Vol.2, No.8, 841-845 (2010) Natural Science
Copyright © 2010 SciRes. OPEN ACCESS
Assessment of biotechnological potential of phosphate
solubilizing bacteria isolated from soils of Southern
Rakhilya Aipova1*, Svetlana A. Aitkeldiyeva1, Askar A. Kurmanbayev1,
Amangeldy K. Sadanov2, Olga B.Topalova3
1Institute of Microbiology and Virology, Almaty, Republic of Kazakhstan; *Corresponding Author: Rakhilya_73@mail.ru
2Center of biological investigations, Almaty, Republic of Kazakhstan
3Kazakh Nationality pedagogical university, Almaty, Republic of Kazakhstan
Received 22 March 2010; revised 25 May 2010; accepted 29 May 2010.
Phosphorus (P) is a vital plant nutrient, avail-
able to plant roots only in soluble forms that are
in short supply in the soil. Adding phosphate-
based fertilizers to increase agricultural yields
is a widely used practice; however, the bio-
availability of P remains low due to chemical
transformations of P into insoluble forms. Thus,
phosphate solubilizing bacteria (PSB) play an
important role in reducing P deficiency in soil.
The goal of this study was to assess biotech-
nological potential of phosphate-solubilizing
bacterial strains. In this study, phosphate solu-
bilizing microorganisms (PSM) were isolated
from different soil samples of Southern regions
of Kazakhstan. The biological activity of PSM
was studied based on their effect on the growth
of wheat seeds. The different taxonomic genera
of these PSM were identified: Arthrobacter spp.,
Aureobacterium spp., Azotobacter spp., Bacte-
rium spp., Baccillus spp. Finally, phosphate-
solubilizing activity of isolated strains of PSM
was assessed.
Keywords: Soil; Phosphate Solubilizing Bacteria;
Identification; Labile Phosphorus; Fertilizers;
Phosphate; Phosphorus
Phosphorus (P) is the second most important plant nu-
trient after nitrogen. However, most Phosphorus in soil
(up to 95-99%) is part of insoluble compounds, which
makes P unavailable for plant nutrition [1]. In order to
increase crop yields, mineral phosphate fertilizers are
regularly incorporated into the soil. However, immedi-
ately after fertilizer application is done, most of the ap-
plied phosphorus transforms into an insoluble form [2].
As a result, most P in the soil is found in poorly soluble,
highly stable forms with limited availability to plants.
Only 5% or less of the total amount of P in soil is avail-
able for plant nutrition [3]. The vicious cycle continues
as such low bioavailability of P requires regular applica-
tion of phosphate-based fertilizers [4].
According to assessments made by the experts from
the U.S. Geological Survey and the International Asso-
ciation of Fertilizer Producers, the demand for fertilizers
over the next 5 years will increase by 2.5-3% annually
[5]. At such rate of phosphate consumption, all global
phosphate resources would be exhausted within 100-125
years [5]. Taking into account the long-term increase in
demand for P and phosphate production, peaking in 20
years, the importance of partial P recycling continues to
grow. Recovering phosphates from livestock waste is
one of the examples of reusing P for agriculture [5].
Other ways to control the wastage of phosphate re-
sources include reducing P run-off into the oceans.
Considering the anticipated food production crisis as
it relates to phosphate deficit in the future, efforts to
study and apply microbiological phosphate solubiliza-
tion processes are well justified. Phosphate solubilizing
Microorganisms (PSM) play an important role in plant
nutrition and growth promotion, especially when phos-
phate fertilizers are used extensively for long periods of
time. It has been proven that agricultural application of
PSM boosts crop yields [6]. On the other hand, soil ac-
tivity depends on the activity of phosphate solubilizing
bacteria [7].
P solubilization mechanisms include acid formation,
chelating metal ions and exchange reactions. The most
active among PSM are micromycetes of following gen-
era: Aspergillus, Penicillium, Curvularia, and phos-
R. Aipova et al. / Natural Science 2 (2010) 841-845
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phate-solubilizing yeast, which is more active in solubi-
lizing phosphates than bacteria. However, since bacteria
are more suitable for high-volume production of bio-
technology products, the goal of our research was to
assess biotechnological potential of phosphate-solubi-
lizing bacterial strains, isolated over the course of our
The subject of this research was to study microorgan-
isms identified from the soils of Southern Kazakhstan.
