The Effect Of Phosphate Supply On Phosphatase Activity Of Phosphate Solubilizing Microorganisms.
THE EFFECT OF PHOSPHATE SUPPLY ON PHOSPHATASE ACTIVITY OF
PHOSPHATE SOLUBILIZING MICROORGANISMS
BETTY N. FITRIATIN, BENNY JOY*, AND TOTO SUBROTO**
*Department of Soil Science Faculty of Agriculture Padjadjaran University
** Department of Chemistry Faculty of Mathematics and Natural Sciences
Padjadjaran University
E-mail : fitriatin@yahoo.com
ABSTRACT
The phosphate solubilizing microorganisms has capable to pro
duce
extracelluler enzyme, i.e. group of phosphatase enzyme which able to mineralized of
organic P to inorganic P. The objective of this experiment was to examine of
phosphatase enzyme activity from phosphate solubilizing microorganism (bacteria and
fungi) i.e. Pseudomonas mallei, Bacillus subtilis, Aspergilus niger and Penicillium sp.
affected by phosphate supply o( rganic phosphate/ phytic acid and inorganic
phosphate/KH2PO4) in medium.
The result of experiment showed that increasing of organic P (phytic acid )
and inorganic P (KH2PO4) affect phosphatase enzyme activity of phosphate solubilizing
microorganisms and the amount of dissolve P. Furthermore, increasing of organic P
substrate increase phosphatase activity; on the contrary, addition to inorganic P decrease
phosphatase activity. Phosphatase activity of fungi (Aspergilus niger and Penicillium
sp.) were higher than bacteria (Pseudomonas mallei, Bacillus subtilis).
Key words : phosphatase activity, phosphate supply, phospahate solubilizing
microorganisms
INTRODUCTION
Many soil bacteria and fungi have the ability to solubilize P and make it
available to growing plants (Whitelaw, 2000). Microorganisms are central to the soil P
cycle and play a significant role in mediating the transfer of P between different
inorganic and organic soil P fractions, subsequently releasing available P for plant
acquisition. There are two aspects in microbial P solubilization: 1) P released by
solubilization processes (Rodriguez and Fraga, 1999), and 2) P released from
accumulated P in biomass of microorganisms (Oehl, 2001). Inorganic phosphate
solubilizing microorganisms constitute various portions of the soil microbial population
and vary from soil to soil (Yadaf, and Tarafdar. 2003; Kucey et al., 1989). The numbers
Inter national Seminar Biotechnology
1
of PSM are more important in rhizosphere than non-rhizosphere soil (Kucey et al.,
1989). PSM occur in both fertile and P-deficient soils and the fastest initial rates of P
incorporation were observed in P-deficient soils (Oehl, 2001).
Penicillium and Aspergillus niger. were isolated from soil that they are dominant
P solubilizing fungi found in rhizosphere. Pseudomonas mallei and Bacillus subtilis are
phosphate solubilizing bacteria that were isolated from rhizosphere (Fitriatin, 2006).
Furthermore, their ability to mineralize organic P need investigate more. They has
capable to produce extracelluler enzyme, i.e. group of phosphatase enzyme which able
to mineralized of organic P to inorganic P so that prepare high P for plant. Due to the
low availability of inorganic P in soil, the organic P mainly contributes to plant nutrition
and to microbial uptake through its mineralization and subsequent release of inorganic
P. P mineralization rate depends on microbial activity and on he
t activity of
phosphatases (Saparatka, 2003). Consequently, the release of inorganic P through the
destruction of the organic matter is usually divided in biological mineralization and
biochemical mineralization. Biological mineralization involves the release of inorganic
P as a consequence of the carbon oxidation and the microbial growth and turnover,
while in biochemical mineralization the release of inorganic P, independent of microbial
respiration, is controlled by the supply and need for P and involves the hydrolysis of
ester-phosphates by extra-cellular hydrolytic enzymes (phosphatases) both free in
solution and stabilised by sorption to the colloidal fraction (McGill and Cole, 1981).
