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J. Biol. Indon. Vol 6, No.3 (2010)
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ISSN 0854-4425
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BIOLOGI
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Akreditasi: No 816/D/08/2009
Vol. 6, No. 3, Desember 2010
Zingiberaceae of the Ternate Island: Almost A Hundread Years After Beguin’s Collection
Marlina Ardiyani
293
Production of Acid Phosphatase in Bacillus sp. Isolated from Forest Soil of Gunung Salak
National Park
Maman Rahmansyah & I Made Sudiana
313
Eksplorasi Keanekaragaman Aktinomisetes Tanah Ternate Sebagai Sumber Antibiotik
Arif Nurkanto, Febrianti Listyaningsih, Heddy Julistiono & Andria Agusta
325
Komposisi Flora dan Struktur Hutan Alami Di Pulau Ternate, Maluku Utara
Edi Mirmanto
341
Penapisan Mikroba Laut Perombak Senyawa Nitril dan Protein yang Diisolasi Dari Sponge di
Perairan Ternate
Rini Riffiani & Nunik Sulistinah
353
Perbandingan Tiga Metode Transformasi Agrobacterium Untuk Pencarian Gen-gen Terkait
Toleransi Kekeringan Menggunakan Transposon Ac/Ds pada padi cv. Batutegi
E.S.Mulyaningsih, H.Aswidinnoor, D.Sopandie, P.B.F.Ouwerkerk, S. Nugroho,
&I.H. Slamet Loedin
367
Kajian Pakan Bersumber Energi Tinggi pada Pembentukkan Monyet Obes
Ria Oktarina, Sri Supraptini Mansjoer, Dewi Apri Astuti, Irma Herawati Suparto
& Dondin Sajuthi
383
BOGOR, INDONESIA
J. Biol. Indon. Vol 6, No. 3 (2010)
Jurnal Biologi Indonesia diterbitkan oleh Perhimpunan Biologi Indonesia.
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Editor Pengelola
Dr. Ibnu Maryanto
Dr. I Made Sudiana
Deby Arifiani, S.P., M.Sc
Dr. Izu Andry Fijridiyanto
Dewan Editor Ilmiah
Dr. Abinawanto, F MIPA UI
Dr. Achmad Farajalah, FMIPA IPB
Dr. Ambariyanto, F. Perikanan dan Kelautan UNDIP
Dr. Aswin Usup F. Pertanian Universitas Palangkaraya
Dr. Didik Widiyatmoko, PK Tumbuhan, Kebun Raya Cibodas-LIPI
Dr. Dwi Nugroho Wibowo, F. Biologi UNSOED
Dr. Parikesit, F. MIPA UNPAD
Prof. Dr. Mohd.Tajuddin Abdullah, Universiti Malaysia Sarawak Malaysia
Assoc. Prof. Monica Suleiman, Universiti Malaysia Sabah, Malaysia
Dr. Srihadi Agung priyono, F. Kedokteran Hewan IPB
Y. Surjadi MSc, Pusat Penelitian ICABIOGRAD
Drs. Suharjono, Pusat Penelitian Biologi-LIPI
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Jurnal ini telah diakreditasi ulang dengan nilai A berdasarkan SK Kepala LIPI 816/
D/2009 tanggal 28 Agustus 2009.
J. Biol. Indon. Vol 6, No.3 (2010)
KATA PENGANTAR
Jurnal Biologi Indonesia yang diterbitkan oleh PERHIMPUNAN BIOLOGI
INDONESIA edisi volume 6 nomer 3 tahun 2010 memuat 13 artikel lengkap. Penulis
pada edisi ini sangat beragam yaitu dari Departemen Kementerian Pertanian, IPB,
Puslit Biologi LIPI, Bioteknologi-LIPI dan Institute of Biology IBL Leiden University Netherlands. Topik yang dibahas pada edisi ini meliputi 5 topik dalam bidang
Botani, tiga topik tentang mikrobiologI, empat topik tentang zoologi dan satu topik
campuran yang mebahas bidang botani dan zoologi. Pada edisi ini yang menarik 6
makalh merupakan hasil kajian kawaasan pulau-pulau Kecil di Ternate Maluku
Utara. Selanjutnya artikel yang memuat serangga pengunjung bunga raflesia dapat
dipastikan merupakan artikel sangat jarang dijumpai sehubungan dengan populasi
bunganya yang sangat sulit diperoleh.
