Chapter -1

INTRODUCTION
Phosphorus is a major nutrient element for plants and it plays an important role in the
life processes as a constituent of DNA/RNA and in energy transfer mechanism via Adenosine
Tri Phosphate / Adenosine Di Phosphate. Plants take up acids (citric and malic acids) through
the roots which dissolve available soil phosphates. Phosphates that are soluble in water or 2%
citric acid are known as available forms. Only about 25 per cent of the phosphorus applied to
the soil is available for the crops and the rest become unavailable due to chemical fixation with
aluminum and iron in acidic soils. N. TENZING BALIAH, 2016 et. al (1)
http://www.tropecol.com/pdf/open/PDF_57_3/8%20Baliah,%20Pandia&Kumar-f.pdf
Rock phosphate mineral essentially contains tri calcium phosphates which are not water
soluble. Some rock phosphates of sedimentary origin may contain varying amounts of P soluble
in 2% citric acid depending on the geological process setting of the deposit formation. Soil pH
plays an important role in the P take up of plants. Soil pH range of 5.5 to 7 is most favorable
for P up take by plants. Below 5.5 pH soil cations such as Fe, Al and Mn lock up P there by
making it unavailable to the plants and Ca, Mg ions lock up the available P in the soil pH range
above 7. The problem of locking up of available P by the soil cations is known as phosphate
fixation problem to soil scientists and agronomists because of that the use efficiency of P from
the applied mineral fertilizers by plants in agriculture is just 15% in the first year and 1 – 2%
per year in the subsequent years. The water soluble P of the applied mineral fertilizers is
misplaced by leaching, run off with rain/ irrigation water or is fixed by the soils into
unavailable forms. Nutrient use efficiency of N applied to the soils through chemical fertilizers
is said to be 50 – 60% in the first year. (DMR sekhar review report 2012) (2)
It functions as one of the major players in the process of photosynthesis, nutrient
transport, energy transfer, early growth and root formation, cell division, DNA and RNA
formation, flower blooms, seed development, improvement in plant strength and to tolerance
for unfavourable environmental conditions. Phosphorus also affects the plant's structure at a
cellular level. A plant with the proper amount of phosphorus available to it will grow more
vigorously and mature earlier than plants with inadequate phosphorus. A plant with phosphors
deficiency will exhibit stunted growth, lack of fruit or flowers, wilting and leaves may be

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greener or have a purple cast to them due to the photosynthetic process being affected
(CFAITC 2009).(3)
Often organic manures are assessed in terms of their content of nutrient elements such
as N, P, K, etc. Microorganism that decay organic matter produce a variety of useful
compounds such as gibberlins, auxins, vitamins, fulvic and humic acids that are vital for the
plant growth, therefore fertilizers and minerals cannot replace manure in agriculture. Peat or
lignite can partly replace organic manures or composts for they also contain humic acids.
Organic matter in the soil greatly enhances the water holding capacity of the soil in addition to
facilitate the aeration by keeping the soil loose.
Rock phosphate (Phosphorite)
Phosphate rocks should provide a cheap source of phosphorus fertilizer for agricultural
use. India alone has about 260 million tons of rock phosphate (RP) deposits and this material
should provide a cheap source of phosphate fertilizer for crop production (FAI 2002). Out of
total resources, 35% are in Jharkhand, 31% in Rajasthan, 17% in Madhya Pradesh, 9% in Uttar
Pradesh and 8% in Uttarakhand.
Unfortunately, rock phosphate is not plant available in soils with a pH greater than 5.5 - 6.0.
Most RPs have low reactivity in alkaline and neutral soils and therefore its direct application is
not always effective without previous treatment (Vassilev e al. 2001). Due to low availability
of P in this native material, high transportation costs, and small crop responses, very little rock
phosphate is currently used in agriculture (Rehm et al.2010) (4)
Crop Uptake phosphate
Crop response to phosphorus depends on the availability of phosphorus in the soil
solution and the ability of the crop to take up phosphorus. The availability of phosphorus in the
soil solution has already been discussed. The ability of a plant to take up phosphorus is largely
due to its root distribution relative to phosphorus location in soil. Because phosphorus is very
immobile in the soil, it does not move very far in the soil to get to the roots. Diffusion to the
root is only about 1/8 of an inch per year, and relatively little phosphorus in soil is within that
distance of a root. Thus, the roots must grow through the soil and basically go get the
phosphorus the plant needs. Therefore root growth is very important to phosphorus nutrition.
Any factor that affects root growth will affect the ability of plant to explore more soil and get
adequate phosphorus. Soil compaction, herbicide root injury, and insects feeding on roots can

