Solubilization of insoluble phosphates by Aspergillus niger Pelarutan fosfat sukar larut oleh Aspergillus niger

Penggunaan langsung fosfat alam (FA) ke dalam tanah sebagai sumber pupuk P telah dilakukan selama bertahun-tahun melalui beberapa macam cara penggunaan. Kualitas FA di Indonesia umumnya rendah dan ketersediaan bahan baku yang berkualitas untuk produksi pupuk fosfat terlarut relatif terbatas. Beberapa mikroba asal tanah yang dapat melarutkan fosfat anorganik telah banyak dilaporkan. Namun, informasi yang tersedia tentang mekanisme pelarutan P dari FA lokal asal Indonesia dan P anorganik oleh Aspergillus niger BCC F194 belum banyak diteliti. Satu seri penelitian laboratorium telah dilaksanakan untuk mengetahui kemampuan A. niger BCC F194 melarutkan P. Evaluasi agronomi FA lokal (FA Cileungsi dan Madura) di rumah kaca juga telah dilakukan. A. niger BCC F194 dapat melarutkan sumber P sukar larut, yaitu FA Cileungsi dan Madura, serta senyawa Ca3(PO4)2 dan AlPO4. Kelarutan P anorganik tersebut berhubungan dengan peningkatan aktivitas proton (H*) yang menyebabkan penurunan pH medium dan produksi asam organik. Asam organik utama yang dihasilkan oleh A. niger BCC F194 dalam medium cair Pikovskaya yang dimodifikasi adalah asam oksalat (3.75 mM), asam sitrat (2.0 mM), dan asam glukonat (0.9 mM). Kelarutan FA Cileungsi lebih besar dibandingkan dengan FA Madura, dan kelarutan Ca3(PO4)2 lebih besar dibandingkan kelarutan AlPO4. Tidak ada korelasi antara kelarutan P anorganik dengan aktivitas enzim fosfatase, walaupun aktivitas enzim fosfatase cukup tinggi terdeteksi dalam medium. Satu formula biosuperfosfat telah berhasil dirakit dengan mereaksikan FA lokal dengan supernatan kultur cair (SKC) pengganti asam sulfat. Hasil percobaan pada bibit kakao, karet dan kelapa sawit di rumah kaca menunjukkan bahwa prototipe pupuk biosuperfosfat dengan bahan baku FA Cileungsi dan Madura bentuk granul maupun serbuk, memiliki nilai efektivitas agronomi yang relatif menyamai SP-konvensional.


