An e cient microbiological growth medium for screening phosphate solubilizing microorganisms

FEMS Microbiology Letters 170 (1999) 265^270 An e cient microbiological growth medium for screening phosphate solubilizing microorganisms C. Shekhar Nautiyal * Agricultural Microbiology Division, National Botanical Research Institute, Rana Pratap Marg, P.B. No. 436, Lucknow 226 001, India Received 30 June 1998; received in revised form 30 October 1998; accepted 15 November 1998 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. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Microbiological growth medium; Phosphate solubilizing growth medium; Phosphate solubilizer; Phosphorus mineralization 1. Introduction 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 xed 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 xed soil phosphorus and applied phosphates resulting in higher * Tel.: +91 (522) 271031-35; Fax: +91 (522) 282849/282881. 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 e ciency 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 modi ed PVK medium using bromophenol blue, to 0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0378-1097(98)00555-2

266 C.S. Nautiyal / FEMS Microbiology Letters 170 (1999) 265^270 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 de ned medium to elucidate the role of microorganisms in phosphorus mineralization. The objectives of the present study were to formulate a de ned medium for screening phosphate solubilizing microorganisms and to establish a procedure for the identi cation of most e cient phosphate solubilizer from soil. 2. Materials and methods 2.1. Bacterial strains, isolation and identi cation Bacterial strains were isolated from the soil and roots of plants growing in elds 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 puri ed on minimal medium based on AT salts [9]. Micro ora associated with the soil and plant rhizosphere were identi ed as described earlier [10,11]. 2.2. Medium and growth conditions Bacteria representative of the predominant morphological types present on the plates were selected at random and puri ed on minimal medium based on AT salts which contained the following ingredients l 31 : glucose, 10.0 g; KH 2 PO 4, 10.9 g; (NH 4 ) 2 SO 4, 1.0 g; MgSO 4 W7H 2 O, 0.16 g; FeS- O 4 W7H 2 O, 0.005 g; CaCl 2 W2H 2 O, 0.011 g; and MnCl 2 W4H 2 O, 0.002 g [9]. Unless or otherwise stated Pikovskaya (PVK) medium contained l 31 : glucose, 10 g; Ca 3 (PO 4 ) 2, 5 g; (NH 4 ) 2 SO 4, 0.5 g; NaCl, 0.2 g; MgSO 4 W7H 2 O, 0.1 g; KCl, 0.2 g; yeast extract, 0.5 g; MnSO 4 WH 2 O, 0.002 g; and FeSO 4 W7H 2 O, 0.002 g [11]. National Botanical Research Institute's phosphate growth medium devoid of yeast extract (NBRIY) medium contained l 31 : glucose, 10 g; Ca 3 (PO 4 ) 2, 5 g; (NH 4 ) 2 SO 4, 0.5 g; NaCl, 0.2 g; MgSO 4 W7H 2 O, 0.1 g; KCl, 0.2 g; MnSO 4 WH 2 O, 0.002 g and FeSO 4 W7H 2 O, 0.002 g. National Botanical Research Institute's phosphate growth medium (NBRIP) contained l 31 : glucose, 10 g: Ca 3 (PO 4 ) 2, 5 g; MgCl 2 W6H 2 O, 5 g; MgSO 4 W7H 2 O, 0.25 g; KCl, 0.2 g and (NH 4 ) 2 SO 4, 0.1 g. Many variations of the PVK, NBRIY and NBRIP media were tested, as indicated in the text and Tables 1^3. The ph of the media was adjusted to 7.0 before autoclaving. 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 28³C. Halo size was calculated by Table 1 E ect of various ingredients of Pikovskaya medium (PVK) broth on tricalcium phosphate solubilization using Pseudomonas sp. 2 Ingredient(s) Control (PVK) 100.0 PVK3glucose 0.0 PVK3Ca 3 (PO 4 ) 2 0.0 PVK3(NH 4 ) 2 SO 4 130.0 PVK3NaCl 79.1 PVK3MgSO 4 34.7 PVK3KCl 87.0 PVK3yeast 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 PVK3yeast extract3(nh 4 ) 2 SO 4 112.5 PVK3MnSO 4 25.2 PVK3FeSO 4 42.0 % phosphate solubilization compared to control a a Control Pikovskaya (PVK) medium contained l 31 : glucose, 10 g; Ca 3 (PO 4 ) 2, 5 g; (NH 4 ) 2 SO 4, 0.5 g; NaCl, 0.2 g; MgSO 4 W7H 2 O, 0.1 g; KCl, 0.2 g; yeast extract, 0.5 g; MnSO 4 WH 2 O, 0.002 g and FeSO 4 W7H 2 O, 0.002 g. The data are means of three experiments.

