BIODECOMPOSITION OF JORDAN PHOSPHORITE BY PHOSPHATE-SOLUBILIZING FUNGI

Abstract

The bio-solubilization of Jordan phosphorite by the phosphate-solubilizing fungus Aspergillus niger has been investigated. The effect of the phosphate concentration in the liquid medium, the duration of biodecomposition, titratable acidity and the effect of preliminary mechanical activation on the process of dissolution have been studied. The investigations indicate that almost complete extraction of P₂O₅ from Jordan Phosphorite in a form utilizable by plants can be achieved. A maximum degree of P₂O₅ extraction 99.10% was obtained on the 15^-th day in a medium containing 0.5% w/v non-activated Jordan phosphorite. The preliminary mechanical activation of the phosphate facilitates the dissolution until a definite period of the bioconversion. Investigations with mechanically-activated Jordan phosphorite showed that a maximum extent of 92.40% of phosphate solubilization was observed on the 10^-th day at a phosphorite concentration of 0.5% w/v.

Keywords:
Rock phosphate; Solubilization; Biodecomposition; Aspergillus niger

INTRODUCTION

Phosphorus is second only to nitrogen in mineral nutrients that most commonly limit the growth of crops. A deficiency in soluble P for many agricultural soils is one of the major factors hampering crop production worldwide. Only 1 to 5% of the total soil P is in a soluble plant-available form (Arcand and Schneider, 2006). Usually it is introduced through the traditional phosphoric fertilizers- the super phos phates. It is known that the utilization of phosphorus from these fertilizers is about 15-20%, because a large portion of soluble inorganic phosphate applied to soil is rapidly immobilized soon after application and becomes unavailable to plants (Bojinova et al., 1997; Rodriguez and Fraga, 1999; Kang et al., 2007; Kang et al., 2008). This phenomenon occurs as a result of complex chemical and biochemical pro cesses in the soil, resulting in fixation and precipitation of P in the soil. This generally depends on pH and the soil type. The fixed forms of P in acidic soils are aluminum and iron phosphates, while in alkaline soils they are calcium phosphates (Rfaki et al., 2014). According to Lindsay (1979) super phosphate con tains a sufficient amount of calcium for the precipitation of its own P as dicalcium phosphate (CaHPO₄) or dicalcium phosphate dihydrate (CaHPO₄.2H₂O).

The second major component of soil P is organic matter (nucleic acids, phospholipids, phosphotriesters, etc.). The organic forms of P may constitute up to 30-50% of the total phosphorus in most soils. Many of these P compounds are materials with high mo lecular mass. They can be assimilated by the cell (Goldstein, 1994) after their bioconversion to either soluble ionic phosphate (Pi, HPO₄^2-, H₂PO₄^-), or low molecular-mass organic phosphate. These Ca-P com pounds are generally resistant to chemical hydrolysis and biodegradation, but recently several reports docu mented microbial release from these sources (Rodriguez and Fraga, 1999).

Based on the current rate of use, it is expected that the worlds, known reserves of high quality rock phosphate (RP) will be depleted within the current century. Consequently, the production of phosphate-based fertilizers will require the processing of low-grade rock phosphates at significantly higher cost and the production costs of P-fertilizers will rise (Mendes et al., 2013).

The development of new nonacid methods, appli cable to both high-quality and low-quality raw mate rials, is important for solving technological and eco logical effectiveness of phosphorus fertilizers pro duction. One of the available non-acid methods of rock phosphate processing is direct application of phosphates as a source of phosphorous in the soil. The phosphorus released from directly applied ground phosphate rock is often too low to provide sufficient P for crop uptake (Vassilev et al., 2001). The direct application of the phosphate as a fertilizer is limited due to its structure, resulting in low solubility. The improvement of phosphate structure through me chanical activation changes the phosphate chemistry and increases its solubility (Ibrahim et al., 2010).

The usage of phosphate-solubilizing microorgan isms (PSM) as a biotechnological alternative for pro ducing soluble P fertilizers from rock phosphate (RP) is the other alternative. Microorganisms are an im portant component in the soil. The ability of PSM to mobilize P from sparingly soluble sources can be a useful tool in P fertilization management. Some stud ies have shown that the product obtained after the treatment of RP with PSM or even the direct application of PSM to soil can improve plant growth and P uptake (Mendes et al., 2013). Therefore, an efficient process including microbial mediated ones able to exploit lower-grade RP and/or after native P sources (Vassilev, et al., 2013) at low cost has to be developed in the near future.

