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 & Taraedar 1984; Venkateswarlu et al. 1984). The population levels of Ecology of microbial communities phosphobacteria were higher in the can be attributed in part to understand rhizosphere soil, and able to produce that these organisms have directly phytohormones and phosphatases effects on ecosystem processes (Beare enzyme under in vitro conditions et al. 1995; Horner-Devine et al. 2004; (Ponmurugan & Gopi 2006). PSB was Fierer & Jackson 2006). Phosphate able to convert the insoluble phosphates solubilizing bacteria (PSB) commonly into soluble forms by acidification, found as adaptive bacteria (Glenn & chelating and exchange reactions, and Mandelstam 1971) in most soils (Chonkar production of gluconic acid (Chen et al.

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2006). The soluble forms also may phosphate and aluminum phosphate as contribute to their stimulatory effect on inorganic phosphate, and in the bacterial plant growth (Hameeda et al. 2006). growth containing para-nitrophenyl Correlation between PSB growth phosphate (pNpp) as organic (artificial) and capacity of phosphatases enzyme phosphate. The results suggest that the activity, as due to availability of phosphate mineralization capability of the phosphorous content in the medium PSB would appropriate to produce plant identified by Barik & Purushothaman nutritive value in subsequent work on (1998). In the other hand, there is biofertilizer function. The presence of increasing evidence that phosphobacteria PSB in Gunung Salak National Park is improve plant caused to biosynthesis of not only important for ecosystem health plant growth substances rather than their but also important genetic resources. action in releasing available phosphorous. Subsequently, PSB is requisite for land MATERIALS AND METHODS reclamation and restoration to improve despoiled land since the genus has useful Surface soil samples (up to 20 cm producing plant growth promoter, depth) collected from fields as a bulk phosphorus availability to plant, and as samples of various places at forest floor the competitor of plant bacterial pathogen. in Gunung Salak National Park; the In the preliminary work, the fastest altitude is 900 m above sea level; situated growing and the widest clearing zone around S 06046’24.3" - 06046’49.8" and formation in selective agar media for the E 106042’09.9" - 106042’25.9"; in June isolate of PSB has deprived from forest 2009. Composite soil sample of each soil soil collected from Gunung Salak case mixed thoroughly, with then air National Park. In the existing dried and passed throughout 100 mesh investigation, culture approach has been sieves for studies. studied whether the fine isolate and then In the preliminary work, based on delineated as gram-positive bacterium, the broadest halozone screen ability named Bacillus sp. Some researcher find growing in selected media, the 21 isolates out that the genus relatively has large recognized as PSB (Tabel 1). Selected number of protein phosphatases (Cohen isolate (GS1) then identified through 1989; Villafranca et al. 1996), it can be analysis of gene 16S RNA using the serine/threonine phosphatases which method of Pitcher et al. (1989), and have showed wide specificities, and also identified as Bacillus sp. tyrosine phosphatases. In the further quantification, the The present work was undertaken strain cultured on liquid media contains to compare the phosphate-repressible of phosphorous substances, and enzyme formed by the Bacillus sp. incubated on a rotary shaker (180 rpm) Activity of phosphatases enzymes at 30ºC. For liquid media, mineral evaluated through the bacterial growth phosphate (5 g·L-1 tri-calcium phosphate within media containing tri-calcium or aluminum phosphate) were sterilized

314 Production of Acid Phosphatase in Bacillus sp.

separately and then mixed with the was washed twice with 10 mM autoclaved medium (10 g·L-1 glucose; potassium phosphate buffer (pH 7.5) and -1 -1 0.27 g·L NH4·NO3; 0.2 g·L KCl; 0.1 resuspended in the same buffer and at -1 -1 g·L MgSO4 ·7H2 O;1mg·L the same concentration as the original -1 MnSO4·6H2O; 1 mg·L FeSO4·7H2O; pre-culture. The suspension (inoculum and 0.1 g·L-1 Yeast extract). mass 5%, v/v) was then transferred to Glucose oxidation to become 100 ml glucose minimal medium gluconic acid is a major mechanism for containing 0.4% glucose and 21 mM mineral phosphate solubilization. When potassium phosphate buffer (pH 6.8); and gluconic acid content and phosphomo- incubated on a rotary shaker (180 rpm) noesterase activity in the culture medium at 30ºC. For determination of organic measured, the strain was prepared for acid produced, the strain was cultured in preculturing in LB medium (10 g·L-1 GMS medium containing 10 g·L-1 glucose; -1 -1 -1 -1 polypeptone, 5 g·L NaCl, 5 g·L Yeast 2 g·L (NH4)2SO4; 0.3 g·L -1 -1 extract, 1 g·L glucose). The precultured MgCl2·6H2O; 1 mg·L MnCl2·4H2O; 6

