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Modified method for

measuring acid phosphatase
activities in forest soils with
high organic matter content
a b
Knut Schneider , Maria‐Belén Turrión &
a
Juan‐Fernando Gallardo
a
Consejo Superior de Investigaciones
Cientificas , Apt. 257, Salamanca, 37071, Spain
b
Area of Soil Science and Soil Chemistry,
E.T.S.I.A. , University of Valladolid , Campus
de Palencia, Palencia, 34004, Spain E-mail:
Published online: 11 Nov 2008.

To cite this article: Knut Schneider , Maria‐Belén Turrión & Juan‐Fernando

Gallardo (2000) Modified method for measuring acid phosphatase activities in
forest soils with high organic matter content, Communications in Soil Science
and Plant Analysis, 31:19-20, 3077-3088, DOI: 10.1080/00103620009370651

To link to this article: http://dx.doi.org/10.1080/00103620009370651

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 COMMUN. SOIL SCI. PLANT ANAL., 31(19&20), 3077-3088 (2000)

Modified Method for Measuring Acid

Phosphatase Activities in Forest Soils with
High Organic Matter Content
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Knut Schneider,a Maria-Belén Turrión,b,l and Juan-Fernando

Gallardoa
a
Consejo Superior de Investigaciones Cientificas, Apt. 257, Salamanca 37071,
Spain
b
Area of Soil Science and Soil Chemistry, E.T.S.I.A., University of Valladolid,
Campus de Palencia, Palencia 34004, Spain

ABSTRACT

The acid phosphatase activity (APA) is a key factor in phosphorus (P),

mineralization, P nutrition of plants, and P cycling. However, the widely used
method of Tabatabai and Bremner for APA determinations is seriously restricted
in soils with high organic matter contents, owing to interference in photometric
detection by dissolved organic matter. The objectives of this study were:A)to
modify the procedure of Tabatabai and Bremner with a view to its application
to forest soils rich in organic carbon, b) to determine APA contents in forest
soils of the Sierra de Gata mountains, and c) to establish relationships between
the APA with different soil parameters. The modified procedure was well

1
Corresponding author (e-mail address: bturrion@agro.uva.es).

3077

Copyright © 2000 by Marcel Dekker, Inc. www.dekker.com

 3078 SCHNEIDER, TURRION, AND GALLARDO

adapted to forest soils with high organic matter contents and had good
reproducibility, while maintaining the relative simplicity and accuracy of the
original Tabatabai and Bremner procedure. APA in the forest soils studied
was high but did not correlate with labile P. The APA in the soils studied
depended principally on availability of readily degradable energy sources and
soil nitrogen (N).

INTRODUCTION

Decomposition by microorganisms is the main process responsible for P release

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from soil organic matter (SOM). Additionally, P is released from SOM through
biochemical mineralization, where roots and microorganisms selectively release P
through the production of ester-hydrolyzing enzymes, often called phosphatases
(Clarholm, 1993). Phosphatases are produced when P is the most growth-limiting
element (Spiers and McGill, 1978; Margesin and Schinner, 1994). Consequently,
increases in phosphatase activities should reflect an increased requirement for P
by plants and/or microorganisms. Acid phosphatases dominate in soils with a low
pH, whereas alkaline phosphatases are dominant in soils with a higher pH, e.g.,
arable soils (Juma and Tabatabai, 1988).
Acid phosphatase activity (APA) is an useful and frequently measured parameter
in the studies of P cycling in ecosystems (Harrison, 1983), the characterization of P
forms in soil (Clarholm, 1993), and the role of organic P forms for plant nutrition
(Häusling and Marschner, 1989; Tarafdar and Marschner, 1994).
Acid phosphatase activity determinations are of special interest in soils in which
organic P (Po ) comprises a large part of total soil P (PT) and where available P (P )
is low. The supply of P to plant depends strongly on P mineralization, which is
principally due to the APA that promotes biochemical mineralization of P (McGill
and Cole, 1981).
One of the simplest and most frequently applied methods to measure APA is
that of Tabatabai and Bremner (1969) which uses p-nitrophenyl phosphate (pNPP)
as the substrate. The p-nitrophenol (pNP) formed after hydrolysis by APA in a soil
is subsequently extracted with sodium hydroxide (NaOH) and is then measured
spectrophotometrically. Unfortunately, in organic-matter rich soils, which often
also have high proportions of Po , NaOH solution also extracts high amounts of
organic carbon (C), interfering with the detection of pNP. For this reason, alternative
methods (Hoffmann, 1968; Sarathchandra and Perrott, 1981) had been used with
forest soils with high organic matter content (Harrison and Pearce, 1979; Trasar-
Cepeda and Gil-Sotres, 1987). These methods are time-consuming and involve
tedious extraction steps. In light of these limitations, we modified the procedure of
Tabatabai and Bremner (1969) in order to enable its application to forest soils rich
in organic carbon, determined the APA in forest soils of the Sierra de Gata mountains,
and studied the relationship between APA and different soil parameters.
 MODIFIED METHOD FOR MEASURING ACID PHOSPHATASE 3079