There are several soil phosphatases (Saparatka, 2003; Cookson, 2002) and the
most commonly determined are: phosphomonoesterases, phosphodiesterases and
phytases. Phosphomonoesterases act on phosphate monoesters and according to their
optimum pH are divided in acid and alkaline phosphomonoesterases. Both are adaptive
enzymes: acid phosphomonoesterase predominates in acid soils while alkaline
phosphomonoesterase predominates in neutral and basic soils (George, et al., 2002).
Activity of phosphatase enzyme is affected by some factors, i.e. the amount
and kind of substrate (organic P), inorganic P, pH, temperature, material of inhibitor
and activator, concentration of enzyme and product, and also the kind of solvent
used.(Saparatka, 2003 and Saparatka, et al., 2004). Besides, soil phosphatase activity
also affected by properties of chemical and physical of soil i.e. soil type, organic matter
Inter national Seminar Biotechnology
2
content, total N content, C/N ratio and total P content (Djordjevic et.al. 2003). The
kind of organic phosphorous of medium and pH affect phosphatase activity of soil
microorganisms (Fitriatin, et al.,2008).
The objective of this experiment was to examine of phosphatase enzyme
activity
from phosphate
solubilizing
microorganism (bacteria and
fungi)
i.e.
Pseudomonas mallei, Bacillus subtilis, Aspergilus niger and Penicillium sp. affected by
phosphate supply (organic phosphate/ phytic acid and inorganic phosphate/KH2PO4) in
medium.
MATERIALS AND METHODS
Some concentration of inorganic P and organic P were used to find out effect
phosphate supply on activity of phosphatase enzyme and phosphate solvent in medium
which cultured of phosphate solubilizing microorganisms (bacteria and fungi) i.e.
Pseudomonas mallei., Bacillus subtilis, Aspergillus niger and Penicillium sp.
The concentration of inorganic P (KH2PO4) which tested consisted of: :
P0 = 0 mg L-1
P1 = 10 mg L-1
P2 = 20 mg L-1
P3 = 30 mg L-1
The concentration of organic P (phytic acid) which tested consisted of :
S1 = 5 mM
S2 = 10 mM
S3 = 15 mM
Phosphatase activity and dissolve P were observed on 5 days after incubation.
Acid phosphatase activity was measured spectrophotometrically by monitoring the
release of para-nitrophenol from para-nitrophenyl phosphate (PNPP) at 400 nm (Eivazi
and Tabatabai, 1977 in Schinner et al., 1996).
RESULTS AND DISCUSSION
Result analysis of phosphatase activity and dissolved P due to the influence of
phosphate supply can be seen in the following table:
Inter national Seminar Biotechnology
3
Table 1. The Effecf of phosphate (P) and phytic acid (S) on phosphatase activity of
phosphate solubilizing microorganisms in medium
P
supply
P0S1
P0S2
P0S3
P1S1
P1S2
P1S3
P2S1
P2S2
P2S3
P3S1
P3S2
P3S3
Pennicillium
A .niger
P.mallei
B.subtilis
phosphatase activity (µg p NP/ml/hour)
7,79
3,67
10,15
5,11
1,63
0,34
1,14
3,86
1,54
0,96
5,15
0,93
e*)
c
f
d
b
a
ab
c
b
ab
d
ab
14,80
19,71
10,38
8,03
0,15
0,38
9,13
7,66
3,60
2,40
7,61
3,20
g
h
f
d
a
a
e
d
c
b
d
c
0,34
0,02
0,43
0,38
2,52
0,83
0,92
0,33
1,45
3,46
0,64
0,60
b
a
d
c
j
g
h
b
i
k
f
e
0,06
1,27
0,73
0,65
1,10
0,34
2,44
0,78
0,16
0,11
0,32
1,27
a
h
f
e
g
d
i
f
c
b
d
h
Note : *) Figures in each column followed by the same letter(s) are not significantly
different (P
PHOSPHATE SOLUBILIZING MICROORGANISMS
BETTY N. FITRIATIN, BENNY JOY*, AND TOTO SUBROTO**
*Department of Soil Science Faculty of Agriculture Padjadjaran University
** Department of Chemistry Faculty of Mathematics and Natural Sciences
Padjadjaran University
E-mail : fitriatin@yahoo.com
ABSTRACT
The phosphate solubilizing microorganisms has capable to pro
duce
extracelluler enzyme, i.e. group of phosphatase enzyme which able to mineralized of
organic P to inorganic P. The objective of this experiment was to examine of
phosphatase enzyme activity from phosphate solubilizing microorganism (bacteria and
fungi) i.e. Pseudomonas mallei, Bacillus subtilis, Aspergilus niger and Penicillium sp.
