MARDI Rel Bull., 7,2 (December, 1979) 35 42

ASSAY OF UREASE ACTIVITY IN SOILS

YA}iYA MOHD. NOR"

Accepted for publication on 14 August, 1979

Keywords.' Urease activity, Urea retension, Urea hydrolysis, Soil pH.

RINGKASAN

Satu cara menguj i akt iv i t i urease te lah dihuraikan. Ianya mel ibatkan inkubasi aerobik 10-g

contoh tanah pada suhu 37'C selama 6 jam selepas di tarnbah substrat urea kepadanya dan

pengukuran urea secara kolor imetr ik selepas diekstrek dengan 2.5M KCI yang Inengandungi

Ag^SO, (100 ug/ml) sebagai penghalang urease. Kesan pengekstrek ke atas warna kornplex dan

per idapl tan semula (recovery) urea dar ipada tanah dengan tatacara tersebut juga din i la ikan.

Akt iv i t i urease di dalam 14 tanah-tanah dar i Malaysia yang te lah dikaj i menunjukkan tanah' tanah

mernpunyai keupayaan menghidrol is iskan urea yang berbeza. p l l tanah dan akt iv i t i urease

rnempunyai korelasi yang baik, tetapi kandungan karbon organik dan muatan penukaran kat ion

(CEC) t idak mempunyai apa-apa hubungan dengannya.

INTRODUCTION

Soil urease rapidly hydrolyzes urea added to soils as a ferti l izer to anmonia and carbon

dioxide (NH2CONH2j92NH3 + CO2). The uti l ization of urea as a source of nitrogen

ferti l izer is not only growing in importance but also expanding faster than any nitrogenproducts (Hanne. ef a i . , 1971). Therefore, there is a s t rong need for research in to the

potential damage to germination and early growth of crops due to urea hydrolysis and

subsequent loss of the fer t i l izer through volat i l izat ion (Volx, 1961: Gasse n, 1964; SIMPSoN,

1968 ; R I InRATNAM and PURUSHOTHAMAN, 1973 . O the r t han supp l i ed as f e r t i l i ze rs , u rea

may also be formed from dung and urine of cattle GASSER, 1964), and as a result of the

act iv i ty of cer ta in rn icroorganisms (VASILENKo, 1962).

Methods of assay of urease activity vary from the estimation of the ammonium evolved( S r o r a N o v r c . 1 9 5 9 ; V A S T L E N K o , 1 9 6 2 ; M c G A R I T Y a n d M v E R S . 1 9 6 1 , T R n e r e s A . t a n d

B R E M N E R . 1 9 7 2 ; N o n o s T A D T . e t a l . , 1 9 ' 1 3 : R A J A R A T N A M a n d P L R U S H o r A t r l a N , 1 9 7 3 ) :

the measurement of carbon d iox ide re leased or urea decomposed (CoNnln. 1942: PoRTER,

1965; SrupsoN, 1968: NoRDSTADT. et a l . , 1973) and f ina l ly . the color i rnetr ic determinat ion

o f u rea (CoNnno . 1942 ; Pon rEn , 1965 ; Douc rns & BREMNER. 1970 ) . So rne i nves t i ga to rs

opted for the use of buf fers (e.g. Wnlr - and L,q, toLrn, 1952: :McLa,RnN er a i . , 1957

S T O J A N O V I C . 1 9 5 9 : M C G n R I T Y , a n d M Y E R S , 1 9 6 7 : T n S A , T A B A I a n d B R l t r l N E n , 1 9 7 2 :

NoRDSTADT, et a l . , i973; ZaNruA and BRL,MNER, 1975), whi le others ut i l ized non-buf fer

methods for determination of urea or ammonium produced (e.g. MCL,+REN. et al., 195'7

P o R r E R , 1 9 6 5 ; P n u r - S o N a n d K u R r z , 1 9 6 9 ; D o u c L A S a n d B R E M N E R , 1 9 7 0 ; Z A N T ' u A

a n d B R E U N E R . 1 9 7 5 ) .

The procedure reported in this paper involves the addition of urea substrate to a

sampie of soil (1840 ug urea-N/g soil) incubation for 6 hours at 37'C as proposedDoUGLAs and BRTUNER (1971) and extract ion of the urea wi th a solut ion of 2.5M

containing 100' ug/ml Ag2 SOa (Tannrnnat and BREMNE,R, 1972). The intensity of

colour developed when as aliquot is reacted with p-dirnethylaminobenzaldehyde reagent

measured at 420 nm. The method adapted is simple, rapid and permits the determination

urea-N concentrations of as low as 10 ppm with adequate accuracy.

1o'gby

KCIthewas

of

School ofUniversitiPenang.

Biological SciencesSains Malaysia

35

MATERIALS AND METHODS

The soils vsed (Table 1) in the experiment were surface (0-15 cm) samples selected togive a wide range in pH (3.4 8.3), organic carbon (0.90 2.90%) and texture. A1l analysesreported were performed on air-dried samples ground to pass through a 2 mm sieve, except fororganic carbon analyses which were done on(l 00 mesh soil. pH determination was accomplisedwith a glass electrode (soil : water, I : 2.5), organic carbon by the method of MERIuS (1960),texture by the hydrometer method (Dnv, 1965), and cation exchange capacity by theammonium acetate nethod (CHApMAN. 1965).

