DESSERTATION ON

COMPARATIVE STUDY OF PHOSPHATE SOLUBILIZING BACTERIA AND COMPATIBILITY CHECKING

AS A PARTIAL REQUIREMENT

FOR FULFILMENT OF THE DEGREE OF

MASTER OF SCIENCE IN BIOTECHNOLOGY(M. Sc. BIOTECHNOLOGY)

YEAR: 2011-2012

CARRIED OUT AT

MITCON BIOPHARMA INSTITUTE, PUNE, MAHARASHTRA

GUIDED BY: SUBMITTED BY:

Miss. PRIYA BANDE PATEL ARPITKUMAR N.

SUBMITTED TO

MITCON BIOPHARMA INSTITUTE, PUNE, MAHARASHTRA

ACKNOWLEDGEMENT

I thank the almighty whose blessings have enabled me to accomplish my

dissertation work successfully.

It is my pride and privilege to express my sincere thanks and deep sense of gratitude

to my Project guidance Miss.Priya Bande, Department of Biotechnology and Environmental

Sciences, MITCON, pune for her valuable advice, splendid supervision and constant patience

through which this work was able to take the shape in which it has been presented. It was her

valuable discussions and endless endeavors through which I have gained a lot. Her constant

encouragement and confidence-imbibing attitude has always been a moral support for me.

My sincere thanks to Miss. Neha Vora and Mr. Chandrashekharkulkarni, Head

Department of Biotechnology and Environmental Sciences, MITCON, pune for his immense

concern throughout the project work.

I also wish to thank all my friends, for providing the mandatory scholastic inputs

during my course venture.

Finally, I wish to extend a warm thanks to everybody involved directly or indirectly

with my work.

The whole credit of my achievements goes to my parents and my brothers who were

always there for me in my difficulties. It was their unshakable faith in me that has always helped

me to proceed further

Patel Arpit n.

Introduction:

Phosphorous is the most limiting nutrient in tropical soil, only 0.1% of the total P present is

available to the plants because of its chemical bonding and low solubility (Tilak et al., 2005).

However, many soil microorganisms have the ability to solubilize and mineralize P from

inorganic and organic pools of total soil P, making the element available for plants.

Phosphorous is essential for growth and productivity of plants. It plays an important role in

plants in many physiological activities such as cell division, photosynthesis, and development of

good root system and utilization of carbohydrate. Phosphorous deficiency results in the leaves

turning brown accompanied by small leaves, weak stem and slow development. In ancient times

the use of animal manures to provide phosphorous for plant growth was common agricultural

practice. Organically bound phosphorous enters in soil during the decay of natural vegetation,

dead animals and from animal excretions. At that time role of micro flora on soil fertility was

hardly understood (1)

Assimilation of phosphate from organic compounds by plants and microorganisms take place

through the enzyme "phosphatase" which is present in a wide variety of soil microorganisms.

Plant can absorb phosphate only in soluble form. The transformation of insoluble phosphate into

soluble form is carried out by a number of microbes present in the soil. A large fraction of soil

microbes can dissolve insoluble inorganic phosphates present in the soil and make them

available to the plants [2]

Phosphorus (P) is sequestered by adsorption to the soil surface and precipitation reaction with

soil cations, particularly iron, aluminium and calcium. Therefore, a large amount of P fertilizer

has been used to increase plant growth, which is likely to cause negative impact in respects to

both environment and economy. Insoluble phosphate compounds can be solubilized by organic

acids and phosphatase enzymes produced by plants and microorganisms For example, PSB have

been shown to enhance the solubilization of insoluble P compounds through the release of

organic acids and phosphatase enzymes[3]

Plants acquire phosphorus from soil solution as phosphate anion. It is the least mobile element in

plants and soil contrary to other macronutrients. In plants Phosphorous increases the strength of

cereal straw, promotes flower formation and fruit production, stimulates root development and

also essential for seed formation. Adequate P fertilization may improve the quality of fruits,

vegetables and grain crops and increase their resistance to diseases and adverse conditions. It is

essential for the development of meristematic tissues, in stimulation of early root growth and in

has tening plant maturity. Because of the negative charge of phosphate ions, they are quickly

absorbed after weathering of clays or detritus particles, forming insoluble forms of aluminum,

calcium, or iron phosphates, all unavailable to mangroves. Fungi and bacteria have the ability to

solubilizing these compounds [4]

