Methods to study the properties of phosphate solubilizers

CONTENTS

Chapter 1: Introduction

Chapter 2: Isolation, screening and identification of phosphate

solubilizing microorganism

Chapter 3: Effect of different concentration of phosphate on

Pseudomonas fluorescens and determination of phosphorus

uptake by the organism

Chapter 4: Effect of different carbon and nitrogen sources on phosphate

solubilization by P. fluorescens

Chapter 5: Effect of NaCl on phosphate solubilization by P. fluorescens

References

Methods to study the properties of phosphate solubilizers

Dr.Pragya Rathore

Associate Professor

Head, Department of Biotechnology

Sanghvi Institute of Management & Science

Pithampur bypass road, behind IIM

Pigdamber, Rau

Indore- 453331, INDIA

Methods to study the properties of phosphate solubilizers

Chapter 1 Introduction

Phosphorus is one of the essential elements for all biological entities. It is associated with several vital functions and is responsible for several characteristics of plant growth such as utilization of sugars and starch, photosynthesis, nucleus formation and cell division, fat and albumin formation, cell organization and the transfer of heredity (Arnon 1956 and Mc Vicker et al. 1963). P has been reported by some workers to stimulate root growth. An adequate supply of P in the early stages of plant growth is important for laying down the primordial for the reproductive parts of plants. It helps in early maturity of crops, particularly cereals, and poor availability of this nutrient markedly reduces their growth. Two symptoms of phosphorus deficiency are reduced plant size and unusually deep green color. P differs significantly from C and N cycles, because it shows no significant fluxes to and from atmosphere and it is not usually involved in oxidation – reduction reactions. In nature, P occurs almost exclusively in phosphate (+5) form and changes in valence state are rare.

If we look at the global P fluxes, we find that most of the P is insoluble or poorly soluble inorganic compounds. About 10¹⁵ metric tons in earth’s crust is present as apatites. A significant amount is contained in oceanic sediment. Estimates of the total amounts of P occurring in certain terrestrial and oceanic reservoirs are shown in table: 1.1. Phosphorus exists mainly as apatites, with the basic formula M₁₀(PO₄)₆X₂. Commonly the mineral (M) is calcium, less often Al or Fe. The anion (X) is F^-, Cl^-, OH^- or CO₃^2- ; thus there exist flour-, chloro-, hydroxy-, and carbonate apatites. Diverse substitutions and combinations of M and X result in some 200 forms of P occurring in nature. Rock phosphates high in carbonate apatite are most commonly mined as fertilizer sources.

In soil, P is found in a multitude of organic and inorganic compounds. Organic fraction of P in soil ranges from 15-85% of total P. Organic fraction of P in soil consists mainly of inositol phosphate, nucleic acids, phospholipids etc.

Organic soil phosphorus:

Inositol phosphates [C₆H₆(OH)₆]:

10-50% are calcium – magnesium salts of phytic acid and are most stable form in soil. Contain from 1-6 phosphorus atoms per inositol units. Phytin is with 6 phosphates. It is synthesized by plants commonly accounts for roughly 40% of the organic P found in soil. Much of the inositol phosphate in soil occurs in polymeric form; as such it accounts for some but not all of the organic P of high molecular weight present in soil. Some of the polymers are believed to be of microbial origin. Some yeast synthesize phosphorylated polymers of mannose and the cell walls of gram positive bacteria contain both techoic acid, a polymer of ribitol phosphate and glycerophosphate, and a 6-phosphate muramic acid. Lower weight inositol ring compounds carrying one to five atoms of P also occur in soil; these probably represent degradation products of inositol hexaphosphate.

Nucleic acids:

0.2- 2.5% are purine and pyrimidine bases combined with a pentose sugar and phosphate. Nucleases are active in soil and should degrade most cell-free DNA contributed by the soil biota and plant litter. Constituent parts of nucleic acid molecules are identifiable in hydrolysates of soil extracts. Among identifiable products are cytosine, adenine, guanine, uracil, hypoxanthine, and xanthine. The last two are decomposition products of guanine and adenine. Of the total organic P in soil, only about 1% can be identified as nucleic acids or their derivatives. The susceptibility of nucleic acids to decomposition, together with lack of incorporation into stable organic matter, is believed to be responsible for their low level of occurrence in soil.

