isolation and characterization of potent phosphate solublizing

ISSN: 2395-6887
Contents lists available at www.iosi.in
ISOI Journal of Microbiology, Biotechnology and Food Science
Volume 1 Issue 1 October 2015; Page No. 23-28
ISOLATION AND CHARACTERIZATION OF POTENT PHOSPHATE SOLUBLIZING BACTERIA
1
MAHANTESH S. P., 2PATIL C S. AND 3HIMANSHU.
1,3
Department of Biotechnology, OPJS University, Churu, Rajasthan
2
Department of Biotechnology, B.V.Bhoomaraddi College of UG and PG Bidar, KARNATAKA
Author Email: [email protected]
ABSTRACT:
Soil Microorganisms play an vital role in maintaining the ecological balance by active participation in Carbon,
Nitrogen, Sulphur and Phosphorous cycles in nature. Phosphate Solubilizing Microbes plays an important role in
plant nutrition through increase in phosphate uptake by plants and used as biofertilizers of agricultural crops.
Phosphate is one of the most vital macronutrient required for the growth and development of plants. A large
number of microorganisms present in the rhizosphere are known to solubilize and make available the insoluble
phosphorus in the available form to the plants.
The present investigation helps for identification, isolation and characterization of phosphate solublizing microbes
which are more efficient in utilization of phosphorous present in soil. In the study 10 different samples were
collected from root rhizosphere of different crop plants of Bidar and Kalaburagi, Karnataka and isolated 29
different phosphate solublizing bacteria’s isolated, in these two potent efficiently phosphate solublizing bacteria’s
were isolated and they are characterized under invitro condition.
Keywords: Phosphorous, Phosphate solublizing bacteria, Rhizosphere etc.
The majority of the isolated organisms are bacterial
organisms, although several fungi are also known to
solubilize phosphates. These bacteria and fungi have
the potential to be used as biofertilizers. Their role in
The objective of this research work is to isolate the PSB
strains from the rhizosphere soil of different crop plants
such as bajra, red gram, maize, and vegetables as bean
and tomato. Further, the isolated strains were
identified and characterized
MATERIALS AND METHODS
Collection of Soil samples:
The soil samples were collected from the depth of 1015cm from the rhizosphere of different crop plants like
Bajra, Redgram, maize and vegetables like bean and
tomato etc. Around the agricultural land of Bidar and
Kalaburagi, Karnataka. Collected soil samples were
carried aseptically in polythene bags and maintained at
the laboratory for the further study.
Isolation of PSM
Isolation of Phosphate Solublizing microbes done with
the suspension of different rhizospere soil samples(1g)
in 100ml of distilled water. Aliquots of 100microlitre
were inoculated of Pikovskaya medium by pour plate
technique and also with swab spread plate technique
and plates were incubated at 30°C. Colonies showing in
plates considers as PSM. Single PSM colony were picked
23
Phosphate solubilizing microorganisms (PSM) play a
significant role in making phosphorus available to plants
by bringing about favourable changes in soil reaction in
the soil microenvironment leading to solubilization of
inorganic phosphate sources. Some microorganisms
associated with different plant rhizosphere are able to
solubilise inorganic insoluble P salts. Pseudomonas and
Bacillus are two important genera of soil bacteria with
promising activity of phosphate solubilisation (Reyes et
al.,1999; Yadav and Tarafdar, 2011).
increasing the soil nutrient value is of utmost
importance. Their application to crop fields has resulted
in an increased yield of several crops, such as cereals,
legumes, fibers, vegetables, oils, and other crop plants
(Silini-Cherif, 2012; Viruel et al., 2011; Khalimi et al.,
2012).
Page
INTRODUCTION
Phosphorus (P) is one of the essential macronutrients
for plant growth and reproduction. However, it is a
limiting factor in many soils, because an important part
of this element is insoluble (Del Campillo et al., 1999).
Mineral P solubilization is a common phenotype in
several rhizobacteria, hence the term "phosphate
solubilizing bacteria" PSB (Pérez et al., 2007). The
application of these bacteria in the soil can increase
plant productivity by improving P nutrition (Hameeda et
al., 2008). These PSB also can stimulate plant growth by
other mechanisms such as the production of
phytohormones, nitrogen fixation, inhibition of
phytopathogenic microorganisms, production of
siderophores and ACC deaminase (Bhattacharyya & Jha,
2012).
MAHANTESH S. P., et al, ISOI Journal of Microbiology, Biotechnology and Food Science
INDOLE TEST
The organisms were grown in tryptone broth for 48 hr
at 370C. Development of a cherry red colour at the
upper layer by adding five drops of Kovac’s reagent
(prepared by adding 10 g of p-dimethyl
aminobenzaldehyde in 150 ml of isoamylalcohol and
then added 50 ml of HCl.) indicated a positive test
METHYL RED (MR) TEST
To 5 ml culture in MR-VP broth added 5 drops of methyl
red indicator solution (0.2% in 50% alcohol).