The bioavailability of soil inorganic phosphorous is rhizosphere varies with nutritional status of

soil, ambient soil conditions and plant species. To circumvent phosphorous deficiency,

phosphate solubilizing bacteria could play an important role in supplying phosphate to plants in

environment friendly and sustainable manner.(Mohamad saghir khan et al 2000),phosphate

solubilizing microorganisms solubilize insoluble form of phosphate as well as scavenges P form

rhizosphere and make it available for plant uptake, hence can enhance plant growth by increasing

the efficiency of phosphate solubilization, enhance the availability of other trace elements and by

producing plant growth promoting substances. Phosphate solubilizing microorganisms improves

or enhances phosphorous uptake and productivity or crops by solubilizing phosphates and

mobilizing the phosphorous to the crop plants.(D. Egamberdiyeva et al 2004).

Crops absorbs phosphorous in the form of soluble orthophosphate. Soil is the main source of

phosphorous for plants, out of added phosphorous fertilizer only 10-20% is available for plants.

The rest remains in the soil as insoluble phosphate in the form of rock phosphate, tri-calcium

phosphate, di-calcium phosphate, hydroxyapatite. However, plants cannot absorb insoluble form

of phosphorous and has to be converted into soluble form by phosphatase enzyme such as acidic

and alkaline phosphatase. Because of their wide applications, phosphate solubilizing

microorganisms are widely applied in agronomic practices to increase the productivity of crops.

(Mohamad saghir khan et al 2000).

Plant can absorb phosphate only in soluble form. The transformation of insoluble phosphate into

soluble form is carried out by a number of microbes present in soil. A large fraction of soil

microbes can dissolve insoluble inorganic phosphatase present in the soil and make available to

the plants.

Mechanisms of Phosphorus Solubilization

“The conversion of insoluble, inorganic phosphate in to solubilized formed by the

phosphatase and other acids is called phosphate solubilization.”

Some bacterial species have mineralization and solubilization potential for organic and

inorganic phosphorus, respectively (Hilda and Fraga, 2000; Khiari and Parent, 2005).

Phosphorus solubilizing activity is determined by the ability of microbes to release metabolites

such as organic acids, which through their hydroxyl and carboxyl groups chelate the cation

bound to phosphate, the latter being converted to soluble forms (Sagoe et al., 1998). Phosphate

solubilization takes place through various microbial processes / mechanisms including organic

acid production and proton extrusion (Surange, 1995; Dutton and Evans, 1996; Nahas, 1996).

General sketch of P solubilization in soil is shown in Figure 1. A wide range of microbial P

solubilization mechanisms exist in nature, and much of the global cycling of insoluble organic

and inorganic soil phosphates is attributed to bacteria and fungi (Banik and Dey, 1982).

Phosphorus solubilization is carried out by a large number of saprophytic bacteria and fungi

acting on sparingly soluble soil phosphates, mainly by chelation-mediated mechanisms

(Whitelaw, 2000). Inorganic P is solubilized by the action of organic and inorganic acids

secreted by PSB in which hydroxyl and carboxyl groups of acids chelate cations (Al, Fe, Ca) and

decrease the pH in basic soils (Kpomblekou and Tabatabai 1994; Stevenson, 2005). The PSB

dissolve the soil P through production of low molecular weight organic acids mainly gluconic

and keto gluconic acids (Goldstein, 1995; Deubel et al., 2000), in addition to lowering the pH of

rhizosphere. The pH of rhizosphere is lowered through biotical production of proton /

bicarbonate release (anion / cation balance) and gaseous (O2/CO2) exchanges. Phosphorus

solubilization ability of PSB has direct correlation with pH of the medium.

Figure 1.

