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Isolation and Characterization of Potential Phosphate Solubilizing Bacteria

from the rhizoplane of Kukrail forest, Lucknow

Faria Fatima1, Neelam Pathak2, Smita Rastogi Verma3

1Integral Institute of Agriculture, Science and Technology, Integral University, Lucknow-226026, India

2Department of Biosciences, Integral University, Lucknow-226026, India

3Department of Biotechnology, Delhi Technological University, Delhi-110042

Abstract

Phosphorus is an important element for seed formation, disease resistance, improvement in fruit

and crops quality. The inoculation of P-solubilizing bacteria is found as a promising technique as

it helps in increase of P availability in soils fertilized with insoluble form of phosphates. A study

has been conducted to isolate and characterize several phosphate solubilizing bacteria form

rhizospheric soil at morphological and biochemical level. Five bacteria having highest

solubilization index, pH reduction and acid phosphatase were isolated which can be used as

biofertilizer in agricultural area.

Introduction

Soil is a dynamic, living matrix that is an important part of the terrestrial ecosystem as it is a

crucial resource for agricultural production, food security and maintenance of a large amount of

life processes. Soil is considered as a depot of microbial activity, though only 5% of living

micro-organisms is estimated. The functions of soil microorganisms are central to the

decomposition processes and cycling of nutrient. They play an important role in soil process that

determines plant growth. For successful functioning of introduced microbial bioinoculants and

their influence on soil health, efforts have been made to investigate soil microbial diversity.

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Rhizospheric bacteria are one of the major groups of microbes, which are plentiful in rhizosphere

soil ranging between 10-6 to 10-8 colony-forming units (cfu) per gram, and some of them have

shown great potential for plant health promotion and as biocontrol agents of nematodes (Siddiqui

and Mahmood, 1999). These plant growth promoting rhizobacteria (PGPRs) are currently being

exploited towards these ends. Antoun and Prevost (2006) gave evidence in the literature

indicating that PGPRs can be a true success story in sustainable agriculture.

Phosphate solubilizing bacteria (PSBs) are important categories of PGPRs which are used in the

form of biofertilizers in various agricultural countries, as significant areas of cultivated soils are

deficient in nutrients, for e.g. phosphorus (P) (Xie, 1996). Phosphorous is the major essential

macronutrient for plant growth and development and hence is commonly added as fertilizer to

optimize yield. PSBs have been used to convert insoluble phosphate into soluble forms available

for plant growth (Nahas et al., 1990). It has been shown that PSBs increased P availability in

soils and increase mineral content in the plant (Sheng et al., 2002).

A combination of application of rock phosphate and bacteria that solubilize them might provide a

faster and permanent supply of phosphorous for optimal plant productivity. This conversion

occurs through acidification, chelation and exchange reactions and results in the production of

organic acids, which have become indicators for routine isolation and selection procedures of

PSBs (Illmer et al., 1995). Collection of soil, isolation and screening of the phosphate

solubilizing bacteria and their morphological and biochemical characterization was done in

present study.

Materials and Methods

Collection of soil sample

The soil samples were collected from different locations of the Kukrail forest, Lucknow, Lawns

of Integral University, Lucknow and Central Institute of Medicinal, and Aromatic Plants

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(CIMAP), Lucknow. The soil samples were taken from two zones viz., upper zone, which is 10-

15 cm deep and lower zone, which is 20-25 cm in depth.

Isolation and screening of phosphate solubilizing bacteria

The soil rhizospheric samples were screened for the presence of phosphate solubilizing bacteria.

The bacteria were grown on nutrient agar media. For this nutrient agar medium comprising of

yeast extract 0.3 %; peptone 0.5 %; CuSO4 100 g/ml was prepared and pH adjusted to 7.0. This

medium was complemented with agar 1.5 % and autoclaved at 15 psi for 15 min. Autoclaved

medium was poured in sterile petriplates (25 ml/plate) under laminar flow hood and allowed to

solidify. Soil rhizospheric samples from various locations were taken and serial dilutions were

made. For this 1 g sample was taken and added in tube containing distilled water and mixed

thoroughly. This represented 10-1 dilution. Under aseptic conditions, 10-2 to 10-9 dilutions of

samples were prepared.