Appearance of red colour indicated a positive test.
VOGES PROSKAUER (VP) TEST
Barritt’s reagent and MR-VP medium were used. In this
test, production of acetoin (acetyl methyl carbinol) is
detected by the addition of few drops of 5% alpha
naphthol in 0.2 ml of 40% aqueous solution of KOH. The
mixture in the tubes were shaken vigorously for few
minutes and allowed to stand for two hours. Positive
reaction was shown by development of crimson ruby
colour.
CITRATE UTILIZATION TEST
Tubes containing Simmon’s citrate agar media were
inoculated by each organism. Following incubation
citrate positive cultures were identified by the presence
of growth on the surface of slant accompanied by blue
colouration.
H2S PRODUCTION TEST
Filter paper strips were saturated with 5% led acetate
solution, air dried and autoclaved at 10 lb pressure for
15 min. Inoculated the nutrient broth tubes with the
desired culture and the led acetate strips were inserted
above the liquid between the plug and inner wall of
tube. Incubated and examined the blackening of paper,
indicating H2S production.
CASEIN HYDROLYSIS TEST
Skim milk agar plates were prepared. Plates were
incubated at 300C for 4 days after spot inoculation and
observations were taken regularly. Formation of clear
zone was taken as positive.
GELATIN HYDROLYSIS TEST
Gelatin agar medium was prepared by adding 12%
gelatine in nutrient broth, dispensed in tubes and
autoclaved at 1210C for 12 minutes. Gelatin tubes were
inoculated by stabbing to the bottom and incubated at
STARCH HYDROLYSIS TEST
Starch agar (prepared by adding 20 ml of 10% starch
solution, steamed for 1 hr in 100 ml of melted nutrient
agar) poured in petriplates was inoculated with test
organisms and incubated for 3-5 days. The plates were
flooded with Gram’s iodine solution. Blue colour of
medium with colourless areas around growth indicated
a positive test.
UREA HYDROLYSIS TEST
Christensen’s urease agar medium containing the
indicator phenol red were inoculated with test cultures
and incubated at 370C for 18-24 hrs. A rose red pink
colour is an indication of positive test for urease.
NITRATE REDUCTION TEST
The cultures were grown in nitrate peptone broth
containing potassium nitrate – 2 g, peptone- 5 g,
distilled water- 1000 ml for five days and five drops of
solution A and B each were added.
Solution A: Sulfanilic acid -8 g; Acetic acid
5N–1
litre
Solution B: α- Napthylamine- 5 g; Acetic acid 5 N – 1
litre
The tubes showing red colour within two minutes were
taken as positive test for nitrate reduction test.
CATALASE TEST
A small portion of microbial growth from solid medium
was taken and emulsified with a drop of H2O2 (3-6%).
Immediate effervescence indicated a positive catalase
reaction.
OXIDASE TEST
A strip of filter paper moistened with 1% solution of
tetra methyl para phenyline diamine dihydrochloride
was placed on the lid of a Petri dish, using a sterile
platinum wire loop small portion of growth was taken
and applied on the filter paper. Immediate
development of deep purple colour indicated the test
organism as oxidase positive.
TWEEN HYDROLYSIS
Medium was prepared by adding 1 ml of Tween, 80
(Sigma) to molten nutrient agar cooled to 550C. mixed
gently several times to disperse the tween and poured
into Pertidish. Spot inoculated the culture under test on
to the plate. Known positive and negative bacteria were
also included. The plates were placed in a plastic bag to
keep them moist and incubated at 370C up to 5 days.
Read as Tween positive those colonies that develop a
clearly visible halo of precipitated fatty acids when
viewed against a dark background.
24
IDENTIFICATION AND CHARACTERIZATION:
Identification of the microbes were done by
morphological and biochemical analysis. The bacterial
isolates
were
identified
by
morphological
characteristics in which gram staining, shapes, motility
test, spore forming test and biochemical analysis as
follows.
370C along with uninoculated controls for 30 days.
Liquefaction at weekly intervals was detected after
placing the tubes in refrigerator for 30 minutes.
Page
and transferred in broth and slants of Pikovskaya
medium for further study.
MAHANTESH S. P., et al, ISOI Journal of Microbiology, Biotechnology and Food Science
pH
Tricalcium phosphate solubilizing efficiency of isolates
were tested at various pH values (6, 6.5, 7, 7.25, and
7.5) after fourteen days of incubation period
Temperature
Solubilization efficiency was examined at different
temperatures (25, 27.5, 30, 32.5 and 35°C).
