An efficient microbiological growth medium for screening phosphate solubilizing microorganisms

Authors

  • C.Shekhar Nautiyal

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    1. Agricultural Microbiology Division, National Botanical Research Institute, Rana Pratap Marg, P.B. No. 436, Lucknow 226 001, India
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Abstract

A novel defined microbiological growth medium, National Botanical Research Institute's phosphate growth medium (NBRIP), which is more efficient than Pikovskaya medium (PVK), was developed for screening phosphate solubilizing microorganisms. In plate assay the efficiency of NBRIP was comparable to PVK; however, in broth assay NBRIP consistently demonstrated about 3-fold higher efficiency compared to PVK. The results indicated that the criterion for isolation of phosphate solubilizers based on the formation of visible halo/zone on agar plates is not a reliable technique, as many isolates which did not show any clear zone on agar plates solubilized insoluble inorganic phosphates in liquid medium. It may be concluded that soil microbes should be screened in NBRIP broth assay for the identification of the most efficient phosphate solubilizers.

1Introduction

Phosphorus is one of the major plant nutrients limiting plant growth. Most of the essential plant nutrients, including phosphorus, remain in insoluble form in soil [1, 2]. A large portion of inorganic phosphates applied to soil as fertilizer is rapidly immobilized after application and becomes unavailable to plants [2]. Thus, the release of insoluble and fixed forms of phosphorus is an important aspect of increasing soil phosphorus availability. Seed or soil inoculation with phosphate-solubilizing bacteria is known to improve solubilization of fixed soil phosphorus and applied phosphates resulting in higher crop yields [1–3]. Several authors attribute the solubilization of inorganic insoluble phosphate by microorganisms to the production of organic acids and chelating oxo acids from sugars [2, 4]. Phosphate solubilizing microorganisms are routinely screened by a plate assay method using Pikovskaya (PVK) agar [5]. The test of the relative efficiency of isolated strains is carried out by selecting the microorganisms which are capable of producing a halo/clear zone on plate due to the production of organic acids into the surrounding medium [6]. However, the reliability of this halo-based technique is questioned as many isolates which did not produce any visible halo/zone on agar plates could solubilize various types of insoluble inorganic phosphates in liquid medium [7, 8]. A modified PVK medium using bromophenol blue, to improve the clarity and visibility of the yellow-colored halo has not necessarily improved the plate assay [7]. Moreover PVK medium contains yeast extract and it is desirable to formulate a defined medium to elucidate the role of microorganisms in phosphorus mineralization.

The objectives of the present study were to formulate a defined medium for screening phosphate solubilizing microorganisms and to establish a procedure for the identification of most efficient phosphate solubilizer from soil.

2Materials and methods

2.1Bacterial strains, isolation and identification

Bacterial strains were isolated from the soil and roots of plants growing in fields with normal and alkaline soils (exposed to high salt, temperature and pH stresses) at Banthra village, Lucknow, India. Roots were thoroughly washed with tap water for two minutes to remove all the loosely adhering soil particles followed by washing with sterile 0.85% (w/v) saline Milli Q water (MQW). The roots were then macerated in 0.85% saline MQW with a mortar and pestle. Serial dilution of the root homogenate and soil (10% soil in 0.85% saline MQW) samples were then individually plated on Pseudomonas isolation agar, Nutrient agar and tryptone-glucose-yeast extract (TGY) agar (from HI-medium Laboratories, Bombay, India) as described earlier [9]. Bacteria representative of the predominant morphologically distinct colonies present on the plates were selected at random and purified on minimal medium based on AT salts [9]. Microflora associated with the soil and plant rhizosphere were identified as described earlier [10, 11].

2.2Medium and growth conditions

Bacteria representative of the predominant morphological types present on the plates were selected at random and purified on minimal medium based on AT salts which contained the following ingredients l−1: glucose, 10.0 g; KH2PO4, 10.9 g; (NH4)2SO4, 1.0 g; MgSO4·7H2O, 0.16 g; FeSO4·7H2O, 0.005 g; CaCl2·2H2O, 0.011 g; and MnCl2·4H2O, 0.002 g [9]. Unless or otherwise stated Pikovskaya (PVK) medium contained l−1: glucose, 10 g; Ca3(PO4)2, 5 g; (NH4)2SO4, 0.5 g; NaCl, 0.2 g; MgSO4·7H2O, 0.1 g; KCl, 0.2 g; yeast extract, 0.5 g; MnSO4·H2O, 0.002 g; and FeSO4·7H2O, 0.002 g [11]. National Botanical Research Institute's phosphate growth medium devoid of yeast extract (NBRIY) medium contained l−1: glucose, 10 g; Ca3(PO4)2, 5 g; (NH4)2SO4, 0.5 g; NaCl, 0.2 g; MgSO4·7H2O, 0.1 g; KCl, 0.2 g; MnSO4·H2O, 0.002 g and FeSO4·7H2O, 0.002 g. National Botanical Research Institute's phosphate growth medium (NBRIP) contained l−1: glucose, 10 g: Ca3(PO4)2, 5 g; MgCl2·6H2O, 5 g; MgSO4·7H2O, 0.25 g; KCl, 0.2 g and (NH4)2SO4, 0.1 g. Many variations of the PVK, NBRIY and NBRIP media were tested, as indicated in the text and Table 1Table 2Table 3. The pH of the media was adjusted to 7.0 before autoclaving.