Schematic diagram of soil phosphorus mobilization and immobilization by

bacteria

Ca3(PO4)2 psppppppppppp H2PO4 + Ca

(insoluble) (soluble)

Release of root exudates such as organic ligands can also alter the concentration of P in the soil

solution (Hinsinger, 2001). Organic acids produced by PSB solubilize insoluble phosphates by

lowering the pH, chelation of cations and competing with phosphate for adsorption sites in the

soil (Nahas, 1996). Inorganic acids e.g. hydrochloric acid can also solubilize phosphate but they

are less effective compared to organic acids at the same pH (Kim et al., 1997). In certain cases

Table 1. Microbial strains producing organic acid

Organic acid Strains

Gluconic acid Pseudomonas sp., Erwinia herbicola, Pseudomonas cepacia,Burkholderia cepacia

2-Ketogluconicacid

Rhizobium leguminosarum, Rhizobium

meliloti, Bacillus firmus

Phosphorous cycle:

Phosphorus enters the environment from rocks or deposits laid down on the earth many years

ago. The phosphate rock is commercially available form is called apatite. Other deposits may be

from fossilizes bone or bird dropping called guano. Weathering and erosion of rocks gradually

releases phosphorous as phosphate ions which are soluble in water. Land plants need phosphate

as a fertilizer on nutrient.

Phosphate is incorporated into many molecules essential for life such as ATP (adenosine

triphosphate), which is important in the storage and use of energy. It is also in the backbone of

DNA and RNA which is involved with coding for genetics. When plant materials and waste

products decay through bacterial action, the phosphate is released and returns to the environment

for reuse.

Much of the phosphate eventually is washed into the water from erosion and leaching. Again

water plants and algae utilize the phosphate as a nutrient. Studies have shown that phosphate is

the limiting agent in the growth of plants and algae. If not enough is present, the plants are slow

growing or stuned. If too much phosphate is present excess growth may occur, particularly in

algae.

A large percentage of the phosphate in water is precipitated from the water is precipitated from

the water as iron phosphate which is insoluble. If the phosphate is in shallow sediments, it may

be readily recycled back into the water for further reuse. In deeper sediments in water , it is

available for use only as part of a general uplifting of rock formation for the cycle to repeat itself

( Chales E. Opharidi 2003).

MATERIALS AND METHOD

MATERIALS REQUIRED

GLASSWARE

Sterile Petri dishes, Glass slides, Glass beakers, Cover slips, Media bottles, Conical flasks,

Pipette, Test tubes, Micro pipette, Beaker , Measuring cylinder, Cavity Slides, Sterile wire

loop ,Sterile centrifuge tube ,Glass spreader.

EQUIPMENT

Microscop ( Labomed )

Laminar air flow ( Micro filt india )

Incubater ( REMI )

Incubater with shaker ( REMI )

Refrigerater

Autoclave ( Meta instrument mumbai )

Centrifuge ( REMI )

Hot air oven ( Meta instrument mumbai )

Water bath ( NEOLAB )

Weighing machine ( ATCO, CITIZEN )

PH meter ( control dynamics )

Isolation of phosphate solubilizer:

Collection of soil samples:

Soil samples were collected from neighboring cultivated area. Collection of soil samples was

made at a depth of 15cm from 6 different points within the area. The samples were than air-

dried, powered and mixed well to represent a single sample. The sample was then taken for the

study.

Preperation of Medium:

Two types of medium were prepared:

(I) Nutrient Agar

(II)) Pikovskaya’s agar medium

PSM were isolated from each sample by serial dilution and spread plate method. One gram (1g)

of soil sample was dispersed in 9 ml of autoclaved distilled water and was thoroughly shaken. 1

ml of the above solution was again transferred to 9ml of sterile distilled water to form 10-2

dilution. Similarly 10-3, 10-4, 10-5, 10-6, 10-7 and 10-8 serials were made for each soil sample.

0.1ml of each dilution was spread on Pikovskaya’s agar medium (PVK) containing insoluble

Tricalcium phosphate and incubated at 27 - 300C for 7 days. Colonies showing halo zones were

picked and purified by 5 times subculture method on Pikovskaya’s (PVK) agar medium for

studying colony morphology. [7]

Detection and estimated of the phosphate solubilization ability of microorganisms have been

possible using plate screening methods. Phosphate solubilizers produce clearing zones around

the microbial colony in media. Insoluble mineral phosphate such as tri-calcium phosphate or

hydroxypatite are contained in the medium.

