To Isolate Phosphate Solubilizing Microorganism

To Isolate Phosphate Solubilizing Microorganism
Word Count: 7500
[Name of the Writer]

[Name of the Institution]
Abstract
Phosphorus has been acquired by the plants in the soluble form which is found in the soil in the
minimum quantity. The available phosphorus is found in the soil, which reacts with the other
minerals available in the soil like calcium, iron and aluminium causing the precipitation of the
compounds leaves the phosphorus in the insoluble form. The role of phosphorus-solubilizingbacteria are found to be very important in the conversion and decomposition in order to produce
a soluble form of phosphorus to the plants so that it can be utilized for plants growth and
development. Moreover, the observable and gradual decrease in the pH indicates the activity of
PSB for phosphorus immobilization by showing the traces of inorganic acids. The current study
aimed to isolate the phosphate solubilizing bacteria by analyzing the four soil samples, i.e.
banana, ground, garden and graveyard soil and analyzing the rhizosphere under the PKV medium
and tricalcium phosphate. The halozone colonies indicated the presence of Pseudomonas
fluorescens and the chemical reaction indicated the production of organic acids and the
experiment indicate the immobilization of phosphorus.
Contents
List of Tables.................................................................................................................. 2
CHAPTER 1: INTRODUCTION......................................................................................... 2
CHAPTER 2: LITERATURE REVIEW.................................................................................2
2.1 Isolation of Strains................................................................................................... 2
2.2 Formation of Phosphate-Solubilizing-Bacteria PSB........................................................2
2.3 Phosphorus Solubilizing Mechanism.............................................................................2
CHAPTER 3: METHODOLOGY........................................................................................ 2
3.1 Sample.................................................................................................................. 2
3.2 Preparation of medium.............................................................................................. 2
3.3 Isolation of PSM...................................................................................................... 2
3.4 Morphological Characterization...................................................................................2
3.5 Gram Staining......................................................................................................... 2
CHAPTER 4: RESULTS AND DISCUSSION.........................................................................2
4.1 Isolation of PSB...................................................................................................... 2
CHAPTER 5: CONCLUSION............................................................................................ 2
References..................................................................................................................... 2
List of Tables
Sr. No
1
Title
Table 1 Prepartion of Medium
Page No.
24
CHAPTER 1: INTRODUCTION
Plants containing adequate amounts of phosphorus entail appropriate growth and development.
Phosphorus plays a vital role by being a component of plant structure which is responsible to
regulate the protein synthesis mechanism in plants. The mechanism of cell division, distribution
and transformation of energy in terms of glucose takes place under the sufficient presence of
phosphorus in plants. Moreover, the process of tissue development is also buffered paving way
for the proper growth in plants. On the contrary, the deficiency of phosphorus can cause revert
the growth mechanism in plants while changing the normal color of leaves to dark-green color or
brown and make the plant vulnerable (Schachtman, et al., 1998).
The detailed work of Holford (1997) infers that the soil may contain high quantity of
phosphorus, but plants, doesn't necessarily absorb the required phosphorus quantity due to its
insoluble form present in soil. Therefore, the potential solubility of phosphorus takes place under
the manifestation of the enzyme known as phosphatase that has been originated in number of
microorganisms found in soil. Phosphorus carries a negative charge which makes it to react with
the minerals found in the soil mainly known as aluminium, calcium, iron and magnesium and
these cations precipitate and form soluble phosphorus that is absorbed by the plants. In the
presence of phosphate solubilizing bacteria, the conversion of phosphorus is enhanced from the
insoluble phosphorus and the process takes place when these bacteria and fungi release organic
acids and enzymes consisting of phosphorus.
According to Chen, et al. (2006), when the adequate amount of phosphorus is available in the
soil then its presence ensure the proper absorption of phosphorus to the plant which causes its
proper development in terms of providing vulnerable to weathering, growth towards maturity
5

stage, production of fruit and increased production of crops. Phosphorus plays a vital role in the
photosynthesis process, providing the essential carbohydrates to the plant. The phosphorus is
assimilated to plant, particularly, as anion but the process of utilization of phosphorus takes place
slowly, which in turn, becomes essentially required to make sure that phosphorus is available
adequately.
The deficiency of phosphorus in plants leads to the adverse effects on the growth and
development of plants. Moreover, insoluble phosphorus that has been already present in soil and
fertilizers can be immersed so that the absorption process of phosphorus can take place
efficiently and this available solution of phosphorus can convert in the soluble minerals essential
for the plants. This process takes place when the microorganisms i.e. Bacteria or fungi
decompose the available insoluble phosphorus in soluble phosphorus and the process is known
as immobilization. The current study aimed to isolate the microorganisms that play vital role in
the solubilizing of phosphate to ensure the maximum uptake of phosphorus in the plants.
CHAPTER 2: LITERATURE REVIEW