Phospholipids:

1-5% are phosphates combined with a lipid. Organic P occurring in alcohol and ether extracts of soil is indicative of the presence of phospholipids. Choline has been identified; it is one of the products of hydrolysis of lecithin. Most of the glycerophosphate found in soil is believed to be of lipid origin. Various studies have shown that phospholipid P accounts for a similarly low fraction of the soil organic P as does nucleic acid P. Even smaller amounts of sugar phosphates are found in soil.

The sizes of the organic P compartments in soil occur in the order inositol phosphate, polymer organic phosphate, nucleic acid P, and phospholipid P. The biota contributing these components or source material contains them in the reverse sequence. Also, the fraction of total P existing as organic P is higher in the biota then in the soil organic matter.

Large fraction of organic P has not been chemically identified.

Inorganic soil phosphorus:

Of inorganic forms much is in minerals where phosphate is part of structure, or as orthophosphates. Phosphate has strong tendency to form insoluble metallic salts with calcium, iron or aluminium. Hundreds of combinations exist in soil. Generally alkaline soils contain calcium and magnesium phosphates while acidic soils are rich in iron and aluminium phosphates. The solubility of inorganic P compounds changes with pH.

Reactions at high pH values: As the pH values shifts towards alkalinity inorganic P converts into relatively less soluble calcium and magnesium compounds.

Ca(H₂PO₄)₂+ CaCO₃+ H₂O ® 2CaHPO₄.2H₂O + CO₂

Very soluble Less soluble

6CaHPO₄.2H₂O + 3CaCO₃® 3Ca₃(PO₄)₂+ 3CO₂+5H₂O

Less soluble

3Ca₃(PO₄)₂+ CaCO₃® 3Ca₃(PO₄)₂.CaCO₃

Very insoluble

This is more serious in calcareous soils of arid regions.

Chemical fixation of phosphates:

Acidic soils: The fixation of Phosphate in acidic soils is due to one or more of the following reasons (Gaur AC, 1990):

(i) Precipitation of insoluble compounds from the soil solution- in many acid soils, added phosphorus is fixed as iron and aluminium orthophosphate.
(ii) Reaction with hydrated sesquioxides- most soils contains appreciable quantities of hydrous oxides of iron, aluminium and manganese. These colloidal oxides and phosphate ions are attracted to each other and held to the surface of particles in the form of basic iron and aluminium phosphate. Soils with a high quantity of this hydrous oxide fix large quantities of phosphorus such as red and yellow podzolic and red-brown latosolic and lateritic soils.
(iii) Reaction with silicate clays- phosphate ions may combine directly with clays such as montmorillonites(2:1 type) and kaolinites (1:1type) by replacing a hydroxyl group in an aluminium compound or by forming a clay – Ca phosphate linkage. Phosphorus is retained to a greater extent by 1:1 than 2:1 type clays (Tisdale and Nelson, 1958).

Neutral to alkaline soils: In alkaline soils, the formation of diphosphate and triphosphate ions takes place and the solubility of calcium orthophosphate decreases in the order mono-, di- and tricalcium phosphate. In most alkaline soils, the activity of calcium is high and thus favours the formation of insoluble di- and tricalcium phosphate. A part of tricalcium phosphates reverts back to form hydroxyapatites. In alkaline soils containing free calcium carbonates, the solid phase carbonates provide surface for precipitation of phosphate ions. Sometimes these phosphate ions are retended by clays saturated with calcium.

Microbial solubilization of inorganic phosphatic compounds is of great economic importance in plant nutrition. Phosphorus solubilizing bacteria and fungi play an important role in converting insoluble phosphatic compounds such as rock phosphate, bone meal and basic slag, and particularly the chemically fixed soil phosphorus, into available form. Such organisms not only assimilate phosphorus but they also cause a large portion of soluble phosphate to be released in quantities in excess of their own requirements. The solubilization is not restricted to calcium salts but iron, aluminium, magnesium, manganese and other phosphates are also acted upon.

