Download An efficient microbiological growth medium for screening phosphate

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: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 5 5 - 2
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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 l31 : glucose, 10.0 g; KH2 PO4 , 10.9 g;
(NH4 )2 SO4 , 1.0 g; MgSO4 W7H2 O, 0.16 g; FeSO4 W7H2 O, 0.005 g; CaCl2 W2H2 O, 0.011 g; and
MnCl2 W4H2 O, 0.002 g [9]. Unless or otherwise stated
Pikovskaya (PVK) medium contained l31 : glucose,
10 g; Ca3 (PO4 )2 , 5 g; (NH4 )2 SO4 , 0.5 g; NaCl, 0.2
g; MgSO4 W7H2 O, 0.1 g; KCl, 0.2 g; yeast extract, 0.5
g; MnSO4 WH2 O, 0.002 g; and FeSO4 W7H2 O, 0.002 g
[11]. National Botanical Research Institute's phosphate growth medium devoid of yeast extract
(NBRIY) medium contained l31 : glucose, 10 g;
Ca3 (PO4 )2 , 5 g; (NH4 )2 SO4 , 0.5 g; NaCl, 0.2 g;
MgSO4 W7H2 O, 0.1 g; KCl, 0.2 g; MnSO4 WH2 O,
0.002 g and FeSO4 W7H2 O, 0.002 g. National Botanical Research Institute's phosphate growth medium
(NBRIP) contained l31 : glucose, 10 g: Ca3 (PO4 )2 ,
5 g; MgCl2 W6H2 O, 5 g; MgSO4 W7H2 O, 0.25 g; KCl,
0.2 g and (NH4 )2 SO4 , 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)
% phosphate solubilization
compared to controla
Control (PVK)
PVK3glucose
PVK3Ca3 (PO4 )2
PVK3(NH4 )2 SO4
PVK3NaCl
PVK3MgSO4
PVK3KCl
PVK3yeast extract (0.0)
PVK+yeast extract (0.1)
PVK+yeast extract (0.25)
PVK+yeast extract (1.0)
PVK+yeast extract (2.5)
PVK+yeast extract (5.0)
PVK3yeast extract3(NH4 )2 SO4
PVK3MnSO4
PVK3FeSO4
100.0
0.0
0.0
130.0
79.1
34.7
87.0
131.4
144.0
125.0
88.0
31.2
24.2
112.5
25.2
42.0
a
Control Pikovskaya (PVK) medium contained l31 : glucose, 10 g;
Ca3 (PO4 )2 , 5 g; (NH4 )2 SO4 , 0.5 g; NaCl, 0.2 g; MgSO4 W7H2 O, 0.1
g; KCl, 0.2 g; yeast extract, 0.5 g; MnSO4 WH2 O, 0.002 g and
FeSO4 W7H2 O, 0.002 g. The data are means of three experiments.
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267
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)
% phosphate solubilization compared to controla
PVK
Control (NBRIY)
NBRIY (glucose, 1)
NBRIY (glucose, 2.5)
NBRIY (glucose, 5)
NBRIY3(NH4 )2 SO4 +KNO3 , 0.5
NBRIY3(NH4 )2 SO4 3KCl+KNO3 , 0.5
NBRIY3(NH4 )2 SO4 +NH4 Cl, 0.5
NBRIY[(NH4 )2 SO4 , 0.1]
NBRIY[(NH4 )2 SO4 , 2.5]
NBRIY3(NH4 )2 SO4 +(NH4 )2 Fe(SO4 )W6H2 O, 0.5
NBRIY3(NH4 )2 SO4 3FeSO4 +(NH4 )2 Fe(SO4 )W6H2 O, 0.5
NBRIY (MgSO4 , 0.25; MnSO4 , 0.0)
NBRIY (MgSO4 , 0.25; MnSO4 , 0.0025)
NBRIY3MgSO4 +MgCl2 , 10
NBRIY+(CuSO4 +ZnCl2 +MoCL4 +CoCl2 W6H2 O, 0.002 of each)
72.4
100.0
15.9
23.6
32.5
27.1
12.1
84.1
112.1
79.9
75.1
56.6
63.2
98.6
109.8
113.5
a
Control NBRIY medium contained l31 : glucose, 10 g; Ca3 (PO4 )2 , 5 g; (NH4 )2 SO4 , 0.5 g; NaCl, 0.2 g; MgSO4 W7H2 O, 0.1 g; KCl, 0.2 g;
MnSO4 WH2 O, 0.002 g and FeSO4 W7H2 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 Insubtracting colony diameter from the total diameter.