Screening of phosphate solubilizing bacteria

The isolates were screened on the basis of plate assay. Two media viz., Pikovskaya’s media

(Pikovskaya, 1948) and bromophenol blue media were used for screening. The Pikovskaya’s

medium consisted of yeast extract 0.50 (g/l), dextrose 10.00 (g/l), calcium phosphate 5.00 (g/l),

ammonium sulphate 0.50 (g/l), potassium chloride 0.20 (g/l), magnesium sulphate 0.10 (g/l),

manganese sulphate 0.0001 (g/l), ferrous sulphate 0.0001(g/l). Bromophenol media consist of

yeast extract 0.50 (g/l), dextrose 10.00 (g/l), calcium phosphate 5.00 (g/l), ammonium sulphate

0.50 (g/l), potassium chloride 0.20 (g/l), magnesium sulphate 0.10 (g/l), manganese sulphate

0.0001(g/l), ferrous sulphate 0.0001 (g/l) and 0.5 % of bromophenol blue dye. Both the media

were prepared and pH adjusted to 7.0. This medium was complemented with agar 1.5 % and

autoclaved at 15 psi for 15 min. Autoclaved medium was poured in sterile petriplates (25

ml/plate) under laminar flow hood and allowed to solidify. Bacterial colonies were inoculated on

petriplates containing medium for plate assay and the plates were incubated in inverted position

in incubator for up to 24 h at 37 C. Positive cultures were screened by observing transparent

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halo zones in Pikovskaya’s medium and yellow halo zone on bromophenol blue medium.

Pure cultures of phosphate solubilizing bacterial species

Positive bacterial colonies were subculture on fresh petriplates containing medium for plate

assay. Axenic cultures were isolated by incubating the plates in inverted position in incubator for

up to 24 h at 37 C. Positive cultures were screened by observing transparent halo zones in

Pikovskaya’s medium which is due to the solubilization of insoluble tricalcium phosphate into

the soluble form and yellow halo zones appears on bromophenol blue medium which is due to

the production of organic acids leading to lowering of pH in the medium.

Morphological characterization of bacterial isolates by Gram staining procedure

The purity and tentative identification of the isolated phosphate solubilizing bacteria was done

by Gram’s staining procedure. The cell shape and Gram’s property of bacteria were examined

after staining with standard Gram staining procedure. A thin smear of bacterial isolate was

prepared on the glass slide, air-dried and heat-fixed. It was stained in the following sequential

order: covered with crystal violet for 30 s, washed with distilled water, covered with Gram’s

iodine solution for 60 s, washed with 95 % ethyl alcohol, washed with distilled water, counter-

stained with safranin for 30 s and finally washed with distilled water. The stained and air-dried

slides were examined under microscope using oil-immersion objective technique. Gram-positive

bacteria retains the color of crystal violet and stain in purple color, while the Gram-negative

takes the color of counter stain safranin appear pink in color.

Biochemical characterization of phosphate solubilizing bacteria

Solubilization index based on colony diameter and halo zone for each PSB indicate the

efficiency of solubilization of insoluble phosphate into the soluble one thus forming a transparent

halo zone around the colony. Catalase production, oxidase production, urease production and

nitrate reduction are valuable criteria for differentiating and identifying various types of bacteria.

Hence, the positive bacterial isolates were also analyzed qualitatively for their nitrate reducing,

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urease, oxidase and catalase producing capabilities.

Solubilization index (SI)

0.1 ml of each PSB culture preserved in sterile distilled water was placed on Pikovskaya’s agar

(Pikovskaya, 1948) plates and incubated for seven days. Solubilization Index was measured

using following formula (Edi-Premono et al., 1996).

Colony diameter + halo zone diameter

SI = Colony diameter

pH change

1 ml of three days old culture of bacteria in sterile distilled water was added to sterile 100 mL

Pikovskaya’s broth (PB) medium and kept on shaker for seven days. Sterile uninoculated

medium served as control. Initial pH and change in pH was recorded on 3rd, 5th and 7th day by

digital pH meter.

Nitrate reduction test

Certain bacteria reduce nitrate to nitrite while others are capable of further reducing nitrite to

form nitrogen or ammonia. The isolates were incubated at 37 C overnight followed by addition

of 0.5 ml each of sulphanilic acid (0.8 % in 5 N acetic acid) and -naphthylamine (0.5 % in 5 N

acetic acid). The appearance of red or pink color indicated the positive test for nitrate reduction.

Urease test

The overnight cultures were inoculated to the test tubes containing sterilized urea broth and

incubated for 24-48 hours at 20ºC ±8ºC. The development of pink color was taken as positive for

the test.