Table 2 E ect 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) subtracting colony diameter from the total diameter. Quantitative estimation of phosphate solubilization in broth was carried out using Erlenmeyer asks (150 ml) containing 10 ml of medium inoculated in triplicate with the bacterial strain (100 Wl inoculum with approximately 1^2U10 9 cfu ml 31 ). Autocleaved uninoculated medium served as control. The asks were incubated for 2 days at 30³C on a New Brunswick Scienti c, 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. 3. Results and discussion 3.1. E ect 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 in uence of each ingredient of the medium, the phosphate solubilization was estimated by deleting one C.S. Nautiyal / FEMS Microbiology Letters 170 (1999) 265^270 267 PVK 72.4 Control (NBRIY) 100.0 NBRIY (glucose, 1) 15.9 NBRIY (glucose, 2.5) 23.6 NBRIY (glucose, 5) 32.5 NBRIY3(NH 4 ) 2 SO 4 +KNO 3, 0.5 27.1 NBRIY3(NH 4 ) 2 SO 4 3KCl+KNO 3, 0.5 12.1 NBRIY3(NH 4 ) 2 SO 4 +NH 4 Cl, 0.5 84.1 NBRIY[(NH 4 ) 2 SO 4, 0.1] 112.1 NBRIY[(NH 4 ) 2 SO 4, 2.5] 79.9 NBRIY3(NH 4 ) 2 SO 4 +(NH 4 ) 2 Fe(SO 4 )W6H 2 O, 0.5 75.1 NBRIY3(NH 4 ) 2 SO 4 3FeSO 4 +(NH 4 ) 2 Fe(SO 4 )W6H 2 O, 0.5 56.6 NBRIY (MgSO 4, 0.25; MnSO 4, 0.0) 63.2 NBRIY (MgSO 4, 0.25; MnSO 4, 0.0025) 98.6 NBRIY3MgSO 4 +MgCl 2, 10 109.8 NBRIY+(CuSO 4 +ZnCl 2 +MoCL 4 +CoCl 2 W6H 2 O, 0.002 of each) 113.5 % phosphate solubilization compared to control a a Control NBRIY medium contained l 31 : glucose, 10 g; Ca 3 (PO 4 ) 2, 5 g; (NH 4 ) 2 SO 4, 0.5 g; NaCl, 0.2 g; MgSO 4 W7H 2 O, 0.1 g; KCl, 0.2 g; MnSO 4 WH 2 O, 0.002 g and FeSO 4 W7H 2 O, 0.002 g. Changes in the concentration of NBRIY components are indicated within brackets. The data are means of three experiments. Table 3 E ect of various ingredients of National Botanical Research Institute's phosphate growth medium (NBRIP) broth on tricalcium phosphate solubilization using Pseudomonas sp. 2 Ingredient(s) PVK 41.6 NBRIY 56.6 Control (NBRIP) 100.0 NBRIP (glucose, 5; MgCl 2, 2.5) 54.3 NBRIP (glucose, 5; MgCl 2, 5.0) 56.8 NBRIP (glucose, 5; MgCl 2, 10) 72.4 NBRIP (glucose, 10 and MgCl 2, 2.5) 96.2 NBRIP (glucose, 10 and MgCl 2, 10) 104.0 NBRIP (glucose, 20; MgCl 2, 2.5) 113.0 NBRIP (glucose, 20; MgCl 2, 5.0) 118.4 NBRIP (glucose, 20; MgCl 2, 10) 121.6 NBRIP3glucose+arabinose 77.5 NBRIP3glucose+fructose 35.0 NBRIP3glucose+galactose 42.2 NBRIP3glucose+sorbitol 4.4 NBRIP3glucose+mannitol 29.0 NBRIP3glucose+xylose 87.4 NBRIP3glucose+sucrose 41.5 NBRIP3glucose+maltose 31.0 NBRIP3glucose+lactose 70.3 NBRIP3glucose+ra nose 28.2 % phosphate solubilization compared to control a a Control NBRIP medium contained l 31 : glucose, 10 g; Ca 3 (PO 4 ) 2, 5 g; MgCl 2 W6H 2 O, 5 g; MgSO 4 W7H 2 O, 0.25 g; KCl, 0.2 g and (NH 4 ) 2 SO 4, 0.1 g. Changes in the concentration of NBRIP components are indicated within brackets. The data are means of three experiments.