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Redox reactions of P

Reduction: P has various valence states. Of most interest biologically are the +5 (orthophosphate), +3 (orthophosphite), +1 (hypophosphite) and –3 (phosphine) redox states.

Oxidation: Reduced forms of P may be aerobically or anaerobically oxidized by bacteria such as Bacillus caldolyticus, a thermophile, which can oxidize hypophosphite to phosphate aerobically.

Interaction of microorganisms with P in soil:

Microorganisms in soil generally interact with P in three different ways:

Solubilization of inorganic phosphorus: Soil organisms and plant roots participate in the solubilization of soil P. The solubilization of insoluble inorganic phosphates from mineral complexes or insoluble salts by microorganisms is basically because of non-enzymic chemical reactions involving ion exchanges caused mainly by the production of organic acids. Many soil microorganisms exhibit phosphate solubilizing efficiency and can solubilize insoluble inorganic phosphates. These insoluble phosphates include tricalcium phosphate, hydroxyapatite, fluorapatite, aluminium phosphate, iron phosphate, bone meal and different rock phosphates.

Microbial involvement in the solubilization of inorganic phosphates was first demonstrated by Stalstrom in 1903. He isolated bacteria from milk and soil infusions and showed solubilization of tricalcium phosphate by these organisms. Subsequently in 1908, Sackett et al used agar plates to demonstrate solubilization of dicalcium phosphate, tricalcium phosphate, bone meal, rock phosphates etc. by various soil organisms.Since then, much evidence has accumulated supporting phosphate solubilizing capability of various groups of soil microorganisms. Most studies on phosphate solubilization were done by isolating microorganisms from soil and studying the extent of solubilization under in vitro conditions. Investigations on solubilization of insoluble phosphates under field conditions however, started later (Kapoor, Mishra et al, 1989).

Several microorganisms responsible for solubilization of insoluble phosphates were found in great numbers in soil rhizosphere isolates (Sperber 1958: Azcon et al. 1976). High concentrations of P, Ca, and other elements were reported to occur in vesicular-arbuscular mycorrhizal (VAM) fungi (Strullu et al. 1981) as a result of oxalic acid production by the VAM fungi. The chelating effects of microbial products and other forms of soil organic matter were thoroughly reviewed by Kononova (1961).

Soviet Union for the first time used the culture of Bacillus megaterium var. phosphaticum as an inoculant for phosphorus solubilization. Canada used Penicillium bilaji with the trade name Provide. Phosphate solubilizing organisms thus were started to be used as inoculants to increase the availability of P to crop in these countries and has led to the development of inoculum preparation which is popularly known as phosphobacterin.

In India, Sundara Rao and Paul first reported significant increase in the yield of berseem (Trifolium alexandrinum) due to inoculation of Phosphobacterin. Microbial inoculants can substitute almost 20-25% of the phosphorus requirement of plants.

Mineralization of organic phosphorus: The complex organic phosphorus compounds present in soil are degraded into relatively simpler forms by microbial enzymes and thereby made available to plants.

Immobilization of soluble phosphate: Phosphate solubilized by microorganisms is seldom absorbed directly by plants as long as a large amount of organic matter remains, because other heterotrophs incorporate phosphate into their biomass. Therefore plant roots cannot absorb enough of soluble phosphate ions and the process is technically termed as immobilization. This is transitory fixation where the P is taken into the cell as inorganic phosphates and are assimilated as organic cellular constituents or precipitated intracellularly as polyphosphate granules. Indirect precipitation of P minerals may occur either authigenically where soluble phosphates react with Ca⁺⁺ forming insoluble Ca phosphate minerals (phosphorite), or diagenetically where phosphate replaces carbonate in insoluble calcium based clay minerals.