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Catalase test

Catalase test was performed by growing bacteria in nutrient broth overnight at 37 C followed by

addition of a drop of 3 % H2O2. The production of effervescence due to catalase-catalyzed

breakdown of H2O2 to molecular oxygen indicated positive reaction.

Oxidase test

To the Trypticase Soy Agar (Himedia) plates, overnight cultures of the test isolates were spotted

and plates were incubated for 24 h at 20ºC ± 8º C. After incubation, two to three drops of

tetramethyl-phenylenediamine dihydrochloride was added to the surface of the growth of each

test organism. The color change to puple or maroon was taken as oxidase positive.

Analysis of acid phosphatase enzyme activity from various isolates

All the bacterial isolates tested positive in plate assay were subjected to analyses of activity of

acid phosphatase enzyme. Bacterial colonies tested positive in plate assay were inoculated in

Pikovskaya’s broth, poured in test tubes (10 ml/tube) and autoclaved at 15 psi for 15 min. The

tubes were incubated in incubator shaker at 120 rpm, 37 C for overnight. 10 ml of above grown

bacterial culture was taken and filtered through Whatman no. 1 filter paper. This was considered

as enzyme or protein sample. The enzyme acid phosphatase was assayed using para nitrophenyl

phosphate (PNP-P) as a substrate. The reaction mixture contained 2.5 ml (0.1 M) sodium acetate

buffer (pH 5.8), 1 ml (1 mM) magnesium chloride, 0.5 ml 1 % PNP-P and 0.5 ml of a suitable

dilution of enzyme preparation. One ml of the reaction mixture was transferred to 2 ml of 0.2 M

sodium hydroxide before and after 15 min incubation at 37 C to stop the reaction. The sodium

hydroxide solution added before incubation acts as a control sample for each analysis. The

amount of para nitro phenol (PNP) liberated was measured by recording the absorbance at 420

nm using an appropriate calibration curve. Activity is expressed as μmol PNP liberated min-1.

The blank was run in a similar manner using distilled water.

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Determination of protein content by Lowry’s method

500 l of bacterial culture was taken in microfuge tube and protein was precipitated with equal

volume of ice-cold 20 % trichloroacetic acid (TCA) and kept at 4 C overnight. The pellet was

recovered by centrifuging at 12,000 rpm for 5 min at room temperature and decanting the

supernatant. The pellet was washed with 0.1 ml ice-cold 10 % TCA and ice-cold acetone.

Depending on the pellet size, it was dissolved in 0.5-1.0 ml of 0.1 N NaOH. The solution was

subjected to heating for 5 min in boiling water bath and vortexed vigorously. The protein content

was determined by Lowry’s method (Lowry et al., 1951). For protein content determination, 0.5

ml of protein solution was taken in test tube and 2.5 ml of alkaline solution [prepared by mixing

2 % Na2CO3 solution (in NaOH), 2 % sodium potassium tartrate and 1 % CuSO4.5H2O in

100:1:1] was added. The contents were mixed well and the tubes were incubated at room

temperature for 10 min. This was followed by addition of 0.25 ml of 1.0 N Folin’s reagent. The

contents in the tube were mixed thoroughly and after 10 min, absorbance at 660 nm against

reagent blank was determined spectrophotometrically using bovine serum albumin fraction V as

standard.

Results and Discussion

Isolation and screening of phosphate solubilizing bacteria

Different types of soil samples, viz., Normal soil, Usar soil and Sandy soil were collected from

various locations. Based on serial dilution 10 bacterial isolates from different soil samples were

isolated, from which 5 bacterial isolates having potential phosphate solubilizing ability were

observed by plate assay. Various bacteria, which were isolated from soil, were preserved on the

nutrient agar media (Fig.1). The shape, color and type of colony were estimated on the nutrient

media and further the Gram’s staining was performed.

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1 2 3 4 5

Figure1. Bacterial isolates having phosphate solubilizing potential growing on nutrient

agar media

The tricalcium phosphate present in Pikovskaya’s medium in insoluble form was converted into

the soluble form by the phosphate solubilizing bacteria (phosphate solubilizers), thereby giving a

clear zone around a positive colony (Fig.2a).