TABLE 1. ANALYSES OF SOI].S

OrganicCarbon Clay*Soil Type pH CEC

(m.e . /1oog)

Lunas

Keranj i

Chuping

Kanggar

Langkawi

Hutan

Kok Diang

Kangkong

Relau

Renggam

Selangor

Melaka

Telok

Guar

ls

scl

ls

s l

scl

ls

scl

scl

ls

ls

scl

sc

scl

8 .3

7 . 1< /

< /

5 .2

) . 1

4 .8

4 .8

4 .3

4.3

4 . I

3 . q3 .4

0.90

0.98

t .28

L . Z J

1.05

0.831 A 1

1.98

0.98

0.90

2.25

1.94

2.m2.U

74

48

74

56

53

63

48

85

u48

534r)

) l

LJ

2'720

4 I

l4

1 i

16

19

4

3

16'7

18

12

3

25

6

3

JJ

'7

21

-l-l

1 1

13

36

40

36

) /

3 . 8

23.6

4 . 4

25.3

9 .5

4 . 0

1 5 . 8

1 3 . 5

.1.8

5 . 1

l - ) . - )

19.0

16.0

19.0* ls , : loamy sand; sc l , : sandy c lay loam; sc, : sandy c lay; s l , : sandy loam.

For the incubation studies, a 10 g sample of soil ((2 mm) was placed in a plasticcontainer with a screw cap (capacity, 200 ml) treated with 4 ml of urea subtrate (4600 ugurea-N/ml) and incubated at 37oC (Doucr ls and Bnr,uNER, 1971) for 6 hours(McGanrrvand Mrypns, 1967). The sample was removed after the specified period, a 50 n-rl solution of2.5M KCl conta in ing 100 ug/ml Ag2SOa as urease inhib i tor (TABATABAT and. BREMNER,1972) added, and the sample shaken on a Gallenkamp Orbital Shaker set at 150 rev./min. for45 mins. The resulting suspension was filtered (Whatman ll42), and as aliquot taken foranalysis.

For the colour development, the procedure of Wn,tt and CRtSp (1954) as outl ined byPORTER (1955) was adopted. It involves taking an aliquot of the soil solution and treating itwith 10 ml of urease reagent in a 25 ml volumetric f lask. This reagent was prepared bydissolving 2.00 g. p-dimethylaminobenzyldehyde (Sigma Chemical Co., St. Louis, Missouri) in100 ml of 95% ethyl alcohol and 10ml concentrated HCl. Deionized water was then added tothe mark. The absorbance of the yellow complex produced was measured using aspectrophotometer (Busch lnmb Spectronic 20 htted with a blue filter) set at a wavelength of

36

420 nm after having allowed the colour to develop in a water bath maintained at 30-C. lt rs

important to ensure that measurement of the optical density be taken at a constant

temperature because the transmittancy has a temperature coefficient of 0.6Vo per 1oC over the

range from 20oC to 40"C (PORTER, 1965). Reference was then made to a calibration curve

constructed using concentrations of 0, 20, 40, 60, 80, 100 and 120 ppm urea-N. In the work

reported here, all incubations were carried out in duplicat6s using (2 mm soil samples and

results are reported as ug urea-N hydrolyzed/hr/g soil.

RESULTS AND DISCUSSION

Table 2 shows the recovery of urea added to soils. This study was carried out because of

the distinct possibility that the urea added might be retained by soils resulting non-quantitative

recoveries of urea. Results shown in this table indicate quantitative recoveries (98.0-101 .9%) of

urea from 9 soils by the procedure adopted. This implies that the urea added is neither

retained in the soil nor hydrolyzed by soil urease. The latter can be attributed to the

effectiveness of Ag2 SOa as an inhibitor of urease as has been shown by other workers

( D o u c l n s a n d B R T U N E R , 1 9 7 1 ; T R s a t l n e t a n d B n r n l N l n , 1 9 7 2 ; N o R , 1 9 7 9 ) . A r e c e n t

study by Non (1979) for example reported 100% inhibit ion by Ag2SOa when tested with

several Malaysian soils at a rate of approximately 120 ppm Ag'.

TABLE 2. RECOVERY OF UREA - N ADDED FROM SOIL*

Urea-N added Urea-N Recoveredugig soil ug/g soil

c/c

recovery

Melaka

Guar

Telok

Lunas

Kangkong

Hutan

Selangor

Kok Diang

Chuping

*1 ml of urea substrate (460 ug Urea-N) was added to a 10-g sample of soil,followed by the prompt addition of 50 ml2.5M KCI + Ag2SO4 and shaking for 1hour.

