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Introduction

Phosphorus (P) and potassium (K) are major essential macronutrients for biological growth and development. However, the concentrations of soluble P and K in soil are usually very low, and a major proportion of P and K in soil are present as insoluble rocks, minerals and other deposits (1). Inspite of that, these sources constitute the biggest reservoirs of P and K in soil because, under appropriate conditions, they can be solubilized and become available for plants (1).

The development of intensively managed agriculture has led to the consumption of increasing amounts of K so that low K supply has become an important yield limiting factor in agriculture (2). However, more than 98% of potassium in soil exists in the form of silicate minerals which K cannot be directly absorbed by plants. Potassium and other minerals can be released when these minerals are weathered. Some microorganisms can play a role in releasing K from minerals (2). Production of organic acids and acidic polysaccharides by microorganism is the mechanism by which K is released. Microorganisms play a central role in the natural P and K cycle. There are considerable populations of P- solubilizing or K-solubilizing bacteria in soil and in plant rhizospheres (3 & 4). Bacteria of the genera Pseudomonas, Agrobacterium, Bacillus, Rhizobium and Flavobacterium have been tested for their ability to solubilize inorganic phosphate compounds, such as tricalcium phosphate, hydroxyapatite and rock phosphate (1 & 5). Potassium solubilizing bacteria were found to release K, silicon and aluminum from insoluble minerals (6). Numerous studies have documented the release of K during the degradation of silicate minerals by bacteria (7- 10) and isolation and identification of K-releasing bacteria has been reported (4, 2, 11- 13). It was postulated that the reactions responsible for microbially promoted K solubilization may involve acidolysis, enzymolysis, capsule absorption and complexation by extracellular polysaccharides (14).

It seems that releasing K from K-containing minerals depends on the type of bacteria, kind of minerals, composition of media and many other factors. In the present study, the potential of seven bacterial species to release K from K-rich minerals was investigated. The major objective of the research was to determine the effect of P sources (soluble or insoluble) on the release of mineral K from biotite and muscovite micas.

Materials and methods

Minerals: Muscovite and biotite were obtained from Zamanabad (Hamadan, Iran) and Qharabagh (Urmia, Iran) Mica Ores, respectively. Micas sheets were powdered to the size <0.5 mm. Owing to the high content of K in mica minerals, soluble K was removed before adding the minerals to Aleksandrov medium (12) by washing with 0.1 M HCl and distilled water. Mica powder (0.4 g) was rinsed in 30 ml of 0.1 M HCl and shaked for 30 min, then the supernatant was removed after centrifuging at 4100 g for 5 min. Further washing was done with 30 ml of water by shaking for 30 min. The mica powder was then dried at 80ºC (4). This pre-treatment should not significantly alter the surface chemistry of the minerals. The content of K in various forms was measured and is shown in Table 1. Soluble K was extracted with dH₂O. Potassium was also extracted from the micas with 1 N NH₄OAc and 1 N HNO₃ by shaking on a reciprocal shaker for 30 min (15). The NH₄OAc extract gave an estimate of the most available fraction of K (soluble and exchangeable K), while extraction with HNO₃ contains nonexchangeable K as well (11). All K in the extracts was determined by flame photometry (Flame photometer 410, Corning).

.Bacterial strains and growth condition: Pseudomonas putida P13 and Pantoea agglomerans P5 were obtained from the Green Biotech company (Tehran, Iran), while P. fluorescens Chao, P. fluorescens Tabriz, P. putida Tabriz, Azotobacter sp. and Bacillus megaterium JK3 were taken from laboratory of soil biology, University of Tabriz (Tabriz, Iran). Nutrient Broth was used to prepare an overnight culture of bacteria to inoculate in Aleksandrov medium (as described by Hu et al. 2006), which was used to study the dissolution of silicate minerals. Aleksandrov medium contained 0.5% glucose, 0.05% MgSO₄.7H₂O, 0.0005% FeCl₃, 0.01% CaCl₂, 0.2% potassium aluminium silicate (muscovite or biotite micas) and 0.2% phosphate source (Ca₃(PO₄)₂ (TCP) or Na₂HPO₄). The initial pH was adjusted to 7.

Experiment 1 (Plate assay): Phosphate solubilizing and K releasing efficiency of seven bacterial strains was determined using Aleksandrov medium in Petri plates containing 0.2% muscovite and 0.2% TCP as sources of K and P respectively. Three replicates of each treatment were included in a completely randomized design. Efficiency was measured based on the diameter of bacterial colonies and halo zone around the colonies. An inoculum of bacteria (10 µl) containing 10⁸ cfu ml^-1 was inoculated on the agar and the inoculated plates were sealed with Parafilm. Dot culture of bacteria was done with three replications. Petri plates were incubated at 26°C and monitored for halo formation for 7 days and the hydrolysis diameter was measured. Solubilizing efficiency was estimated using a solubilizing index (E) according to the following equation: E = (Diameter of hydrolysis zone /Diameter of bacterial growth) (13).

Table 1- Content of K in various forms of muscovite and biotite, before acid wash treatment.

