Agricultural Science Research Journals Vol. 2(7), pp.

412-419, July 2012

Available online at http://www.resjournals.com/ARJ
ISSN-L:2026-6073© 2012 International Research Journals

Full Length Research Paper

Soil phosphatase activity of agricultural land: A

possible index of soil fertility
Anwesha Banerjee1, Sanghamitra Sanyal2 and *Sribir Sen3
1,2,3
Durgapur College of Commerce and Science, Department of Biochemistry, (A unit of Rahul Foundation), G.T. Road,
Rajbandh, Durgapur, Dist. Burdwan, West Bengal, India

*Corresponding Author’s Email: [email protected]

Abstract

Plethora of cellular events is regulated by phosphatases (EC 3.1.3.-). Agricultural soil contains
phosphatases in variable amounts depending on microbial count, amount of organic materials, other
macroscopic living organisms and their activities etc. The amount of phosphate released into the soil
can then be directly co-related to soil fertility. The assay of soil phosphatase was carried out following a
0
standard procedure with a minor modification. The pH and temperature optima were pH 5.0 and 37.0 C,
respectively. Using standard assay conditions kinetic parameters of soil phosphatases such as
maximum reaction rates of phosphatase (V max) and the substrate concentration at which the reaction
rate is half maximal (Km) were determined. It produced an apparent K m value of 1.353mM. The effects of
+ + 2+ 2+
various metal ions like Na , K , Mg and Ca were also carried out using the standard assay procedure.
Phosphatase activity of 10 soil samples, collected from the various agricultural lands, of the district
Burdwan, West Bengal, India, was studied. The maximum unit of activity was 6.393 units in standard
assay conditions. The phosphatases present in a soil sample are heterogeneous and might be utilized
as a major parameter to assess soil fertility in an agricultural land.

Key words: Soil phosphatase, Enzymology, Agricultural field, Available phosphate

INTRODUCTION

In nature, phosphorus cycle plays an important role in the phosphorous and remain in soils in various forms (Tyler,
survival of living organisms (Stewart and Mckercher, 1976). Rock phosphates of various kinds undergo
1982). Phosphatase is an enzyme that release inorganic solubilization due to microbial activities in the soil (Hysek
phosphate from organic moiety and complex inorganic and Sarapatka, 1997). In particular, phosphatases play a
materials. It is known to play an essential role in key role in phosphorous cycle by solubilizing organic and
phosphorus cycle, even though; roles of other various inorganic phosphates into available forms that support
physical factors cannot be ignored. Phosphorus is the growth of crop plants (Wyszkowska and Wyszkowski,
maker of the energy currency and it plays important roles 2010). Released inorganic form of phosphate is readily
in enumerable metabolic pathways in living systems soluble in soil and plant system can easily uptake it as
(Rasol and Reshi, 2010). Cellular signaling events nutrient source. However, phosphorus also comes from
cascaded with phosphorylation and dephosphorylation, death and decay of all living organisms that reside in soils
are associated with an enzyme called phosphatase (EC (Anderson and Ingram, 1993; Hu and Cao, 2007). Soil
3.1.3.-). Soil receives various phosphatases from living phosphatase activity depends heavily on moisture
organisms that play important roles in the solubilization of content and environmental temperature of the soil
inorganic phosphates (Acosta-Mortinez and Tabatabai, (Huang et al., 2011). Phosphatases are usually classified
2000). Enzymatic activities of a soil sample are critical based on pH optima; these are neutral (EC 3.1.3.-), acid
index of soil fertility because enzymes play an important [EC 3.1.3.2] and alkaline [EC 3.1.3.1] (Hoffmann, 1968;
role in nutrient cycles (Dick et al., 1996). Various minerals Akanji and Adesokan, 2005; Raghav et al., 2011).
such as Al, Fe, Mg and Ca are complexes with However, there are several subclasses; this classification
 Banerjee et al. 413