Mechanism of phosphate solubilization by microorganisms:

Different microorganisms cause solubilization of P by different methods. The microbial metabolites like organic acids bring about changes in soil pH in most cases towards acidic sides. Almost all phosphate solubilizing microorganisms when grown on simple carbohydrates produce acetic, formic, lactic, gluconic, glycolic, 2-ketogluconic, oxalic, succinic, malic, and maleic acids in growth medium. These organic acids are intermediary products of metabolic pathways and some amount is always released outside the cell surface by feedback inhibitions and or catabolic repressions. The type of organic acids produced and their amounts differs with different microorganisms. The type of organic acid has a significant effect on the solubilization. Tri- and dicarboxylic acids are more effective as compared to monobasic and aromatic acids. The extent of solubilization and ultimate release of soluble phosphate also depends on the accessory minerals present in rock phosphates. The solubilized phosphate may react with calcium or magnesium present in rock phosphate as soon as the pH of the growth medium increases. Pareek and Gaur studied the extent of solubilization of tricalcium phosphate and rock phosphate by organic acids. Aliphatic acids were found to be comparatively more effective in phosphate solubilization than phenolic acids and citric acid and fumaric acid had highest P solubilizing ability.

As indicated by Pohlman and McColl (1986), several factors are important in determining the degree or rate of dissolution of soils in organic acids. These are (i) rate of diffusion of organics from bulk solution and diffusion of products from the site of reactivity,(ii) contact time between the organic acids and mineral surface, (iii) degree of dissociation of organic acids, (iv) type and position of functional groups, and (v) chemical affinities of chelating agents for the metals.

Many soil bacteria and fungi are known to solubilize phosphate from inorganic phosphatic compounds (Sen and Paul 1957, Gaur et al. 1973, Arora and Gaur 1979, Asea et al. 1988). Most of the reports on solubilization of insoluble phosphate by microorganisms suggest that organic acid metabolites excreated by microorganisms are solely responsible for the process (Sperber 1957, Bajpai and Sundara Rao 1971, Banik and Dey 1982).Organic acids, e.g., 2- ketogluconic acid, act as good chelators of divalent cations besides their acidifying effects. Duff and Webley (1959) documented marked chelation of calcium accompanying release of phosphate from insoluble phosphatic compounds by microorganisms. Earlier, Rose (1957) demonstrated that Aspergillus niger could bring ferric phosphate into solution by producing H₂S gas. Sperber (1958) suggested that H₂S production by soil bacteria could also be a factor in releasing phosphate from ferric phosphate. Thus, inorganic phosphate solubilization by microbial means has been attributed to the processes of acidification, chelation, H₂S production and exchange reaction in the growth environment (Sperber 1958, Ahmad and Jha 1968, Molla and Chowdhury 1984). However, Roos and Luckner (1984) observed that an isolate of Penicillium cyclopium employed an NH₄⁺ /H⁺ exchange mechanism in presence of NH₄Cl and glucose in the medium resulting in the generation of inorganic acid which caused reduction of medium pH.

Four alternative methods have commonly been used to increase P availability in rock phosphate amended soils. These include mixing rock phosphates with various soil amendments like nitrogen sources, farmyard manures and elemental sulphur before incorporation into soils (Lipman et al. 1916), partial acidulation of rock phosphate with sulphuric and phosphoric acids (Hammond et al. 1986), compaction of rock phosphate with water soluble phosphorus fertilizers (Kpomblekou et al. 1991), and microbiological methods (Louw and Webley 1959).

This last method is based on fact that certain microorganisms produce organic acids that complex metals in rock phosphate thus altering the release of Phosphorus. The involvement of chelating compounds produced from microbial growth in weathering processes was reviewed by Berthelin (1983). Weathering of silicate minerals in the presence of weakly and strongly complexing acids was studied by Huang and Keller (1972). The effect of organic acids on P release from P-bearing minerals was investigated by Johnston (1959), Moghimi and Tate (1978), and Bradley and Sieling (1953).

Lipman et al.(1961) advocated the use of rock phosphate and sulphur for increasing P availability in soil. Swaby and Sperber reported that the population of phosphate dissolving microorganisms is more in the rhizosphere (20-40% of the total population) compared to non-rhizosphere (10-15% of the total population). Raghu and McRae studied the incidence of phosphate dissolving bacteria in submerged rice soils and in the rhizosphere of rice plant growing in one of these soils.