Editor
J. Biol. Indon. Vol 6, No. 3 (2010)
UCAPAN TERIMA KASIH
Jurnal Biologi Indonesia mengucapkan terima kasih dan penghargaan kepada
para pakar yang telah turut sebagai penelaah dalam Volume 6, No 3, Juni 2010:
Prof.Dr. Woro.A.Noerdjito Puslit Biologi-LIPI
Drs. M. Noerdjito, Puslit Biologi-LIPI
Dr Yulin Lestari F MIPA-IPB
Awal Riyanto, Puslit Biologi-LIPI
Drs. Roemantyo, Puslit Biologi-LIPI
Ir. Titi Juhaeti MSi, Puslit Biologi-LIPI
Dr. Nuril Hidayati, Puslit Biologi-LIPI
Edisi ini dibiayai oleh DIPA Puslit Biologi-LIPI 2010
J. Biol. Indon. Vol 6, No.3 (2010)
DAFTAR ISI
Zingiberaceae of the Ternate Island: Almost A Hundread Years After Beguin’s Collection
Marlina Ardiyani
293
Production of Acid Phosphatase in Bacillus sp. Isolated from Forest Soil of Gunung Salak
National Park
Maman Rahmansyah & I Made Sudiana
313
Eksplorasi Keanekaragaman Aktinomisetes Tanah Ternate Sebagai Sumber Antibiotik
Arif Nurkanto, Febrianti Listyaningsih, Heddy Julistiono & Andria Agusta
325
Komposisi Flora dan Struktur Hutan Alami Di Pulau Ternate, Maluku Utara
Edi Mirmanto
341
Penapisan Mikroba Laut Perombak Senyawa Nitril dan Protein yang Diisolasi Dari Sponge di
Perairan Ternate
Rini Riffiani & Nunik Sulistinah
353
Perbandingan Tiga Metode Transformasi Agrobacterium Untuk Pencarian Gen-gen Terkait
Toleransi Kekeringan Menggunakan Transposon Ac/Ds pada padi cv. Batutegi
E.S.Mulyaningsih, H.Aswidinnoor, D.Sopandie, P.B.F.Ouwerkerk, S. Nugroho,
&I.H. Slamet Loedin
367
Kajian Pakan Bersumber Energi Tinggi pada Pembentukkan Monyet Obes
Ria Oktarina, Sri Supraptini Mansjoer, Dewi Apri Astuti, Irma Herawati Suparto
& Dondin Sajuthi
383
Pengaruh Laju Eksploitasi Terhadap Keragaan Reproduktif Ikan Tembang (Sardinella gibbosa)
di Perairan Pesisir Jawa Barat
Yunizar Ernawati & Mohammad Mukhlis Kamal
393
Keragaman Genetik Amfibia Kodok (Rana nicobariensis) di Ecology Park, Cibinong
Berdasarkan Sekuen DNA dari Mitokondria d-loop
Dwi Astuti & Hellen Kurniati
405
Model Pemanfaatan Lahan Pulau Moti, Kota Ternate, Maluku: Suatu Analisis Tata Ruang
Berbasis Vegetasi
Roemantyo
415
Komunitas Serangga pada Bunga Rafflesia patma Blume (Rafflesiaceae) di Luar Habitat
Aslinya Kebun Raya Bogor Kota Bogor Provinsi Jawa Barat Indonesia
Sih Kahono, Sofi Mursidawati & Erniwati
429
J. Biol. Indon. Vol 6, No. 3 (2010)
Kajian Hubungan Tutupan Vegetasi dan Sebaran Burung di Pulau Moti, Ternate, Maluku Utara
Hetty I.P. Utaminingrum & Eko Sulistyadi
443
Pengujian 15 Genotipe Kedelai pada Kondisi Intensitas Cahaya 50% dan Penilaian Karakter
Tanaman Berdasarkan Fenotipnya
Gatut Wahyu Anggoro Susanto & Titik Sundari
459
Jurnal Biologi Indonesia 6 (3): 313-323 (2010)