Table 1.  Effect of various ingredients of Pikovskaya medium (PVK) broth on tricalcium phosphate solubilization using Pseudomonas sp. 2
Ingredient(s)% phosphate solubilization compared to controla
  1. aControl Pikovskaya (PVK) medium contained l−1: glucose, 10 g; Ca3(PO4)2, 5 g; (NH4)2SO4, 0.5 g; NaCl, 0.2 g; MgSO4·7H2O, 0.1 g; KCl, 0.2 g; yeast extract, 0.5 g; MnSO4·H2O, 0.002 g and FeSO4·7H2O, 0.002 g. The data are means of three experiments.

Control (PVK)100.0
PVK−glucose0.0
PVK−Ca3(PO4)20.0
PVK−(NH4)2SO4130.0
PVK−NaCl79.1
PVK−MgSO434.7
PVK−KCl87.0
PVK−yeast extract (0.0)131.4
PVK+yeast extract (0.1)144.0
PVK+yeast extract (0.25)125.0
PVK+yeast extract (1.0)88.0
PVK+yeast extract (2.5)31.2
PVK+yeast extract (5.0)24.2
PVK−yeast extract−(NH4)2SO4112.5
PVK−MnSO425.2
PVK−FeSO442.0
Table 2.  Effect of various ingredients of National Botanical Research Institute's phosphate growth medium devoid of yeast extract (NBRIY) broth on tricalcium phosphate solubilization using Pseudomonas sp. 2
Ingredient(s)% phosphate solubilization compared to controla
  1. aControl NBRIY medium contained l−1: glucose, 10 g; Ca3(PO4)2, 5 g; (NH4)2SO4, 0.5 g; NaCl, 0.2 g; MgSO4·7H2O, 0.1 g; KCl, 0.2 g; MnSO4·H2O, 0.002 g and FeSO4·7H2O, 0.002 g. Changes in the concentration of NBRIY components are indicated within brackets. The data are means of three experiments.

PVK72.4
Control (NBRIY)100.0
NBRIY (glucose, 1)15.9
NBRIY (glucose, 2.5)23.6
NBRIY (glucose, 5)32.5
NBRIY−(NH4)2SO4+KNO3, 0.527.1
NBRIY−(NH4)2SO4−KCl+KNO3, 0.512.1
NBRIY−(NH4)2SO4+NH4Cl, 0.584.1
NBRIY[(NH4)2SO4, 0.1]112.1
NBRIY[(NH4)2SO4, 2.5]79.9
NBRIY−(NH4)2SO4+(NH4)2Fe(SO4)·6H2O, 0.575.1
NBRIY−(NH4)2SO4−FeSO4+(NH4)2Fe(SO4)·6H2O, 0.556.6
NBRIY (MgSO4, 0.25; MnSO4, 0.0)63.2
NBRIY (MgSO4, 0.25; MnSO4, 0.0025)98.6
NBRIY−MgSO4+MgCl2, 10109.8
NBRIY+(CuSO4+ZnCl2+MoCL4+CoCl2·6H2O, 0.002 of each)113.5
Table 3.  Effect of various ingredients of National Botanical Research Institute's phosphate growth medium (NBRIP) broth on tricalcium phosphate solubilization using Pseudomonas sp. 2
Ingredient(s)% phosphate solubilization compared to controla
  1. aControl NBRIP medium contained l−1: glucose, 10 g; Ca3(PO4)2, 5 g; MgCl2·6H2O, 5 g; MgSO4·7H2O, 0.25 g; KCl, 0.2 g and (NH4)2SO4, 0.1 g. Changes in the concentration of NBRIP components are indicated within brackets. The data are means of three experiments.