Chelating substances have also an important role in solubilization of insoluble phosphates. The acids chelate Ca⁺⁺ ions and the chelation depends on the hydroxyl ions of the acid. The Ca is chelated to a small extent with a-hydroxy aliphatic monobasic acid like lactic acid, more strongly with dibasic acids like malic and tartaric and more strongly with tribasic like citric acid. Among the dibasic aliphatic acids, hydroxy derivatives like malic acid form strongest complexes and a-substitutions by hydroxyl group of an aliphatic acid exhibit greater effect than b-substitutions of the same acid. Dibasic aromatic acids also chelate Ca ions but not the monobasic aromatic acids. Under acidic soil pH conditions the phosphate ions are precipitated by Fe³⁺ and Al³⁺. The organic acids prevent such precipitation by chelation, forming metallo organic molecules e. g. Ferric citrate by citric acid. Dibasic acids also form chelates hydroxy phosphates and form hydroxy salts thereby releasing the phosphate ions. Some bacteria produce 2- ketogluconic acid which is a strong chelator of Ca. It can also solubilize insoluble phosphates like hydroxyapatite, fluorapatite and aluminium phosphate.

The major end product of organic matter decomposition is CO₂. Carbon dioxide evolved by microorganisms in rhizosphere region help plants to absorb soluble phosphorus. P may react directly with CO₂or it may form carbonic acid which reacts with Ca₃(PO₄)₂to form CaHPO₄or Ca(H₂PO₄)₂with formation of CaCO₃.

Fermentative microorganisms produce H₂S from sulphur containing amino acids. Some anaerobic sulphate respiring bacteria like Desulfovibrio and Desulfotomaculum also produce H₂S. This H₂S reacts with soil mineral phosphates to form sulphides and releases phosphate ions.

The autotrophic bacteria use inorganic salts for their metabolism and the end products are mainly the inorganic acids. These include the nitrifying bacteria Nitrosomonas and Nitrobacter producing HNO₃from ammonium salts and the iron bacteria Ferrobacillus ferrooxidans and Thiobacillus ferrooxidans which oxidize iron and forms H₂SO₄or convert Fe(OH)₂.H₂PO₄to Fe(OH)₃releasing H₂PO₄ions in solution. The third group comprises sulphur bacteria Thiobacillus which oxidize reduced sulphur compounds to suphate resulting in the formation of H₂SO₄. These end products as HNO₃and H₂SO₄bring down soil pH, releasing phosphorus ions by chemical reactions.(Yadav and Dadarwal, 1997).

Humic acid and fulvic acid are the other chelating substances produced during decomposition of organic matter. These form stable complexes with calcium, iron and aluminium and release phosphate. Mishra et al. reported the release of P from phosphates by humic acid isolated from compost. A 5% solution of humic acid in alkali could solubilize 362 mgP/g rock phosphate. Sadik et al. have also reported the solubilization of phosphates by humic acid but the presence of calcium carbonate was found to decrease solubilization. The action of humic and fulvic acid appears to be due to the functional groups such as carboxylic, phenolic hydroxyl and alcoholic hydroxyl.(Kapoor, Mishra, Kukreja,1989).

Some fungi and some plants are known to accumulate calcium- oxalate internally and externally. This might be increasing phosphorus uptake.(Paul and Clark,1990).

Mechanism of mineralization of organic compounds: Microbial mineralization of organic P is strongly influenced by environmental parameters.Mild alkaline conditions favour mineralization. Extracellular enzymes like phosphatases and phytases are responsible for mineralization.

Phosphatases are collective name for enzymes that cleave phosphate from organic compounds like phospholipids nucleic acids etc.Depending on the optima of enzymes they have been classified as alkaline and acid phosphatases. Phosphatase activity is widespread in soil with larger percentage in rhizosphere.

Phytase is enzyme that cleaves phosphate from phytin with accumulation of inositol. However, phytin is not readily metabolized in soil and is probably limited by low solubility of phytic acid. Certain mycorrhizae have phytases as well as phosphatases. Rate of mineralization of phospholipids, nucleic acids and sugar phosphates is rapid. Phosphate liberation from nucleic acid requires the action of nucleases. Phosphate release from phosphoproteins, phospholipids requires the action of phosphomonoesterases and phosphodiesterases which attack the mono-ester and di-ester linkages respectively. Mineralization of inositol phosphate is relatively slow.