Also the bromophenol blue method is used that produce yellow hallows following pH drop

through the release of organic acid is more reproducible and has greater correlation in

comparison with the simple hallow method. Pikovskays’s medium is generally used for isolation

of phosphate solubilizer (4. Phosphate solubilizers).

The test of the relative efficiency of isolated strains is carried out by selecting the

microorganisms which are capable of producing a halo/ clear zone on plate due to the producing

of organic acid into the surrounding medium [ pikovskays’s R.J(1948)]. However, as the

reliability of this halo based technique is questioned as many isolates which did not produce any

visible halo/zone on agar plates could solubilise various types of insoluble inorganic phosphates

in liquid medium a modified PVK medium using Bromophenol blue (BPB 0.025 gm/lit), to

improve the visibility of the yellow colored halo has not necessary improved the plate assay ( US

patent issued 2008).

Morphological Characterization

Morphological characteristics of isolates viz. shape, size, elevation, surface form, margins and

surface texture, color were observed for their characterization. [8]

Gram staining

The isolate was characterized for its gram staining characteristics as per the following standard

procedure:

Take the smear on the glass slide with the help of inoculating loop let it be air dry.

After this with the help of flame fix it with the heat. Add crystal Voilet for 3o seconds.

Wash it with the distilled water, let it be dry.

After that add Gram’s iodine for 60 seconds.

Wash it with 95% Ethyl alcohol, Add saffranin for 30 seconds after this wash it with the

distilled water.

Air dry it with the help of blotting paper.

Observe in the microscope.

The pink colonies will show the gram negative bacteria and the purple colonies will show

the gram positive bacteria.

REQUIREMENTS:

Yong culture of microorganism

Crystal violets

Gram’s iodine

Alcohol

Distill water

Saffranin

BIOCHEMICAL TEST:

CARBOHYDRATES FERMENTATION TEST

REQUIREMENTS:

1. Test culture

2. Nutrient sugar broth

PROCEDURE:

1. Inoculate a loopful of culture into the sugar broth and incubate 37 0c for overnight

2. Observe the tube for acid and gas production

METHYL RED (M-R) TEST

REQUIREMENTS:

1. Glucose phosphate broth (GPB) , methyl red indicator.

2. Test culture

PROCEDURE:

1. Inoculate GPB with the test culture and incubate the broth at 37 0c for 48-72 hr.

2. After incubation add about 5 drops of methyl red indicator to the medium

3. Observe for development of the red color

VOGES-PROSKAUER (V-P) TEST

REQUIREMENTS:

1. Glucose phosphate broth (GPB)

2. 5% alcoholic alfa napthol and 40% KOH solution.

3. Test culture

PROCEDURE:

1. Inoculate the medium (GPB) with culture and incubate the medium at 37 0c for 24-48 hr.

2. After incubation add 0.6 ml of alfa nephthol and 0.2 ml of KOH solution per ml of

culture broth

3. Shake well after addition of each reagent and slope the tube to increase the aeration. Read

results after 15-60 minutes.

CITRATE UTILIZATION TEST

REQUIREMENTS:

1. Simmon’s citrate agar slant

2. Test culture

PROCEDURE:

1. Streak heavily on the surface of agar slant and incubate the slant at 370c for 24-48 hr.

2. Record the color change of the slant after incubation

INDOL PRODUCTION TEST

REQUIREMENTS:

1. 1% Tryptone broth and Erlich’s or Kovac’s reagent

2. Test culture.

PROCEDURE:

1. Incubate the tryptone broth with of test culture and incubate at 37 0c for 24 hr.

2. After incubation add 3-4 drops of xylene in the medium and shake it vigorously.

3. Allow the two layers to seprate.

HYDROGEN SULPHIDE PRODUCTION TEST

REQUIREMENTS:

1. Standard thiosulphate iron agar stab medium

2. Test cultur

PROCEDURE:

1. Stab the medium with the test culture and incubate the medium at 37 0c for 24hr.

2. After incubation look for the black color in the lower portion of the stab agar medium.

UREA HYDROLYSIS TEST

REQUIREMENTS:

1. Stuart’s urea broth

2. Test culture.

PROCEDURE:

1. Inoculate a loopful of test culture in urea broth add incubate at 37 oc for 24 hr.

2. Observe for the change in color of the after incubation

NIRATE REDUCTION TEST

REQUIREMENTS:

1. Peptone nitrate broth (PNB).

2. Test culture

3. Zinc dust

4. α-napthylamine reagent (reagent A)

5. Sulphanilic acid reagent (reagent B)

PROCEDURE;

1. Inoculate PNB with a loopful of test culture and incubate the medium at 37 0c.

2. Add 0.5 ml of the reagent A and B each to the test medium in this order.

3. Observe the development of color within 30 seconds after adding test reagent.

4. If no color develops add a pinch of Zinc dust mix them well and observe the development

of red color.

GELATIN HYDROLYSIS TEST

REQUIREMENTS

1. Two nutrient gelatin agar tube

2. Test culture

3. Refrigerator

PROCEDURE:

1. Inoculate a loopful of test culture into one of the tube and the second tube is left

uninoculated incubate both the tubes at 37 0c for 24-72 hr.

2. After incubation place both the at 5-10 0c either in refrigerator or in ice water bath for

30-60 min.

3. After refrigeration slightly tilt tubes so as to check the liquefaction of gelatin.

CATALASE TEST

REQUIREMENTS:

1. Microscopic glass slide

2. 3% H2O2

3. Test culture

PROCEDURE:

1. Place one or two drops of hydrogen peroxide solution on a glass microscopic slide.

2. With a nicrome wire loop pick up cells from the of a well isolated colony of the test.

3. Observe for the production of the gas bubbles of effervescence.

OXIDASE TEST

REQUIREMENTS:

1. Nutrient agar plate

2. Filter paper, platinum wire loop

3. Test culture

4. 1% tetramethyl-p-phenylenediamine dihydrochloride solution

PROCEDURE:

1. Grow the test organism under aerobic condition on nutrient agar medium for 18-24 h.

2. Take a filter paper strip and moisten it with 3-4 drops of tetramethyl-p-

phenylenediamine dihydrochloride solution

3. With the help of platinum wire pick up a colony and make a compact smear on moistened

filter paper.

4. Wait for 10-15 seconds and observe for formation of violet color.

TRIPAL SUGAR IRON ( TSI ) AGAR TEST

REQUIREMENTS:

1. TSI agar slant

2. Test culture

PROCEDURE:

1. Streak a loopful of test culture on slant and stab the same culture into butt of the slant.

2. Incubate the TSI slant at 37 0c for 24 hr.

3. After incubation observe the medium for presence of acid/gas/H2S in butt as well in the

slant.

PREPARATION OF INOCULUM

Inoculum were used in order to obtain maximum solubilizatuon of phosphate and best inoculum

was used for further studies. Inoculum was prepared by a loopful organism into 5 ml Normal

saline or nutrient broth and incubated at 28 0c for 48hr. and transfer a 2 ml of old culture into

respective fermentation broth .

QUALITATIVE ASSAY

Enrichment of organisms done

incubation for 5 days

at room temp

0.1 ml of culture was inoculated in wells of pikovskaya’s agar plate

containing bromophenol blue.

STUDY PHOSPHATE SOLUBILIZATION BY THE ORGANISMS AT DIFFERENT

PARAMETER:

EFFECT OF CARBON SOURCES ON PHOSPHATE SOLUBILIZATION:

Prepare different pvk agar plate containing different carbon source, like glucose, sucrose,

lactose ,and inoculate 0.1 culture in to well and incubate plate 37 c for 4 days. Observe the

colour change occur due bromophenol blue (blue colour turn in to yellow) and measure the clear

zone.

EFFECT OF PH ON PHOSPHATE SOLUBILIZATION:

Prepare different pvk agar plate containing different Ph 5,7,9, and inoculate 0.1 culture in to well

and incubate plate 37 c for 4 days. Observe the colour change occur due bromophenol blue (blue

colour turn in to yellow) and measure the clear zone.

EFFECT OF TEMPERATURE ON PHOSPHATE SOLUBILIZATION

Prepare different pvk agar plate and inoculate 0.1 culture in to well and incubate plate at

different temperature. Observe the colour change occur due bromophenol blue (blue colour turn

in to yellow) and measure the clear zone.

