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 / StrainsGluconic acid / Pseudomonas sp., Erwinia herbicola, Pseudomonas cepacia,
Burkholderia cepacia
2-Ketogluconic
acid / 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
ü Microscop ( Labomed )
ü Laminar air flow ( Micro filt india )
ü Incubater ( REMI )
ü Incubater with shaker ( REMI )
ü Refrigerater
ü Autoclave ( Meta instrument mumbai )
ü Centrifuge ( REMI )
ü Hot air oven ( Meta instrument mumbai )
ü Water bath ( NEOLAB )
ü Weighing machine ( ATCO, CITIZEN )
ü 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.