The role of mycorrhizae: The mycorrhizal fungi play a more important role in P transfers than the general soil population. Four mechanisms are probably involved. First, the mycorrhizal hyphae contribute to the solubilization of mineral phosphate by their production of respiratory CO₂and by excreation of organic acids. Thus there is a mycorrhizosphere effect that supplements the rhizosphere effect. Second, the mycorrhizal hyphae extend soil exploration over and beyond the soil exploration accomplished by the plant root themselves. Fungal hyphae penetrate soil organic matter particles and macroaggregates more thoroughly Many soil microorganisms exhibit phosphate solubilizing efficiency and can solubilize insoluble inorganic phosphates. These insoluble phosphates include tricalcium phosphate, hydroxyapatite, fluorapatite, aluminium phosphate, iron phosphate, bone meal and different rock phosphates.

Microbial involvement in the solubilization of inorganic phosphates was first demonstrated by Stalstrom in 1903. He isolated bacteria from milk and soil infusions and showed solubilization of tricalcium phosphate by these organisms. Subsequently in 1908, Sackett et al used agar plates to demonstrate solubilization of dicalcium phosphate, tricalcium phosphate, bone meal, rock phosphates etc. by various soil organisms.Since then, much evidence has accumulated supporting phosphate solubilizing capability of various groups of soil microorganisms. Most studies on phosphate solubilization were done by isolating microorganisms from soil and studying the extent of solubilization under in vitro conditions. Investigations on solubilization of insoluble phosphates under field conditions however, started later (Kapoor, Mishra and Kukreja, 1989).

Several microorganisms responsible for solubilization of insoluble phosphates were found in great numbers in soil rhizosphere isolates (Sperber 1958: Azcon et al. 1976). High concentrations of P, Ca, and other elements were reported to occur in vesicular-arbuscular mycorrhizal (VAM) fungi (Strullu et al. 1981) as a result of oxalic acid production by the VAM fungi. The chelating effects of microbial products and other forms of soil organic matter were thoroughly reviewed by Kononova (1961).

Soviet Union for the first time used the culture of Bacillus megaterium var. phosphaticum as an inoculant for phosphorus solubilization. Canada used Penicillium bilaji with the trade name Provide. Phosphate solubilizing organisms thus were started to be used as inoculants to increase the availability of P to crop in these countries and has led to the development of inoculum preparation which is popularly known as phosphobacterin.

In India, Sundara Rao and Paul first reported significant increase in the yield of berseem (Trifolium alexandrinum) due to inoculation of Phosphobacterin. Microbial inoculants can substitute almost 20-25% of the phosphorus requirement of plants than do root extentions. Third, the hyphae may take up P at lower levels of concentration in the soil solution than can the roots. Fourth, some mycorrhiza are known to produce extracellular enzymes that cause mineralization of organic P which is not otherwise available.

Phosphorus reservoirs: Major phosphate reservoir is in rocks and sediments where phosphate is bound with a variety of cations especially Al and Fe, two abundant elements in the earth’s crust, as well as other cations such as Ca, Mg, metals. Although this reservoir is large but it is very slowly cycled because it is inaccessible to physical and chemical weathering and it is unavailable to organisms for biological cycling.

Smaller phosphate reservoirs are present in waters, surface soils and living or dead matter. These are more actively cycled.