RESULT AND DISCUSSION
Isolation of PSM
Ten soil samples were collected from different crop
plants of Bidar and Kalaburagi (Table 5). All samples
showed acidic pH. Twenty nine potential PSBs were
isolated from different rhizosphere soil samples. All the
isolates were subjected to in vitro tricalcium phosphate
solubilization. The isolates showed variations in their P
solubilizing ability. Their extents of P solubilization
ranged between 11.38 – 73.52 mg/100 ml of soluble P
in liquid medium. In general, P solubilizations of all the
isolates were accompanied by reduction in pH of the
culture filtrate. Maximum solubilization efficiency (SE%)
was detected in PSB 13.
Among the isolates, 05 PSBs showed promising results
and were re-evaluated for their phosphate solubilizing
activity.
Quantitative evaluation of soluble P in the liquid
medium showed that PSB 12 and 29 were the most
efficient P solubilizing isolates. These two bacteria
along
with
standard
phosphate
solubilizing
Pseudomonas striata were utilized for further studies.
The selected isolates were subjected to morphological,
cultural physiological and biochemical analysis for
identification.
Both isolates were aerobic, Gram negative, nonsporulating and motile rods arranged in single and
pairs. PSB 29 found to possess diffusible brownish
pigment. These isolates were able to grow over a wide
range of temperature (25-35°C) and pH (6-7.5), PSB 29
found to be metabolically more active compared to PSB
12.
Table 1: Morphological Tests
Test
PSB 12
Colony Morphology
Configuration Circular
Margin
Entire
Surface
Smooth and shiny
Pigment
Grams
Negative
PSB 29
Circular
Lobate
Pale brownish
Pale brown
Negative
Rod
Single and pair
+
Rod
Single and pair
+
Table 2: Physiological Tests
Tests(Growth)
Temperature
10°C
15°C
20°C
25°C
30°C
37°C
42°C
52°C
pH
pH 4.0
pH 5.0
pH 6.0
pH 7.0
pH 8.0
pH 9.0
NaCl
2.0
4.0
5.0
7.0
10.0
Anaerobic
PSB 12
PSB29
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
NG
NG
+
+
+
+
NG
NG
NG
NG
NG
NG
NG
-
NG
NG
NG
NG
NG
-
Table 3: Biochemical tests
Tests
Growth on MacConkey
agar
Inole test
Voge Proskauer test
Citrate utilization
H2S Production
Casein hydrolysis
Esculin Hydrolysis
Gelatin Hydrolysis
Starch hydrolysis
Urea hydrolysis
Nitrate reduction
Catalase test
Oxidase test
Lysine decarboxylase
Arginine dihydrolase
Ornithine decarboxylase
Tween 80 hydrolysis
Tyrosine degradation
Hippurate hydrolysis
NG: No growth
PSB12
NG
PSB 29
NG
NG
NG
NG
NG
+
NG
-
NG
NG
+
+
+
-
25
(pH and Temperature):
The Phosphate solublizing microbes isolates were
analyzed using various pH(6-7.5) and Temperature(2535°C) and the results were recorded.
reaction
Cell Shape
Arrangement
Spore
Motility
Page
Optimization of Physiological conditions
MAHANTESH S. P., et al, ISOI Journal of Microbiology, Biotechnology and Food Science
Out of 95 substrates utilized in BIOLOG GN2 plate PSB
12 was able to utilize 19 different substrates while PSB
29 assimilated 40 distinct substrates as their sole source
of carbon. Both the isolates were able to utilize glucose,
mannitol, mannose and arabinose as their C source. But
assimilation of inositol and galactose was detected only
in PSB 29.
The isolate PSB 12 was from Maize rhizosphere soil
collected from Bidar, Karnataka. On the other hand PSB
29 was isolated from rhizosphere soil of Bajra collected
from Kalaburagi disctrict, Karnataka (Plate I). Both the
isolates showed considerably greater P solubilizing
activity compared to standard P solubilizing
Pseudomonas striata (50.12 mg/100 ml of soluble P).
On the basis of above tests, the organism(s) has been
identified as follows:
CONCLUSION:
All the isolated bacterial strains were found to exhibit
phosphate solubilizing capacity in varying degrees of
culture filtrate. The strains isolated were gram negative
rod shaped, non-spore forming, motiles arranged in
single and pairs. The isolates able to grow over a wide
range of temperature (25-35°C) and pH (6-7.5), the
isolate PSB29 found to be more metabolically active
compared to PSB 12.
The PSB 29 solubilised significantly higher amount of
inorganic phosphate and demonstrating their higher
potential to be used as soil ino-inoculants to enhance
soil fertility and plant growth.
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Page
Fig 1: Growth of Phosphate solublizing microbes on Pikovaskaya’s
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