During the preliminary course of the investigation it was observed that the optical

density of standards in the presence of KCI was higher than in the presence of only water. It

was felt necessary, therefore, to evaluate any interference from the extracting solution. The

result of this study is shown in Fig. 1. As can be seen from the graph, the colour of the

yellow complex was more intense in the presence of 2.5M KCI solution compared to that of

water, thus resulting in a positive interference which may be due to the rather concentrated salt

solution. Inspite of this, no problem whatsoever was encountered in the analyses performed

since reference was made to a standard solution prepared in the presence of KCl.

For valid assay of any enzymatic activity, it is important to ensure that the substrate is

not limiting. In this investigation, a substrate concentration of 1840 ug urea-N/g soil was used.

This concentration was presumed adequate on the basis of the work done by TlsaTes,{t and

460

460

460

460

460

460

460

460

460

A < 1 1

451.7n < < A

454.9

461.3

99.5

98.2

99.0

98.9

r00.3

461.3 100.3

4s0.8 98.0

453.6 98.6

468.7 101.9

37

{

=

\!

Y . !

T \

: \

^ ! i{ > . :

= _ i :7 . { " -

^ - r < : f

v : :- > A

: * t

! r

: 1 -

: \e i 5I . S : :- : y

* <

. s !> N

;:-

AJNVSUOSgV

aN

? . t: ! !26ai =

o o

38

BREMNER (1972) and ZANTUI and BRr l tNan (1975) who used concentrat ions of 1120 and

1000 ug urea-N/g soil, respectively. Recent word by Ncln (1979) also indicate that a substrate

concentration of 4 mg urea (1840 ug urea-Nig soil) per soil was sufficient even for soils

that have very high activity. This category of soils, however, generally constitute only a small

proportion of Malaysian soils (Non, 1979).

FiS. 2 i l lustrates the urease activity of l4 soils studied. The abil ity of the soils to

hydrolyze urea varies considerably - ranging from a low of about 5 ug/hour/g soil in Lunas soil

to a high of (l 50 in Keranji soil. Two soil series - Telok and Guar which have been identif ied

as acid sulphate soils exhibited low capacity to hydrolyze urea in cornparison to others, This

may be related to the tremendous acidity produced as a result of oxidation of pyrit ic materials

(KlNarerHy, 1976). McGanrry and MysRs (1967) expla ined that the increase in urease

activity with increasing pH could be due to increased accumuiation, release and stabil ity of

urease from imcrobial source at higher pH. This could explain the high activity observed in

Keranji soil which has a pH of 7.1 , but not so in Lunas soil, pH 8.3. In this investigation,

however, urease activity and soil pH correlated significantly, r = 0.81 when Lunas soii is

excluded, a l though MCGARITy and MyeRS ( .1967) concluded that ' the ro le of pH is only a

minor one in their study.

pg UREA hydrolyzed/hr/g soil

KERANJI

CHUPING

KANGAR

KODIANG

RENGGAM

HUTAN

LANGKAWI

MELAKA

RELAU

KANGKONG

SELANGOR

TELOK

GUAR

LUNAS

Figure of urea hydrctlyzed (W urea-Nlhrlg soil) in 14 soils.

NJ (^( . , | o

2. The amounts

39

The urease activity and the cation exchange capacity of the soils dcl not seern to becorrelated a negative non-significant correlation coefficient was obtained. The organic carboncontent however, when correlated with the urease activity resulted in a correlation coefficientof - 0.51 which is not significant at both 1% and 5Vo levels.

The differences in the amount of urea hydrolyzed can be attributed to changes in thecondi t ions of the soi ls and var iat ions due to seasons (SrornNovtc, 1959; Vnst lEN Ko,1962). More importantly, variations in urease activity are caused by the rnicrobial populationsin the soil. PeulsoN and KURTZ (1969) for example reported a correlation coefficient of0.94 between urease activity and ureolytic organisms. Many genera of bacteria, fungi andactinoltlycetes have the abil ity to synthesize urease and therefore can use urea as a nitrogensou rce fo r g row th (A laxnNnrR , 1961 ) .

ACKNOWLEDGEMENTS

The author wishes to express his appreciation to Y.B. Tan Sri Datuk Haji Hamdan binSheikt Tahir, Vice Chancellor, Universit i Sains Malaysia for his permission to publish the paperand the financial support through the short terrn research grant; Encik David Chuah for theinit ial work on this study and Cik Lai Yen Choo for some of the analvses performed.

SUMMARY

A method for assay of urease act iv i ty is descr ibed. I t involved aerobic incubat ion of a10-g sample of soi l at 37'C for 6 hours af ter addi t ion of urea substrate to i t , and color imetr icdeterrn inat ion of the urea rernain ing af ter extract ion wi th 2.5M KCI contain ing Ag SO (100ug/nrl) as urease inhibitor. The effect of extractant on the colour of the cornplex and reJoveriesof urea added to soils by the method were also evaluated. The urease activity in 14 Malaysiansoils studied indicated that the soils have varying capacity to hydrolyzed urea. Soil pH andurease activity correlated well, but neither organic carbon content nor cation exchange capacitybore any significant relationship.

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A 1