Production of Acid Phosphatase in Bacillus sp. Isolated from Forest Soil of
Gunung Salak National Park
Maman Rahmansyah & I Made Sudiana
Research Center for Biology, Indonesian Institute of Sciences, Jl. Raya Jakarta Bogor km 46,
Cibinong Science Center, Cibinong 16911, E-mail: [email protected]
ABSTRAK
Produktivitas Fosfatase Asam pada Bacillus sp. yang Diisolasi dari Tanah Hutan Taman
Nasional Gunung Salak. Pada pengamatan ini dilakukan karakterisrik bakteri pelarut fosfat
yang diisolasi dari tanah hutan Taman Nasional Gunung Salak. Sebanyak 21 koloni hasil isolasi
diuji terhadap produktivitas enzim fosfatase berdasar pelarutan media mengandung fosfat.
Isolat yang terkuat melarutkan fosfat diidentifikasi sebagai Bacillus sp. Pada pengamatan
lanjutan terhadap strain teruji dilakukan penumbuhan pada media cair selama 90 jam inkubasi,
dan hasilnya ternyata mampu melarutkan fosfat inorganik (Pi) dari sumber trikalsium fosfat
(Ca-Pi) dan alumunium fosfat (Al-Pi) masing-masing pada kisaran 1,2 sampai 152 dan 0.8 sampai
25 mg.L-1; dan menunjukkan aktifitas enzim fosfomonoesterase antara 0.2 sampai 1.01 unit
pada media yang mengandung larutan para-nitrophenylphosphate sebagai media fosfat
organik (Po) artifisial. Konsumsi glukosa pada media yang diukur selama pertumbuhan sejalan
pula dengan produk ortofosfat sebagai akibat adanya aktifitas enzim fosfatase. Peningkatan
fosfatase juga sejalan dengan bertambahnya biomassa sel bakteri dan penambahan produk
asam glukonat. Penurunan pH dari 7 menjadi 5 diakibatkan peningkatan produk asam glukonat
di dalam media tumbuh. Bakteri pelarut fosfat yang berasal dari tanah hutan Taman Nasional
Gunung Salak dapat memproduksi fosfatase asam untuk memineralisasi sumber-sumber fosfat
menjadi sumber nutrisi yang siap digunakan oleh akar tumbuhan, dan itu merupakan prediksi
kuat untuk menjadikan isolat bakteri pelarut fosfat sebagai sumber bahan pupuk hayati.
Kata kunci: Bacillus sp., tanah hutan, fosfatase asam, Ca-Pi, Al-Pi.
INTRODUCTION
Ecology of microbial communities
can be attributed in part to understand
that these organisms have directly
effects on ecosystem processes (Beare
et al. 1995; Horner-Devine et al. 2004;
Fierer & Jackson 2006). Phosphate
solubilizing bacteria (PSB) commonly
found as adaptive bacteria (Glenn &
Mandelstam 1971) in most soils (Chonkar
& Taraedar 1984; Venkateswarlu et al.
1984). The population levels of
phosphobacteria were higher in the
rhizosphere soil, and able to produce
phytohormones and phosphatases
enzyme under in vitro conditions
(Ponmurugan & Gopi 2006). PSB was
able to convert the insoluble phosphates
into soluble forms by acidification,
chelating and exchange reactions, and
production of gluconic acid (Chen et al.
