , J. J., Enzymes in soil. In Soil Biochemistry, eds. A. D. M c L a r e n and G.
H. P e t e r s o n . Marcel Dekker, Inc., New York (1967).
12 S u m n e r , J. B. and S o m e r s , G. F., Chemistry and Methods of Enzymes, 3rd ed.
Academic Press, New York (1953).
13 V o l k , G. M., Volatile loss of a m m o n i a following surface application of urea to turf or
bare soils. Agron. J. 51, 746-749 (1959).
14 Volk, G. M., Gaseous loss of a m m o n i a from surface-applied nitrogenous fertilizers. J.
Agr. Food Chem. 9, 280-283 (1961).