Berita Biologi, Volume 6, Nomor I, April 2002, Edisi Khusus

"Biodiversitas Taman Nasional Gunung Halimun (II) "

PHOSPHATASE ACTIVITY OF Bacillus sp. ISOLATED FROM FOREST SOIL

OF GUNUNG HALIMUN NATIONAL PARK
[Aktifitas Fosfatase Bacillus sp. yang Diisolasi dari Tanah Hutan
di Taman Nasional Gunung Halimun]

I Made Sudiana

Research Center for Biology, The Indonesian Institute of Science

Jl. Juanda 18 Bogor 16122, Telp 251-324006, Fax. (251) 325854
E-mail: [email protected]

ABSTRACT
Bacillus sp diisolasi dari tanah Taman Nasional Gunung Halimun. Dalam media tumbuh yang mengandung Ca3(PO4)2 bakteri membentuk
zona bening disekitar koloni. Glukosa digunakan dengan cepat selama kultivasi. Tinggi konsentrasi fosfat terlarut yang dibebaskan selama
fase pertumbuhan menunjukkan bahwa bakteri mampu memacu pelarutan Ca3(PO4)2. Selama fase pertumbuhan terjadi hidrolisa organik
fosfat (phenyl phosphate) menghasilkan phenol dan fosfat hal ini menunjukkan, bahwa Bacillus sp mampu menggunakan organik fosfat.
Selama kultivasi terjadi penurunan pH sejalan dengan pelarutan Ca3(PO4)2.

Kata kunci: Bacillus, aktifitas fosfat, Taman Nasional Gunung Halimun.

INTRODUCTION Phospholiplase-C has capacity to hydrolyze

Soil microflora play significant role in lecithin to 1,2 digleceraldehyde, choline
transformation of both either organic and inorganic phosphorine, many bacteria has capacity to produce
phosphorous (Alexander, 1961). Soil that enzyme. Many works have been devoted to
microorganisms produce phosphatase enzymes verify the kinetic and characteristic of
enable the microorganism hydrolyze phosphorous phosphomonoesterase (PME). Categorized as the
and becomes available for plant growth. To hydrolase group enzyme owing to its capacity to
determine the activity of phosphatase in soil some introduce H2O molecule substituting ester bonded
methods were proposed (Tabatabaii, 1982). phosphate. Mainly produced when soil limited in
Basically phosphate ester bound compound such as ionic phosphorous, of which excreted mainly by
p-naftilphosphate, phenylphosphate, p- microbes and soil animal but rare by plant (Schiner
nitrophenylphosphate, bis p-nitrophenylphosphate, et al, 1996). Bacterial cell P is mineralized quickly,
tris p-nitrophenylphosphate, and p-nitrophenyl phospholipid, and DNA are dephosphorelated in a
phosphorylcholin. Of which P-naftilphosphate, short period. P in microbial RNA is released more
phenylphosphate, p-nitrophenylphosphate is mostly slowly. Our present work aimed at studying the
used for determining phosphomonoesterase characteristic of PME produced by soil bacteria.
activity. The presence of those substances in
solution stimulate the microbe produce enzyme and
MATERIALS AND METHODS
finally hydrolize the ester bound phosphate, and
thereby the hydrolysis product is quantified. Isolation and identification
Phosphodiesterase hydrolyze nucleic acids and Bacillus sp was isolated from soil of Gunung
found in microorganism, plant and animal. The Halimun National Park using Pivoskaya medium
activity phosphotiestearse in soil was found in contained : 5 g I"1 Ca3(PO4)2 I"1 , 10 g I"1 glucose,
1976, but not much attention is given further (Dick 0.2 g I"1 NaCl, 0.2 g I"1 KC1, 0.0025 g 1"'
et al., 2000). Phospholipid is a component of cell MnSO4.H2O, 0.1 g I"1 MgSO4 7H2O, 0.0025 g 1"'
membrane of most organism containing cholin. FeSO4 7H2O, 0.5 g I"1 yeast extract. The isolated