Soil bacteria and fungi mediate soil process such as decomposition, nutrient mobilization and mineralization, storage release of nutrients and water (Rashid et al., 2004). Several reports have indicated that some microorganisms are able to solubilize rock phos phates and to release soluble P (Sharma et al., 2012; Sanjotha et al., 2011; Yadav et al., 2011; Deepa et al., 2010; Pradhan and Sukla, 2005; Kang et al., 2002). A wide range of microorganisms able to solubilize inorganic P have been cultivated from soil, including bacteria (e.g Actinomycetes, Pseudomonas and Ba cillus spp.) and fungi (e.g. Aspergillus and Peniciluus spp.). It is generally accepted that the major mecha nism of mineral phosphate solubilization is the action of organic acids synthesized by soil microorgan isms. The production of organic acids such as citric, oxalic, gluconic, malonic, succinic, etc. by phosphate-solubilizing microorganisms has been well docu mented and seems to be most frequent agent for mine ral phosphate solubilization (Khan et al., 2007). Such organic acids can either directly dissolve the mineral phosphate as a result of anion exchange of PO₄^3- by acid anion or can chelate both iron and aluminum ions associated with phosphate (Omar, 1998). Some elements that may be released during RP solubilization could affect these mechanisms by promoting changes in microbial metabolism (Gadd, 1993). The amount of P solubilized is dependent on the form of inorganic P precipitate used (including various sources of RP, pure Ca, iron and aluminum phos phates) along with culture and sampling procedures (Whitelaw et al. 1999; Barroso et al., 2006; Richardson and Simpson, 2011). In general, phosphate-solu bilizing fungi produce more acids and consequently exhibit greater phosphate-solubilizing activity than bacteria in both liquid and solid media (Venkateswarlu et al., 1984). The fact that all identified P-solubilizing fungi belong to the Aspergillus or Pene cillium genus agrees with reports of several authors (Nahas et al. 1994; Ghosh and Banik, 1998; Rashid et al. 2004; Seshadri et al., 2004; Deepa et al., 2010). Fungal diversity affects soil agglomeration, thereby increasing the soil quality and fertility (Tallapragada and Seshachala, 2012). Species of fungi, particularly Aspergillus, are capable to produce citric acid and form non-ionizable association with calcium. Asper gillus niger, used in the industrial production of citric acid, has been reported as one of the most effective organisms for rock phosphate solubilization (Arcand and Schneider, 2006).

The dissolution of different types of P-contained resources (including Ca, iron and aluminum phosphates and various sources of rock phosphate) by Aspergillus niger has been demonstrated earlier (Vassilev et al. 2005; Nahas, 1996; Bojinova et al., 1997; Goenadi et al. 2000; Barroso et al., 2006; Bojinova et al., 2008; Mendes et al., 2013). The amount of P solubilized in culture is also depend on the composition of the medium (carbon and nitrogen composition), medium volume, pulp density, particle size, initial pH of the medium, temperature, inoculum concentration, along with culture and sampling procedures for the solubilization of the phosphates.

Solubilization of inorganic phosphate by microorganisms involves a wide range of processes concern ing the secretion of organic acids, lowering of the pH as a result of acid production, ion chelating and ex change reactions which are a part of the phosphorus cycle (Akuntokin et al., 2007).

The aim of the present study is to investigate the biodecomposition of Jordan phosphorite using the phosphate-solubilizing fungus Aspergillus niger. We use in our investigation Jordan phosphorite, which has not been studied with this objective. Jordan phosphorite is imported into Bulgaria for the phosphoric fertilizer industry. The effects of the phosphate con centration in the liquid medium, the duration of bio decomposition, the concentration of citric acid generated from the fungus and results with/without preliminary mechanical activation of the phosphorite, were studied.

MATERIALS AND METHODS

Natural Phosphate

The chemical composition of the initial Jordan phosphorite (JP) is shown in Table 1. The fraction below 0.2 mm was used. The total content of phosphorus was determined by dissolving in 25% HCl, the citric-soluble and water-soluble phosphorus was determined after extracting with 2% citric acid or water followed by spectrophotometric analysis as a vanadate-molybdate complex (Jackson, 1967). Ca was determined complexometrically, Si by weight and the other elements using Atomic Absorption Spectro photometry (AAS).