PVK41.6
NBRIY56.6
Control (NBRIP)100.0
NBRIP (glucose, 5; MgCl2, 2.5)54.3
NBRIP (glucose, 5; MgCl2, 5.0)56.8
NBRIP (glucose, 5; MgCl2, 10)72.4
NBRIP (glucose, 10 and MgCl2, 2.5)96.2
NBRIP (glucose, 10 and MgCl2, 10)104.0
NBRIP (glucose, 20; MgCl2, 2.5)113.0
NBRIP (glucose, 20; MgCl2, 5.0)118.4
NBRIP (glucose, 20; MgCl2, 10)121.6
NBRIP−glucose+arabinose77.5
NBRIP−glucose+fructose35.0
NBRIP−glucose+galactose42.2
NBRIP−glucose+sorbitol4.4
NBRIP−glucose+mannitol29.0
NBRIP−glucose+xylose87.4
NBRIP−glucose+sucrose41.5
NBRIP−glucose+maltose31.0
NBRIP−glucose+lactose70.3
NBRIP−glucose+raffinose28.2

Bacterial strains were tested by plate assay using PVK and NBRIP media supplemented with 1.5% Bacto-agar (Difco Laboratories, Detroit, MI, USA). Four strains per plate were stabbed in triplicate using sterile toothpicks. The halo and colony diameters were measured after 14 days of the incubation of plates at 28°C. Halo size was calculated by subtracting colony diameter from the total diameter. Quantitative estimation of phosphate solubilization in broth was carried out using Erlenmeyer flasks (150 ml) containing 10 ml of medium inoculated in triplicate with the bacterial strain (100 μl inoculum with approximately 1–2×109 cfu ml−1). Autocleaved uninoculated medium served as control. The flasks were incubated for 2 days at 30°C on a New Brunswick Scientific, USA, Innova Model 4230 refrigerated incubator shaker at 180 rpm. The cultures were harvested by centrifugation at 10 000 rpm for 10 min, using Sorvall RC 5C centrifuge, Dupont, USA. Phosphate in culture supernatant was estimated using the Fiske and Subbarow method [12]. The data are means of three experiments.

3Results and discussion

3.1Effect of yeast extract on phosphate solubilization

Quantitative estimation of solubilization was carried out using strain Pseudomonas sp. 2 grown on PVK liquid medium for 2 days. To elucidate the influence of each ingredient of the medium, the phosphate solubilization was estimated by deleting one component at a time (Table 1). It was observed that glucose and Ca3(PO4)2 were essential and yeast extract and (NH4)2SO4 non-essential components of the medium. Moderately required ingredients in the PVK were in the decreasing order MnSO4, MgSO4, FeSO4, NaCl and KCl. Phosphate solubilization ability of Pseudomonas sp. 2 increased by about 30% in the absence of either yeast extract or (NH4)2SO4. In the absence of both yeast extract and (NH4)2SO4, the phosphate solubilization ability of Pseudomonas sp. 2 was enhanced by 12.5%. It was interesting to note that by simply omitting yeast extract from PVK consistently higher phosphate solubilization levels were obtained. When the yeast extract was used in the range of 0.1–5.0 (g l−1), it was observed that at the concentration of 0.1 g l−1 the phosphate solubilization ability of Pseudomonas sp. 2 increased by 44%. On the contrary, increasing the concentration of yeast extract to more than 0.5 g l−1 resulted in the reduction of phosphate solubilization (Table 1). This further proved that the presence of yeast extract in the PVK medium was inhibitory to the phosphate solubilization. Therefore, the yeast extract was omitted from PVK medium, to formulate a new medium with defined components. This medium devoid of yeast extract was designated as NBRIY.

3.2Effect of carbon and nitrogen sources on phosphate solubilization

The amount of glucose as a carbon source played an important role in the phsophate solubilization. The rate of the phosphate solibilization was increased with increasing concentrations of glucose (Table 2). The ability of the nitrogen source to influence the phosphate solubilization by NBRIY was checked by replacing (NH4)2SO4 with KNO3. When used as sole source of nitrogen, KNO3 was 27.1% less effective compared to (NH4)2SO4. When KCl was excluded from NBRI to test the ability of KNO3 to act both as a sole source of nitrogen and potassium, the phosphate solubilization ability of Pseudomonas sp. 2 further declined to 12.1%. NH4Cl could be used as a nitrogen source. However, phosphate solubilization ability improved when (NH4)2SO4 was used at a lower concentration of 0.1 instead of 0.5 g l−1 (Table 2). (NH4)2Fe(SO4) could be used both as the source of nitrogen and iron albeit less efficiently (Table 2). The ability of phosphate solubilization improved when the concentration of MgSO4 was increased from 0.1 to 0.25 g l−1 (Table 2). MgCl2 had a better synergistic effect on phosphate solubilization activity, in the presence of MgSO4 compared to MnSO4 (Table 2). Addition of trace amounts of CuSO4, ZnCl2, MoCl4 and CoCl2 had no significant effect on phosphate solubilization (Table 2).