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 Sudiana - Phosphatase Activity of Bacillus sp. from Forest Soil

bacterium was the n growth maintaining media NA PME activity is correlated with population
for further physiological studies. of phosphate solubilizing organism implying tha:
Enzyme assay most of the phosphate mineralization in soil is
Several methods were available for PME conducted by microorganism .
activity assay (Tabatabai, 1978; Dick and Tabatai,
pH
1986). Our present study used p-nitrophenyl
During culture growth pH was fluctuatec
phosphate as a model substrate and released p-
and they may affect the phosphate species in
nitrophenol was measured spectrophotometrically.
solution. At the beginning of incubation the pH o:
The basic principle of PME assay, P-nitrophenyl
culture decreased dramatically (Figure 2).
phosphate is added into supernatant culture and
incubated for 1 h at 37°C. P-nitrophenol released is
extracted with NaOH, and then measured
spectrophotometrically at 400 nm. Enzyme activity °oooooOOo°O
is expressed as umoll p-nitophenol released per 1
supernatant. PME assay also can be conducted
using phenylphosphate as enzim substrate, phenol
released are then quantified spectrophotometrically. 0 2 4 6 8 1U 12 14

Incubation time (d)

Glucose consumption
Amount of glucose utilized was Figure 2. Profile of pH during culture incubation ir
determined by dinitrosalycycilic method (Miller, Pivoskaya Medium
1959), and culture growth was measured
Release of proton have remarkabK
spectrophoto-metrically
decreased pH value (equation 3). pH has potentia
effect on the activity of enzymes by altering the
RESULT functional active site of enzyme, altering solubilin
The activity PME ase and the population of substrate, changing the adsorption rate, anc
of PSB in soil of GHNP is shown Figure 1 altering the substrate enzyme binding.
indicating that PME ase play significant role in
Growth of culture
accelerating phosphate mineralization in soil, and
Rapid growth of culture was observed ir
those enzymes are mainly produced by PSO
Pivoskaya medium. The carbon sources utilizec
(Tabatabai, 1978).
mainly from glucose. After glucose was limited,
cell growth was suppressed (Figure 3).

0 2 4 6 8 10
Log cell number, g-l.soil; microgram p-NPP/g-l.soil.h-l

Figure 1. PME-ase activity and the population of phos- Incubation time (d)
phate solubilizing bacteria (Rahmansyah et al.,
2000). Figure 3. Growth of culture

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 Berita Biologi, Volume 6, Nomor I, April 2002, Edisi Klnisus
"Biodiversitas Taman Nasional Gunung Halinmn (II) "

Glucose into ester phosphate bound. The activity of PMEase

Many author used glucose as main carbon was determined at pH 6.5, 38 C and incubated for
sources for phosphate solubilizing bacteria. 45 minutes. CaCl2 was added to stop enzyme
Glucose was easily used by microorganism as reaction and the activity is expressed as microgram
indicated by a rapid decrease of glucose during />-NP/ml enzyme/ incubation time. And the specific
cultivation. About 90 % of glucose was utilized activity is expressed as unit enzyme per mg protein.
(Figure 4). Glucose could be converted into reserve The Activity of PME-ase is shown in Figure 6.
materials or for electron donor in fermentation Maximum activity was observed when culture
processes for production of organic acids growth was maximum implying that enzyme
(Cosgrove, 1967). activity was linked to cell growth.

• * • • » •
±6
• • •
I 2
c
an
Incubation lime (d) =• 0

0 5 10 15
Figure 4. Glucose consumption during culture growth
Incubation Time (d)

Phosphate solubilization
Figure 6. Activity of PME-ase
Maximum P-released was after 2 days
incubation was 2003 |ig/L (Figure 5). The relation
between pH and P-dissolution is in consistent
DISCUSSION
Many factor affecting availability of
45
phosphorous to plant growth (Tabatabai, 1982).
_ 4
The presence of phosphate solubilizing organism
—. IS
~ 3
(Table 1) which have capacity to solubilize P than
it is necessary for its metabolism will enhance
•-•-•
mineralization of phosphate in soil. Soil of GHNP
contained a number of bacteria that are able to
05
solubilize Ca3(PO4 )2 which was dominated by
0 * T Bacillus sp is able to grow rapidly in Pivoskaya
0 2 4 6 8 10 12
medium and forming clearing zone implying they
hteubaiion time{d) are abfe to sofubiTi'ze inorganic phosphate
Figure 5. Phosphate released during culture growth (Alexander, 1961). Table 1 also indicates that PSO
is quite diverse in soil consisted of yeast, bacteria
Activity PME ase and fungi.
PME ase is a group of hydrolase enzyme
which responsible for addition of H2O molecule

51
Sudiana - Phosphatase Activity of Bacillus sp. from Forest Soil

Table 1. List of Phosphate Solubilizing organism ( Rao, 1982).