Table 1
Chemical composition of Jordan phosphorite.

Al2O3	CaO	Fe2O3	SiO2	TiO2	MgO	P2O5	Pb	Cu	Zn	Cd	Ni	Ag	As	Mo
%							mg/kg
0.26	53.69	0.27	2.60	0.02	0.26	35.37	18	32	183	4	10	1	7	4

The mechanical activation was performed for 4 hour using a "Pulverisite 5" planetary mill. Metal balls of 20 mm diameter were used and the rotation applied was 320 rpm. The weight ratio of phosphorite to milling bodies was 1:20.

The phosphorus content was determined in the non-activated Jordan phosphorite (NAJP) as well as after its mechanical activation (MAJP). It was analyzed as total P₂O₅ (P₂O_{5 t.}), citric-soluble (P₂O_{5 c.s.}) and water-soluble (P₂O_{5 w.s}). The phosphate P₂O_{5 t} value was 35.37% for the both examined phosphate types (NAJP and MAJP). The values of P₂O_{5 c.s.} were 11.75% (NAJP) and 16.99% (MAJP) and those of P₂O_{5 w.s.} were 0.01% and 0.03%, respectively.

Microorganisms and Nutritive Medium

Investigations were performed using the Aspergil lus niger strain obtained from the Institute for Microbiology, Bulgarian Academy of Sciences. The bioconversion was studied through deep incubation of the microorganisms in a liquid nutritive medium contain ing (in g/L): Glucose - 120; (NH₄)₂SO₄ - 3; KH₂PO₄ - 1; K₂HPO₄ - 1; MgSO₄.10H₂O - 0.5; MnSO₄.5H₂O - 0.02; FeCl₃.6H₂O - 0.01.

The initial pH value of the nutritive medium was 6.8.

Experimental Methods

Incubation with Aspergillus niger was carried out in 300 ml Erlenmeyer flasks containing 100 ml of sterilized nutritive medium. After cooling to 30 ºC, 1 mL inoculums with a concentration of spores of 1×10⁷/mL and JP (MAJP and NAJP) were introduced into the reaction medium. The flasks were incubated in a shaking water bath at 31±1 ºC for different pe riod of time with a rotational speed of 150 rpm. The concentrations of NAJP and MAJP in the nutrient medium were 0.5, 1 and 2% w/v. After different time intervals, the samples were filtered and pH, sugar content (Bernfeld, 1959), titratable acidity through titration with 0.1 N NaOH and the content of water-soluble P₂O_{5 w.s.} (c₁) in this first filtrate were deter mined. The precipitate (biomass and remaining min eral mass) was treated for 2 hours with 2% citric acid at room temperature. After filtration the solution ob tained was analyzed for citrate-soluble P₂O_{5 c.s..}(c₂). The precipitate, which contained residual mineral mass and biomass was dried to a constant weight at 60 ºC and was ashed to a constant weight at 500 ºC. The loss of weight during heating is equal to the bio mass produced during cultivation. The P₂O₅ content in the residue of mineral mass after thermal treat ment was also determined - P₂O_{5 m.m}. The P₂O₅ con tent in the biomass (P₂O_{5 b.}) was determined using material balance for P₂O₅ based on the quantity of P₂O₅ input in the system (with phosphorite and nutri tive medium) and, at the exit, after biodecomposition, (P₂O_{5 w.s.}, P₂O_{5 c.s.}, and P₂O_{5 m.m}).

The process was analyzed by using two parallel samples at various times of incubation and the results obtained were averaged.

Investigations of solubilization of Jordan phospho rite in the nutritive medium without microorganisms were made for the longest incubation period of 15 days at the studied concentrations of Jordan phos phorite. The obtained α value for 0.5, 1.0 and 2.0% JP was nearly 2%.