268 C.S. Nautiyal / FEMS Microbiology Letters 170 (1999) 265^270 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 Medium Bacteria Treatment PVK Agar (halo size (mm)) Pseudomonas sp. 1 ^ 8 Pseudomonas sp. 2 5 35 P. uorescens 6 13 P. aerogenes ^ 14 P. aeruginosa 8 14 Bacillus polymyxa 2 8 B. subtilis 3 11 Bacillus sp. 1 5 17 Broth (Wg ml 31 P solubilized) NBRIP Pseudomonas sp. 1 2 26 Pseudomonas sp. 2 6 90 P. uorescens 7 42 P. aerogenes ^ 31 P. aeruginosa 6 28 Bacillus polymyxa 2 21 B. subtilis 4 35 Bacillus sp. 1 5 60 The data are means of three experiments. component at a time (Table 1). It was observed that glucose and Ca 3 (PO 4 ) 2 were essential and yeast extract and (NH 4 ) 2 SO 4 non-essential components of the medium. Moderately required ingredients in the PVK were in the decreasing order MnSO 4, MgSO 4, FeSO 4, NaCl and KCl. Phosphate solubilization ability of Pseudomonas sp. 2 increased by about 30% in the absence of either yeast extract or (NH 4 ) 2 SO 4. In the absence of both yeast extract and (NH 4 ) 2 SO 4, 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 31 ), it was observed that at the concentration of 0.1 g l 31 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 31 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 de ned components. This medium devoid of yeast extract was designated as NBRIY. 3.2. E ect 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 in uence the phosphate solubilization by NBRIY was checked by replacing (NH 4 ) 2 SO 4 with KNO 3. When used as sole source of nitrogen, KNO 3 was 27.1% less e ective compared to (NH 4 ) 2 SO 4. When KCl was excluded from NBRI to test the ability of KNO 3 to act both as a sole source of nitrogen and potassium, the phosphate solubilization ability of Pseudomonas sp. 2 further declined to 12.1%. NH 4 Cl could be used as a nitrogen source. However, phosphate solubilization ability improved when (NH 4 ) 2 SO 4 was used at a lower concentration of 0.1 instead of 0.5 g l 31 (Table 2). (NH 4 ) 2 Fe(SO 4 )

C.S. Nautiyal / FEMS Microbiology Letters 170 (1999) 265^270 269 Based on the observations obtained as described above, a new medium, NBRIP, was de ned. Concentrations of glucose and MgCl 2 played an important role in phosphate solubilization ability. Maximum phosphate solubilization activity was obtained when 20 and 10 g l 31 of glucose and MgCl 2, respectively, were used (Table 3). However, 10 and 5 g l 31 of glucose and MgCl 2, respectively, were used in the nal formulation of NBRIP, keeping in mind the cost of the end product. 3.4. E ciency of phosphate solubilization by NBRIP Fig. 1. Phosphate solubilization by Pseudomonas sp. 2. Solubilization of phosphate (Wg ml 31 ) by Pseudomonas sp. 2 in broth was determined using Pikovskaya medium (PVK; a), National Botanical Research Institute's phosphate growth medium devoid of yeast extract (NBRIY; b) and National Botanical Research Institute's phosphate growth medium (NBRIP; E) medium. E ciency of phosphate solubilization in NBRIP medium was signi cantly 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. could be used both as the source of nitrogen and iron albeit less e ciently (Table 2). The ability of phosphate solubilization improved when the concentration of MgSO 4 was increased from 0.1 to 0.25 g l 31 (Table 2). MgCl 2 had a better synergistic e ect on phosphate solubilization activity, in the presence of MgSO 4 compared to MnSO 4 (Table 2). Addition of trace amounts of CuSO 4, ZnCl 2, MoCl 4 and CoCl 2 had no signi cant e ect on phosphate solubilization (Table 2). 