Introduction
Most rock phosphate deposits found in the world are classified as low reactive RPs and therefore, it cannot be used successfully as phosphorus (P) sources for crop production.The composition of these rock phosphates varies from one deposit to another.Most of the world rock phosphates are of sedimentary origin.Large rock phosphate deposits estimated 1-2 million tonnes, are distributed in different parts of Indonesia (Moersidi, 1999).Since these deposits are low in phosphorous content (<25% P 2 O 5 ) and contain some impurities like oxides of Si, Fe, Al, and Ca, they are unsuitable for the manufacture of superphosphate (Narsian et al., 1993).The conventional method for enhancing the rock phosphate availability is to increase its solubility by treating with inorganic acids, mainly sulphuric acid and phosphoric acid; but, this approach is not applicable because of high capital production (Hammond et al., 1989, Goenadi et al., 2000).A very attractive approach for rock phosphate solubilization is the application of microbes capable of excreting organic acids (Gerke, 1992).
Many soil microbes are known to solubilize insoluble forms of inorganic phosphatic compounds.In vitro studies with microbial isolates from soil indicated that fungi were more efficient in the solubilization of inorganic phosphate as compared to bacteria (Thomas et al., 1985;Nahas 1996;Goenadi et al., 1999).Filamentous fungi are widely used as producers of organic acids, particularly A. niger and some Penicillium sp, which have been tested in fermentation systems or inoculated directly into soil in order to solubilize rock phosphate (Vassilev et al., 1997;Vasileva et al., 1998).
Acidification of soil is a consequence of natural and anthropogenic processes (Ulrich & Sumner, 1991).One of the outstanding changes in soil due to acidification is the mobilization of Al 3+ ions, which are toxic to plants on one hand and cause chemical fixation of plant-available P on the other hand.Hardly-soluble aluminum phosphates are formed and become the largest P-fraction in many acidic soils (McLean, 1976).AlPO 4, in the contrary to calsium phosphate, will never play an important role in ameliorating soils.Leaving that out of consideration, the importance of AlPO 4 solubilization for soil formation, mineral transformation and Al-toxicity is obvious (Illmer et al., 1995).In this investigation, pure insoluble inorganic phosphates i.e. aluminum phosphate (AlPO 4 ) and calcium phosphate (Ca 3 (PO 4 ) 2 ) have been used for expressing phosphate solubilizing activity of A. niger BCC F194.
Natural RPs has been recognized as a valuable alternative source for P fertilizer, especially for acid soils.The economic value of the rocks increases considerably along with the increasing costs of SP production (Goenadi et al., 2000).Consequently, there is a growing interest in ways of manipulating such rock to obtain a more valuable product, i.e. partially acidulating RPs (Goenadi, 1990;Rajan and Ghani, 1997), reacting with synthetic organic acids (Sagoe et al., 1998) or natural organic acids (Singh & Amberger, 1998), and decreasing particle size (Babare et al., 1997).Goenadi et al. (2000) reported that by using Liquid Culture Supernatant (LCS) instead of H 2 SO 4 in superphosphate (SP) production and using both with lower H 3 PO 4 concentration to bioactivated Morrocan RP as raw materials was found to increasing the solubility of P in 2% citric acid.Indications were obtained that LCS could replace H 2 SO 4 in the production of SP , and believed to yield a more eco-friendly P fertilizer than conventional SP.
The objectives of this study were: (i) determining phosphate solubilizing (PS) activity by A.niger BCC F194 on four types of inorganic P compounds, (ii) effect of organic acids and phosphatase enzyme on RPs dissolution, and (iii) agronomic evaluation of bioactivated CRP and MRP for direct application in greenhouse experiment with cocoa, rubber, and oil palm seedlings as test crops.

Phosphate solubilizing (PS) activity
Phosphate solubilizing activity was assayed in 50 mL aliquots of standard Pikovskaya's broth and modified Pikovskaya's broth prepared by replacing Ca 3 (PO 4 ) 2 (19.9%P) with inorganic phosphate i.e.AlPO 4 (50.8%P) and different rock phosphates, i.e.Cileungsi (8.9% P) and Madura (6.9% P).Cileungsi rock phosphate (CRP) and Madura rock phosphate (MRP) were ground (200 mesh) and dry-oven.For this purpose, P sources were sterilized separately and then mixed with the sterile Pikovskaya medium.Two pieces of inocula (Ø 8 mm) were inoculated into Pikovskaya liquid medium containing 0.125 %(w/v) respective phosphorus sources, incubated on a mechanical shaker at 100 rpm, 28 o C for nine dyas.The dissolved P was then determined by applying the molybdenum-blue method of Olsen & Sommers (1982).Absorbance was measured using a Spectronic 21 spectrophotometer at 693 nm.

Solubilization of RPs in Pikovskaya medium
Modified Pikovskaya liquid medium supplemented with 0; 0.125; 0.25; 0.50; and 1.0 % (w/v) CRP and MRP respectively was used.Inoculation was performed by transferring two pieces of inocula (Ø 8 mm) on to 100 mL Erlenmeyer flask containing 50 mL culture medium shaker at 100 rpm (28 ο C) for nine days.The cultures were incubated on a mechanical.
The growth of the fungus was measured in standard procedure by drying the decanted mycelium in an oven at 70 ο C to constant weight at the end of incubation.Phosphorus solubilizing ability was determined by using the molybdenum-blue method of Olsen & Sommers (1982).pH of the filtrates was measured with Methrom pH meter and organic acids concentration in the culture determined by using HPLC (0.01 N H 2 SO 4 mobile phase, 210 nm UV detector, 0.5 mL.min -1 flowrate, at 50 ο C) (Cunningham & Kuiack, 1992).

Effect of organic acids on RPs dissolution
This experiment was carried out to clarify the relative strength of different types of organic acids in solubilizing P from the CRP and MRP by using a method of Illmer et al. (1995).Citrate, oxalate and gluconate were added separately in different concentration (0; 0.05; 0.50; 1.0; 3.0 and 6.0 mM) to 50 mL Pikovskaya sterile medium containing 0.125% (w/v) CRP or MRP, then incubated for seven days on a mechanical shaker, at 100 rpm (28 ο C).P-concentration in supernatant solutions was determined using the method of Olsen & Sommers (1982).

