JURNAL BIOLOGI INDONESIA

J. Biol. Indon. Vol 6, No.3 (2010) ISSN 0854-4425 ISSN 0854-4425

JURNAL JURNAL BIOLOGI BIOLOGI INDONESIA INDONESIA 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. Jurnal ini memuat hasil penelitian ataupun kajian yang berkaitan dengan masalah biologi yang diterbitkan secara berkala dua kali setahun (Juni dan Desember). 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 Dr. Tri Widianto, Pusat Penelitian Limnologi-LIPI Dr. Witjaksono Pusat Penelitian Biologi-LIPI Alamat Redaksi

Sekretariat Oscar efendi SSi MSi d/a Pusat Penelitian Biologi - LIPI Jl. Ir. H. Juanda No. 18, Bogor 16002 , Telp. (021) 8765056 Fax. (021) 8765068 Email : [email protected]; [email protected] Website : http://biologi.or.id 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


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


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


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


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


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


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 REFERENCES Barik, SK. & CS. Purushothaman. 1998. Phosphatase activity of two strain of bacteria on orthophosphate enrichment. Proc. Natl. Frontiers in Appl. Environ. Microbial. Dec.11-13, SES, CUSAT. Cochin. pp.165-170. Beare, MH., DC. Coleman, DA. Crossley Jr., PF. Hendrix & EP. Odum. 1995. A hierarchical approach to evaluating the significance of soil biodiversity to biogeochemical cycling. Plant and Soil. 170: 5-22. Chen,YP., PD. Rekha, AB. Arun, FT. Shen, WA. Lai & CC. Young. 2006. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. App. Soil Ecol. 34:33-41. Chonkar, PK & JC. Tarafdar. 1984. Accumulation of phosphatase in soils. J. Indian Soc. Soil Sci. 32: 266–272.

321


Cohen, P. 1989. The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58: 453-508. Cunningham, JE., & C. Kuiack. 1992. Production of citric and oxalic acid and solubilization of calcium phosphate by Penicillium bilail. Appl. Environ. Microbial. 58:1451-1458. Falih, AMK. & TM. Wainwrigh. 1996. Microbial and enzyme activity in soils amended with natural source and easily available carbon. Biol Fertil Soils. 21: 177–183. Fierer, N., & RB. Jackson. 2006. The diversity and biogeography of soil bacterial communities. PNAS 103 (3): 626–631. Glenn, AR. & J. Mandelstam. 1971. Sporulation in Bacillus subtilis 168, comparison of alkaline phosphatase from sporulating and vegetative cells. Biochem. J. 123:129-138. Gyaneshwar, P., GN. Kumar, LJ. Parekh & PS. Poole. 2002. Role of soil microorganism in improving P nutrition of Plants. Plant soil. 245: 83-93. Hameeda, B., OP. Rupela, G. Reddy & K. Satyavani. 2006. Application of plant growth promoting bacteria associated with compost and macrofauna for growth promotion of Pearl Millet (Pennisetum glaucum L.). Biol. Fertil. Soils. 43: 221-227. Horner-Devine, MC., M. Lage, JB. Hughes & BJM. Bohannan. 2004. A taxa-area relationship for bacteria. Nature 432: 750-753.

322

Hysek, J. & B.Sarapatka. 1998. Relationship between phosphatase active bacteria and phosphatase activities in forest soils. Biol Fertil Soils. 26: 112–115. Kelly, CT., AM Nash & MW. Fogarty. 1984. Effect of manganese on alkaline phosphatase production in Bacillus sp. RK11. Applied Microbial Biotechnology. 19: 6166. Lal, L. 2002. Phosphatic biofertilizers. Agrotechnology Publication Academy, Udaipur, India, 224p. Landeweert, R., E. Hoffland, RD Finlay, TW. Kuyper, N. Van Breemen. 2001. Linking plants to rocks, ectomycorrhizal fungi mobilize nutrients from minerals. Trends Ecol. Evol. 16: 248-253. Margesin, R. 1996. Enzymes Involved in Phosphorus Metabolism: Acid and Alkaline Phosphomonoesterase Activity with the Substrate pNitrophenyl Phosphate. In: Eds. F. Schinner, R. Ohlinger, E. Kandeler & R. Margesin. Methods in Soil Biology. Springer-Verlag, Berlin Heidelberg. p. 213-217. Nomoto, M., M. Ohsawa, H. Wang, C. Chen & K.Yeh. 1988. Purification and characterization of extracellular alkaline phosphatase from an alkalophilic bacterium. Agri. Biol.Chem.52(7): 16431647. Parker, RE.1979. Introductory Statistics for Biology. Studies in Biology No. 43. Edward Arnold Ltd. London. Pitcher, DG. & RJ. Owen. 1989. Rapid extraction of bacterial genomic