Tabel 1. PSB was common bacteria isolated from Gunung Salak National Park

No Strain aGrowth bCa-P rate dissolution capacity 1 GS1 +++ 3.1 2 GS2 ++ 2.8 3 GS3 ++ 2.8 4 GS4 + 1.9 5 GS5 + 1.8 6 GS6 + 1.8 7 GS7 + 1.8 8 GS8 + 1.8 9 GS9 + 1.7 10 GS10 + 1.7 11 GS11 + 1,7 12 GS12 + 1,7 13 GS13 + 1,6 14 GS14 + 1.7 15 GS15 + 1.7 16 GS16 + 1,7 17 GS17 + 1,7 18 GS18 + 1,6 19 GS19 + 1.7 20 GS20 + 1.7 21 GS21 + 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

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-1 -1 mg·L FeSO4·7H2O; 6 mg·L NaMoO4; C18 column (250 x 4.6mm). Organic thiamine (20 ì·L-1); tri-calcium phosphate acids were monitored using a UV (20 mg·mL-1); and incubated on a rotary detector at 220 nm. The mobile phase shaker (180 rpm) at 37ºC. consisted of 50 mM sodium phosphate Cells were collected by and 5 mM tetra-butyl-ammonium centrifugation at different growth periods hydrogen sulfate, pH 6.5 (95%), plus in order to verifications the change in pH acetonitrile (5%) and a flow rate of 0.25 and P-concentration in the medium. ml·min-1. Samples were centrifuged 6000 rpm for The protein phosphatases activity 10 minutes to receive clear solutions for was tested by the ability to hydrolyze p- analysis. The P-concentration and pH nitrophenyl phosphate (pNpp) in a buffer were determined throughout superna- containing 50 mM Tris HCl (pH 7.2). tants in each investigation. The P- Phosphomonoesterase activity (PME) concentration estimated with ascorbic was measured after incubation at 37ºC acids methods. The 50 ~ 200 micro-liter (Margesin1996). Phosphatases activity of the culture filtrate was mixed with 900 was determined by measurement of p- micro-liter of phosphorus mixed reagents, nitrophenol in a spectrophotometer at a keep at room temperature for 10 minutes. wavelength of 400 nm. PME activity was The P-concentration was measured in expressed in unit and defined as spectrophotometer at 880 nm. In order micromoles nitrophenol produced by 1 ml to observe the effect of cultural enzyme per hour. conditions for mineral phosphate Three replicate of flasks and tubes solubilizing, bacterial strains were cultured were used for all in each treatment of at various insoluble phosphates (tri- examination; and also from which the calcium phosphate, and aluminum other samples were collected at 4 to 96 phosphate) at defined concentrations of hours in various sampling interval of glucose conditions. incubation; those were analyzed for the Five ml of a precultured solution parameters studies. Analysis of was inoculated in 50 ml of medium in a correlation and some other statistical 250-ml Erlenmeyer flask. After requirement among mean values inoculation, the flasks were placed on a calculated at various variables amongst shaker and the bacteria were grown at the confidence level of the degree of 370C for 24 h. The supernatant of each freedom (Parker 1979). culture was obtained by centrifugation at 10,000 rpm for 10 min. For the RESULTS experiment to determine gluconic acid, a 0.5 ml aliquot of the culture filtrate passed The isolated bacterium had a through 0.2 ìm Whatman membrane marked of insoluble phosphate solubilizing filter. The organic acids in filtrates were activities, because the culture grows to identified by high-performance liquid visualize clear zone upward in the region chromatography with a Thermo hypersil of the colony forming after 3 days

316 Production of Acid Phosphatase in Bacillus sp.

incubation at 30ºC. In estimating the PSB live activity in culture was closely efficacy of phosphate source utilization followed by gluconic acid production as by the PSB, result in phosphatases assay caused by glucose incorporation. The of Bacillus sp. in liquid cultures containing phenomenon gives sequence that the insoluble phosphate analytically option culture incubation for Bacillus sp. measured. Result of the studies have should be after 36 hours and reaching correlated in the parameters measure- the limit action in 60 hours incubation ment of Ca- and Al-phosphate metabo- (Figure 1). That information becomes lism as inorganic phosphate incorporation useful to find out the maximum incubation to glucose reduction in the substrate, but period for that bacterial in the propagation it does not proper to organic phosphate culture for biotechnological purpose. of para-nitrophenylphosphate. The Kinetic potential of unit phosphatase highest production of phosphorus activities belonging to Bacillus sp. was substance during incubation was measured in tri-calcium phosphate and measured, and it found in the medium alumunium phosphate solution culture containing tri-calcium phosphate. substance; and subsequently those Gluconic acid production was increase measurement acquiesced phosphate followed by pH reduction in the culture quantity of culture in the yield of 150 and medium (Table 2). This indicates the 25 mg·L-1 respectively, at 88 hours evenness of phosphatic source utilization, incubation (Figure 2). The result assumed a prominent phenotypic characteristic of the PSB effectively capable to increase phosphate solubilizing bacterial isolates soluble phosphate in their habitat (in soil in relation to carbon augmentation. of forest floor in Gunung Salak National Most bacterial (PSB) population Park), because of phytase commonly was stable after 24 hours incubation.