TABLE 1. Sites of the rainfall gradient with the respective abbreviation

symbols, annual amounts of precipitation, altitudes and types of
geological substrate. The symbol of each site represents the type of
geological substrate (G: Granite, Sch: Schist) and geographical exposure
(N: North, S: South).

Location Symbol Altitude Precipitation Litology

(m) (mm)
Fuenteguinaldo GN1 850 750 Granite
Casilla de Flores GN2 880 850 ««
Navasfrias GN3 900 1200
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Navasfh'as GN4 980 1600

Hoyos GS1 450 1000 »
u
San Martin GS2 480 1100
It
San Martin GS3 500 1200
M
VjJlaipieJ . „ GS4 J220
Robleda SchNl 850 800 Schist
Villasrubias SchN2 880 900 «I

Navasfrias SchN3 980 1600

«4
Villamiel ScbSl 850 1250

MATERIALS AND METHODS

Site Description
Study sites were selected along a strong orographic precipitation gradient located
in the 'Sierra de Gata' mountains, 40°2'N; 3°0'W (western side of the Central
Iberian mountainous System, Spain). Two transepts across the 'Sierra de Gata'
mountains, one on granites and the other on schists litology, were studied (Table
1). Sites oriented towards the northern plateau of Castilla (with a cooler and more
Continental type of climate) are distinct from those oriented southwards to the
Extremaduran plain (with a warmer and more Mediterranean type of climate). The
altitudinal gradient is more pronounced on the southern side of the range.
Precipitation data for the transepts were interpolated from data of the closest
pluviometric stations and maps of isohyets from Forteza del Rey (1986) and De
Leon (1991). Forests of deciduous oak {Quercus pyrenaica Willd.) form the
autochtonous vegetation of these mountains, but large extensions of degraded
oak forests and abandoned-cultivated fields have been replaced by pine (Pinus
pinaster L.) plantations. Soils were classified as humic Cambisols (FAO, 1988).

Soil and Leaf Litter Samples

Soil samples were taken from the Ah horizon. Some properties of the examined
Ah horizons are shown in Table 2.
 3080 SCHNEIDER, TURRIÖN, AND GALLARDO

TABLE 2. Chemical soil properties of the A^ horizons of the

climosequence. Effective cation exchange capacity (CEC), saturation
of base cations on the exchange complex (V) and Al on the exchange
complex (Al ).

Site pH pH CEC V Al»,

(HjO) (KC1) (mmolc kg-l) (%) (%ofV)
GN1 5.9 4.9 79.0 98 2
GN2 6.3 5.2 90.4 100 0
GN3 5.6 4.3 53.0 81 19
GN4 4.9 4.2 53.8 20 80
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GS1 5.8 5.3 104.0 98 1

GS2 5.7 4.3 49.6 86 14
GS3 5.2 4.0 68.3 83 16
GS4 5.3 4.1 60.7 84- 15
SchNl 5.0 4.0 54.7 71 28
SchN2 5.4 4.1 53.8 57 42
SchN3 5.1 4.2 43.2 26 74
SchSl 4.7 4.0 65.6 20 79

The amount of litter produced was determined for each site collecting leaf litter
in October and November, when almost all leaves had fallen, with a steel frame of
0.5 m x 0.5 m laid at the soil surface. Eight samples for each plots were collected,
placing the traps at random positions throughout the plots.