Calculation of the Conversion of Jordan Phosphorite

On the basis of the results obtained, the extent of the JP solubilization and conversion of P₂O₅ to wa ter-soluble (α₁), citrate-soluble (α₂) forms and P₂O₅ in biomass (α₃) were determined and expressed as follows:

The total degree of extraction is:

where:

P₂O₅ w.s. - P₂O₅ content in the first filtrate (g)

P₂O₅ c.s - P₂O₅ content in the second filtrate (g)

P₂O₅ b - P₂O₅ content in the dry biomass (g)

P₂O₅ t. - total P₂O₅ content in the system, with the phosphorite and nutritive medium (g).

A simple correlation was run to determine correlation coefficients (r) by the method of Ordinary Least Squares (OLS).

RESULTS AND DISCUSSION

Figure 1 presents the change in the concentration of glucose and citric acid (A) and the extents of P₂O₅ extraction α₁, α₂, α₃, α (B) in nutritive medium containing 0.5% w/v NAJP.

Microorganisms consume glucose as a carbohy drate source and its concentration decreased from 120 g/L at the beginning of the experiment to 11.5 g/L on the 15^th day. The titratable acidity slowly in creased up to the 12^th day of incubation when it reached a maximum of 10.24 µE.mL^-1; it then de creased to the end of the experiment, when its value was 7.5 µE.mL^-1. As seen from the data, the extent of P₂O₅ extraction in water-soluble form (α₁) increased with time and achieved 73.70% on the 15^th day. The extent of P₂O₅ extraction in citric-soluble form (α₂) slowly decreased from 20.80% on the 3^rd day to 12.20% at the end of the period. The total extent of P₂O₅ extraction (α) achieved a maximum of 99.10% on the 15^th day.

Figure 1
Change in the concentration of glucose and the titratable acidity (A) in the culture medium containing 0.5% w/v NAJP, and extents of P₂O₅ ex traction, α₁, α₂, α₃, α (B), after 3, 5, 9, 12 and 15 days of incubation. The values are the average ± SD (p ˂ 0.05) from duplicate experiments.

When 0.5% w/v MAJP was added to the nutritive medium the titratable acidity increased and reached a value of 10.66 µE.mL^-1 on the 12^th day (Figure 2A).

The extents of P₂O₅ extraction are insignificantly lower than those in the investigation with 0.5% w/v NAJP, but these values were achieved for a shorter period of incubation (Figure 2B). For example, using 0.5% NAJP the Σ (α₁+ α₂) and α reached values of 82% and 92.7% on the 12^th day. If MAJP was used under the same conditions the value for the Σ (α₁+ α₂) and α were 84% and 92.4% on the 10^th day. α₂ de creased in the range of 23.4 to 21.25 at the end of the incubation period. The quantity of P₂O_5, separated together with biomass (α₃) increased from the first day up to the 15^th day for (NAJP) and 12^th day for (MAJP) with the values in the range from 4.7 to 13.2% and from 1.29 to 10%, respectively.

Figure 2
Change in the concentration of glucose and the titratable acidity (A) in the culture medium containing 0.5% w/v MAJP, and extents of P₂O₅ extraction, α₁, α₂, α₃, α (B), after 2, 4, 6, 8, 10 and 12 days of incubation. The values are the average ± SD (p ˂ 0.05) from duplicate experiments.

With increasing concentration of JP (NAJP and MAJP) in the nutritive medium the extents of P₂O₅ extraction fall (Figure 3 and Figure 4). The result shows that the higher rate of phosphate dissolution was achieved in the investigations with 1% w/v NAJP - α reached a maximum of 80.80% on the 15^th day (Figure 3B).

The maximum of α in nutritive medium containing 1% w/v MAJP was 69.29% on the 12^th day (Figure 4B). If we compare the results for Σ (α₁+ α₂) and α, the same tendency can be seen. In the investigations with NAJP Σ (α₁+ α₂) had a value of 56% on the 9^th day, but for MAJP this value was 59% on the 8^th day. In the same conditions, the α values were 61.5% and 64%. The reason is the higher values of α₃ for MAJP compared with those for NAJP. The titratable acidity is higher in the experiments with MAJP - 13.88 µE.mL^-1 on the 10^th day (Figure 4A) compared with 11 µE.ml^-1 on the 12^th day for NAJP (Figure 3A). The results show that there is no correlation between citric acid produced and the phosphate solubilization after the 12^th day (NAJP) and 10^th day (MAJP).