Effect of phosphatase enzyme on RPs and inorganic P dissolution
The isolate of A. niger BCC F 194 was grown in 50 mL modified Pikovskaya liquid medium with various levels of CRP or MRP (0; 0.125; 0.25;0.50and 1.0%(w/v)).The P sources were sterilized separately and then mixed with the sterile Pikovskaya medium.The cultures were incubated for nine days on a mechanical shaker 100 rpm, (28 o C) for the production of extracellular phosphatase.Correlation between activity of phosphatase (Souciet et al., 1980) and P-solubilizing was determined.One enzyme unit is the amount which catalyses the hydrolysis of 1 μmol of pNPP per min.under the experimental condition.Inactivation of phophatase in liquid culture supernatant (LCS) of A. niger BCC F194 was studied at temperature of 40, 50, 60, 70, 80, and 90 o C. Further, 50 mL LCS of A.niger BCC F194 contained inactivated phosphatase was added with Santi et al. 0.125% (w/v) AlPO 4 sterile and incubated on mechanical shaker at 100 rpm, 28 o C for 24 h.Activity of phosphatase enzyme (Souciet et al., 1980), P-solubilization, and concentration of organic acid (mainly citric acid) were determined.

Agronomic evaluation of bioactivated CRP and MRP
Bioactivation was conducted by reacting 8.5 mL LCS with the highest contents of organic acids (mainly citric acid from A.niger BCC F194 isolate) and 28 mL H 3 PO 4 52% (v/v) on 55 g RPs (200 mesh) (Goenadi et al., 2000).The most efficient bioactivation of CRP and MRP were evaluated on the basis of soluble P contents in water and citric acid, as well as perchlorate-extractable P content (SII 0029-73, 1984).Effectiveness of the both granular and powder biosuperphosphate prototypes in substituting conventional P fertilizer, i.e.SP-36, was determined on the basis of relative agronomic effectiveness (RAE) (Mackay et al., 1984) in a completely random design experiment with four levels of P dosages, i.e. 0, 50, 100 and 150% to that of recommended SP-36 dosage.The RAE (%) was evaluated on the basis of seedling's dry weight that calculated from a greenhouse experiment using cocoa, rubber, and oil palm seedlings grown on Cikopomayak Ultisols, West Java (pH 4.7, 18.1 ppm P 2 O 5 (Olsen test), total N 0.10%, organic matter 2.9 % (C), and 0.1 % (N)).

Phosphate solubilizing (PS) activity
A. niger BCC F194 could solubilize Ca 3 (PO 4 ) 2 , CRP, and MRP, but showed poor in AlPO 4 solubilization in nine days incubation period.The amount of P solubilized by A. niger BCC F194 was directly related to the decrease in pH of the medium, except AlPO 4 treatment.These phenomena suggested that PS activity depends on the types and constanta solubility product (Ksp) of insoluble phosphate (Ksp for AlPO 4  10 -30 and Ca 3 (PO 4 ) 2  2.0 x 10 -20 ) -as supposed by many investigators (Mc Lean, 1976;Narsian et al. 1993;Illmer et al., 1995;Nahas, 1996).

Solubilization of RPs in Pikovskaya medium
Employing a modified Pikovskaya medium, A. niger BCC F194 produced oxalic acid (3.75 mM), citric acid (2.0 mM), and gluconic acid (0.9 mM) as the main organic acids from MRP and CRP as P source of Pikovskaya medium at 0 -1.0%(w/v) level.Figure 1.shows the relationship among P-solubilizing ability and pH of the medium, or organic acid produced in a modified Pikovskaya medium.
Regression analysis indicates that P-solubilization was highly positive correlated with organic acid concentration ( r MRP =0.92** and r CRP =0.85**) and negative correlated with the pH of the medium (r MRP =-0.87* and r CRP =-0.99**) (Figure 1).Organic acid concentrations were also strongly negative correlated with the pH of the medium (r MRP =-0.99** and r CRP =-0.87*).

