Lesson: Phosphate Solubilising Microorganisms
What are they?
Phosphate solubilizing microorganisms are organisms which solubilize the insoluble phosphates present in/ added to the soil and make them available to the plants in a soluble or easily accessible form.
What is their importance?
Phosphorus is an important component of the cell. It is a constituent of nucleic acids (DNA and RNA), cell membranes (phospholipids) and ATP (the energy currency of the cell). It is important for plant growth. Availability of phosphorus dictates root development, strength of the stem, flower and seed formation, crop quality, and resistance to plant diseases. It is present in the soil in both organic and inorganic forms but unavailable to the plants since it is insoluble. Phosphorus is added as a fertilizer to soil in the form of rock phosphates or inorganic phosphates. These phosphates are insoluble and thus also unavailable to plants/ crops. Phosphate solubilizing microorganisms produce organic acids which solubilize the phosphates present in the soil and make them available to the plants[1,4]. Inoculation of seeds or soil with phosphate solubilizing microorganisms increases crop yield by 5-10 %.
What are the examples of Phosphate Solubilizing Microorganisms?
Phosphate Solubilizing Microorganisms are of the following types:
- Bacteria: e.g. Pseudomonas sp., Bacillus sp., Streptomyces sp.
- Fungi: e.g. Aspergillus sp., Penicillium sp., Trichoderma sp.
On which nutrient media would you grow them?
They can be grown on various media but their activity is detected [1,2] using:
- Pikovskaya Agar medium
- National Botanical Research Institute’s phosphate growth medium (NBRIP)
How would you detect their presence ?
The media contain tricalcium phosphate in the form of an insoluble precipitate, making the media appear turbid. Upon growth, the organisms produce organic acids (which diffuse into the surrounding medium) causing the solubilization of phosphate resulting in the formation of a halo/ zone of clearance around the colonies. The organisms which show a halo/clear zone around their colonies are considered as phosphate solubilizing microorganisms.
What is the composition of Pikovskaya Agar medium ?
- Composition of Pikovskaya Agar medium: glucose, 10 g; Ca3(PO4)2, 5 g; (NH4)2SO4, 0.5 g; NaCl, 0.2 g; MgSO4.7H2O, 0.1 g; KCl, 0.2 g; Yeast extract, 0.5 g; MnSO4.H2O, 0.002 g; and FeSO4.7H2O, 0.002 g; Distilled water, 1 L
- Bromothymol blue is a pH indicator dye and it may be added (as 0.01 %) to the medium to improve visualization of organic acid production .
- The pH of the medium is adjusted to 7.0. Agar-agar 25 g/ L is added to the medium before autoclaving it.
What is the composition of National Botanical Research Institute’s phosphate growth medium (NBRIP) :
Composition of National Botanical Research Institute’s phosphate growth medium (NBRIP): glucose, 10 g; Ca3(PO4)2, 5 g; MgCl2.6H2O, 5 g; MgSO4.7H2O, 0.25 g; KCl, 0.2 g and (NH4)2SO4, 0.1 g; Distilled water, 1 L.
How do you sterilize these media?
Autoclave the media at 15 PSI (ie lbs/ in2), at 121.6°C for 15-20 min. Cool the media to room temperature before use. Pour agar containing media into sterile petri plates when they are moderately hot ie 55-60°C. Allow the media to solidify or set.
How do you measure the quantity of phosphate solubilized?
Semi-quantitative method [1,2]:
- The petri plates containing inoculated media are incubated at 28°C and check daily upto a period of 14 days for the development of a halo (zone of phosphate solubilization) around the colonies. The diameters of halos around the colonies and diameters of the colonies themselves are measured. Ratio of zone diameter to colony diameter indicates the ability to solubilize phosphate. More the ratio higher is the ability.
Quantitative estimation of phosphate solubilization in broth can be carried out as follows :
- Inoculate 10 ml of medium in a conical flask (of 100 ml capacity) with 100 μl of the culture (containing inoculum density approximately 1–2×109 cfu ml−1). Use autoclaved uninoculated medium as a control. Incubate the flasks for 2 days at 30°C on a shaker incubator set at 180 rpm. Harvest the cultures by centrifugation at 10,000 rpm for 10 min, at 4°C. Estimate the phosphate in culture supernatant using the Fiske and Subbarow method .
- The Fiske–Subbarow method basically involves the measurement of phosphomolybdate complex formed by interaction of phosphate and ammonium molybdate in the presence of sulfuric acid[3,5]. It was originally developed to estimate the inorganic and organic phosphate in different types of biological samples such as blood, urine, and so on and is widely used even today with reproducible and reliable results all around the world by biochemists, pathologists, and analytical chemists. In the absence of interfering factors, phosphate present at lower (0.2–1 mM) or higher concentration (2–10 mM) can be estimated without any problem. However, in the presence of phytic acid, the complex formed was not solely due to the phosphate but also due to phytate. Phytate can also influence the phosphomolybate complex formed, leading to elevated values even at a very low concentration (0.8 mM) of phosphate.
1. C. Shekhar Nautiyal, An efficient microbiological growth medium for screening phosphate solubilizing microorganisms, FEMS Microbiology Letters, Volume 170, Issue 1, January 1999, Pages 265–270, https://doi.org/10.1111/j.1574-6968.1999.tb13383.x
2. Sneha Ogale, Karan Singh Yadav and Shrutika Navale. Screening of endophytic bacteria from the pharmacologically important medicinal plant Gloriosa superba for their multiple plant growth promoting properties. The Pharma Innovation. 2018; 7(1): 208-214.
3. Fiske C.H. Subbarow Y. (1925) A colorimetric determination of phosphorus. J. Biol. Chem. 66, 375–400.
4. Sharma, S.B., Sayyed, R.Z., Trivedi, M.H. et al. Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils SpringerPlus (2013) 2: 587. https://doi.org/10.1186/2193-1801-2-587
5. https://www.researchgate.net/figure/Phosphate-estimation-by-Fiske-and-Subbarow-inthe- absence-or-presence-of-phytic-acid_fig1_258920206