Increasing the JP concentration to 2% w/v in liquid medium the titratable acidity increases (Figure 5 and Figure 6). In the experiment with 2% w/v NAJP it reached a maximum of 13 µE.mL^-1 on the 12^th day and slowly decreased to the end of the period when its value was 11.2 µE.mL^-1 (Figure 5A). In the nutritive medium containing 2% w/v MAJP the titratable acidity achieved a value of 15.7 µE.mL^-1 on the 12^th day (Figure 6A).

Figure 3
Change in the concentration of the glucose and the titratable acidity (A) in the culture medium containing 1.0% w/v NAJP, and extents of P₂O₅ extraction, α₁, α₂, α₃, α (B), after 3, 5, 9, 12 and 15 days of incubation. The values are the average ± SD (p ˂ 0.05) from duplicate experiments.

Figure 4
Change in the concentration of the glucose and the titratable acidity (A) in the culture medium containing 1.0% w/v MAJP, and extents of P₂O₅ extraction, α₁, α₂, α₃, α (B), after 2, 4, 6, 8, 10 and 12 days of incubation. The values are the average ± SD (p ˂ 0.05) from duplicate experiments.

Figure 5
Change in the concentration of the glucose and the titratable acidity (A) in the culture medium containing 2.0% w/v NAJP, and extents of P₂O₅ extraction, α₁, α₂, α₃, α (B), after 3, 5, 9, 12 and 15 days of incubation. The values are the average ± SD (p ˂ 0.05) from duplicate experiments.

Figure 6
Change in the concentration of glucose and the titratable acidity (A) in the culture medium containing 2.0% w/v MAJP, and extents of P₂O₅ extraction, α₁, α₂, α₃, α (B), after 2, 4, 6, 8, 10 and 12 days of incubation. The values are the average ± SD (p ˂ 0.05) from duplicate experiments.

There is no significant difference between the extent of P₂O₅ extraction in both experiments (2% w/v NAJP and 2% w/v MAJP). The total degree of phosphate solubilization (α) had a maximum value of 46.02% in the experiments with NAJP on the 12^th day (Figure 5B) and 49.735% in the investigations with MAJP on the 10^th day (Figure 6B), respectively, and these values were preserved to the end of the incubation period. The same tendency was observed for the lower concentrations connected with higher α value and shorter time: for the study with 2% NAJP, α had a value of 33.4% on the 9^th day, but for MAJP a value of 39.5% was obtained on the 8^th day.

The culture pH and the concentration of P₂O₅ extracted from the phosphate detected in the first filtrate, the second filtrate and in biomass expressed as P₂O₅ (C₁, C₂, C₃) and their sum (C) in the investigation with NAJP in the culture are presented in Table 2.

Table 2
Change in culture pH and P concentra­tions (C, g/L) in different forms, after 3, 5, 9, 12 and 15 days of incubation in nutritive medium containing 0.5, 1.0 and 2.0% NAJP.

Day	pH	Concentration of P2O5 (g/L)
		C1	C2	C3	C
0. 5% NAJP
3	3.30	1.34	0.52	0.12	1.98
5	3.30	1.32	0.58	0.17	2.07
9	3.30	1.57	0.45	0.25	2.27
12	3.30	1.66	0.39	0.27	2.32
15	3.31	1.86	0.31	0.33	2.50
1% NAJP
3	3.30	1.38	0.88	0.19	2.45
5	3.30	1.45	0.90	0.18	2.53
9	3.30	1.45	0.90	0.23	2.58
12	3.30	1.94	0.80	0.26	3.00
15	3.31	2.18	0.81	0.41	3.40
2% NAJP
3	3.90	0.58	0.98	0.15	1.71
5	3.30	0.85	1.00	0.16	2.01
9	3.30	1.10	1.05	0.38	2.53
12	3.30	2.14	1.09	0.43	3.66
15	3.30	1.97	1.06	0.41	3.44

• ^*
  c₁ - P₂O_{5 w.s.} ; c₂ - P₂O_{5 c.s.} ; c₃ - P₂O_{5 b.}; c – ∑ (c₁+c₂+c₃)
  The values are the average ± SD (p ˂ 0.05) from duplicate experiments

The culture pH of the initial nutritive medium de creased from 6.8 to 3.3 immediately on the 3^rd day and preserved this value to the end of the period for all the experiments. The concentration of the P₂O₅ extracted from the phosphate (C) increased with in creasing concentration of NAJP and had a constant value of 3.66 g/L from the 12^th day to the end of the incubation period at the highest concentration of 2% w/v NAJP in the liquid.

