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Phosphate solubilizing bacteria thesis paper

Apr 26, 2018

Vol.2, No.8, 841-845 (2010)

Natural Science

http://dx.doi.org/10.4236/ns.2010.28105

Copyright © 2010 SciRes.

OPEN ACCESS

Assessment of biotechnological potential of phosphate

solubilizing bacteria isolated from soils of Southern

Kazakhstan

Rakhilya Aipova

1

*, Svetlana A. Aitkeldiyeva

1

, Askar A. Kurmanbayev

1

,

Amangeldy K. Sadanov

2

, Olga B.Topalova

3

1

Institute of Microbiology and Virology, Almaty

, Republic of Kazakhstan; *Corresponding Author:

[email protected]

2

Center of biological investigati

ons, Almaty, Republic of Kazakhstan

3

Kazakh Nationality pedagogical universit

y, Almaty, Republic of Kazakhstan

Received 22 March 2010; revised 25 May 2010; accepted 29 May 2010.

ABSTRACT

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

1. INTRODUCTION

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

Copyright © 2010 SciRes.

OPEN ACCESS

842

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

research.

2. MATERIAL AND METHODS

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,

К

2

SO

4

–

0.2, MgSO

4

–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

Ca

3

(PO

4

)

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

them.

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

ï‚´

10

6

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

РО

3-

4

mg/L format using the following formula:

X = (1000

ï‚´

a)/(V

ï‚´

1000) mkg/L = 0.1

ï‚´

а

(mg/L),

where:

a

―

phosphate ion content in the sample, defined by

calibration chart, mkg;

V

―

aliquot of a sample, 10 cubic centimeters.

3. RESULTS AND DISCUSSIONS

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 orthophos

phate). 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

strains.

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

Copyright © 2010 SciRes.

OPEN ACCESS

843

Figure 1.

Phosphate dissolution zones around phos

phate 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 bact

erial treatment on whea

t seed germination.

Strain of PSB Germination capacity , % Average le

ngth 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

Copyright © 2010 SciRes.

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844

Table 2.

Morphological, physiological, and biochemical characteristics

of phosphate solubilizing bacteria strains in the study.

Strains

Characteristics

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

А

s AG AG

А

s

Glucose A AG A A

Xylose

А

s AG A

А

s

Lactose

А

s AG

А

А

s

Fructose A AG A A

Arabinose A AG A

А

s

Saccharose A AG A A

Maltose A AG

А

s A

Glycerin

А

s AG

А

s

А

s

Mannitol A AG

А

s

А

s

Growth under different temperatures

-3

℃

+ – + +

22

℃

+ + + + +

28

℃

+ + + +

30

℃

+ + + + +

37

℃

+ + – +

Comment:

А

s-assimilates, A-acid formati

on, AG–acid and gas formation

Table 3.

Phosphate solubilization activity with the following

PSB strains.

Strains:

Concentration of

Р

2

О

5

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

А

z 1 47 300/0,005

P1 + P8 +

А

z 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 +

А

z 1) allowed the highest level of phosphorus mobiliza-

tion.

4. CONCLUSIONS

The results obtained over the course of this study let us

theorize that phosphate solubilizing bacteria positively

affect wheat seed germinat

ion in a multifaceted way.

Identification of PSB in our study has demonstrated that

R. Aipova

et al.

/ Natural Science 2 (2010) 841-845

Copyright © 2010 SciRes.

OPEN ACCESS

845

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.

REFERENCES

[1]

Corona, M.E.P., Klundert, I.V.D. and Verhoeven, J.T.A.

(1996) Availability of organic and inorganic phosphorus

compounds as phosphorus sources for carex species.

New

Phytologist

,

133(2)

, 225-231.

[2]

Pundarikakshudu, R. (1989) Studies of the phosphate

dynamics in a vertisol in relation to the yield and nutrient

uptake of rainfed cotton.

Experimental Agriculture

,

25(4)

,

39-45.

[3]

Boronin,

А

.M. (1998) Rhizosphere bacteria of the genus

Pseudomonas enabling plant growth and development.

Sorovsky Educational Magazine

,

10

, 25-31.

[4]

Omar, S.A. (1998) The role of rock-phosphate-solubi-

lizing fungi and vesicular-arbusular-mycorrhiza (VAM)

in growth of wheat plants fertilized with rock phosphate.

World Journal of Microbiology and Biotechnology

,

14(2)

,

211-218.

[5]

Gilbert, N. (2009) Environment: The disappearing nutri-

ent.

Nature

,

461(7265)

, 716-718.

[6]

Khan, M.S., Zaidi, A. and Wani, P.A. (2007) Role of

phosphate-solubilizing microorganisms in sustainable

agriculture

―

A review.

Agronomy for Sustainable De-

velopment

,

27(1)

, 29-43.

[7]

Rimkevich, O.V. (2006) Environmental and functional

roles of microorganisms in technogenically disturbed

soils (based on samples from Dambukinsk gold-mining

site). Dissertation Thesis

for Ph.D. Degree, Blago-

veshensk.

[8]

Tepper, E.Z., Shilnikova, V.K. and Pereverzeva, G.I.

(2004) Microbiology practicum: Study aid for universi-

ties. Drofa, Moscow.

[9]

Schroth, M.N. and Hancock, J.G. (1982) Disease-suppre-

ssive soil and root-colonizing bacteria.

Science

,

216(4553)

,

1376-1381.

[10]

Rodina, A.G. (1965) Aquatic microbiology methods.

Nauka, Moscow; Leningrad.

[11]

Namsaraev, B.B., Barkhutova, D.D. and Khakhinov, V.V.

(2006) Field practicum in aquatic microbiology and

aquatic chemistry. Ulan-Ude.

[12]

Gromovyh,

Т

.I

., Litovka, Y.

А

., Sadykova, V.S. and Gab-

dulina,

И

.G.-

М

. (2005) Perspective for applicationincrop

science/biological features of a new strain of streptomy-

ces lateritius 19/97-

М

, promising for use in crop science.

Biotechnology

,

5

, 37-40.

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