0
is based on the type of substrate where it acts such as substrate used in the assay within 15.0min at 37.0 C. It
tyrosine specific phosphatase, serine-threonine specific produced 0.61 optical densities at 430nm, which was the
phosphatase, dual specificity phosphatases, histidine degradation product of 10.0mM substrate present in the
phosphatase and lipid phosphatases (Seger and Krebs, assay. One unit of enzyme activity was described as the
1995; Zhang, 2002). Soil phosphatases are degradation of 1mM substrate in the standard assay
heterogeneous in nature and the enzymes have tribal conditions (Plummer, 1987).
names, according to their substrates (Alvear et al., 2005), The pH optimum was determined using 100.0mM
such as phosphoric monoester hydrolases, (EC 3.1.3.-) buffer with specific pH values (1.0, 3.0, 5.0, 7.0, 9.0 and
and phosphoric diester hydrolases (EC 3.1.4.-). 11.0, respectively). Buffers were made according to
Importantly the first group is composed of phytase, standard protocol, as by Gomori (1955). In order to
nucleotidases, sugar phosphatases, glycerophosphatase determine the temperature optima, the following
0
(Cohen, 1989). However, the second group contains temperatures; 17.0, 27.0, 37.0, 47.0, 57.0 and 67.0 C,
nucleases and phospholipases (Speir and Ross, 1978). were used. In these assays 100.0mM acetate buffer pH
The present study reports for the first time phosphatase 5.0 was included. Other assays of the phosphatase were
activity determination of agricultural soil taken from the carried out as per the standard procedure, unless
district Burdwan, West Bengal, India. This study otherwise stated. Three replicates for each sample were
introduces a simple, less expensive and reproducible carried out.
procedure of estimation of soil phosphatase activity, Determination of pH of various soil samples: The pH of
which is very easy to implement in a field laboratory. a soil sample was determined using 1:2 wt /v mixture of
Quick examination of many soil samples can be carried soil and water, following a method by Anderson and
out using this method. Ingram (1993). 10.0g of soil was mixed with 20.0ml of
distilled water. The mixture was kept at room temperature
Materials and methods for 15min. The water part was poured in a beaker. Then
the pH was measured using a pH meter with a
Alluvium type of soil samples were collected from combination of glass electrode (Labtronics Digital pH
various agricultural fields of the district Burdwan, West meter, Table model LT-11) and readings were recorded
Bengal, India. These soil samples were collected from 0- to two decimal places.
15cm depth of the agricultural land during spring season Scientific data analysis and graphing were done using
0
(March and April). Temperature was around 30.0 ± 2.0 Sigma plot 12 scientific graph system. Microsoft office
0
C. 10.0g of soil sample was collected, stored in glass excel 2007 was also used to make various graphs. Other
tubes and kept in a glass container with sufficient results were expressed as mean ± standard error of
moisture which was controlled by distilled water addition mean (SEM).
inside the container. The major crops under cultivation
were rice, wheat, various kinds of vegetables, like:
potato, tomato, cabbage etc. Results
The following materials were purchased from the
suppliers indicated. p-nitro phenyl phosphate, NaOH, Assay of soil phosphatase activity: The assay tube was
KH2PO4, K2HPO4, KCl, NaCl, CaCl2, Merck. Germany taken out from the incubation and centrifuged. The clear
(India). Other chemicals those were used in this study supernatant was treated with 2.0ml of 1.0M NaOH. It
were from Glaxo Smith Co, Mumbai, India. showed yellow color which was produced due to the p-
Assay of soil phosphatase activity: 100.0 mg of soil nitro phenyl released from the substrate used in the
sample was taken in a micro centrifuge tube and 0.5ml of assay. The control sets demonstrated no change of color.
100.0mM phosphate buffer was included. 10.0mM of p- Degradation of the substrate resulted by the presence of
nitro phenyl phosphate (p-NPP) in 100.0µl solution was phosphatase activity in the soil sample. The color
used as substrate. The final volume of the reaction intensity was estimated by using a colorimeter at
mixture was adjusted to 1.0ml with the addition of λ=430nm. It produced a value of 0.35. Conditions of
requisite amount of distilled water. The tube was vortexed assays were as follows: time of incubation 60min,
0
(2.0min.) at room temperature. It was then incubated at temperature 37.0 C and 100.0mM sodium phosphate
0
37.0 C for 60min in shake condition (100 rpm). After the buffer. The assay was carried out for various time
incubation period, the sample was centrifuged at 10000 periods, 30.0., 60.0., 90.0., 120.0., and 240.0min,
rpm (5.0min). The clear supernatant was taken in a clean respectively. Linear increase of phosphatase activity as
test tube and 2.0ml of 1.0M NaOH was added. The per the time of incubation period was noted (Figure 1).
yellow colored filtrate was analyzed using a colorimeter
(Klett colorimeter, Clinical model, 800-3, 115 VAC) at
λ=430nm. Goat liver phosphatase activity was also Effect of pH on phosphatase activity of a soil sample
determined, as a reference using the same conditions.
The goat liver phosphatase degraded 10.0mM pH optima was determined over a pH range from 1.0 to
414 Agric. Sci. Res. J.