313
Rahmansyah & Sudiana
2006). The soluble forms also may
contribute to their stimulatory effect on
plant growth (Hameeda et al. 2006).
Correlation between PSB growth
and capacity of phosphatases enzyme
activity, as due to availability of
phosphorous content in the medium
identified by Barik & Purushothaman
(1998). In the other hand, there is
increasing evidence that phosphobacteria
improve plant caused to biosynthesis of
plant growth substances rather than their
action in releasing available phosphorous.
Subsequently, PSB is requisite for land
reclamation and restoration to improve
despoiled land since the genus has useful
producing plant growth promoter,
phosphorus availability to plant, and as
the competitor of plant bacterial pathogen.
In the preliminary work, the fastest
growing and the widest clearing zone
formation in selective agar media for the
isolate of PSB has deprived from forest
soil collected from Gunung Salak
National Park. In the existing
investigation, culture approach has been
studied whether the fine isolate and then
delineated as gram-positive bacterium,
named Bacillus sp. Some researcher find
out that the genus relatively has large
number of protein phosphatases (Cohen
1989; Villafranca et al. 1996), it can be
serine/threonine phosphatases which
have showed wide specificities, and also
tyrosine phosphatases.
The present work was undertaken
to compare the phosphate-repressible
enzyme formed by the Bacillus sp.
Activity of phosphatases enzymes
evaluated through the bacterial growth
within media containing tri-calcium
314
phosphate and aluminum phosphate as
inorganic phosphate, and in the bacterial
growth containing para-nitrophenyl
phosphate (pNpp) as organic (artificial)
phosphate. The results suggest that the
phosphate mineralization capability of the
PSB would appropriate to produce plant
nutritive value in subsequent work on
biofertilizer function. The presence of
PSB in Gunung Salak National Park is
not only important for ecosystem health
but also important genetic resources.
MATERIALS AND METHODS
Surface soil samples (up to 20 cm
depth) collected from fields as a bulk
samples of various places at forest floor
in Gunung Salak National Park; the
altitude is 900 m above sea level; situated
around S 06046’24.3" - 06046’49.8" and
E 106042’09.9" - 106042’25.9"; in June
2009. Composite soil sample of each soil
case mixed thoroughly, with then air
dried and passed throughout 100 mesh
sieves for studies.
In the preliminary work, based on
the broadest halozone screen ability
growing in selected media, the 21 isolates
recognized as PSB (Tabel 1). Selected
isolate (GS1) then identified through
analysis of gene 16S RNA using the
method of Pitcher et al. (1989), and
identified as Bacillus sp.
In the further quantification, the
strain cultured on liquid media contains
of phosphorous substances, and
incubated on a rotary shaker (180 rpm)
at 30ºC. For liquid media, mineral
phosphate (5 g·L-1 tri-calcium phosphate
or aluminum phosphate) were sterilized
Production of Acid Phosphatase in Bacillus sp.
separately and then mixed with the
autoclaved medium (10 g·L-1 glucose;
0.27 g·L-1 NH4·NO3; 0.2 g·L-1 KCl; 0.1
g · L -1M g S O 4· 7 H 2O ; 1 m g · L -1
MnSO4·6H2O; 1 mg·L-1 FeSO4·7H2O;
and 0.1 g·L-1 Yeast extract).