Figure 2. (a)Bacteria showing phosphate solubilization leading to formation of clear zone in

Pikovskaya's medium (b) Bacteria showing yellowing of bromophenol blue medium

In bromophenol blue medium (Fig.2b) all the positive bacterial isolates were capable of

producing organic acids, which led to change in pH and thereby color change from blue to

yellow.

Bacterial isolates were characterized by analyzing their shape, presence of catalase, oxidase,

urease and nitrate reduction abilities. The isolates were examined and the results are given in

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Morphological characterization of PSBs by microscopic examination

All the 5 isolates were studied for their Gram reaction property, cell morphology using standard

Gram’s staining procedure. The isolates, as shown (Fig.3) were found to be purple and pink

colored, Gram-positive and Gram-negative respectively, of varying size under microscope using

oil immersion technique.

1 2 3 4 5

Figure 3. Gram’s staining slides of bacterial isolates

Biochemical characterization of isolates

Solubilization index (SI)

All the 5 isolates were able to solubilize tricalcium phosphate (TCP) in Pikovskaya’s agar

medium and the diameter of the zones of solubilization indicated wide variations among the

isolates (2.5 to 3.4).

Figure 4 Histogram showing solubilization indices of bacterial isolates

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pH change

All the 5 isolates were allowed to grow on Pikovskaya’s broth (PB) supplemented with TCP (0.5

% w/v). These bacterial isolates showed decrease in pH with time. Minimum pH was observed

after 7th day. The pH lowered down due to the liberation of the organic acids in liquid media

(Nahas, et al., 1996). The minimum pH of 3.2 was shown by isolate PKN 3 after 7th day. The

bacterial isolates, PKS 4, PBS 4, PKS 3 and PKU 5 significantly decreased the pH of media up

to 3.3, 3.6, 3.8, 3.6 and 3.4 respectively after 7th day of growth (Fig.5). Therefore, these bacterial

isolates were considered of exhibiting high phosphate solubilizing efficiency.

Figure 5 Histogram showing comparative analysis of bacterial isolates on the pH on

different days of growth

Nitrate reductase test

Nitrate, present in the broth, is reduced to nitrite, which may then be reduced to nitric oxide,

nitrous oxide, or nitrogen. The nitrate reduction test is based on the detection of nitrite and its

ability to form a red compound when it reacts with sulfanilic acid to form a complex (nitrite-

sulfanilic acid) which then reacts with α-naphthylamine to give a red precipitate (prontosil). The

isolates, which reduce nitrate into nitrite and further reduce nitrite into ammonia, indicates the

presence of pink color, showing positive results (table 1).

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Urease test

Microorganism, which can produce a urease enzyme, attacks the carbon and nitrogen bond

amide compound with the liberation of ammonia gas. Phenol red acts as an indicator dye. Due to

the production of ammonia, the yellow media turns deep pink in color indicating hydrolysis of

urea (table 1).

Catalase and oxidase test

The production of catalase enzyme breaks the hydrogen peroxide into water and oxygen, which

comes in the form of effervescence, indicating positive catalase reaction (Fig.6). The oxidase test

is a test which helps in determining if bacteria can produce certain cytochrome c oxidases which

reduces N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) or N,N-dimethyl-p-

phenylenediamine (DMPD) results in dark-blue to maroon color when oxidized, and colorless

when reduced. Oxidase-positive bacteria possess cytochrome oxidase or indophenol oxidase (an

iron-containing hemoprotein) (Fig. 7) (table 1).

Figure 6 Positive catalase test Figure 7 Positive oxidase test

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Table 1. Tentative identification of bacterial isolates

Bacterial

isolates

Gram

positive

or

negative

Shape Nitrate

reduction

test

Urease

test

Catalase

production

test

Oxidase

test

Tentative

identification

PKU 5 Negative Rod Positive Positive Positive Positive Pseudomonas

brassicacearum

PBS 4 Negative Rod Positive Negative Positive Negative Pantoea

agglomerans

PKN 3 Positive Rod Positive Positive Positive Negative Bacillus cereus

PKS 4 Positive Rod Positive Positive Positive Positive Bacillus

anthracis

PKS 3 Positive Spherical Positive Positive Positive Negative Staphylococcus

succinus

Quantitative analysis of acid phosphatase enzyme activities

The tentative PSBs were grown on Pikovskaya’s broth containing 0.5 % TCP and their

phosphatase activities were measured. The phosphatase activity was estimated in the supernatant

of broth taken after centrifugation at 10,000 X g at 4° C. The main mechanism for the

solubilization of insoluble organic and inorganic phosphate was due to production of an enzyme

acid phosphatase, which catalyzes hydrolysis of phosphate to liberate inorganic phosphorus (Pi).