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all dramatically reduce the ability of the plant to get adequate phosphorus. Young seedlings can
suffer from phosphorus deficiency even in soils with high available phosphorus levels because
they have very limited root systems that are growing very slowly in cold, wet, early early-
season soil conditions. This is why some crops respond to phosphorus applied at planting in
starter fertilizers even in relatively high phosphorus soils. (Starter fertilizer management is
discussed later in this fact sheet. See also Penn State Agronomy Facts 51: Starter Fertilizer.)(5)
Phosphate solubilizing microorganisms
For a sustainable agriculture system, it is imperative to utilize renewable inputs, which can
maximize the ecological benefits and minimize the environmental hazards. Microbial
solubilization of rock phosphate and its use in agriculture is receiving greater attention.
Phosphate solubilizing microorganisms (PSMs) can be useful to reverse the process of
phosphate fixation. PSMs is a group of heterotrophic microorganisms capable of solubilizing
inorganic P from insoluble sources through the process of acidification, chelation and exchange
reaction (Gerke 1992).(6)
Phosphate solubilizing microorganisms are ubiquitous and their numbers vary from soil to soil.
In soil, P solubilizing bacteria constitute 1–50% and fungi 0.5–0.1% of the total respective
population. In general, P solubilizing bacteria generally outnumber P solubilizing fungi by 2–
150 folds (Banik and Dey 1982; Kucey 1983; Kucey et al. 1989).(7) These PSMs include the
following bacteria, fungi and yeast. They are:
Bacteria: Bacillus megaterium, B. circulans, B. subtilis, Pseudomonas straita, P.
rathonis;
Fungi: Aspergillus awamori, Aspergillus niger, Aspergillus tubingensis Penicillium
digitatum, Penicillium rugulosum, Trichoderma sp.
Yeast: Schwanniomyces occidentails
Mainly the solubilization of P by these microorganisms is attributed to excretion of
organic acids like citric acid, glutamic acid, succinic acid, lactic acid, oxalic acid, glyoxalic
acid, malic acid, fumaric acid, tartaric acid and ketobutyric acid (Hegde 1998; Reyes et al.
2006). (8) The role of organic acids in dissolving mineral phosphates and phosphorylated
minerals can be attributed to the lowering of pH which helps in formation of stable complexes
with such cations as Ca++, Mg++, Fe+++ and Al+++. These complexes are more stable than

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original inorganic phosphate compounds (Struthers and Sieling 1950; Dalton et al. 1952,
Bradley and Sieling 1953; Mortensen 1963). (9)
The reaction of organic acid with calcium phosphate is one of acid dissolution, the
amount of phosphate released depends upon the strength of acid. These acids form unionized
association compounds with Ca++, thus removing it from solution and soluble phosphate
concentration is increased. Ca++ is also chelated to small extent with alpha hydroxyl aliphatic
acid (most strongly with tribasic like citric acid) (Johnston and Miller1959). (11)Phosphate
solubilization is a complex phenomenon, which depends on many factors such as the
nutritional, physiological, and growth conditions of the cultures (Cunningham and Kuiack
1992). Since PSMs are heterotrophs and solubilize insoluble phosphates by secreting organic
acids, the role of carbon in this context is very important. 20 Apart from carbon, nitrogen is
also an important element needed by microorganisms for synthesis of amino acids, proteins,
purines and pyrimidine nitrogenous bases. It may be taken in the form of ammonia, nitrate, and
nitrite or in amino form (Nahas 1996; Reyes et al. 1999b; Whitelaw et al. 1999; Seshadri 2004).
(12)
There are two components of P in soil, organic and inorganic phosphates. Mineralization
of phosphate from soil organic P by phosphatase enzymes is of particular significance, as
organic P accounts for a major proportion (generally 40 to 80%) of the total P in most soils,
occurring primarily as inositol phosphates (Turner et al. 2002). Phosphorus can be released
from organic compounds in soil by three groups of enzymes: (1) Nonspecific phosphatases,
which perform dephosphorylation of phospho-ester or phosphoanhydride bonds in organic
matter, (2) Phytases, which specifically cause P release from phytic acid and (3)
Phosphonatases and C–P Lyases, enzymes that perform C–P cleavage in organophosphonates.
The main activity apparently corresponds to the work of acid phosphatases and
phytases because of the predominant presence of their substrates in soil (Rodriguez et al.
2006).(13) These include changes to root structure, association with mycorrhizal fungi and
other soil microorganisms and biochemical processes at the root-soil interface (Jakobsen et al.
2005). The P solubilizing microorganisms also produce fungistatic and growthpromoting
substances, which influence plant growth. The performance of these microorganisms is affected
by availability of a carbon source, P concentration, type of phosphate, particle size of
phosphate and other factors like temperature and moisture (Hegde 1998).