Glucose oxidation to become
gluconic acid is a major mechanism for
mineral phosphate solubilization. When
gluconic acid content and phosphomonoesterase activity in the culture medium
measured, the strain was prepared for
preculturing in LB medium (10 g·L-1
polypeptone, 5 g·L-1 NaCl, 5 g·L-1 Yeast
extract, 1 g·L-1 glucose). The precultured
was washed twice with 10 mM
potassium phosphate buffer (pH 7.5) and
resuspended in the same buffer and at
the same concentration as the original
pre-culture. The suspension (inoculum
mass 5%, v/v) was then transferred to
100 ml glucose minimal medium
containing 0.4% glucose and 21 mM
potassium phosphate buffer (pH 6.8); and
incubated on a rotary shaker (180 rpm)
at 30ºC. For determination of organic
acid produced, the strain was cultured in
GMS medium containing 10 g·L-1 glucose;
2 g·L -1 (NH 4 ) 2 SO 4 ; 0.3 g·L -1
MgCl2·6H2O; 1 mg·L-1 MnCl2·4H2O; 6
Tabel 1. PSB was common bacteria isolated from Gunung Salak National Park
No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Strain
GS1
GS2
GS3
GS4
GS5
GS6
GS7
GS8
GS9
GS10
GS11
GS12
GS13
GS14
GS15
GS16
GS17
GS18
GS19
GS20
GS21
a
Growth
rate
+++
++
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
b
Ca-P
dissolution
capacity
3.1
2.8
2.8
1.9
1.8
1.8
1.8
1.8
1.7
1.7
1,7
1,7
1,6
1.7
1.7
1,7
1,7
1,6
1.7
1.7
1,7
Remarks: +++ fast growing; ++ medium growing; + slow growing, a Colonies appear after 24 h (+++);
after 48 h (++); and after 72 h (+). b Ratio of clear zone areas per colony areas
315
Rahmansyah & Sudiana
mg·L-1 FeSO4·7H2O; 6 mg·L-1 NaMoO4;
thiamine (20 ì·L-1); tri-calcium phosphate
(20 mg·mL-1); and incubated on a rotary
shaker (180 rpm) at 37ºC.
Cells were collected by
centrifugation at different growth periods
in order to verifications the change in pH
and P-concentration in the medium.
Samples were centrifuged 6000 rpm for
10 minutes to receive clear solutions for
analysis. The P-concentration and pH
were determined throughout supernatants in each investigation. The Pconcentration estimated with ascorbic
acids methods. The 50 ~ 200 micro-liter
of the culture filtrate was mixed with 900
micro-liter of phosphorus mixed reagents,
keep at room temperature for 10 minutes.
The P-concentration was measured in
spectrophotometer at 880 nm. In order
to observe the effect of cultural
conditions for mineral phosphate
solubilizing, bacterial strains were cultured
at various insoluble phosphates (tricalcium phosphate, and aluminum
phosphate) at defined concentrations of
glucose conditions.
Five ml of a precultured solution
was inoculated in 50 ml of medium in a
250-ml Erlenmeyer flask. After
inoculation, the flasks were placed on a
shaker and the bacteria were grown at
370C for 24 h. The supernatant of each
culture was obtained by centrifugation at
10,000 rpm for 10 min. For the
experiment to determine gluconic acid, a
0.5 ml aliquot of the culture filtrate passed
through 0.2 ìm Whatman membrane
filter. The organic acids in filtrates were
identified by high-performance liquid
chromatography with a Thermo hypersil
316
C18 column (250 x 4.6mm). Organic
acids were monitored using a UV
detector at 220 nm. The mobile phase
consisted of 50 mM sodium phosphate
and 5 mM tetra-butyl-ammonium
hydrogen sulfate, pH 6.5 (95%), plus
acetonitrile (5%) and a flow rate of 0.25
ml·min-1.
The protein phosphatases activity
was tested by the ability to hydrolyze pnitrophenyl phosphate (pNpp) in a buffer
containing 50 mM Tris HCl (pH 7.2).
Phosphomonoesterase activity (PME)
was measured after incubation at 37ºC
(Margesin1996). Phosphatases activity
was determined by measurement of pnitrophenol in a spectrophotometer at a
wavelength of 400 nm. PME activity was
expressed in unit and defined as
micromoles nitrophenol produced by 1 ml
enzyme per hour.
Three replicate of flasks and tubes
were used for all in each treatment of
examination; and also from which the
other samples were collected at 4 to 96
hours in various sampling interval of
incubation; those were analyzed for the
parameters studies. Analysis of
correlation and some other statistical
requirement among mean values
calculated at various variables amongst
the confidence level of the degree of
freedom (Parker 1979).