Experiment 2 (liquid assay): In order to quantify the amount of released K in liquid medium a factorial completely randomized design with three replications was used with three factors of (1) the seven bacterial strains plus an uninoculated control, (2) two phosphorus sources (insoluble Ca₃(PO₄)₂ and soluble Na₂HPO₄) and (3) two K-containing minerals (muscovite and biotite). Quantitative estimation of K solubilization was carried out in 100 ml Erlenmeyer flasks containing 25 ml Aleksandrov. Each flask was inoculated with 0.5 ml of bacterial inoculum (approximately 10⁸ cfu ml^-1) (4). Autoclaved, uninoculated medium served as the control to which was added 0.5 ml of sterile NB. The flasks were incubated for 7 days at 26 °C on an incubator with 150 rpm. At the end of incubation 2 ml of cultures were diluted 1:5 (v:v) using dH₂O and centrifuged 4100 g for 4 min. The supernatants were used to assay the solubilized K.

It should be mentioned that Aleksandrov medium was used to determine the amount of released P from TCP while muscovite was added to the medium as a sole source of potassium. Concentration of P was determined spectrophotometrically by ammonium-vanadate-molybdate method and K was determined by flame photometry (16). In presence of V⁵⁺ and Mo⁶⁺, orthophosphates form a yellow colored phosphor-vanado-molybdayte complex which shows an optimal absoption at wavelength 430 nm (16). In order to determine P, 2 ml of diluted supernatant mixed with 4 ml nitrovanadomolybdate reagent and 4 ml dH₂O. Standard series were made between 0 to 50 ppm P. After one hour, the absorption was measured at wavelength 430 nm with a spectrophotometer. To determine K, after dilution of the original diluted supernatant to 1/5, the K-emission is measured in an air-propane flame at wavelength 768 nm. A calibration curve was made with a standard series of 0-50 ppm K. The pH value was measured at the end of incubation with a pH meter.

Statistical analysis: Analysis of variance and mean comparison by Tukey's Significant Difference Test were carried out using MSTATC software. Values are given as means ± S.E. and differences were considered to be significant at the P value< 0.01 level.

Results

.P solubilization in solid and liquid media: In the plate assay only three strains produced a clear zone and highest ratio of clear zone to the diameter of colony (1.47) was for Azotobacter sp. which was followed by P. putida P13 and P. fluorescens Tabriz ratios of 1.40 and 1.39, respectively. A strong positive correlation (r = 0.76; P value< 0.01) was found between this ratio in solid medium and soluble-P concentration in liquid medium.

The solubilization of TCP in Aleksandrov liquid medium by different strains was accompanied by a significant drop in pH to 4.4 from an initial pH of 6.8–7.0 after one week. The soluble-P concentration in the medium, ranged between 67.2 and 348.8 mg l^-1 with variations among different bacterial strains (Fig. 1). In the control treatment 71.5 mg l^-1 soluble-P was detected as well and no drop in pH was observed. The greatest P solubilization was recorded by P. putida P13 (348.4 mg/l) followed by Azotobacter sp., P. putida Tabriz and P. fluorescens Tabriz respectively. Among the isolates, the lowest concentration of soluble-P (67.2 mg/l) was observed in the cultures of Bacillus megaterium JK3. Statistically a strong negative correlation (r = -0.77; P value< 0.001) between pH and soluble-P concentration was observed (Table 4). Results showed that there is no difference between other strains (Pantoea agglomerans P5, P. fluorescens Chao and Bacillus megaterium JK3)and unioculated liquid medium of Aleksandrov.

.K releasing under soluble or insoluble source of P: Potassium release was significantly (P value< 0.001) influenced by bacterial strains, type of K-containing mineral and P sources. The effect of seven strains on biotite and muscovite (as the sole K source) dissolution in Aleksandrov medium is shown in Table 2 and Table 3. The experiments indicated that there was considerable enhancement of K release in the bacterial treatment in the culture fluid with minerals as the sole K source.

The influence of P source (TCP or sodium phosphate) on releasing K is shown in Table 2. It can be seen that when TCP was added to the medium, the content of K in solutions inoculated with the bacterial strains were superior than those media contained sodium phosphate. In presence of TCP the highest released K is obtained by P. putida P13 (8.25 mg/g), which is 4.87 mg/g in the presence of sodium phosphate. In this condition, strain P13 compared with control increased content of K in solution by 12.8 and 62.5%, respectively (Table 2).

Fig. 1- Effect of bacterial strains on solubilizing of P from tricalcium phosphate (Pseudomonas putida P13, P. putida Tabriz, P. fluorescens Tabriz, P. fluorescens Chao, Pantoea agglomerans P5, Azotobacter sp., Bacillus megaterium JK3).

Table 2- Effect of source of phosphate (soluble or insoluble) on releasing K by bacterial strains (P value< 0.001).

Table 3- Effect of type of Micas on releasing K by bacterial strains (P value < 0.05).

Table 4- Correlation coefficient between P and K solubility and pH and H/C.

** Significant at the 1% level, * significant at the 5% level, ^ns non-significant