The result from the investigation with MAJP showed that the culture pH also decreased insignificantly with the incubation period (Table 3). The con centrations of P₂O₅ in the filtrates are about two times higher than those obtained when the culture contained NAJP. The concentration of P₂O₅ released (C) achieved a value of 6.31 g/L on the 15^th in the experiment with 2% w/v MAJP.

Table 3
Change in culture pH and P concentra­tions (C, g/L) in different forms, after 2, 4, 6, 8, 10 and 12 days of incubation in nutritive medium containing 0.5, 1.0 and 2% MAJP.

Day	pH	Concentration of P2O5 (g/L)
		C1	C2	C3	C
0. 5% MAJP
2	3.22	1.22	0.67	0.05	1.94
4	3.02	1.64	0.58	0.15	2.37
6	3.00	1.77	0.66	0.20	2.63
8	2.83	1.96	0.71	0.26	2.93
10	2.61	2.28	0.77	0.29	3.34
12	2.74	2.31	0.77	0.38	3.46
1% MAJP
2	3.28	1.33	1.19	0.06	2.58
4	3.14	1.66	1.00	0.17	2.83
6	3.14	1.94	1.00	0.21	3.15
8	3.00	2.09	1.09	0.26	3.44
10	2.86	2.55	1.05	0.36	3.96
12	2.79	2.61	0.91	0.41	3.93
2% MAJP
2	3.37	1.39	1.53	0.15	3.07
4	3.22	1.72	1.67	0.25	3.64
6	3.22	1.83	1.65	0.26	3.74
8	2.96	2.92	1.59	0.47	4.98
10	2.89	3.27	1.50	0.52	5.29
12	2.91	3.86	1.89	0.56	6.31

• ^*
  c₁ - P₂O_{5 w.s.} ; c₂ - P₂O_{5 c.s.} ; c₃ - P₂O_{5 b} ; c - ∑ (c₁+c₂+c₃)
  The values are the average ± SD (p ˂ 0.05) from duplicate experiments

The results from the present as well as earlier (Ivanova et al., 2006; Bojinova et al., 2008a) studies indicate that the lower the quantity of phosphate applicable, the greater is the conversion percentage, independent of the type of JP used (NAJP or MAJP). These results are in conformity with those reported by others (Nahas et al., 1990; Ghosh and Banic, 1998; Reddy et al., 2002).

In our earlier study we also documented that pre liminary mechanical activation facilitated phosphate dissolution during bioconversion of Morocco phos phorite (MP) by Aspergillus niger up to the 3^rd day, when the total P₂O₅ extraction had higher values using AMP (Bojinova et al., 2008b). In conformity with these investigations a positive correlation be tween the solubility of nonactivated (NMP) and activated Morocco phosphorite (AMP) and titratable acidity was observed to the 12^th day of the incubation period with 1% w/v in the liquid culture. When the incubation time was prolonged to the 15^th day under the same conditions this correlation was negative. If the concentration of NMP and AMP was 2% the total P₂O₅ extraction increased with increasing production of citric acid for all investigated incubation periods of time. According to the present studies the data showed that in the investigations with NAJP (0.5%, 1% and 2%) the tendency is similar. The titratable acidity achieved a maximum on the 12^th day of incubation and decreased to the 15^th day (Figure 1, Figure 3 and Figure 5), but the extent of P₂O₅ extraction into water-soluble forms (α₁) and the total degree of extraction (α) increased for all periods of time investigated. The decrease of the titratable acidity after the 12^th day (Figure 3), 10^th day (Figure 4) and 12^thday (Figure 5), respectively, can be explained by the partial neutralization as Ca-citrate of the citric acid produced with the Ca²⁺ ions liberated as a result of de composition of the phosphate structure. As seen from the data (Figure 2, Figure 4 and Figure 6) for experiments with MAJP, α₁ and α increased together with increasing titratable acidity, independent of the con centration of MAJP used. The correlation coefficients (r) between the quantities of extracted P (C, g/L) and the titratable acidity for different concentrations of NAJP were: (0.64 at 0.5% w/v; 0.92 at 1% w/v and 1.0 at 2% w/v, p_< 0.001), and: (1.0 at 0.5% w/v; 0.96 at 1% w/v and 0.98 at 2% w/v, p_< 0.001) for MAJP. This is in accordance with the results obtained by others (Nahas et al., 1996; Goldstein, 2000).