affected by phosphate supply o( rganic phosphate/ phytic acid and inorganic
phosphate/KH2PO4) in medium.
The result of experiment showed that increasing of organic P (phytic acid )
and inorganic P (KH2PO4) affect phosphatase enzyme activity of phosphate solubilizing
microorganisms and the amount of dissolve P. Furthermore, increasing of organic P
substrate increase phosphatase activity; on the contrary, addition to inorganic P decrease
phosphatase activity. Phosphatase activity of fungi (Aspergilus niger and Penicillium
sp.) were higher than bacteria (Pseudomonas mallei, Bacillus subtilis).
Key words : phosphatase activity, phosphate supply, phospahate solubilizing
microorganisms
INTRODUCTION
Many soil bacteria and fungi have the ability to solubilize P and make it
available to growing plants (Whitelaw, 2000). Microorganisms are central to the soil P
cycle and play a significant role in mediating the transfer of P between different
inorganic and organic soil P fractions, subsequently releasing available P for plant
acquisition. There are two aspects in microbial P solubilization: 1) P released by
solubilization processes (Rodriguez and Fraga, 1999), and 2) P released from
accumulated P in biomass of microorganisms (Oehl, 2001). Inorganic phosphate
solubilizing microorganisms constitute various portions of the soil microbial population
and vary from soil to soil (Yadaf, and Tarafdar. 2003; Kucey et al., 1989). The numbers
Inter national Seminar Biotechnology
1
of PSM are more important in rhizosphere than non-rhizosphere soil (Kucey et al.,
1989). PSM occur in both fertile and P-deficient soils and the fastest initial rates of P
incorporation were observed in P-deficient soils (Oehl, 2001).
Penicillium and Aspergillus niger. were isolated from soil that they are dominant
P solubilizing fungi found in rhizosphere. Pseudomonas mallei and Bacillus subtilis are
phosphate solubilizing bacteria that were isolated from rhizosphere (Fitriatin, 2006).
Furthermore, their ability to mineralize organic P need investigate more. They has
capable to produce extracelluler enzyme, i.e. group of phosphatase enzyme which able
to mineralized of organic P to inorganic P so that prepare high P for plant. Due to the
low availability of inorganic P in soil, the organic P mainly contributes to plant nutrition
and to microbial uptake through its mineralization and subsequent release of inorganic
P. P mineralization rate depends on microbial activity and on he
t activity of
phosphatases (Saparatka, 2003). Consequently, the release of inorganic P through the
destruction of the organic matter is usually divided in biological mineralization and
biochemical mineralization. Biological mineralization involves the release of inorganic
P as a consequence of the carbon oxidation and the microbial growth and turnover,
while in biochemical mineralization the release of inorganic P, independent of microbial
respiration, is controlled by the supply and need for P and involves the hydrolysis of
ester-phosphates by extra-cellular hydrolytic enzymes (phosphatases) both free in
solution and stabilised by sorption to the colloidal fraction (McGill and Cole, 1981).