Microorganism Phosphate source

Bacteria Mineral
Bacillus sp., B. fulvifaciens, B. megaterium, B. circulans, B. subtilis, Tricalcium phosphate
B. mycoides, B. mesenlericus, B. fluorescence, B. circulans Calcium phosphate
Pseudomonas sp., P. Putida, P. liquifaciens, P. calcis, P. rathonia Iron phosphate
Escherichia freundii, E. intermedia hydroxyapatite
Xanthomonas spp. Fluoroapatit
Flavobacterium spp. Rock phosphate
Brevibacterium spp.
Serratia spp.
Alcaligenes spp.
Achromobacter spp. Organic
Aerobacter aerogenes Calcium phytate
Erwinia spp.
Nitrosomonas spp. Phytin
Thiobacillus thiooxidans Lecithin
Phenyl phosphate
Other organic phosphate
Fungi
Aspergillus sp.. A niger, A. jlavus, A. fumigatus, A. lerreus,
awamori
Penicillum sp., P. lilacinum, P. digitatum
Fusarium sp.. F. oksisporum
Curvularia lunata, Humicola sp., Sderotium rolfsii
Pythium sp., Acrothecium sp., Phoma sp.
Mortierella sp., Paecilomyces sp.
Cladosporium sp. Rhizoctonia sp.
Cunninghamella sp., Rhodotulla sp.
Candida sp.
Schwanniomyces occidentalis
Oideodendron sp.
Pseudogymnoascus sp.

Actinomycetes
Streptomycts sp.

One important aspect of soil pH is colloid, via cation exchange. When exchanged Ca
determining the oxidation state of phosphate in increased result in increases of basic saturation, and
soil. The fluctuation of pH is chemically controlled thus increase pH values (Tabatabai, 1982). P-
by several mechanisms (Kpomblekou and dissolution correlated with pH of culturing medium
Tabatabai, 1994). Lowering pH value in solution as was observed by (Rao, 1982). Oxidation of
a result of production of organic acids such as ammonium in soil caused soil acidification
citric, glutamic acids, and oxalic acids (reaction 1)
(Kpomblekou and Tabatabai, 1994). Those organic 22 NH4+ + 37 O2 +4 CO2 + HCO3 --> C5H7N02+
acids bound with Ca2+, Mg2+, Fe3+ and Al3+ (Rao, 21NO 3 + 20H 2 O + 42H + (1)
1982) result in released of ionic P. Released P will
a part bound to Fe, Al and a part will be water Beside soil acidity, several mechanisms are
soluble and thus available for plant. Released Ca, a reported to affect phosphorous dissolution.
part will bound with organic acids forming organic Cosgrove, 1967 proposed that proton (H+) released
complex, and the remaining will bound with soil from cytoplasm to outer membrane as an exchange

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 Berita Biologi, Volume 6, Nomor I, April 2002, Edisi Khusus
"Biodiversitas Taman Nasional Gunung Halimun (II) "