RESULTS
The isolated bacterium had a
marked of insoluble phosphate solubilizing
activities, because the culture grows to
visualize clear zone upward in the region
of the colony forming after 3 days
Production of Acid Phosphatase in Bacillus sp.
incubation at 30ºC. In estimating the
efficacy of phosphate source utilization
by the PSB, result in phosphatases assay
of Bacillus sp. in liquid cultures containing
insoluble phosphate analytically
measured. Result of the studies have
correlated in the parameters measurement of Ca- and Al-phosphate metabolism as inorganic phosphate incorporation
to glucose reduction in the substrate, but
it does not proper to organic phosphate
of para-nitrophenylphosphate. The
highest production of phosphorus
substance during incubation was
measured, and it found in the medium
containing tri-calcium phosphate.
Gluconic acid production was increase
followed by pH reduction in the culture
medium (Table 2). This indicates the
evenness of phosphatic source utilization,
a prominent phenotypic characteristic of
phosphate solubilizing bacterial isolates
in relation to carbon augmentation.
Most bacterial (PSB) population
was stable after 24 hours incubation.
PSB live activity in culture was closely
followed by gluconic acid production as
caused by glucose incorporation. The
phenomenon gives sequence that the
option culture incubation for Bacillus sp.
should be after 36 hours and reaching
the limit action in 60 hours incubation
(Figure 1). That information becomes
useful to find out the maximum incubation
period for that bacterial in the propagation
culture for biotechnological purpose.
Kinetic potential of unit phosphatase
activities belonging to Bacillus sp. was
measured in tri-calcium phosphate and
alumunium phosphate solution culture
substance; and subsequently those
measurement acquiesced phosphate
quantity of culture in the yield of 150 and
25 mg·L -1 respectively, at 88 hours
incubation (Figure 2). The result assumed
the PSB effectively capable to increase
soluble phosphate in their habitat (in soil
of forest floor in Gunung Salak National
Park), because of phytase commonly
Table 2. Probability levels for statistical significance value in observation
Parameters studied of in-vitro culture of Bacillus
sp.
Correlation
1. Relationship of Ca-phosphate solubilization vs.
glucose reduction in the substrate:
y = -24.04x + 3832.7
(r = - 0.91 S)*
2. Relationship of Al-phosphate solubilization vs.
glucose reduction in the substrate:
y = -199.07x + 4890.1
(r = - 0.95 S)*
3. Relationship of Ca-phosphate to Al-phosphate
solubilization in the substrate:
y = 23x + 0.38
(r = 0.37 NS)*
4. Relationship of gluconic acid production to pH
level for the period of incubation:
y = -0.78x + 7.27
(r = - 0.97 S)**
Remarks: * (S) Significant and (NS) non-significant at p0.001 = 0.597; df = 25, with 26 samples **
(S) Significant at p0.001 = 0.872; df = 8, with 9 samples
317
Rahmansyah & Sudiana
have specify requirement to Ca 3+ and
Al2+ for its enzyme activity.
Phytate is the major component of
organics forms of P in soil (Richardson
1994). Phytase activity can be important
for stimulating growth under limited P in
soil, and supports to improve or transfer
the P-solubilizing trait to plant-growthpromoting bacteria (Rodrý´guez et al.
2006). Most phytases (myo-inositol
hexakisphosphate phosphohydrolases)
belong to high molecular weight acid
phosphatases, which has capability to
hydrolyze of phytic acid to be orthophosphate inorganic and phosphate esters
from lower mio-inositol. Phytic acid is a
phosphate ester that usually establishes
in soil, and it could bond the important
minerals and protein. Phosphomonoesterase (PME) examined as acid
phosphatase under Tris-HCl (pH 7.2)
buffer in the medium culture solution, but
only low activity of the enzyme detected
in the investigation.