3.3. Novel phosphate solubilization medium 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 e cient 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 identi able as the most e cient strain in an NBRIP broth assay (Table 4). Phosphate solubilization of the strain Pseudomonas sp. 2 was studied using PVK, NBRIY and NBRIP broth up to 10 days (Fig. 1). E ciency of phosphate solubilization by the strain Pseudomonas sp. 2 in NBRIP medium was signi cantly 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 e cient phosphate solubilization medium over PVK. 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 di usion rates of di erent 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

270 C.S. Nautiyal / FEMS Microbiology Letters 170 (1999) 265^270 screened in NBRIP broth assay for the identi cation of most e cient phosphate solubilizers. Thus, one advantage of using the present formulation is that NBRIP can be used as a de ned medium because it excludes the use of yeast extract. Secondly, NBRIP is more e cient in a broth assay compared to PVK. Furthermore, the present work indicates that soil microbes should be screened in NBRIP broth assay for the identi cation of the most e cient phosphate solubilizers. Acknowledgments 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 Scienti c and Industrial Research, New Delhi. References [1] Abd-Alla, M.H. (1994) Phosphatases and the utilization of organic phosphorus by Rhizobium leguminosarum biovar viceae. Lett. Appl. Microbiol. 18, 294^296. [2] Yadav, K.S. and Dadarwal, K.R. (1997) Phosphate solubilization and mobilization through soil microorganisms. In: Biotechnological Approaches in Soil Microorganisms for Sustainable Crop Production (Dadarwal, K.R., Ed.), pp. 293^308. Scienti c Publishers, Jodhpur. [3] Jones, D.L. and Darrah, P.R. (1994) Role of root derived organic acids in the mobilization of nutrients from the rhizosphere. Plant Soil. 166, 247^257. [4] Leyval, C. and Barthelin, J. (1989) Interactions between Laccaria laccata, Agrobacterium radiobacter and beech roots: in- uence on P, K, Mg and Fe mobilization from mineral and plant growth. Plant Soil. 17, 103^110. [5] Pikovskaya, R.I. (1948) Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17, 362^370. [6] Katznelson, H., Peterson, E. and Rouatt, J.W. (1962) Phosphate dissolving microorganisms on seed and in the root zone of plants. Can. J. Bot. 40, 1181^1186. [7] Gupta, R., Singal, R., Shankar, A., Kuhad, R.C. and Saxena, R.K. (1994) A modi ed plate assay for screening phosphate solubilizing microorganisms. J. Gen. Appl. Microbiol. 40, 255^260. [8] Louw, H.A. and Webley, D.M. (1959) A study of soil bacteria dissolving certain phosphate fertilizers and related compounds. J. Appl. Bacteriol. 22, 227^233. [9] Nautiyal, C.S. (1997) A method for selection and characterization of rhizosphere-competent bacteria of chickpea. Curr. Microbiol. 34, 12^17. [10] Nautiyal, C.S. (1997) Rhizosphere competence of Pseudomonas sp. NBRI9926 and Rhizobium sp. NBRI9513 involved in the suppression of chickpea (Cicer arietinum L.) pathogenic fungi. FEMS Microbiol. Ecol. 23, 145^158. [11] Surange, S., Wollum, A.G. II, Nikhil Kumar and Nautiyal, C.S. (1997) Characterization of Rhizobium from root nodules of leguminous trees growing in alkaline soils. Can. J. Microbiol. 43, 891^894. [12] Fiske, C.H. and Subbarow, Y. (1925) A colorimetric determination of phosphorus. J. Biol. Chem. 66, 375^400. [13] Johnston, H.W. (1952) The solubilization of phosphate: the action of various organic compounds on dicalcium and tricalcium phosphate. New Zealand J. Sci. Technol. 33, 436^444.