COMPARATIVE STUDY OF PHOSPHATE SOLUBILIZING BACTERIA:

Individual organisms inoculation in pikovskaya’s broth (200ml)

Kept on shaker for 5 days

checking total viable count on fifth day

centrifuge 20 ml at 4000 rpm for 20 min (on sixth day)

Phosphate estimation of supernatant was done on alternate days up to 18 days of incubation.

COMPATIBILITY STUDY BETWEEN TWO ORGANISMS:

Individual organisms inoculation in pikovskaya’s broth (200ml) and combinationin

Kept on shaker for 5 days

checking total viable count on fifth day

centrifuge 20 ml at 4000 rpm for 20 min (on sixth day)

Phosphate estimation of supernatant was done on alternate days up to 18 days of incubation.

PHOSPHATE ESTIMATION

To check the phosphatase activity, inoculated culture was centrifuged and used to estimate

phosphatase activity. For this, 20 ml of culture media was taken and centrifuged at 4000 rpm for

20 minutes. The supernatant was then used for eatimation.

PROCEDURE:

The 50 ml of filtered, clear and colorless sample in a conical flask. If the sample is having any

color and colloidal impurities remove them by adding a spoonful activated charcoal and filtering.

Now add 2 ml of ammonium molybdate solution and 5 drops of stannous chloride reagent. A

blue color will appeared in the presence of phosphate. Take optical density reading at 690 nm on

a spectrophotometer using a distilled water blank with the same amounts of chemicals. Reading

on the spectrophotometer should be taken after 5 minutes but before 12 min of the addition of

last reagent. Find out the available phosphate in the sample.

REAGENT PREPARATION:

(A) Ammonium molybdate solution:

a. Dissolved 25.0 gm of ammonium molybdate in 175 ml of distilled water.

b. Add the 280 ml of concentrated H2SO4 to 400 ml of distilled water.

Mix the two solution (a) and (b) and make up the volume to 1 lit.

(B) Stannous chloride solution:

Dissolved 2.5 gm of stannous chloride in 100 ml glycerol by heating on the water bath.

(C) Standard phosphate solution:

Dissolved 4.388 gms of pre dried anhydrous potassium hydrogen phosphate K2HPO4 in

distilled water and make up the volume up to 1 lit. Dilute the standard solution 200

times. This is standard phosphate solution containing 10 mg/lit.

samples Sample

(ml)

Distilled

water

(ml)

Ammonium

molybdate

(ml)

Stannous

chloride

(ml)

Absorbance

at 690 nm

standard 0.1 2 4 0.5 Add

distilled

water and

make

volume up

to 50 ml

keep for

incubation

for 12

minuets

Reagent

blank

00 2 4 0.5

Test

sample

00 2 4 0.5

RESULTS & DISCUSSION

Physio- chemical characteristics of isolates:- There were total four bacteria, from soil sample isolated.

Table-7 Colony Characteristics of isolates:-

CharacteristicsPSB1

Size SmallShape CircularColor Yellow

Margin EntireElevation ConvexOpacity Opaque

Consistency Dry

CharacteristicsPSB2

Size MediumShape CircularColor Yellow

Margin EntireElevation ConvexOpacity Opaque

Consistency Moist

Characteristics PSB3

Size MediumShape CircularColor Colorless

Margin EntireElevation ConvexOpacity Opaque

Consistency Moist

CharacteristicsPSB4

Size SmallShape CircularColor Yellow

Margin EntireElevation ConvexOpacity Opaque

Consistency Dry

Test A1 A2 A3 A1

1.Carbohydrate hydrolysis

Glucose + + + +

Sucrose + – + +

Maltose + + + +

Mannitol + – + +

Lactose – – + –

Xylose + – + +

2. Urea utilization test

– – - –

3. H2S Production test

– – - –

4. Gelatin hydrolysis test

– – - –

5 Citrate utilization test

+ + + (Blue color) +

6. Nitrate reduction test

- + + -

7. Oxidase test + + + +

8. Catalase test + + + +

9. M-R test – – + –

10. V-P test – – – –

11. Iodole production test

– – – –

12. TSI slant No color change No color change Slant/butt- Yellow

No gas prods.