DISCUSSION
Microbial communities in forest soil
play important role in maintaining global
ecosystem health. PSB is one of the
important soil microbes which stimulate
dissolution of less soluble-P into soluble
form and that available for plant growth.
The availability of P in soil is generally
low, and in forest ecosystem most of
phosphate in the form of organic bound
phosphate. Those organically bound
phosphates should be hydrolyzed by
phophatase enzyme produced by soil
microorganism. Soluble phosphate
released (H2 PO4 -, HPO4= and PO4=),
are then undergoes several biochemical
5
Gluc.103(g.L‐1)
4
G.Ac.101(mg.L‐1)
3
2
Biom.CFU.109
1
0
0
20
40
60
80
100
120
Incubation (hour)
Figure 1. Bacillus sp. ( ) increased which indicate apart of glucose ( ) consumed; and it
was converted into biomass followed by gluconic acid ( ) increasing in the media
318
Production of Acid Phosphatase in Bacillus sp.
transformation, or again bounded to other
soil minerals (Cunningham & Kuiack
1992). The availability of phosphate in
soil is greatly dependent on soil pH,
carbon content, redox potentials, and soil
physical structures. The Strain of GS1
identified as Bacillus sp., was able to
solubilize both Ca-P and Al-P. Their ability
to solubize those less soluble phosphate
may indicate that the strain is important
in stimulating phosphate dissolution. The
physiological mechanism by which P
dissolved is very complex.
Phosphobacteria (PSB) have been
found to produce some organic acids such
as monocarboxylic acid (acetic, formic),
monocarboxylic hydroxy (lactic, glucenic,
glycolic), monocarboxylic, ketoglucenic,
decarboxylic (oxalic, succinic),
dicarboxylic hydroxy (malic, maleic), and
tricarboxylic hydroxy (citric) acids in
180
Ca‐Pi 1.2
28
Al‐Pi p‐Npp
24
150
Phosphatases (Unit) order to solubilize inorganic phosphate
compounds (Lal 2002). Phosphobacteria
deprive from rhizosphere soils when was
tested under in-vitro condition on their
production capacity of growth regulators
and phosphatase enzyme (Ponmurugan
& Gopi 2006). Several soil bacteria
posses the ability to solubilizing insoluble
inorganic phosphate and make it available
to plants. The effect is generally due to
the production of organic acids by these
organisms, and also produce amino acids,
vitamins, and growth promoting
substances like indole acetic acid (IAA)
and gibberellic acid (GA3), which help in
better growth of plants (Richardson
2001;Gyaneshwar et al. 2002). The
results of enzyme activities, including soil
phosphatase activity, could be compared
not only with soil physical and chemical
properties, but also with other biological
1
20
0.8
120
16
0.6
90
12
0.4
60
8
30
0.2
4
y = ‐0.01x2 + 2.804x ‐ 16.59
R² = 0.944
0
0
0
0
25
50
75 100
y = ‐0.000x2 + 0.024x + 0.030
R² = 0.938
y = ‐0.002x2 + 0.453x + 1.370
R² = 0.939
0
25
50
75
100
0
25
50
75 100
Incubation (hour)
Figure 2. Trend of kinetic potential of phosphatase released with Bacillus sp. altering
phosphate inorganic (Ca- and Al-phosphate; Figure 2A & 2B), and phosphate organic
(p-nitrophenyl-phosphate; Figure 2C)
319
Rahmansyah & Sudiana
factors such as microbial biomass, the
level of adenosine triphosphate, etc.
(Chhonkar & Tarafdar 1984). Falih &
Wainwright (1996) found that the activity
of phosphatase enzymes increased when
a carbon source was added to the soil.
Evaluate to the result of experiment here,
inform that glucose incorporation proved
to cause increasing cell biomass of
Bacillus sp. in culture, and gluconic acid
increment (Figure 1); and in the different
way, the medium acidity is plunge to 5
from pH 7 as before during 96 hours
incubation.