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Mechanisms of phosphorus solubilisation
Phosphorus solubilizing activity is determined by the ability of microorganisms 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 et al. 1995; Dutton and Evans
1996; Nahas 1996). Some bacterial species have mineralization and solubilization potential for
organic and inorganic phosphorus, respectively (Hilda and Fraga 2000; Khiari and Parent
2005). General sketch of P solubilization by bacteria in soil is shown in figure 2.1. 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 PSMs in which hydroxyl and carboxyl groups of acids chelatecations (Al, Fe,
Ca) and decrease the pH in basic soils (Kpomblekou and Tabatabai 1994; Stevenson 2005).
The PSMs 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 PSMs has direct correlation with pH of the
medium. 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 PSMs 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 phosphate solubilization is induced by phosphate starvation
(Gyaneshwar et al. 1999).(14)

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Figure 2.1: Schematic diagram of soil phosphorus mobilization and immobilization by bacteria
(Khan et al. 2009). (7)
Biological Nitrogen Fixation
Nitrogen is a critical limiting element for plant growth and production. It is a major
component of chlorophyll, the most important pigment needed for photosynthesis, as well as
amino acids, the key building blocks of proteins. It is also found in other important
biomolecules, such as ATP and nucleic acids. Even though it is one of the most abundant
elements (predominately in the form of nitrogen gas (N2) in the Earth’s atmosphere), plants can
only utilize reduced forms of this element. Plants acquire these forms of “combined” nitrogen
by:
1) The addition of ammonia and/or nitrate fertilizer (from the Haber-Bosch process) or manure
to soil,
2) The release of these compounds during organic matter decomposition,
3) The conversion of atmospheric nitrogen into the compounds by natural processes, such as
lightning, and
4) Biological nitrogen nfixation (Vance2001).Biological nitrogen fixation (BNF), discovered
by Beijerinck in 1901 (Beijerinck 1901), is carried out by a specialized group of
prokaryotes. These organisms utilize the enzyme nitrogenase to catalyze the conversion of
atmospheric nitrogen (N2) to ammonia (NH3). Plants can readily assimilate NH3to produce

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 the aforementioned nitrogenous biomolecules. These prokaryotes include aquatic
organisms, such as cyanobacteria, free-living soil bacteria, such as Azotobacter, bacteria
that form associative relationships with plants, such as Azospirillum, and most importantly,
bacteria, such as Rhizobiumand Bradyrhizobium, that form symbioses with legumes and
other plants (Postgate 1982).
The Process
The reduction of atmospheric nitrogen is a complex process that requires a large input
of energy to proceed (Postgate 1982). The nitrogen molecule is composed of two nitrogen
atoms joined by a triple covalent bond, thus making the molecule highly inert and nonreactive.
Nitrogenase catalyzes the breaking of this bond and the addition of three hydrogen atoms to
each nitrogen atom.