Chemical Analyses
Soil pH was measured in water and 1 Mpotassium chloride (KG) with an Ingold
pH electrode at a soil-solution ratio of 1:2.5. Soil organic C (Co ) was determined
by dry combustion and conductimetric detection using a Wostöff Carmhograph.
Total nitrogen (NT) was measured by microKjeldahl digestion followed by steam
distillation and titration of ammonia (NH3). Total and organic P (PT and P^)
concentrations were determined by the method of Saunders and Williams (1955).
Available P (Pav) was extracted with anion exchange membranes according to the
method of Saggar et al. (1990). Final P determination was made by
spectrophotometry following a modified molybdenum-blue method (Murphy and
Riley, 1962). The effective cation exchange capacity (CEC) was determined with
the method of Hendershot and Duquette (1986) and the percentage of base (V) and
saturation in aluminum (AlMt) were calculated after determination of cations by
atomic absortion spectrometry (AAS) with a Varian 1200 apparatus (Kerven et al.,
1989).
 MODIFIED METHOD FOR MEASURING ACID PHOSPHATASE 3081

TABLE 3. Kinetic parameters for acid phosphatase activity for

the transformed Michaelis-Menten equation.

Transformation km V^ r2
(mM) (nmol g ' h"')
Hanes-Wolf 13.7 13.5 0.998***
Lineweaver-Burk 12.8 13.2 0.995***
***Significantatap<0.001 level.
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Kinetic Parameters
Kinetic parameters were calculated to determine the optimal substrate
concentration for the highly organic soils used in this study. A final substrate
concentration assay in the 5-100 mM range of pNPP was performed. The kinetic
parameters of the enzymatic reaction were calculated from the Michaelis-Menten's
equation (Villar-Palasi and Santos Ruiz, 1962).
V = V S / ( k m + S)
where: V is the reaction rate, S is the substrate concentration, VmM is the maximum
rate (enzymatic activity) at which the enzyme acts on the substrate, and km (the
Michaelis' constant) is the substrate concentration where the reaction rate is one
half of Vmax. Hanes-Woolf and Lineweaver-Burk transformations of the Michaelis-
Menten equation for APA in soils was used to estimate km and Vimx, representing
S/V versus S, and 1/V versus 1/S (Villar-Palasi and SantosRuiz, 1962).

Procedure for APA Analyses

One gram of fresh field soil was weighed into a 100-mL Erlenmeyer flask. Four
replicates including one blank were used for each soil. One mL 100 mM pNPP and
4 mL modified universal buffer (MUB) were added to adjust the solution to the pH
of the soil, resulting thus a substrate concentration of 20 mM in the reaction
mixture. The flasks were then sealed and incubated at 30°C for 30 min. When
incubation had finished, the flask were immediately placed on ice and then 1 mL 2
Mcalcium chloride (CaCL,) and 4 mL 0.2 Msodium hydroxide (NaOH) were added
to stop the reaction and to extract the pNP formed. For the blanks, pNPP was
added after (instead before) the incubation. Ninety mL of deionized water was
added, and the resulting solution was mixed and filtered through Whatman-42
filter paper. Where necessary, further dilutions were made when APA levels were
too high. A 100 ug pNP mL*1 working solution was prepared from a stock solution
of 1,000 ug pNP mL"1. For standard preparations, aliquots between 0 and 8 mL were
pipetted into 100 mL volumetric flasks; 1 mL CaC^, and 4 mL NaOH were added and
 3082 SCHNEIDER, TURRIÖN, AND GALLARDO

volume was brought up. Final standard concentrations thus ranged from 0-8 ug
ml/ 1 . These were filtered in the same way as the samples because precipitation of
Ca(OH)2 may occur. The concentrations of pNP in standards and samples were
determined spectrophotometrically at 400 nm. The absorptions of the blanks were
subtracted from those of the samples. Solutions of pNP were linear in the range of
0-8 ug ml/ 1 range. Enzymatic activity was expressed in umol pNP g-1 soil Ir1 or
enzyme units (EU). The modified method was tested with a set of the selected acid
forest soils.
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Statistical Analyses
The reproducibility of the proposed method was checked by one way analysis
of variance (ANOVA). Correlations between variables were established applying
regression analysis.