The results show a negative correlation between the final pH value and titratable acidity. However, the results with MAJP showed values for r (-0.96; -0.96; -0.90, p_< 0.001) distinct from the results with NAJP (r = -0.001; -0.60; -0.60, p_< 0.001), respectively for 0.5, 1.0 and 2% w/v RP.

The result obtained by Rashid et al. (Rashid et al., 2004; Deepa et al., 2010) show a positive correlation between organic acid excretion and P solubilization and a negative correlation between pH and P solubilization. Our results indicate a negative correlation between the quantity of P extracted from the phosphate (C, g/L) and the change of pH. The values of the correlation coefficients (r) were (-0.74; -0.85 and -0.65, p_< 0.001) for NAJP and (-0.96; -0.97 and -0.94, p_< 0.001) for MAJP at similar conditions.

Many of the calcium phosphates, including rock phosphate ores (fluorapatite, francolite), are insoluble in soil with respect to the release of inorganic P at rates necessary to support agronomic levels of plant growth (Goldstein, 2000). Gerretsen (1948) first showed that pure cultures of soil bacteria could increase the P nutrition of plants through increased solubility of Ca phosphates. Their solubility in creases with a decrease of soil pH. Phosphate solubilization is the result of the combined effect of pH decrease and organic acid production (Fankem et al., 2006). Obviously, various chemical elements contained in phosphates are liberated concurrently with P during microbial solubilization. The fungus Asper gillus niger used in our investigations produced mainly citric acid and low concentrations of other organic acids. The dynamic variations of the medium conditions due to changes in Aspergillus niger metabolism and in chemical equilibria are probably the reason for the variations in the solubilized P concentrations (P₂O_{5 c.s.} and P₂O_{5 b.}) observed throughout the incubation (Mendez et al., 2013). Vassilev et al. (1995) observed that decreases in soluble P in the fermentation medium were accompanied by de creases in titratable acidity and suggested that this resulted from the consumption of organic acid by fungus under conditions of C depletion. The data obtained in our work support this hypothesis since the decreases in titratable acidity apparently occurred after the 12^th day of incubation (Figures 1, 2 and 3) in response to the beginning of a new growth cycle, when the fungus may have used part of the organic acid in the metabolism. The fact that the concentration of biomass increased constantly from the 1^st to the 15^th day (NAJP) or from the 1^st to the 12^th day (MAJP) may be a proof for this. At the same time the organic acids form complexes with metalions like Ca, Fe and Al, liberating soluble phosphate. This fact is the other reason for the increasing titratable acidity.

The extent of P₂O₅ extraction in citric soluble form (α₂) decreased from 20.8% to 12.2% (NAJP) and from 23.4% to 20.5% (MAJP), from the 3^rd to the 15^th day and from the 2^nd to the 12^th day, respectively, when the concentration was 0.5% (Figure 1 and Figure 2). The same tendency can be seen for the other concentrations of RP used in the study (Figure 3 - Figure 6). Obviously, the bioconversion was per formed in a complex heterogeneous system with simul taneously occurring biosynthetic and chemical reactions, with different velocities depending on the continuously changing concentrations of macro- and microelements, diffusion velocity, etc. Phosphorus-containing compounds and other ions (Ca²⁺, Mg²⁺, Fe²⁺, Al³⁺, K⁺, Na⁺, etc.) move to the liquid medium face after a defined period of bioconversion. As a result of the phosphate solubilization and a high Ca²⁺ ion concentration, a process of partial reprecipitation of P as slowly soluble and insoluble phosphates in citric acid together with biomass may possibly occur.

Gyaneshwar et al. (2002) suggested that the organic acid secreted can either directly dissolve the mineral phosphate as a result of anion exchange of PO₄^3- by the acid anion or canchelate both Fe³⁺ and Al³⁺ ions associated with phosphate. Complexing of cations is an important mechanism in P solubilization if the organic acid structure favors complexation (Fox et al., 1990). Organic acids may form soluble complexes with metalions associated with insoluble P and thus P is released (Rashid et al., 2004; Kim et al., 2005).