Table 2. Probability levels for statistical significance value in observation

Parameters studied of in-vitro culture of Bacillus Correlation sp.

1. Relationship of Ca-phosphate solubilization vs. y = -24.04x + 3832.7 (r = - 0.91 S)* glucose reduction in the substrate: 2. Relationship of Al-phosphate solubilization vs. y = -199.07x + 4890.1 (r = - 0.95 S)* glucose reduction in the substrate: 3. Relationship of Ca-phosphate to Al-phosphate y = 23x + 0.38 (r = 0.37 NS)* solubilization in the substrate: 4. Relationship of gluconic acid production to pH y = -0.78x + 7.27 (r = - 0.97 S)** level for the period of incubation:

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

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have specify requirement to Ca3+ and buffer in the medium culture solution, but Al2+ for its enzyme activity. only low activity of the enzyme detected Phytate is the major component of in the investigation. organics forms of P in soil (Richardson 1994). Phytase activity can be important DISCUSSION for stimulating growth under limited P in soil, and supports to improve or transfer Microbial communities in forest soil the P-solubilizing trait to plant-growth- play important role in maintaining global promoting bacteria (Rodrý´guez et al. ecosystem health. PSB is one of the 2006). Most phytases (myo-inositol important soil microbes which stimulate hexakisphosphate phosphohydrolases) dissolution of less soluble-P into soluble belong to high molecular weight acid form and that available for plant growth. phosphatases, which has capability to The availability of P in soil is generally hydrolyze of phytic acid to be orthophos- low, and in forest ecosystem most of phate inorganic and phosphate esters phosphate in the form of organic bound from lower mio-inositol. Phytic acid is a phosphate. Those organically bound phosphate ester that usually establishes phosphates should be hydrolyzed by in soil, and it could bond the important phophatase enzyme produced by soil minerals and protein. Phosphomo- microorganism. Soluble phosphate - = = noesterase (PME) examined as acid released (H2PO4 , HPO4 and PO4 ), phosphatase under Tris-HCl (pH 7.2) 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 20406080100120 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 order to solubilize inorganic phosphate soil minerals (Cunningham & Kuiack compounds (Lal 2002). Phosphobacteria 1992). The availability of phosphate in deprive from rhizosphere soils when was soil is greatly dependent on soil pH, tested under in-vitro condition on their carbon content, redox potentials, and soil production capacity of growth regulators physical structures. The Strain of GS1 and phosphatase enzyme (Ponmurugan identified as Bacillus sp., was able to & Gopi 2006). Several soil bacteria solubilize both Ca-P and Al-P. Their ability posses the ability to solubilizing insoluble to solubize those less soluble phosphate inorganic phosphate and make it available may indicate that the strain is important to plants. The effect is generally due to in stimulating phosphate dissolution. The the production of organic acids by these physiological mechanism by which P organisms, and also produce amino acids, dissolved is very complex. vitamins, and growth promoting Phosphobacteria (PSB) have been substances like indole acetic acid (IAA) found to produce some organic acids such and gibberellic acid (GA3), which help in as monocarboxylic acid (acetic, formic), better growth of plants (Richardson monocarboxylic hydroxy (lactic, glucenic, 2001;Gyaneshwar et al. 2002). The glycolic), monocarboxylic, ketoglucenic, results of enzyme activities, including soil decarboxylic (oxalic, succinic), phosphatase activity, could be compared dicarboxylic hydroxy (malic, maleic), and not only with soil physical and chemical tricarboxylic hydroxy (citric) acids in properties, but also with other biological