RESULTS AND DISCUSSION

Optimization of the Substrate Concentration
Preliminary attempts to determine the APA of this soils studied here with the
original procedure of Tabatabai and Bremner (1969) failed. Apart from the high
organic matter dilution, this was due to the very high enzymatic activity observed,
giving pNP concentration outside of the linear range for spectrophotometric
detection. This can be overcome by appropriate dilution of the reaction mixture
with water when incubation has finished (Schinner etal., 199I) and a reducing the
incubation time. However, with such high activities, it is essential to test whether
the substrate concentration of the incubation is sufficient to reach an adequate
reaction rate as recommended by Malcolm (1983) for determination of enzymatic
activities.
The kinetic parameters of the enzymatic reaction were calculated from the linear
regressions of data obtained by the Michaelis-Menten's equation (Table 3).
Substrate concentrations plot and the linearization of the Michaelis-Menten's
equation by the Hanes-Woolf s transformation, from which km (Michaelis' constant)
can be estimated, are shown in Figures la and lb.
Burns (1978, 1986) recommended that the optimal concentration of pNPP for
enzymatic activity analysis should be at least five times km, 68 mM in the case of
the forest soils used in this study (Figure lb). Therefore, in all subsequent assays
a substrate concentration of 100 mM (corresponding to a concentration of 20 mM
in the reaction mixture) was applied. Although the original procedure of Tabatabai
and Bremner (1969) used a 23 mM pNPP substrate, in most subsequent work the
substrate concentration for soil assays has been considerably reduced, with no
prior examination ofenzyme kinetics (Adams, 1992). For many studies where the
kinetic parameters had not been determined, APA has been measured under limiting
substrate conditions (Eivazi and Tabatabai, 1977; Trasar-Cepeda and Gil-Sotres,
 MODIFIED METHOD FOR MEASURING ACID PHOSPHATASE 3083

B
"JC 15
800

s/v* 100
600 /

400 /
200
n
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20 40 60 80 100 •20 0 20 40 60 80100

p-NPP [mM] p-NPP [mM]

FIGURE 1. a) Acid phosphatase activity (APA) versus substrate concentration; b)

corresponding Hanes-Woolf s transformation; S: substrate concentration; V: reaction rate;
km: Michaelis' constant.

1987). Accordingly, the data of these studies cannot be used for comparisons of
enzyme activities.

Factors Affecting the Extraction Process

Organic matter dissolution was avoided by applying a 2 M instead of a 0.5 M
CaCl2 solution which is normally used to prevent clay dispersion. However, the
calcium (Ca2+) ion is also a good agent for the coagulation of SOM. Furthermore,
the concentration of the added NaOH was reduced from 0.5 to 0.2 M. Schinner et
al. (1991) mentioned the possibility that the NaOH solution can be added when soil
had already been filtered from the pNP extract. This alternative however fails to
take into account that NaOH is not only necessary for the coloring of the pNP
chromophore, but also for ensuring good extraction from the soil. Under these
conditions, only 75% of the total pNP was extracted. Consequently, NaOH solution
was included in the extraction process. Despite all precautions, a small degree of
coloring of the extracts due to the presence of dissolved organic C (DOC) cannot
be avoided. Therefore, blanks in which pNPP is added after incubation had finished
should be included. The color of blanks was subtracted from that of the samples
incubated with the substrate. Often, a few drops of toluene are added to avoid the
occurrence of microbial activity during the incubation (Eivazi and Tabatabai, 1977),
but this step is not necessary if the incubation time is short (Schinner et al., 1991).

Precision and Reproducibility

The high degree of precision of our modified method (determined with
independently repeated measurements of six replicates of the same soil sample) is
 3084 SCHNEIDER, TURRIÖN, AND GALLARDO

TABLE 4. Precision of the modified method for acid phosphatase activity (in (ig
pNP g-' h1).

Set of samples 1 2 3 4 5 6 Mean value

Mean 11.5 11.4 11.5 11.4 11.0 11.1 11.3
S.D.* 0.46 0.06 0.25 0.30 0.17 0.16 0.26
*S.D.=standard deviation (n=3).
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illustrated in Table 4. There was little variation in the APA between measurements,
pointing to the good reproducibility of the method.

Acid Phosphatase Activity

Table 5 shows the APA of 12 forest soils with varying contents in Co and soil
P fractions. No problems owing to background coloration from DOC were observed.
The enzymatic activities varied over a wide range (2.9-14.8 umol pNP g-1 h'1) although
the substrate concentration was assumed to be sufficient for all soils.
In comparison with the data obtained by Clarholm (1993) in the humus layer of a
spruce forest, and Adams (1992) and Pang and Kolenko (1986) in forest soils, the
APA was high at all sites, in particular at GS1. Phosphatases are inducible enzymes,
and hence their excretion by microorganisms and plant roots is regulated by
end-product inhibition. Therefore, high APA levels in soils indicate a low content
of available P (Margesin and Schinner, 1994). However in this study, the APA was
not directly related either to the soil P status or to the rainfall gradient (Table 6). No
signs of a possible inhibition of the enzyme activity attributable to high
concentrations of labile ?t were observed. In contrast, the highest APA coincided
on a site, GS1, with a relatively high concentration of available P as extracted by
anion exchange membranes (Pay). The APA was not higher in extracts from strongly
acid soils than in those from only moderately acid soils (Tables 2 and 5).
In forest soils, Adams (1992) reported that APA was not directly related to soil P
fractions or P r Negative correlations between APA and available P in soils were
obtained by Gerritse and van Dijk (1978) andNannipieri et al. (1978) in sandy soils,
and Pang and Kolenko (1986) in forest soils. However, the decrease in APA is not
entirely related to the increase in available P in limed forest soils (Badalucco et al.,
1992).
Among the parameters determined at the different soil samples the Co and NT
concentrations and the C/P ratio and the amount of leaf litter had a strong positive
correlation with APA (Table 6). The variation in APA was best explained by the
concentration of NT in the Ah horizon and the quantity of leaf litter supplied to the
soil (rM).75). Despite the fairly high APA at all sites, maximum activities were not
 MODIFIED METHOD FOR MEASURING ACID PHOSPHATASE 3085