DNA with guanidium thiocyanate. Lett.appl. Microbial. 8 : 151-156. Ponmurugan, P. & C. Gopi. 2006. In vitro production of growth regulators and phosphatase activity by phosphate solubilizing bacteria. Afri. J. Biotech. 5(4): 348-350. Richardson, AE. 1994. Soil microorganisms and phosphorous availability. In Eds. CE Pankhurst, BM Doube and VVSR Gupta . Soil Biota: Management in Sustainable Farming Systems. . pp. 50–62. CSIRO, Victoria, Australia. Rodrý´guez, H., R. Fraga, T. Gonzalez & Y. Bashan. 2006. Genetics of phosphate solubilization and its potential applications for improving

plant growth-promoting bacteria. Plant and Soil. 287:15–21. Richardson, AE. 2001. Prospect for using soil microorganisms to improve the quisition of phosphorus by plants. Aust. J. Plant Physiol. 58: 797906. Venkateswarlu, B., AV. Rao & P. Raina. 1984. Evaluation of phosphorus solubilization by microorganisms isolated from arid soils. J. Indian Soc. Soil Sci. 32: 273–277. Villafranca, JE., CR. Kissinger & HE. Parge.1996. Protein serine/ threonine phosphatases. Curr. Opin. Biotechnol. 7: 397-402.

Memasukkan: November 2009 Diterima: Maret 2010

323

J. Biol. Indon. Vol 6, No.3 (2010) PANDUAN PENULIS

Naskah dapat ditulis dalam bahasa Indonesia atau bahasa Inggris. Naskah disusun dengan urutan: JUDUL (bahasa Indonesia dan Inggris), NAMA PENULIS (yang disertai dengan alamat Lembaga/ Instansi), ABSTRAK (bahasa Inggris, maksimal 250 kata), KATA KUNCI (maksimal 6 kata), PENDAHULUAN, BAHAN DAN CARA KERJA, HASIL, PEMBAHASAN, UCAPAN TERIMA KASIH (jika diperlukan) dan DAFTAR PUSTAKA. Naskah diketik dengan spasi ganda pada kertas HVS A4 maksimum 15 halaman termasuk gambar, foto, dan tabel disertai CD. Batas dari tepi kiri 3 cm, kanan, atas, dan bawah masingmasing 2,5 cm dengan program pengolah kata Microsoft Word dan tipe huruf Times New Roman berukuran 12 point. Setiap halaman diberi nomor halaman secara berurutan. Gambar dalam bentuk grafik/diagram harus asli (bukan fotokopi) dan foto (dicetak di kertas licin atau di scan). Gambar dan Tabel di tulis dan ditempatkan di halam terpisah di akhir naskah. Penulisan simbol α, β, χ, dan lain-lain dimasukkan melalui fasilitas insert, tanpa mengubah jenis huruf. Kata dalam bahasa asing dicetak miring. Naskah dikirimkan ke alamat Redaksi sebanyak 3 eksemplar (2 eksemplar tanpa nama dan lembaga penulis). Penggunaan nama suatu tumbuhan atau hewan dalam bahasa Indonesia/Daerah harus diikuti nama ilmiahnya (cetak miring) beserta Authornya pada pengungkapan pertama kali. Daftar pustaka ditulis secara abjad menggunakan sistem nama-tahun. Contoh penulisan pustaka acuan sebagai berikut : Jurnal : Hara, T., JR. Zhang, & S. Ueda. 1983. Identification of plasmids linked with polyglutamate production in B. subtilis. J. Gen. Apll. Microbiol. 29: 345-354. Buku : Chaplin, MF. & C. Bucke. 1990. Enzyme Technology. Cambridge University Press. Cambridge. Bab dalam Buku : Gerhart, P. & SW. Drew. 1994. Liquid culture. Dalam : Gerhart, P., R.G.E. Murray, W.A. Wood, & N.R. Krieg (eds.). Methods for General and Molecular Bacteriology. ASM., Washington. 248-277. Abstrak : Suryajaya, D. 1982. Perkembangan tanaman polong-polongan utama di Indonesia. Abstrak Pertemuan Ilmiah Mikrobiologi. Jakarta . 15 –18 Oktober 1982. 42. Prosiding : Mubarik, NR., A. Suwanto, & MT. Suhartono. 2000. Isolasi dan karakterisasi protease ekstrasellular dari bakteri isolat termofilik ekstrim. Prosiding Seminar nasional Industri Enzim dan Bioteknologi II. Jakarta, 15-16 Februari 2000. 151-158. Skripsi, Tesis, Disertasi : Kemala, S. 1987. Pola Pertanian, Industri Perdagangan Kelapa dan Kelapa Sawit di Indonesia.[Disertasi]. Bogor : Institut Pertanian Bogor. Informasi dari Internet : Schulze, H. 1999. Detection and Identification of Lories and Pottos in The Wild; Information for surveys/Estimated of population density. http//www.species.net/primates/loris/ lorCp.1.html.

J. Biol. Indon. Vol 6, No. 3 (2010)

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

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

Recommend Documents