Effect of organic acids on RPs dissolution
Production of organic acids is an important mechanism for solubilizing inorganic phosphate, although not the only possible one.Gluconic acid concentrations in Pikovskaya medium contained CRP and MRP as P source were comparatively constant, whereas the increased citrate and oxalate concentrations were apparently related to the increase in CRP and MRP concentrations in solution (Figure 2).It indicates that citrate and oxalate have strong influences to the solubilization of P-RPs, r CRP =0.96**, and r MRP =0.99** (citric acid) and r CRP =0.93** and r MRP = 0.86* (oxalic acid).On the other hand, no correlation between gluconic acid concentration and dissolution of CRP and MRP (r CRP =0.6 ns and r MRP = 0.1 ns ) were found.These phenomena lead to the assumption that citric acid and oxalic acid produced by A. niger BCC F194 isolate are responsible for lowering the pH of medium providing protons (H  ) to increase the P-RP solubilization.In this case, there was relationship between the pKa values of the acids and the amounts of P released.Citric acid has a higher dissociation constant (pKa=3.14)than oxalic acid  (pKa = 1.25).Pohlman & McColl (1986) reported that several factors are important in determining the degree or rate of dissolution of RPs i.e. : (i) rate of diffusion of organic acid from bulk solution and diffusion of products from the site of reactivity, (ii) contact time between the organic acids and mineral surface, (iii) degree of dissociation of organic acids, (iv) type and position of functional groups, and (v) chemical affinities of chelating agents for the metals.

Effect of phosphatase enzyme on RPs and inorganic P dissolution
Phosphate solubilization mediated by phosphatase enzyme is believed to be taken place for organic P sources (Bishop et al., 1994).There was no correlation between activity of phosphatase enzyme and P solubilization (r CRP =0.39 ns and r MRP =0.22 ns ), as well as with P concentration in (r MRP =0.39 ns and r CRP =0.23 ns ).The phosphatase activity Pikovsakaya medium decreased at 80 o C (15 U/mL) and 90 o C (5.8 U/mL) (Figure 3A).7 .9U/mL (H), respectively (Figure 3B).On the other hand, the citric acid concentration was relatively stable at 0-20 minute of heating periods (Figure 3C).These results were in agree with assumption that phosphate solubilization mediated by phosphatase enzyme is taken place for organic P sources (Traina et al., 1986;Bishop et al., 1994;Sigh & Amberger, 1998).

Agronomic evaluation of bioactivated CRP and MRP for direct application in greenhouse experiment
Application of bioactivated CRP and MRP resulted in significant increase of leaf number and height of the three-month old cocoa and oil palm seedlings compared to the control pot.For rubber seedlings, significant responses have been observed starting at four months after treatment (P<0.05).There were no significant differences between conventional SP and biosuperphosphate (SPab) applied on height, leaf number, girth, and dry weights of cocoa, rubber, and oil palm seedling (unpublished data).At the end of the experiment (four months after treatment for cocoa and rubber, and six months for oil palm seedling), CRP and MRP originating SPab in granular and powder form applied at 100% rate equivalent to standard SP application indicated a higher value of relative agronomic effectiveness (RAE) (Table 1).

Conclusions
A. niger BCC F194 performed high abilities in solubilizing inorganic phosphate (>50%), i.e.Ca 3 (PO 4 ) 2 , CRP, and MRP, but not for AlPO 4 .Citric and oxalic acids were important components responsible for phosphate solubilization by A. niger BCC F194.Oxalic acid was the main organic acid produced by A. niger BCC F194 in modified Pikovskaya medium, for nine days incubation at 100 rpm, 28 o C. Citrate and oxalate had strong influences to the solubilization of CRP and MRP.There was no correlation between phosphatase activity and solubilization of inorganic phosphate.
Results of cocoa, rubber, and oil palm seedlings experiments in greenhouse showed that the prototype products from bioactivation (biosuperphosphate) had a comparable relative agronomic effectiveness value to that of the conventional SP (SP-36).

Figure 2 .
Figure 2. Relationship between concentration of selected organic acids (citric, oxalic, and gluconic) and solubilization of rock phosphate from Madura and Cileungsi in modified Pikovskaya medium.

Figure 3 .
Figure 3.Effect of temperature (A), and heating period (minute) at 80 o C on activity of phosphatase enzyme (B), and effect of heating period at 80 o C on organic acid (citric acid) concentration (C).Santi et al.