Alkaline phosphatase of several
bacterial species has been investigated
by Landeweert et al. (2001), and the
genus have differ one from another in
certain respects. The genus seems in
general to share the property of being
repressed by phosphate inorganic in
Escherichia coli, Bacillus subtilis,
Pseudomonas fluorescens and
Staphylococcus aureus. PSB is capable
of solubilizing accumulated insoluble
phosphate compound sources in soil by
the production of organic acid, phenolic
compounds, protons, and siderophores.
According to Kelly et al. (1984)
investigation’s, maximum phosphates
(inorganic pyrophosphate) formed at 12
hours incubation in Bacillus sp. when
divalent metal Mn 2+ occurred in the
medium culture, and the inorganic
pyrophosphates activity found as
intracellular enzyme. In the other study
on the extracellular phosphates activity,
Nomoto et al. (1988) found the optimum
alkaline phosphate excreted into broth
culture fairly stable in pH 5 ~ 12.
Extracellular enzymes, including
phosphatases, are important for the
degradation of organic substances in the
soil for organic phosphate mineralization
(Hysek & Sarapatka 1998). The
functionalities of PSB communities differ
on the basis of phosphate sources present
in the culture medium in this investigation.
Phosphatases activities expressed
optimum in 75 to 100 hours incubation
(Figure 2) may cause the MnCl 2
containing in media culture.
8
10
7
7
= Ca-Pi unit-1
= Al-Pi
8
unit-1
= p-NPP unit-1
6
4
6
0
75
100
3
Gluconic acid
2
0
50
4
3
1
25
5
4
2
0
6
pH
5
2
Acidity level (pH)
Gluconic Acid (mg.L-1)
8
Carbon Used (10-4)
12
1
0
0
25
50
75
100
Incubation (hour)
Incubation (hour)
Figure 3. Incorporation of glucose along with phosphatases activities and carbon used by Bacillus
sp. (left), followed by increasing of gluconic acid and pH 7 reduce to pH 5 (right)
320
Production of Acid Phosphatase in Bacillus sp.
Gluconic acid is organic acid
compound arise from the oxidation of
glucose. It is produce by the fermentation
of glucose by bacteria, and in aqueous
solution at neutral pH the gluconic acid
forms the gluconate ion. Chen et al.
(2006) investigate the isolates of
phosphobacteria collected from
agriculture soil. The isolates belong to
genus of Bacillus, Rhodococcus,
Arthrobacter, Serratia, Chryseobacterium, Delftia, Gordonia and
Phyllobacterium were identified. In
other finding, four strains namely
Arthrobacter ureafaciens, Phyllobacterium myrsinacearum, Rhodococcus
erythropolis and Delftia sp. reported for
the first time as PSB after confirming
their capacity to solubilized considerable
amount of tricalcium phosphate in the
medium by secreting organic acids. Ten
isolates of Bacillus megaterium can do
not producing any gluconic acid in the
culture along 72 hours incubation. In the
contrary, 96 hour’s incubation of Bacillus
sp. in this experiment was able to produce
gluconic acid, and negatively correlated
with glucose incorporation but positively
correlated to cell biomass (Figure 3).
CONCLUSION
Isolate of Bacillus sp. demonstrate
clearly in solubilizing tri-calsiumphosphate
more than alumuniumphosphate;
Increasing cell population of Bacillus sp.
was following in gluconic acid yield as
due to glucose metabolism as carbon
source in culture; Bacillus sp. was
certainly characterized on the basis of
biochemical reaction, and as due to the
cultural performance in the medium
containing inorganic phosphate.
ACKNOWLEDGEMENTS
We acknowledge to the Directorate
General for Higher Education, Ministry
of National Education, Republic of
Indonesia, for the financial assistance
provided for this study in the year 2009.
Also, we are grateful to our acquaintance
for critically reading this manuscript
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Naskah dapat ditulis dalam bahasa Indonesia atau bahasa Inggris. Naskah disusun dengan urutan:
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