Figure 3. Effect of temperature on phosphatase activity of an

Figure 1. Effect of prolongation of time of incubation on agricultural soil. The assays were carried out using 100.0mM
phosphatase activity of a soil sample. The assay included 100mM buffer pH 5.0. Other conditions were the same as described in
phosphate buffer, pH 5.0, 10mM p-NPP, 100mg of soil sample. materials and methods.
Other parameters were the same as in materials and methods.

Effect of temperature on phosphatase activity of a

soil sample

Determination of p-nitro phenyl phosphate degradation

was measured over a temperature range from 17.0 to
0
67.0 C (Figure 3). The degradation of p-nitro phenyl
phosphate demonstrated a broad range of activity.
However, the maximum enzyme activity was attained at
0
temperature 37.0 C. Inhibitory effect on phosphatase
activity was observed above or below this temperature.

Effect of substrate concentration on phosphatase

activity of a soil sample

Degradation of p-nitro phenyl phosphate increased

linearly with increase of substrate concentrations and
reached a plateau at 15.0mM substrate concentration
(Figure 4.a). It produced an apparent Km value of
1.353mM, what was almost similar that was obtained
from Lineweaver-Burk plot (Figure 4.b).
Figure 2. Effect of pH on phosphatase activity. The assay was
carried out using various buffers with specific pH. Other
Effect of metal ions on phosphatase activity of a soil
parameters were the same as described in materials and + + 2+
methods. sample: Monovalent and divalent cations (Na , K , Mg ,
2+ 2+ 2+
Ca , Mn and Fe ) were used separately in the assays.
Results are presented in Figure 5-8. The optimum
+
concentration for Na was 40.0mM where the enzyme
11.0 (Figure 2). The maximum of phosphatase activity activity was 5.245 units. Below or above the optimum
was at pH 5.0. The enzyme activity above or below of this concentration the phosphatase activity was inhibited
+
pH showed less degradation of the substrate used in (Figure 5). The optimum K concentration equaled
these assays. 5.0mM, where the enzyme activity was 4.426 units.
 Banerjee et al. 415

Figure 4(a): Effect of substrate concentration on

phosphatase activity. The assays were carried out using
variable concentrations of substrate. Other parameters were
the same as described in materials and methods.

Figure 4 (b): Double reciprocal plot of substrate concentration [S] v/s reaction rate (V) of
phosphatase activity showing the effect of substrate concentration on the velocity of phosphatase
activity of a soil sample collected from an

+
Figure 5. Effect of Na on phosphatase activity of
an agricultural land. The assays were carried out at
standard assay conditions. Variable concentrations
+
of Na were used separately in each assay.
416 Agric. Sci. Res. J.

Figure 6. Effect of K+ concentration on phosphatase

2+
activity of soil. The assays were carried out using Figure 8. Effect of Ca on phosphatase activity of a soil
2+
variable concentrations of K+. Other parameters were sample. Variable concentrations of Ca were added separately
remained the same as described before. in each assay. Other parameters as were the same as
described before.

Phosphatase activity in 10 soil samples collected

from various agricultural fields

Phosphatase activity of 10 soil samples was estimated

following the same procedure (Table 1). The soil samples
were collected from the agricultural lands, where
predominant crops were rice, wheat and different kinds of
vegetables. 9 soil samples were collected from the fields,
where various crops were under cultivation. Another soil
sample was collected from an open land, where
harvesting was completed. The phosphatase activity was
the highest in the sample collected from the wheat field. It
was 6.393 units in standard assay conditions.