180 28 1.2 Ca‐Pi Al‐Pi p‐Npp

24 150 1

20 120 0.8

16 (Unit) 90 0.6 12

60 0.4 8

Phosphatases 30 0.2 4 y = ‐0.01x2 + 2.804x ‐ 16.59 y = ‐0.002x2 + 0.453x + 1.370 y = ‐0.000x2 + 0.024x + 0.030 R² = 0.944 R² = 0.939 R² = 0.938 0 0 0 0 255075100 0 255075100 0 255075100 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 the production of organic acid, phenolic level of adenosine triphosphate, etc. compounds, protons, and siderophores. (Chhonkar & Tarafdar 1984). Falih & According to Kelly et al. (1984) Wainwright (1996) found that the activity investigation’s, maximum phosphates of phosphatase enzymes increased when (inorganic pyrophosphate) formed at 12 a carbon source was added to the soil. hours incubation in Bacillus sp. when Evaluate to the result of experiment here, divalent metal Mn2+ occurred in the inform that glucose incorporation proved medium culture, and the inorganic to cause increasing cell biomass of pyrophosphates activity found as Bacillus sp. in culture, and gluconic acid intracellular enzyme. In the other study increment (Figure 1); and in the different on the extracellular phosphates activity, way, the medium acidity is plunge to 5 Nomoto et al. (1988) found the optimum from pH 7 as before during 96 hours alkaline phosphate excreted into broth incubation. culture fairly stable in pH 5 ~ 12. Alkaline phosphatase of several Extracellular enzymes, including bacterial species has been investigated phosphatases, are important for the by Landeweert et al. (2001), and the degradation of organic substances in the genus have differ one from another in soil for organic phosphate mineralization certain respects. The genus seems in (Hysek & Sarapatka 1998). The general to share the property of being functionalities of PSB communities differ repressed by phosphate inorganic in on the basis of phosphate sources present Escherichia coli, Bacillus subtilis, in the culture medium in this investigation. Pseudomonas fluorescens and Phosphatases activities expressed Staphylococcus aureus. PSB is capable optimum in 75 to 100 hours incubation of solubilizing accumulated insoluble (Figure 2) may cause the MnCl2 phosphate compound sources in soil by containing in media culture.

12 8 8

) 7 7 10 -1 -1 ) = Ca-Pi unit Acidity level (pH) -4 6 6 = Al-Pi unit-1 8 pH = p-NPP unit-1 5 5 6 4 4 3 3 4 2 Gluconic acid 2 Gluconic Acid (mg.L Carbon Used(10 2 1 1 0 0 0 0 25 50 75 100 0 255075100 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 cultural performance in the medium compound arise from the oxidation of containing inorganic phosphate. glucose. It is produce by the fermentation of glucose by bacteria, and in aqueous ACKNOWLEDGEMENTS solution at neutral pH the gluconic acid forms the gluconate ion. Chen et al. We acknowledge to the Directorate (2006) investigate the isolates of General for Higher Education, Ministry phosphobacteria collected from of National Education, Republic of agriculture soil. The isolates belong to Indonesia, for the financial assistance genus of Bacillus, Rhodococcus, provided for this study in the year 2009. Arthrobacter, Serratia, Chryseobac- Also, we are grateful to our acquaintance terium, Delftia, Gordonia and for critically reading this manuscript Phyllobacterium were identified. In other finding, four strains namely REFERENCES Arthrobacter ureafaciens, Phyllobac- terium myrsinacearum, Rhodococcus Barik, SK. & CS. Purushothaman. 1998. erythropolis and Delftia sp. reported for Phosphatase activity of two strain the first time as PSB after confirming of bacteria on orthophosphate their capacity to solubilized considerable enrichment. Proc. Natl. Frontiers amount of tricalcium phosphate in the in Appl. Environ. Microbial. medium by secreting organic acids. Ten Dec.11-13, SES, CUSAT. Cochin. isolates of Bacillus megaterium can do pp.165-170. not producing any gluconic acid in the Beare, MH., DC. Coleman, DA. culture along 72 hours incubation. In the Crossley Jr., PF. Hendrix & EP. contrary, 96 hour’s incubation of Bacillus Odum. 1995. A hierarchical sp. in this experiment was able to produce approach to evaluating the gluconic acid, and negatively correlated significance of soil biodiversity to with glucose incorporation but positively biogeochemical cycling. Plant and correlated to cell biomass (Figure 3). Soil. 170: 5-22. Chen,YP., PD. Rekha, AB. Arun, FT. CONCLUSION Shen, WA. Lai & CC. Young. 2006. Phosphate solubilizing bacteria Isolate of Bacillus sp. demonstrate from subtropical soil and their clearly in solubilizing tri-calsiumphosphate tricalcium phosphate solubilizing more than alumuniumphosphate; abilities. App. Soil Ecol. 34:33-41. Increasing cell population of Bacillus sp. Chonkar, PK & JC. Tarafdar. 1984. was following in gluconic acid yield as Accumulation of phosphatase in due to glucose metabolism as carbon soils. J. Indian Soc. Soil Sci. 32: source in culture; Bacillus sp. was 266–272. certainly characterized on the basis of biochemical reaction, and as due to the

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Memasukkan: November 2009 Diterima: Maret 2010

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