Soil samples were taken in accordance with the aseptic
regulations and recommendations and were stored in
sterile parchment paper bags [8].
Calcium orthophosphate-dissolving bacteria were
identified in the Muromcev medium of the following
composition (g/l): glucose–10.0, asparagines–1.0, К2SO4
0.2, MgSO4–0.2, corn extract–0.02; agar–20.0, tap water,
pH = 6.8, sterilized at 0.5 atm. for 20 minutes. The salts
were added in dry form, which provided gradual interac-
tion with the medium over the course of sedimentation.
The proportion of dissolving agent was 1.5 grams of
Ca3(PO4)2 per liter of liquid medium. Nistatin was in-
troduced additionally to suppress the growth of micro-
mycetes, in proportion 500 000 units per 250 ml of ster-
ilized medium. Mediums prepared by this method were
distributed in 25 ml amounts per Petri dish. After cooling
down, the agar medium was added the soil suspensions
(in 10-4-10-7 dilution) in 0.1 ml volume amounts. Petri
dishes with soil suspension on the surface were incu-
bated at 28ºC for 3-9 days. We counted only colonies
that had zones of calcium phosphate dissolution around
To evaluate phosphate solubilizing microorganisms’
influence on seed germination, we used wheat seeds.
Laboratory research was conducted according to the
Schroth, Hancock method 1982 [9]. The surface of the
seeds was sterilized with 10% sodium hypochloride so-
lution for 20 minutes, after which the seeds were washed
with 70% ethanol and triple sterile distilled water.
Cultures of microorganisms under the study were in-
cubated in 250 ml flasks, containing 100 ml of medium
for cultivation (beef extract broth) at 28ºC until they
reached steady stage of growth (titer is 1 106 cells/mL).
Culture-containing liquid was obtained by centrifuging
bacterial suspension for 10 minutes at 5000 RPM. Bac-
terial biomass sediment was washed three times with
physiological saline solution, later diluted with sterile
distilled water. We used 10 ml of test subculture per 20
seeds for 2 hours in each seed treatment round.
In accordance with aseptic regulations, inoculated
seeds were placed onto dampened filter paper in Petri
dishes. For control specimens, we used seeds treated
with sterile water and sterile medium used for bacteria
cultivation. Incubation was done at 28ºC.
Phosphate solubilizing activity of bacterial strains was
determined using the Novogrudsky medium [10]. We
added 1% suspension of phosphate-solubilizing bacterial
(PSB) strains into the flasks containing 100 ml of me-
dium, and incubated the flasks on the shake flask propa-
gator for 14 days at 180 RPM. After 14 days, we calcu-
lated the amount of labile phosphorus in the cell culture
liquid by colorimetric method using blue phosphorus-
molybdenum complex [11].
The active mass concentration was converted into
4 mg/L format using the following formula:
X = (1000 a)/(V 1000) mkg/L = 0.1 а (mg/L),
aphosphate ion content in the sample, defined by
calibration chart, mkg;
Valiquot of a sample, 10 cubic centimeters.
The form or type of phosphorus compounds in soil de-
pends on oxidation-reduction conditions of the medium.
The main component of mineral phosphates is tricalcium
phosphate (calcium orthophosphate). In all of the soil
samples assessed in our study using Muromcev medium,
we observed visible zones of phosphate dissolution
(Figure 1).
Inoculation of plants seeds by active strains of micro-
organisms is often beneficial to their growth and devel-
opment. Such effect can be defined by different mecha-
nisms: by intensification of nitrogen fixation and phos-
phate solubilization, by production of physiologically
active substances, by an increase in root absorption ca-
pacity, or by improved solubility of highly immobile
compounds of plant nutrients [12].
Table 1 and Figure 2 illustrate the influence of phos-
phate solubilizing bacterial treatment on wheat seed
germination. Our research demonstrates that applying
PSB in seed treatment increases wheat germination ca-
pacity (see strains P3-P6) and stimulates root growth as
observed in all specimens (P1-P6).