Some authors indicate that acid production is not the only reason for phosphate release into the medium (Pradhan and Sukla, 2005; Gyaneshwar et al., 2002). In certain cases phosphate solubilization is induced by phosphate starvation (Gyaneshwar et al., 1999). Buffering capacity of the medium reduces the effectiveness of PSB in releasing P from tricalcium phosphates (Stephen and Jisha, 2009).

CONCLUSIONS

The production of chemical phosphoric fertilizers is a highly energy-intensive process. On the other hand most of these fertilizers are transformed into insoluble compounds in the soil, unavailable for plants,. Thus, the dependence of fertilizer production on fossil energy, and the prospects of diminishing availability of costly input material for fertilizer production in years to come have obviously brought the subject of mineral phosphate solubilization to the forefront (Khan et al., 2007). It is well known that the high-grade rock phosphate reserves deplete con tinuously. Hence, the exploration of an alternative phosphate source like low-grade phosphate at low cost should be developed in the near future. These are important reasons to use phosphate-solubilizing organisms in agronomic practice as advocated by several researchers.

According to our investigations and the arguments mentioned above, the study with MAJP can be considered to be an alternative for intensification of P-utilization. The investigations indicate that almost complete extraction of P₂O₅ from Jordan Phosphorite in a form utilizable by plant can be achieved by us ing the phosphorus-solubilizing fungus Aspergillus niger. The results obtained can be employed to reduce the fertilizer used if a half dose of P-fertilizer mixed with biofertilizer is introduced into the soil. It is possible to achieve a double effect: the production cost is minimized and the net return maximized.

C total concentration of soluble P2O5 (g/L) C1 concentration of soluble P2O5 in the first filtrate (g/L) C2 concentration of soluble P2O5 in the second filtrate (g/L) C3 concentration of soluble P2O5 in the biomass (g/L) P2O5 b content of P2O5 in the dry biomass (g) P2O5 c.s. content of citric-soluble P2O5 (g) P2O5 m.m. content of P2O5 in the residue of mineral mass (g) P2O5 t. content of total P2O5 (g) P2O5 w.s. content of water-soluble P2O5 (g) rpm rotational speed (revolutions per minute) Greek Letters α total extent of P2O5 extraction (% w/w) α1 extent of P2O5 extraction in water-soluble forms (% w/w) α2 extent of P2O5 extraction in citric-soluble forms (% w/w) α3 extent of P2O5 extraction in biomass (% w/w) Abbreviations JP Jordan Phosphorite MAJP Mechanically Activated Jordan Phosphorite NAJP Non-Activated Jordan Phosphorite OLS Ordinary Least Squares RP Rock Phosphate PSM Phosphate-Solubilizing Microorganisms PSB Phosphorus-Solubilizing Bacteria

C	total concentration of soluble P2O5 (g/L)
C1	concentration of soluble P2O5 in the first filtrate (g/L)
C2	concentration of soluble P2O5 in the second filtrate (g/L)
C3	concentration of soluble P2O5 in the biomass (g/L)
P2O5 b	content of P2O5 in the dry biomass (g)
P2O5 c.s.	content of citric-soluble P2O5 (g)
P2O5 m.m.	content of P2O5 in the residue of mineral mass (g)
P2O5 t.	content of total P2O5 (g)
P2O5 w.s.	content of water-soluble P2O5 (g)
rpm	rotational speed (revolutions per minute)
	Greek Letters
α	total extent of P2O5 extraction (% w/w)
α1	extent of P2O5 extraction in water-soluble forms (% w/w)
α2	extent of P2O5 extraction in citric-soluble forms (% w/w)
α3	extent of P2O5 extraction in biomass (% w/w)
	Abbreviations
JP	Jordan Phosphorite
MAJP	Mechanically Activated Jordan Phosphorite
NAJP	Non-Activated Jordan Phosphorite
OLS	Ordinary Least Squares
RP	Rock Phosphate
PSM	Phosphate-Solubilizing Microorganisms
PSB	Phosphorus-Solubilizing Bacteria

• *
  To whom correspondence should be addressed

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Publication Dates

• Publication in this collection
  Jan-Mar 2016

History

• Received
  07 Feb 2014
• Reviewed
  11 Dec 2014
• Accepted
  08 Feb 2015