Figure 7. Effect of Mg
2+
on phosphatase activity of an
DISCUSSION
2+
agricultural land. Variable concentrations of Mg were added
separately in each assay. Other parameters were the same as Soil is the source of nutrients for crop plants and also the
described in materials and methods. natural matrix for cultivation. Phosphorus is an important
element for all living organisms including crop plants. It
comes from soil and cyclic rotation of this element in
various forms is a common natural phenomenon. The
phosphorus cycle has been described in both ecological
Inhibitory effect was noted below or above this
and pedological literature. The differences between an
concentration (Figure 6). Phosphatase activity was
2+ unmanaged ecosystem and agro ecosystem have been
determined also by using various concentrations of Mg .
2+ also reported (Tivey, 1990). Plants acquire this element
The maximum activity was obtained at 5.0mM Mg
through the uptake of soluble organic or inorganic forms
concentration, above or below which phosphatase activity
of phosphate containing various materials. There are
showed decreasing tendency (Figure 7). The enzyme
2+ various organic and inorganic molecules that contain
activity in presenceof Ca ion increased with increasing
phosphorus, however mostly are unavailable to the plant
metal concentration and showed its optimum value at
2+ system. Therefore release of phosphorous from these
80.0mM Ca ion concentration. Following which further
2+ materials is greatly needed. The enzyme that plays
addition of Ca ions exhibited inhibitory effect (Figure 8).
2+ 2+ critical role in the process of solubilization of phosphate is
Mn and Fe produced precipitation in the reaction
phosphatases of multiple types. Phosphatases, which are
mixture even at very low concentrations.
 Banerjee et al. 417

1
Table 1. Phosphatase activity of various agricultural lands

No. of Soil Types of Units of

soil sample Type of soil pH Crop phosphatase activity
1. alluvium 7.05 rice 4.426
2. alluvium 6.56 wheat 4.098
3. alluvium 7.56 open 2.622
4. alluvium 6.76 rice 6.229
5. alluvium 7.73 wheat 6.393
6. alluvium 6.70 wheat 3.950
7. alluvium 7.58 rice 3.459
8. alluvium 6.78 rice 5.081
9. alluvium 6.71 wheat 2.786
10. alluvium 7.55 vegetable 3.934
1
Assay used 100.0 mg of soil sample; other conditions were the same as described in material and method section.
Open land was the post harvest land; other samples were collected when crops were under cultivation.

present in a soil, must be heterogeneous. The (Figure 1). The assay conditions were standardized and
0
phosphatases come from various macro and microscopic the optimum pH (5.0) and optimum temperature (37.0 C)
organisms. However, bacteria are the main sources of were determined (Figure 2, 3). The pH optimum indicates
phosphatases in a soil environment. There are several that the soil phosphatase released maximum phosphate
methods of estimation of soil phosphatases (Hoffmann, at acidic pH. However, it showed activity even at pH 11.0.
1968; Speir and Ross, 1978; Amador et al., 1997; Hysek This results from the heterogeneity of phosphatases
and Sarapatka, 1997). More recently HPLC, HPLC-UV present in the soil. Therefore, it is a mixture of acid,
detectors are commonly recommended for estimation of neutral, alkaline and several other kinds of phosphatases
phosphatase activity (Freeman et al., 2008). Method with that are associated in the soil sample. Using above
application of 4-methylumbelliferyl phosphate, where a conditions Km value of the enzyme was calculated. It
fluoro meter is used was also suggested (Agboola and produced an apparent Km value of 1.353mM. The Km
Afolayan, 2008). However, all signalized procedures used value is variable depending on type of substrate used in
sophisticated instruments and expensive chemicals. The the study. Phosphatase activity was also estimated in
present study was relied on synthetic chemical presence of various metal ions. Both divalent and
supplement, as a substrate (p-nitro phenyl phosphate). monovalent cations were used in the assays. The results
+ + 2+ 2+
As a matter of fact, various researchers used different showed that presence of Na , K , Mg , and Ca ,
substrates to estimate phosphatase activity (Hoffmann, enhanced enzyme activity. Similar results have been
1968; Tabatabai and Bremner, 1969; Schinner et al., reported previously (Dick and Tabatabai, 1983; Gabbrielli
1993; Tabatabai, 1994). Kroll et al. (1950) introduced the et al., 1989). The optimum concentrations were as
+ + 2+
use of artificial model substrate phenyl phosphate follows: 40.0mM for Na , 5.0mM for K , 5.0mM for Mg
2+
(Freeman et al., 2008). Presently, p-nitro phenyl and 80.0mM for Ca . The variations of phosphatase
phosphate is recommended as the most convenient activity in 10 soil samples might be caused by many
substrate for the assay of phosphatase activity reasons, like: variations of microbial content, amount of
(Schneider et al. 2000; Agboola and Afolayan, 2008; bio-molecules, amount of organic and inorganic
Huang et al., 2011). Some other substrates used by phosphates and others (Chen et al., 2004). The
investigators, are: di-sodium phenyl phosphate (Samuel phosphatase activity of a soil sample where wheat was
32
et al., 2010), [ P] labeled phosphate (Plummer et al., under cultivation showed highest activity (6.393 units). It
2005). p-nitro phenyl phosphate hydrolyses by the soil might be due to various reasons, such as soil pH, macro-
enzyme releases phosphate. The assay mixture included and micro elements present in soil , microbial biomass,
a buffer, 10.0mM substrate and 100.0 mg of soil sample. organic and inorganic phosphates of soil, fertilizer used,
Our results showed degradation of the substrate in the seasonal variations, as previously reported by various
assay condition as evident by the formation of yellow investigators ( Dick et al., 2000; Khodaeijaejoghan et al.,
coloration in alkaline condition, whereas the control set 2010; Mohammadi, 2011; Richardson et al., 2011; Zhou
demonstrated no such kind of chromogenic product. The et al., 2012). The phosphatase activity in wheat cultivated
assay was continued for many hours and it showed land is highly affected by the agricultural practices and
increased of color intensity as per the time of incubation acid phosphatase activity is positively correlated with
418 Agric. Sci. Res. J.