Strains that positively affected wheat seed germina-
tion were identified at the species level. Table 2 presents
morphological and biochemical characteristics of studied
Based on the obtained data on strain characteristics,
phosphate-solubilizing bacterial strains were identified
as follows: P1–Artrobacter sp., P3–Bacillus sp.,
P6–Bacterium sp., P8–Aureobacterium sp., Az 1–Azoto-
bacter sp. In order to evaluate PSB strains’ ability to
mobilize phosphorus from insoluble soil phosphates, a
R. Aipova et al. / Natural Science 2 (2010) 841-845
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Figure 1. Phosphate dissolution zones around phosphate solubilizing bacteria colonies.
Figure 2. Germinated wheat seeds after treatment with PSB cell culture liquid. Left: control; Right: treatment
with the P6 strain.
Table 1. The influence of phosphate solubilizing bacterial treatment on wheat seed germination.
Strain of PSB Germination capacity , % Average length of sprout, mm Average length of root, mm
Control 65,00 40,69 5,02 34,34 3,93
P1 46,65 32,40 3,03 40,88 2,66
P2 61,65 43,25 9,84 44,73 7,52
P3 66,65 29,40 8,79 43,15 4,69
P4 68,35 20,63 7,94 35,88 5,99
P5 76,65 24,58 17,55 38,92 3,26
P6 66,65 32,76 0,23 41,84 9,16
P7 51,65 23,29 8,12 33,63 5,50
P8 60,00 25,79 11,77 40,71 7,49
further experiment was conducted using strains P1, P3,
P6, P8, Az1, which were grown over a 14-day period. In
the obtained cell culture liquid, the assessment of soluble
(labile) phosphorus concentration was done.
The assessment of phosphate solubilization activity by
PSB strains (Table 3) has demonstrated that phosphorus
mobilization activity has increased more than 100 times
in comparison to control sample. The most active strains
R. Aipova et al. / Natural Science 2 (2010) 841-845
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Table 2. Morphological, physiological, and biochemical characteristics of phosphate solubilizing bacteria strains in the study.
P-1 P-3 P-6 P-8 Az1
Morphology rods rods rods rods rods
Gram Staining + - – + –
Mobility Immobile Mobile Mobile Mobile Mobile
Acid-Fastness – + – –
Catalase + + + + +
Oxidase + + + –
Growth on Beef/Peptone Broth + + + +
Relationship with oxygen P.anaerobe P.anaerobe P.anaerobe Aerobes Aerobes
Dilution of gelatin + + – +
Hydrogen sulphide formation – + – +
Nitrate formation – + + –
Starch Hydrolysis + – – – +
Indole formation – + – –
Ammonia formation + – – –
Spore formation – + – – –
Growth on Beef/Peptone Broth
with NaCl
3% + + + +
6% + + + +
Starch conversion + – – – +
Carbohydrate uptake:
Glucose A AG A A
Galactose Аs AG AG Аs
Glucose A AG A A
Xylose Аs AG A Аs
Lactose Аs AG А Аs
Fructose A AG A A
Arabinose A AG A Аs
Saccharose A AG A A
Maltose A AG Аs A
Glycerin Аs AG Аs Аs
Mannitol A AG Аs Аs
Growth under different temperatures
-3+ – + +
22+ + + + +
28+ + + +
30+ + + + +
37+ + – +
Comment: Аs-assimilates, A-acid formation, AG–acid and gas formation
Table 3. Phosphate solubilization activity with the following
PSB strains.
Strains: Concentration of Р2О5 in mkg per 1000 ml of
medium/phosphate mobilization
P8 75 000/0,008
P1 70 000/0,007
P3 44 000/0,004
P6 20 000/0,002
Аz 1 47 300/0,005
P1 + P8 +
Аz 1 146 000/0,015
Control 800/0,00008
were P8, P1 and Az1 strains, whereas strain P6 was least
active. Growing three strains simultaneously (P1 + P8 +
Аz 1) allowed the highest level of phosphorus mobiliza-
The results obtained over the course of this study let us
theorize that phosphate solubilizing bacteria positively
affect wheat seed germination in a multifaceted way.
Identification of PSB in our study has demonstrated that
R. Aipova et al. / Natural Science 2 (2010) 841-845
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they belong to different taxonomic bacterial genera:
Azotobacter sp., Artrobacter sp., Bacterium sp., Bacillus
sp., and Aerobacterium sp. We believe that the strains
obtained in samples P8, P1 and Az 1 are of particular
interest for further research.
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