stitute's phosphate growth medium (NBRIP) broth on tricalcium
Quantitative estimation of phosphate solubilization
phosphate solubilization using Pseudomonas sp. 2
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 1^2U109 cfu ml31 ). 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
Ingredient(s)
% phosphate solubilization
compared to controla
PVK
NBRIY
Control (NBRIP)
NBRIP (glucose, 5; MgCl2 , 2.5)
NBRIP (glucose, 5; MgCl2 , 5.0)
NBRIP (glucose, 5; MgCl2 , 10)
NBRIP (glucose, 10 and MgCl2 , 2.5)
NBRIP (glucose, 10 and MgCl2 , 10)
NBRIP (glucose, 20; MgCl2 , 2.5)
NBRIP (glucose, 20; MgCl2 , 5.0)
NBRIP (glucose, 20; MgCl2 , 10)
NBRIP3glucose+arabinose
NBRIP3glucose+fructose
NBRIP3glucose+galactose
NBRIP3glucose+sorbitol
NBRIP3glucose+mannitol
NBRIP3glucose+xylose
NBRIP3glucose+sucrose
NBRIP3glucose+maltose
NBRIP3glucose+lactose
NBRIP3glucose+ra¤nose
41.6
56.6
100.0
54.3
56.8
72.4
96.2
104.0
113.0
118.4
121.6
77.5
35.0
42.2
4.4
29.0
87.4
41.5
31.0
70.3
28.2
a
Control NBRIP medium contained l31 : glucose, 10 g; Ca3 (PO4 )2 ,
5 g; MgCl2 W6H2 O, 5 g; MgSO4 W7H2 O, 0.25 g; KCl, 0.2 g and
(NH4 )2 SO4 , 0.1 g. Changes in the concentration of NBRIP components are indicated within brackets. The data are means of three
experiments.
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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
Agar (halo size (mm))
Broth (Wg ml31 P solubilized)
Pseudomonas sp. 1
Pseudomonas sp. 2
P. £uorescens
P. aerogenes
P. aeruginosa
Bacillus polymyxa
B. subtilis
Bacillus sp. 1
^
5
6
^
8
2
3
5
8
35
13
14
14
8
11
17
Pseudomonas sp. 1
Pseudomonas sp. 2
P. £uorescens
P. aerogenes
P. aeruginosa
Bacillus polymyxa
B. subtilis
Bacillus sp. 1
2
6
7
^
6
2
4
5
26
90
42
31
28
21
35
60
PVK
NBRIP
The data are means of three experiments.
component at a time (Table 1). It was observed that
glucose and Ca3 (PO4 )2 were essential and yeast extract and (NH4 )2 SO4 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 )2 SO4 . In the absence of both yeast extract
and (NH4 )2 SO4 , 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 l31 ), it was
observed that at the concentration of 0.1 g l31 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
l31 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 formu-
late 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 (NH4 )2 SO4 with KNO3 .
When used as sole source of nitrogen, KNO3 was
27.1% less e¡ective compared to (NH4 )2 SO4 . 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%. NH4 Cl
could be used as a nitrogen source. However, phosphate solubilization ability improved when
(NH4 )2 SO4 was used at a lower concentration of
0.1 instead of 0.5 g l31 (Table 2). (NH4 )2 Fe(SO4 )
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269
tively, were used (Table 3). However, 10 and 5 g l31
of glucose and MgCl2 , 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 ml31 ) 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 MgSO4 was increased from 0.1 to 0.25 g
l31 (Table 2). MgCl2 had a better synergistic e¡ect
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 signi¢cant e¡ect on phosphate solubilization (Table 2).
3.3. Novel phosphate solubilization medium NBRIP
Based on the observations obtained as described
above, a new medium, NBRIP, was de¢ned. Concentrations of glucose and MgCl2 played an important
role in phosphate solubilization ability. Maximum
phosphate solubilization activity was obtained
when 20 and 10 g l31 of glucose and MgCl2 , respec-
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
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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.
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