TABLE 5. Total organic C (Corg), total N (Nt), total P (P), organic P (Porg), available
P (Pav), acid phosphatase activity (APA) of forest soils of Ah horizons of the
climosequence, and the supply of leaf litter to the soil.

Site c N, P, p ptv APA Leaf litter

1 1 1
(mg g"1) (mg g 1 ) (mgkg" ) (rag kg" ) (mg kg ) (nmol pNP (gm 2 )

GN1 28.3 2.2 602 451 9.28 5.0 156

GN2 22.4 2.4 430 328 15.01 6.2 163
GN3 19.9 0.8 354 249 3.09 2.9 244
GN4 71.0 4.0 629 479 1.28 7.1 156
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GS1 62.3 3.5 389 283 22.19 14.8 330

GS2 13.1 1.6 755 310 27.45 8.9 281
GS3 44.2 3.1 399 302 11.68 8.0 215
GS4 24.9 1.8 655 298 27.14 5.6 236
SchNl 45.0 3.0 633 520 2.90 11.1 174
SchN2 68.6 4.3 745 628 2.20 11.4 148
SchN3 96.0 4.6 730 301 0.40 11.3 163
SchSl 73.9 4.4 1001 840 0.88 7.9 170

found where humus content was greatest and the labile P least. This may be
because APA also depends on the availability of readily decomposable
carbohydrates and of N in the soil. A dependence of soil APA on the availability
of readily degradable energy sources and N was reported by Spiers and McGill
(1978) who attributed the positive effect of N to an increase in phosphatase-protein
synthesis by soil microorganisms. As in this study, Harrison (1983) and Baligar et
al. (1988) also found a high correlation between forest soil APA and soil Co and NT
contents. Harrison (1983) attributed the positive influence of high humus contents
to a possible binding of phosphatases in protein-humus complexes, protecting the
phosphatases from microbial decomposition.

CONCLUSIONS

Our procedure for measuring APA has been adapted to forest soils with high
SOM contents. In the relative simplicity and accuracy of the original Tabatabai
and Bremner procedure (1969) is preserved, and the modified procedure is, therefore,
less tedious than those of Hoffmann (1968) or Sarathchandra and Perrott (1981)
which were also adapted for organic matter-rich forest soils. When the magnitude
of the soil enzymatic activity is unknown, the optimum substrate concentration
should be determined. The APA in the forest soils studied here was high, but
maximum activities were not observed when the humus content was greatest and
that the labile P least. The APA in these soils did not seem to be a function of the
 3086 SCHNEIDER, TURRIÖN, AND GALLARDO

TABLE 6. Correlation values of the activity of acid phosphatase (APA) with different
soil parameters and the supply of leaf litter to the soil (n=12).

pH C^ N, P| P^ P,v OS
C/P CEC Precipitation Leaf
litter
APA N.s. 0.48 0.60 N.s. N.s. N.s. N.s. 0.51 N.s. N.s. 0.51
Significance - • • • • - - - - • * - - *•
**Significantatap<0.01 level.
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P demand of soil organisms and plants nor of the P deficit in the soil, but rather on
the availability of readily degradable energy sources and N.

ACKNOWLEDGMENTS

The authors wish to thank the 'Junta de Castilla y Leon' for allowing the use of
forest plots, the European Union (MEDCOP/AIR and CAST/ENVIRONMENT
Projects), and the Spanish Fund C.ICY.T. for financial support. The English version
was revised by N. Skinner. We are indebted to Dr. Pauline Grierson, Department of
Botany (University of Western Australia) for reviewing the manuscript.

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