3.3Novel phosphate solubilization medium NBRIP

Based on the observations obtained as described above, a new medium, NBRIP, was defined. Concentrations of glucose and MgCl2 played an important role in phosphate solubilization ability. Maximum phosphate solubilization activity was obtained when 20 and 10 g l−1 of glucose and MgCl2, respectively, were used (Table 3). However, 10 and 5 g l−1 of glucose and MgCl2, respectively, were used in the final formulation of NBRIP, keeping in mind the cost of the end product.

3.4Efficiency of phosphate solubilization by NBRIP

Comparative studies on NBRIP and PVK with eight bacteria in a plate assay showed similar results when compared for phosphate solubilization ability (Table 4). However, in broth assay NBRIP was about 3-fold more efficient compared to PVK broth for all the eight strains. Thus the strain Pseudomonas sp. 2 which was otherwise indistinguishable from other strains in its ability to solubilize phosphate on a plate assay was easily identifiable as the most efficient strain in an NBRIP broth assay (Table 4).

Table 4.  Comparison of tricalcium phosphate solubilization by bacterial isolates in agar and broth using Pilovskaya medium (PVK) and National Botanical Research Institute's phosphate growth medium (NBRIP) medium
MediumBacteriaTreatment
  Agar (halo size (mm))Broth (μg ml−1 P solubilized)
PVK   
  1. The data are means of three experiments.

 Pseudomonas sp. 18
 Pseudomonas sp. 2535
 P. fluorescens613
 P. aerogenes14
 P. aeruginosa814
 Bacillus polymyxa28
 B. subtilis311
 Bacillus sp. 1517
    
NBRIP   
 Pseudomonas sp. 1226
 Pseudomonas sp. 2690
 P. fluorescens742
 P. aerogenes31
 P. aeruginosa628
 Bacillus polymyxa221
 B. subtilis435
 Bacillus sp. 1560

Phosphate solubilization of the strain Pseudomonas sp. 2 was studied using PVK, NBRIY and NBRIP broth up to 10 days (Fig. 1). Efficiency of phosphate solubilization by the strain Pseudomonas sp. 2 in NBRIP medium was significantly higher compared to PVK and NBRIY. The ability of the strain to solubilize phosphorus in NBRIP was also maintained at a higher level throughout the duration of 10 days (Fig. 1). This further augments well for the use of NBRIP as an efficient phosphate solubilization medium over PVK.

Figure 1.

Phosphate solubilization by Pseudomonas sp. 2. Solubilization of phosphate (μg ml−1) by Pseudomonas sp. 2 in broth was determined using Pikovskaya medium (PVK; ◯), National Botanical Research Institute's phosphate growth medium devoid of yeast extract (NBRIY; •) and National Botanical Research Institute's phosphate growth medium (NBRIP; □) medium. Efficiency of phosphate solubilization in NBRIP medium was significantly higher compared to PVK and NBRIY. The ability of the strain to solubilize phosphorus in NBRIP was also maintained at a higher level up to 10 days.

Among the various bacteria tested, P. aerogenes did not produce a halo on PVK and NBRIP plate assay, while Pseudomonas sp. 1 and P. aerogenes did not produce a halo on PVK plates (Table 4). However, all eight bacteria could solubilize tricalcium phosphate in broth (Table 4). The data indicated that the criterion for isolation of phosphate solubilizer based on the formation of a visible halo/zone on agar plates is not an infallible technique. It has been reported that many isolates which did not show any clear zone on agar plates solubilized insoluble inorganic phosphates in liquid medium [4, 8]. Thus, the existing plate assay fails where the halo is inconspicuous or absent. This may be because of the varying diffusion rates of different organic acids secreted by an organism [13]. Contrary to indirect measurement of phosphate solubilization by plate assay, the direct measurement of phosphate solubilization in broth assay resulted into reliable results. Therefore, it is hereby suggested that microbes from soil may be screened in NBRIP broth assay for the identification of most efficient phosphate solubilizers.

Thus, one advantage of using the present formulation is that NBRIP can be used as a defined medium because it excludes the use of yeast extract. Secondly, NBRIP is more efficient in a broth assay compared to PVK. Furthermore, the present work indicates that soil microbes should be screened in NBRIP broth assay for the identification of the most efficient phosphate solubilizers.

Acknowledgements

I am grateful to P.V. Sane for his valuable encouragement, many useful discussions and critical comments on the manuscript. This investigation was supported by a Super Special Grant from the Director General, Council of Scientific and Industrial Research, New Delhi.

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