Microorganisms that fix nitrogen require 16 moles of adenosine triphosphate (ATP) to

reduce each mole of nitrogen (Hubbell & Kidder, 2009). These organisms obtain this energy by
oxidizing organic molecules. Non-photosynthetic free-living microorganisms must obtain these
molecules from other organisms, while photosynthetic microorganisms, such as cyanobacteria,
use sugars produced by photosynthesis. Associative and symbiotic nitrogen-fixing
microorganisms obtain these compounds from their host plants’ rhizospheres (National
Research Council 1994, Hubbell & Kidder 2009)

Industries use the Haber-Bosch process to reduce nitrogen essentially in the same way.
Conventional agriculture has depended upon this process to produce the commercial fertilizer
needed to grow most of the world’s hybrid crops. But this approach comes with many
consequences, including using fossil fuels for the energy needed to produce this fertilizer, the
resulting carbon dioxide emissions and pollution from burning these fuels, and adverse affects
on human health (Vitousek 1997).

Nitrogen Fixation by Free-Living Heterotrophs

Many heterotrophic bacteria live in the soil and fix significant levels of nitrogen without
the direct interaction with other organisms. Examples of this type of nitrogen-fixing bacteria
include species of Azotobacter, Bacillus, Clostridium, and Klebsiella. As previously noted,
these organisms must find their own source of energy, typically by oxidizing organic molecules
released by other organisms or from decomposition. There are some free-living organisms that

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have chemolithotrophic capabilities and can thereby utilize inorganic compounds as a source of
energy.

Because nitrogenase can be inhibited by oxygen, free-living organisms behave as

anaerobes or microaerophiles while fixing nitrogen. Because of the scarcity of suitable carbon
and energy sources for these organisms, their contribution to global nitrogen fixation rates is
generally considered minor. However, a recent study in Australia of an intensive wheat rotation
farming system demonstrated that free-living microorganisms contributed 20 kilograms per
hectare per year to the long-term nitrogen needs of this cropping system (30-50% of the total
needs; Vadakattu & Paterson 2006). (19) Maintaining wheat stubble and reduced tillage in this
system provided the necessary high-carbon, low-nitrogen environment to optimize activity of
the free-living organisms.

Associative Nitrogen Fixation

Species of Azospirillum are able to form close associations with several members of
the Poaceae (grasses), including agronomically important cereal crops, such as rice, wheat,
corn, oats, and barley. These bacteria fix appreciable amounts of nitrogen within the
rhizosphere of the host plants. Efficiencies of 52 mg N2 g-1 malate have been reported
(Stephan et al. 1979). The level of nitrogen fixation is determined by several factors, including
soil temperature (Azospirillum species thrive in more temperate and/or tropical environments),
the ability of the host plant to provide a rhizosphere environment low in oxygen pressure, the
availability of host photosynthates for the bacteria, the competitiveness of the bacteria, and the
efficiency of nitrogenase (Vlassak & Reynders, 1979).

Symbiotic Nitrogen Fixation

Many microorganisms fix nitrogen symbiotically by partnering with a host plant. The
plant provides sugars from photosynthesis that are utilized by the nitrogen-fixing
microorganism for the energy it needs for nitrogen fixation. In exchange for these carbon
sources, the microbe provides fixed nitrogen to the host plant for its growth one example of this
type of nitrogen fixation is the water fern Azolla’s symbiosis with a cyanobacterium Anabaena
azollae. Anabaena colonizes cavities formed at the base of Azolla fronds. There the
cyanobacteria fix significant amounts of nitrogen in specialized cells called heterocysts. This
symbiosis has been used for at least 1000 years as a biofertilizer in wetland paddies in

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Southeast Asia. Rice paddies are typically covered with Azolla “blooms” that fix up to 600 Kg
N ha-1 yr-1 during the growing season (Postgate 1982, Fattah 2005).

Legume Nodule Formation

The Rhizobium bacteria colonize the host plant’s root system and cause the roots to
form nodules to house the bacteria (Figure 4). The bacteria then begin to fix the nitrogen
required by the plant. Access to the fixed nitrogen allows the plant to produce leaves fortified
with nitrogen that can be recycled throughout the plant. This allows the plant to increase
photosynthetic capacity, which in turn yields nitrogen-rich seed. The consequences of legumes
not being nodulated can be quite dramatic, especially when the plants are grown in nitrogen-
poor soil. The resulting plants are typically chlorotic, low in nitrogen content, and yield very
little seed ref. 8