viewLand 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 ...
Dessertation onCOMPARATIVE STUDY OF PHOSPHATE SOLUBILIZING BACTERIA AND COMPATIBILITY CHECKINGAS A PARTIAL REQUIREMENTFOR fulfilment of THE DEGREE OFMASTER OF SCIENCE IN Biotechnology(M. Sc. BIOtechnology)YEAR: 2011-2012Carried out atMITCON BIOPHARMA INSTITUTE, PUNE, MAHARASHTRA GUIDED BY: SUBMITTED BY:Miss. priya bande Patel ARPITKUMAR N.Submitted toMITCON BIOPHARMA INSTITUTE, PUNE, MAHARASHTRAACKNOWLEDGEMENT 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 ofgood 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 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 organicacids 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 enzymesPlants acquire phosphorus from soil solution as phosphate anion. It is the least mobile element inplants and soil contrary to other macronutrients. In plants Phosphorous increases the strength ofcereal straw, promotes flower formation and fruit production, stimulates root development andalso 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 isessential for the development of meristematic tissues, in stimulation of early root growth and inhas 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 tosolubilizing these compounds 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 andinorganic 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 bacteriaCa3(PO4)2 psppppppppppp H2PO4 + Ca (insoluble) (soluble)Release of root exudates such as organic ligands can also alter the concentration of P in the soilsolution (Hinsinger, 2001). Organic acids produced by PSB solubilize insoluble phosphates bylowering 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 acidOrganic acidStrainsGluconic acidPseudomonas sp., Erwinia herbicola, Pseudomonas cepacia,Burkholderia cepacia2-KetogluconicacidRhizobium 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 GLASSWARESterile 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)) Pikovskayas agar mediumPSM 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. 1ml 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 Pikovskayas 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 Pikovskayas (PVK) agar medium for studying colony morphology. 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. Pikovskayss 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 [ pikovskayss 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 CharacterizationMorphological characteristics of isolates viz. shape, size, elevation, surface form, margins andsurface texture, color were observed for their characterization. Gram stainingThe 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 Grams 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 Grams iodine Alcohol Distill water Saffranin BIOCHEMICAL TEST: CARBOHYDRATES FERMENTATION TESTREQUIREMENTS:1. Test culture2. Nutrient sugar brothPROCEDURE:1. Inoculate a loopful of culture into the sugar broth and incubate 37 0c for overnight2. Observe the tube for acid and gas production METHYL RED (M-R) TEST REQUIREMENTS:1. Glucose phosphate broth (GPB) , methyl red indicator.2. Test culturePROCEDURE: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 medium3. Observe for development of the red color VOGES-PROSKAUER (V-P) TESTREQUIREMENTS:1. Glucose phosphate broth (GPB)2. 5% alcoholic alfa napthol and 40% KOH solution.3. Test culturePROCEDURE: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 broth3. Shake well after addition of each reagent and slope the tube to increase the aeration. Read results after 15-60 minutes. CITRATE UTILIZATION TESTREQUIREMENTS:1. Simmons citrate agar slant 2. Test culturePROCEDURE: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 TESTREQUIREMENTS:1. 1% Tryptone broth and Erlichs or Kovacs reagent2. 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 culturPROCEDURE: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. Stuarts 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 TESTREQUIREMENTS:1. Peptone nitrate broth (PNB).2. Test culture 3. Zinc dust 4. -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 TESTREQUIREMENTS 1. Two nutrient gelatin agar tube 2. Test culture 3. RefrigeratorPROCEDURE: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 slide2. 3% H2O23. Test culturePROCEDURE: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 TESTREQUIREMENTS:1. Nutrient agar plate 2. Filter paper, platinum wire loop3. Test culture4. 1% tetramethyl-p-phenylenediamine dihydrochloride solutionPROCEDURE: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 solution3. 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 TESTREQUIREMENTS:1. TSI agar slant 2. Test culturePROCEDURE: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 INOCULUMInoculum 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 temp0.1 ml of culture was inoculated in wells of pikovskayas 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 SOLUBILIZATIONPrepare 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 pikovskayas 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 pikovskayas 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 ESTIMATIONTo 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 nmstandard0.1240.5Add distilled water and make volume up to 50 ml keep for incubation for 12 minuetsReagent blank00240.5Test sample00240.5RESULTS & DISCUSSIONPhysio- chemical characteristics of isolates:- There were total four bacteria, from soil sample isolated.Table-7 Colony Characteristics of isolates:- CharacteristicsPSB1SizeSmallShapeCircularColorYellowMarginEntireElevationConvexOpacityOpaqueConsistencyDryCharacteristicsPSB2SizeMediumShapeCircularColorYellowMarginEntireElevationConvexOpacityOpaqueConsistencyMoistCharacteristicsPSB3SizeMediumShapeCircularColorColorlessMarginEntireElevationConvexOpacityOpaqueConsistencyMoistCharacteristicsPSB4SizeSmallShapeCircularColorYellowMarginEntireElevationConvexOpacityOpaqueConsistencyDryTestA1A2A3A11.Carbohydrate hydrolysisGlucose++++Sucrose+++Maltose++++Mannitol+++Lactose+Xylose+++2.Urea utilization test-3.H2S Production test-4. Gelatin hydrolysis test-5Citrate utilization test+++ (Blue color)+6. Nitrate reduction test-++-7.Oxidase test++++8.Catalase test++++9.M-R test+10.V-P test11. Iodole production test12.TSI slantNo color changeNo color changeSlant/butt- YellowNo gas prods.No color change13. Macconkey`s Agar plateNo growth obtainedYellowish color colony GrownPink colored colony grown With pink centreNo growth obtained14.Gram`s staininingGram positive, CocciGram negative, Rod shapeGram negative, Short rod ShapedGram positive, Cocci15.MotilityNon-motileMotileNon-motileNon-motileDifferent phZone of diameter of colour change (mm)PSB1PSB2PSB3PSB4511791571512159991097Different Sugar conc.Zone of diameter of colour change (mm)PSB1PSB2PSB3PSB4110111417212121512314151616Different Sugar Zone of diameter of colour change (mm)PSB1PSB2PSB3PSB4GLUCOSE12151416SUCROSE14161413FRUCTOSE15181617COMPARATIVE ANALYSIS OF INDIVIDUAL ORGANISMS:Days of incubationIsolated organismsPSB1(ppm)PSB2(ppm)PSB3(ppm)PSB4(ppm)6444.19214.51379.03112.588663.97919.35596.37561.7110379.03338.7663.97338.0612401.3596.37401.3248.2614446.9510.75510.75229..8316228.33252.39444.19198.2718895.781060.17895.78848.62COMPATIBILTIY ANALYSIS OF PSB1 AND PSB3Days of incubationPSB1PSB1& PSB3PSB36444.19214.51112.588663.97919.35561.7110379.03338.7338.0612401.3596.37248.2614446.9510.75229..8316228.33252.39198.2718895.781060.17848.62CONCLUSIONPhosphate 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 . 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