No color change

13. Macconkey`s Agar plate No growth

obtained

Yellowish color colony Grown

Pink colored colony grown

With pink centre

No growth

obtained

14. Gram`s stainining

Gram positive, Cocci

Gram negative, Rod shape

Gram negative, Short rod Shaped

Gram positive,

Cocci15. Motility Non-motile Motile Non-motile Non-

motile

Different

ph

Zone of diameter of colour change (mm)

PSB1 PSB2 PSB3 PSB4

5 11 7 9 15

7 15 12 15 9

9 9 10 9 7

Different

Sugar conc.

Zone of diameter of colour change (mm)

PSB1 PSB2 PSB3 PSB4

1 10 11 14 17

2 12 12 15 12

3 14 15 16 16

Different

Sugar

Zone of diameter of colour change (mm)

PSB1 PSB2 PSB3 PSB4

GLUCOSE 12 15 14 16

SUCROSE 14 16 14 13

FRUCTOSE 15 18 16 17

COMPARATIVE ANALYSIS OF INDIVIDUAL ORGANISMS:

Days of

incubation

Isolated organisms

PSB1

(ppm)

PSB2

(ppm)

PSB3

(ppm)

PSB4

(ppm)

6 444.19 214.51 379.03 112.58

8 663.97 919.35 596.37 561.71

10 379.03 338.7 663.97 338.06

12 401.3 596.37 401.3 248.26

14 446.9 510.75 510.75 229..83

16 228.33 252.39 444.19 198.27

18 895.78 1060.17 895.78 848.62

COMPATIBILTIY ANALYSIS OF PSB1 AND PSB3

Days of

incubation

PSB1 PSB1& PSB3 PSB3

6 444.19 214.51 112.58

8 663.97 919.35 561.71

10 379.03 338.7 338.06

12 401.3 596.37 248.26

14 446.9 510.75 229..83

16 228.33 252.39 198.27

18 895.78 1060.17 848.62

CONCLUSION

Phosphate activity of PSB1 and PSB3 individualy checked and found good activity for

phosphate solubilization and combination of both show additive effect of phosphate

solubilization and can be used for plant growth.

From the qualitative analysis when sugar conc increase the phosphate activity of all organisms is

increased .

And all organism increase its phosphate activity with help of sucrose sugar. Ph 7 is optimum for

all this organisms.

REFERENCES

Ames B (1998). Micronutrients prevent cancer and delay aging. Toxicol. Lett., 102: 5-18.

Aruoma OI, Cuppett SL (1997). Antioxidant methodology Arivazhagan S, Balasenthil S, Nagini

S (2000). Garlic and neem leaf extracts enhance hepatic glutathione and glutathione dependent

enzymes during N-methyl-Nnitrosoguanidine (MNNG)-induced gastric carcinogenesis,

Phytother Res., 14: 291-293

Kannaiyan,S., Kumar,K. and Govindarajan, K. Scientific pub. (India), Jodhpur 2004.

Yosef, B.B., Rogers, R.D., Wolfram, J.H. and Richman, E. J.Soil sci of America, 1999 :

1703-1708 .

Sharma, K. In: Manual of Microbiology. Isolation, Purification and Identification of

Bacteria. Ane Books Pub. New Delhi, p. 41 2005

Bisen, P.S. and Verma, K. In: “Handbook of Microbiology.” CBS publishers and distributors,

New Delhi (1996).

Tilak., K.V.B.R., Ranganayaki, N., Pal, K.K., De, R., Saxena, A.K., Nautiyal, C.S., Mittal,

S., Tripathi, A.K. and Johri, B.N. (2005) Diversity of plant growth and soil health

supporting bacteria. Current Science 89, 136-150.

Pikovskaya, R.I.. Microbiologia, 1948; 17: 362-370 .

Ponmurugan, P. and Gopi, C., African J. Biotechnol., 5(4): 348-350:2006

Goenadi, D.H., Siswanto, Y and Sugiarto, Y. Soil science society of America journal,

64:927-932 ;2000

Lal, L. In: Agrotech Pub. Academy, Udaipur, p. 224 : 2002.

Yahya, A. and S. K. A. Azawi. Plant Soil. 1998. 117:135-141.