Phosphorus fertilizers in Agriculture: Indian agriculture is mainly dependent on the extensive use of chemical fertilizers. Application of chemical fertilizers is essential to boast up the crop yields. Fertilizers have played a key role in the modernization of Indian agriculture and in making the country self sufficient in food grain production. Superphosphate is applied in fields as P fertilizer. Industrial production of superphosphate is highly expensive and energy intensive process as it requires addition of sulfuric acid to acidulate rock phosphates. Since 70 to 90 % of the supplemented superphosphate is not available for crops as a result of mineralization (Vig and Dev, 1984), the requirements for this fertilizer are high. Per unit of P, rock phosphate is about one third the price of simple superphosphate, but the ineffectiveness of phosphate rocks as compared to superphosphate is the result of little or no water solubility of the rocks. So it is very important , particularly for poor countries often gravely short of phosphate in their soils, to know in what conditions it may be profitably used (Nye and Kirk, 1987). Phosphorus is frequently a limiting nutrient in some soils. Rock phosphates are the nonrenewable resources for phosphate fertilizers. Besides, much of the applied phosphate as fertilizers is fixed by the soil and rendered less available to plants. The long term application of P fertilizers has resulted in the accumulation of total soil P, most of which is poorly soluble. For this reason, the possibility of practical use of rock phosphate as a fertilizer has received significant interest in recent years. Recent researches in this regard are diverted towards more practical approach, i.e. the application of phosphate solubilizing microorganisms for solubilizaton of rock phosphates. Phosphate solubilizing microorganisms are capable of solubilizing tricalcium, aluminium and iron phosphates, as well as rock phosphate making the phosphorus present in the soil available to plants. Soils also contain organic phosphorus, which can be used by crops if it is mineralized. Microorganisms play active part in the decomposition and mineralisation of organic matter and release of nutrients. Organisms that cause increases in plant available phosphorus in the soil system belong to a diversified group including bacteria, actinomycetes and several group of fungi. Huge investments would have to be made in fertilizer factories to meet growing demand of phosphatic fertilizers. The fertilizers so produced are not only high price, but the factories have an adverse effect on the environment. Phosphate rock deposits to the tune of 260 million tones are estimated in India, but most of these are low grade materials (<30% P₂O₅) unsuitable for production of conventional P fertilizers (Narayanasamy G and Biswas DR, 2000). Direct application of these materials to soil as ground mineralphosphate is not advisable in India, as the reactivity (as adjudged by carbonate substitution for phosphate in apatite structure) is very low. Hence there is need to increase the effectiveness of low grade Indian phosphate rocks. Phosphatic biofertilizers offer a viable alternative. Direct use of even low grade rock phosphate as fertilizer is feasible in neutral to alkaline soils if phosphate solubilizing microorganisms are used as inoculants.

Status of Phosphorus fertilizers in India: At present, there are 64 large sized fertilizer units in the country. Of these, 18 units manufacture diammonium phosphate (DAP) along with other fertilizers. DAP is the dominant phosphatic fertilizer accounting for 58% of consumption, followed by superphosphate with a 20% share. In the year 1998-1999 overall P fertilizers consumed were 4.0 million tons (India Infoline Ltd.2000). Against an all India average consumption of 90 Kg/hectare of NPK fertilizers, states like Punjab and Andhra Pradesh comsume over 135 Kg/hectare, while economically underdeveloped states like Madhya Pradesh and Orissa consume about 30 Kg/hectare. In India, there are reserves of high grade rock phosphates at Kaliapatnum, Mussourrie, Purulia and Udaipur. Low grade rock phosphate reserves are at Jhabua, Jhamarkotra, Marton, Mussourrie and Purulia.

Phosphate solubilizing microorganisms (PSM):

PSM include different groups of microorganisms such as bacteria and fungi which convert insoluble inorganic phosphatic compounds into soluble form. The species of Pseudomonas, Micrococcus, Bacillus, Flavobacterium, Penicillium, Fusarium, Sclerotium, Aspergillus and others have been reported to be active in bio-conversion. Such bacteria and fungi can grow in media where calcium phosphate, iron phosphate, aluminium phosphate,apatite, bone meal, rock phosphate or similar insoluble compounds are the sole source of phosphate. Besides bacteria and fungi, mycorrhizal fungus like Glomus fasciculatum has also been studied for its capacity to solubilize insoluble phosphates.

In the present study the organism, Pseudomonas fluorescens has been screened from the indigenous soil samples and an attempt has been made to study the effects of various physiological parameters on solubilization of phosphates.

Table: 1.1

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Many soil microorganism exhibit phosphate solubilizing efficiency and can solubilize insoluble inorganic phosphates. These insoluble phosphates include tricalcium phosphate, hydroxyapatite, fluorapatite, aluminium phosphate, iron phosphate, bone meal and different rock phosphates.