of NH4+ absorption, or releasing H+ by ATP-ase environment. Thereby activity of acid PME is high
utilizing energy derived from hydrolysis of ATP. in acid soil and alkaline PME is high in alkaline
Other mechanism could be direct solubilization of environment.
P took place in cell surface and produced soluble P The finding of phytin derivatives as well as
in the form of H2PO4" and HPO42" (Joner et al, as phytin itself in the soil organic fraction suggest
2000) Carbon dioxide produced during aerobic that a breakdown of the inositol hexaphosphate
respiration may also affect P-dissolution follow take place. The enzyme phytase liberates phosphate
reaction 2 and 3 from phytic acid or its calcium magnesium salt,
CO2 + H2O — -»HCO3- + H+ (2) phytin, with the accumulation of inositol.
Ca3(PO4)2 + HCO3- --> 3 Ca(HCO3)2 + 2 PO43- (3) Phytase activity is widespread and is
enhanced by carbonaceus materials that increase
Those reactions took place in plant
the size of the microbial population. Species of
rhizosphere and released P is in excess than its
Aspergillus, Penicillium, Rhizopus,
necessary for bacterial growth (Rao, 1982).
Cunninghamella, Arthrobacter, and Bacillus can
Most of soil composed of more of organic-P than
synthesize the enzyme (Rahmasyah et al., 2000).
that of inorganic-P. Mineralization of organic-P
Yet, despite the great phytase potential, phytin is
executed by a complex of phosphatase enzyme that
not readily metabolized in soil (Kpomblekou and
has capacity to hydrolyze ester phosphate and
Tabatabai, 1994). The hydrolyses apparently, is not
found both and intra-and extracelluler. Most of this
limited by phytase producing capacity of
enzyme is produced when the soil contain less of
microorganism, which is appreciable, but by the
soluble phosphorous. Phosphatase is produced by
small amount of phytic acid in the soil solution.
microorganism and plant, but dominantly by
The fact that phytate-phosphorous is relatively
microorganism. Tabataibai (1982) proposed 5
unavailable to crop growing in acid soils where the
group of enzymes belonged to phosphatase:
substrate is bound into iron and aluminum
Phosphomonoesterase include Phytase, glycerin
complexes.
phosphatase, nucleotidase and sugar phosphatase.
If the major reserves of P in humus were
Phosphodiesterase such as nuclease and
phytin and nucleic acids, it would seem that the
phospholipase. Phosphotriesterase. Poliphosphatase
later is the more active fraction in mobilization and
include ATPase and pirofostase inorganic.
immobilization reaction because phytin is relatively
Phosphoamidase which breakdown phosphorous
resistance to decay while nucleic acids are highly
and nitrogen bound.
susceptible to microbial attack.
Phytic acids as the major component of soil
Attempt has been made to exploit
organic-P hydrolyzed enzymaticaly by
microbiological phosphate release. When
phosphomonoesterase especially phytase (Michael
microorganism known as phosphobacteria is
et al., 1994). Based on its optimum pH activity and
inoculated into soil or on the seeds of several crop
substrate specificity phosphomonoesterase enzyme
plants, there allegedly is remarkable increase in
is grouped into acid and alkaline PME. Acid PME
yield and in P-content of the crop harvested.
found in rhizosphere and plant tissue, in contrast
alkaline PME only produced by microbes and soil Oxidation-Reduction Reaction
animal. Fungi and bacteria produced both acid and P like N, may exist in a number of oxidation
alkaline PME (Joner et al., 2000). The activity of states ranging from the -3 of Phosphine, PH3, to
intracellular PME is higher in alkaline environment the oxidized state, +5, of orthophosphate
than that of neutral and acid. In contrast higher (Kpomblekou and Tabatabai (1994). In contrast to
activity of extra-cellular PME is observed in acidic nitrogen little attention has been given to the

53
Sudiana - Phosphatase Activity of Bacillus sp. from Forest Soil

inorganic transformation of P, but there is some phosphite and hypophosphite from orthophosphate
evidence for biologically catalyzed changes in the (Rao, 1982). The process seems analogous
oxidation state of this element too (Willet, 1989). biochemically to denitrification or to bacterial
Biological oxidation of reduced P conversion of sulfate to sulfide. It is unlikely that
compound was demonstrated by Alexander, (1961) the reduction takes place in well-aerated
who noted that phosphite added to soil disappear environments. The mineralization and
with a corresponding increase in the concentration immobilization of P are related to the analogous
of phosphate, reaction 5. reaction of N (Dick and Tabatabai, 1987). As a
rule, P release is more rapid under condition
HPO3" ---> HPCV (5)
favoring ammonification (Tisdale et al., 1985).
The conversion is brought about Thus, highly significant correlation is observed
microbiologically since the reaction is eliminated between the rate of N and P conversion to
upon the addition of a biological inhibitor such as inorganic form, the nitrogen mineralized being
toluene. A number of heterothrophic bacteria, from 8 to 15 times amount of P made available.
fungi, and actinomycetes utilize phospohite within There is also correlation between and P
the cell to organic phosphate compounds. Bacteria mineralization, a ratio of C:N:P mineralized
utilize phosphite as sole P source in culture media microbiologically at the equilibrium condition is
and oxidize the phosphite within the cell to organic similar to the ratios of these there element in humus
phosphate compounds. Bacteria utilize Phosphate (Garcia-Gil et al., 2000).
in preference to phosphite so that, in media
containing both anions, the former disappears first. ACKNOWLEGMENT
There is no evidence that the oxidation is capable We would to express our sincerely gratitude
of providing energy for the development of to JICA for research grant and management staff of
chemoautothrophic bacteria. Gunung Halimun National Park for good research
The possibility of the reverse process, a collaboration.
reductive pathway, has received somewhat more
attention. When a soil sample is incubated
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