grain yield (Zhang et al., 2010; Wang et al., 2011). 128-131.

However these parameters were not considered in this Akanji MA, Adesokan AA (2005). Effect of repeated
study. Soil phosphatases solubilize inorganic and organic administration of aqueous extract of Enantia chloranta stem
bark on selected enzyme activities of rat liver. Nig. Soc.
phosphates and make them available for plant growth Exptl. Biol.17: 13-18.
and the soil becomes rich in soluble phosphate which Alvarez M, Huygens D, Diaz LM, Villanueva CA, Heyser W,
behaves like an index of soil fertility (Cookson, 2002; Boeckx P (2012).The spatial distribution of acid phosphatase
Roldan et al., 2005; Garcia-Ruiz et al., 2008; Alvarez et activity in ectomycorrhizal tissue depend on soil fertility and
al., 2012). Our study is the first report about phosphatase morphotype, and relates to host plant phosphorus uptake.
activity in soil samples from agricultural fields of the Plant. Cell. Environ. 35: 126-135.
district Burdwan, West Bengal, India. The alluvium soil Alvear M, Rosas A, Rouanet JL, Borie F (2005). Effects of three
was used in this investigation. This soil contains about soil tillage systems on some biological activities in an Ultisol
50% sand, 20% silt, 30% clay and smectite is the from southern chile. Soil. Tillage. Res. 82: 195-202.
Amador JA, Glucksman AM, Lyons JB, Gorres JH (1997).
dominant mineral in the clay followed by illite (Acharyya Spatial distribution of soil phosphatase activity within a
and Shah, 2007; Pal et al., 2009). Presented results riparian forest. Soil Sci. 162: 808-825.
clearly suggest that by using the simple assay procedure Anderson JM, Ingram JSI (1993). Tropical soil biology and
one may estimate the level of phoshatases in a soil fertility: A hand book of methods. London, CAB International.
sample that might then be co-related to assess the level Chen CR, Condron LM, Davis MR, Sherlock RR (2004). Effect
of solubilization of inorganic and organic phosphates in of plant species on microbial biomass phosphorous and
an agricultural land. It is also suggested that huge phosphatase activity in a range of grassland soils. Biol. Fertil.
number of soil samples can be analyzed within a short Soils. 40: 313-322.
time period following this procedure. Cohen P (1989). The structure and regulation of protein
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Cookson P (2002). Variation in phosphatase activity in soil: A
Conclusion case study. Agric. Sci. 7: 65-72.
Dick RP, Breakwell DP, Turco RF (1996). Soil enzyme activities
Multiple procedures of estimation of soil phosphatase and biodiversity measurements as integrative microbiological
activity are presented and available in literature. indicators. In: Methods for Assessing Soil Quality, vol.9. Soil
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Acknowledgement Gabbrielli R, Grossi L, Vergnano O (1989). The effects of nickel,
calcium and magnesium on the acid phosphatase activity of
Thanks to Mr. R. N. Majumder, Chairman of ‘Rahul two alyssum species. New Phytol. 111: 631-636.
Foundation’ for providing laboratory and other facilities. Garcia-Ruiz R, Ochoa V, Hinojosa MB, Carreira JA (2008).
Suitability of enzyme activities for the monitoring soil quality
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Durgapur College of Commerce and Science for Biochem. 40: 2137-2145.
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