Thus, the isolates were evaluated for their acid phosphatase producing ability by measuring Pi

ability. Among all the positive isolates, six bacteria exhibited significantly higher amount of acid

phosphatase enzyme activity, including bacteria PKN 3, PKU 5, PKS 4, PBS 4, and PKS 3 (Fig.

8). Moreover, these isolates also showed relatively higher solubilization index. Thus, the

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solubilization index and the acid phosphatase enzyme activity are directly proportional to each

other indicating that high enzymatic activity results in the formation of large halo zone.

Figure 8 Histogram showing acid phosphatase activity of bacterial isolates

Conclusion

The results indicated existence of variation among the isolated PSBs at morphological and

biochemical levels. Some of the isolates showed white colored pigmented colonies while others

formed light green and light orange colonies. The variation in colony color could be due to the

production of different types of pigments. Several bacterial species are known to produce various

kinds of pigments, the allocation of these pigments in the genus is uncertain (Palleroni et al.,

1970). In addition to this, these isolates also showed diversity in shape from round to irregular

shaped colonies. Furthermore, the round shaped colonies were non-spreading where as the

irregular shaped colonies were spreading type. PSBs also exhibited diversification in cell shape,

Gram property, nitrate reductase, urease oxidase, and catalase activities.

Prominent halo zones were found in case of positive PSB isolates on Pikovskaya’s agar.

Based on transparent halo zone the isolates that exhibited higher SI also exhibited higher acid

phosphatase activity. However, on the basis of morphological and biochemical characterization

of isolates these PSBs were tentively identified as Pseudomonas brassicacearum, Pantoea

agglomerans, Bacillus cereus, Bacillus anthracis, Staphylococcus succinus.

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Acknowledgements

The authors are highly thankful to Vice Chancellor, Integral University for his support

and encouragement. The grant of Uttar Pradesh Biodiversity Board is gratefully acknowledged.

References

1. SIDDIQUI, ZA, MAHMOOD I, (1999), Role of bacteria in the management of

plant parasitic nematodes: a review, Bioresource Technol 69, 167–179.

2. ANTOUN H, PRÉVOST D, (2006), Ecology of plant growth promoting

rhizobacteria. In: PGPR: Biocontrol and Biofertilization, Siddiqui, Z.A. (ed.),

Springer, pp.1–38.

3. XIE, H, PASTERNAK J. J, GLICK B. R, (1996), Isolation and characterization of

mutants of the plant growth promoting rhizobacterium Pseudomonas putida GR12-

2 that overproduce indoleacetic acid, Curr. Microbiol, 32, 67-71.

4. NAHAS E, BANZATTO D.A, ASSIS L.C, (1990), Fluorapatite solubilization by

Aspergillus niger in vinasse medium. Soil Biol. Biochem, 22, 1097–1101.NAHAS,

E, (1996), Factors Determining Rock Phosphate Solubilization by Microorganisms,

World Journal of Microbiology and Biotechnology, Vol. 12, No. 6, (November

1996), pp. 567-572.

5. SHENG X.F, HUANG W.Y, (2002), Mechanism of potassium release from feldspar

affected by the strain NBT of silicate bacterium, Acta Pedol. Sin, 39, 863–871.

6. ILLMER P, BARBATO A, SCHINNER F, (1995), Solubilization of hardly-soluble

AlPO4 with P-solubilizing microorganism, Soil Biol. Biochem, 27, 265–270.

7. EDI PREMONO M, MOAWAD A. M, AND VLEK P. L. G, (1996), Effect of

phosphate-solubilizing Pseudomonas putida on the growth of maize and its survival

in the rhizosphere, Indones. J. Crop Sci, 11, 13Ð23.

8. LOWRY O.H, N.J. ROSEBROUGH, A.L. FARR, R.J. RANDALL, (1951), Protein

Measurement with the Folin Phenol Reagent., J. Biol. Chem. 193, 265-275.

Faria,Neelam, Smita Page 15

9. NAHAS, E, (1996), Factors Determining Rock Phosphate Solubilization by

Microorganisms, World Journal of Microbiology and Biotechnology, Vol. 12, No. 6,

(November 1996), pp. 567-572.