There are several soil phosphatases (Saparatka, 2003; Cookson, 2002) and the
most commonly determined are: phosphomonoesterases, phosphodiesterases and
phytases. Phosphomonoesterases act on phosphate monoesters and according to their
optimum pH are divided in acid and alkaline phosphomonoesterases. Both are adaptive
enzymes: acid phosphomonoesterase predominates in acid soils while alkaline
phosphomonoesterase predominates in neutral and basic soils (George, et al., 2002).
Activity of phosphatase enzyme is affected by some factors, i.e. the amount
and kind of substrate (organic P), inorganic P, pH, temperature, material of inhibitor
and activator, concentration of enzyme and product, and also the kind of solvent
used.(Saparatka, 2003 and Saparatka, et al., 2004). Besides, soil phosphatase activity
also affected by properties of chemical and physical of soil i.e. soil type, organic matter
Inter national Seminar Biotechnology
2
content, total N content, C/N ratio and total P content (Djordjevic et.al. 2003). The
kind of organic phosphorous of medium and pH affect phosphatase activity of soil
microorganisms (Fitriatin, et al.,2008).
The objective of this experiment was to examine of phosphatase enzyme
activity
from phosphate
solubilizing
microorganism (bacteria and
fungi)
i.e.
Pseudomonas mallei, Bacillus subtilis, Aspergilus niger and Penicillium sp. affected by
phosphate supply (organic phosphate/ phytic acid and inorganic phosphate/KH2PO4) in
medium.
MATERIALS AND METHODS
Some concentration of inorganic P and organic P were used to find out effect
phosphate supply on activity of phosphatase enzyme and phosphate solvent in medium
which cultured of phosphate solubilizing microorganisms (bacteria and fungi) i.e.
Pseudomonas mallei., Bacillus subtilis, Aspergillus niger and Penicillium sp.
The concentration of inorganic P (KH2PO4) which tested consisted of: :
P0 = 0 mg L-1
P1 = 10 mg L-1
P2 = 20 mg L-1
P3 = 30 mg L-1
The concentration of organic P (phytic acid) which tested consisted of :
S1 = 5 mM
S2 = 10 mM
S3 = 15 mM
Phosphatase activity and dissolve P were observed on 5 days after incubation.
Acid phosphatase activity was measured spectrophotometrically by monitoring the
release of para-nitrophenol from para-nitrophenyl phosphate (PNPP) at 400 nm (Eivazi
and Tabatabai, 1977 in Schinner et al., 1996).
RESULTS AND DISCUSSION
Result analysis of phosphatase activity and dissolved P due to the influence of
phosphate supply can be seen in the following table:
Inter national Seminar Biotechnology
3
Table 1. The Effecf of phosphate (P) and phytic acid (S) on phosphatase activity of
phosphate solubilizing microorganisms in medium
P
supply
P0S1
P0S2
P0S3
P1S1
P1S2
P1S3
P2S1
P2S2
P2S3
P3S1
P3S2
P3S3
Pennicillium
A .niger
P.mallei
B.subtilis
phosphatase activity (µg p NP/ml/hour)
7,79
3,67
10,15
5,11
1,63
0,34
1,14
3,86
1,54
0,96
5,15
0,93
e*)
c
f
d
b
a
ab
c
b
ab
d
ab
14,80
19,71
10,38
8,03
0,15
0,38
9,13
7,66
3,60
2,40
7,61
3,20
g
h
f
d
a
a
e
d
c
b
d
c
0,34
0,02
0,43
0,38
2,52
0,83
0,92
0,33
1,45
3,46
0,64
0,60
b
a
d
c
j
g
h
b
i
k
f
e
0,06
1,27
0,73
0,65
1,10
0,34
2,44
0,78
0,16
0,11
0,32
1,27
a
h
f
e
g
d
i
f
c
b
d
h
Note : *) Figures in each column followed by the same letter(s) are not significantly
different (P