2.1 Isolation of Strains
The comprehensive work of Frageria (2010) suggests that phosphorus is an important nutrient
for the plants which causes them to grow healthy and develop ideally with the proper absorption
of phosphorus in plants. The availability of inorganic phosphorus in soil makes plants unable to
absorb the phosphorus for development mechanism which can cause adverse effects on plant
growth. The presence of certain minerals i.e. Aluminium in rich soil can help in stimulating the
absorption of phosphorus in plants. The process takes place when the absorption of inorganic

phosphorus in the form of hydrogen phosphate in rich soil where aluminium prevails and
reaction between these minerals causes the precipitation of phosphorus ions on Aluminium.
These minerals are available in the soil which causes the fixation of phosphorus in the form of
hydrogen sulfate and precipitates of Aluminium. The chemical equation for the process is as
follows:
Al3+ + H2PO4- (soluble) + 2H2O
Al(OH)2H2PO4(insoluble) +2H+
The study also exhilarates that the lack of phosphorus in plants under the circumstances
in which soils are not found to be rich in minerals affects the overall yield of the crops by
postponing the maturity stage of the plants, producing low quality of crops and reduction in the
vulnerability of plants to encounter diseases.
The meta-study conducted by Richardson and Simpson (2011) confers that the large numbers of
microorganisms have the tendency of solubilizing the inorganic form of phosphorus being the
part of the soil. These microbes are found to be isolated while prevailing around rhizosphere and
able to possess the tendency to convert inorganic phosphorus in organic form. This study
determines that the microorganisms existing in basic or alkaline soil when examined in such
manner that they are evaluated by using calcium phosphates which makes them to discharge
soluble phosphorus. The absorption of phosphorus also depends on the lack of soil capacity for
absorption and capability of the soil to provide phosphorus to the plants. The phosphorus fixation
process is stimulated at the higher pace when soil is composed of coarse texture having its
contents highly patterned with clay. The fixation process takes place when the hydrogen
phosphate is met with calcium ions and the reaction of both chemicals results in the reversible
formation of calcium phosphate and hydrogen ions and the reaction.

Ca(H2PO4)2 (soluble) + 2Ca2+
Ca3(PO4)2 (insoluble) + 4H+
The study also advocates that the absorption of phosphorus in the process is relatively at
the fastest pace and considerably reduces during the passage of time. Furthermore, the
availability of phosphorus should be made when the crops or plants are at their growth cycle and
if the phosphorus is applied at the beginning when the seeds are sown then the huge proportion
of phosphorus will be fixed even before the plant requirement which will ultimately cause the
deficiency of phosphorus in plants.
Fankem, et al. (2006) infers that the unavailability of phosphorus has become a crucial problem
because the phosphorus by means of fertilizers provided to the crops become fixed after its
application making the phosphorus insoluble for the plants. The phosphorus available in the soil
reacts with the soil minerals mainly consisting of calcium, aluminium and iron, which ultimately
results in the fixation of phosphorus and converts it in the insoluble form. It has been emphasized
that the presence of microorganisms which helps in the solubilization of insoluble phosphorus
has taken the utmost importance in order to enhance the plant growth and overall yield. These
microorganisms solubilize the inorganic or insoluble form of phosphorus so that it can be
absorbed the plants as required. The experiment was conducted in this regard by analyzing the
seven samples from the rhizosphere of oil-palm-tree. The soil samples were found to be highly
acidic in nature and the presence of iron and aluminium was highly noted. The dilution of
samples took place and isolates were formed which were observed in order to find the PSM and
further tests to calculate the pH were conducted which determined the considerable drop in pH,
which indicated the presence of organic acids which resulted in the formulation of carboxylic
acid at the highest pace. The PSM were identified as the Pseudomonas and were found to be
responsible for the decomposition of insoluble phosphorus into the soluble phosphorus.
8

Therefore, the study concluded by postulating that by using PSM in agricultural processes can be
beneficial to increase the crop yields by the activity of PSM the availability to phosphorus to the
plant increases which leads to better agricultural yields.
The brief work of Pradhan and Sukla (2009) in respect of characterizing the fungal strains was
conducted in order to analyze their ability to convert insoluble phosphorus to a soluble form. The
minerals found in soil are composed of enriched amount of phosphorus and despite of its
availability; the phosphorus availability is incorporated as insoluble in state which proves to be
useless in accordance with the plant uptake and growth. Therefore, Fungi existing in the
rhizosphere has proved its highest solubility to convert insoluble phosphorus in the soluble form.
To analyze the discussed phenomena, the study conducted an experiment in which the
rhizosphere from rice field was collected and out of which fungal strains were collected by their
isolation using the PVK medium followed by its reaction with tricalcium phosphate. The media
were analyzed for seven days and the morphology of fungal colonies proved the presence of
Aspergillus and Penicillium, which were found to be active in the production of organic acids
and phosphorus solubilization. The results of the experiment indicated the halozone which were
significant enough to be identified as fungal colonies. Furthermore, the continuous drop in pH
was observed during the overall process indicating the presence of multiple organic acids
produced by the PSM. The study also concluded that the higher amount of phosphorus was
released and the production of organic acids was not the only reason behind the conversion of
phosphorus. The study postulated that use of PSM in agricultural processes will not only proved
to be cost effective, but the immobilization of phosphorus assimilated with soil-fertilizers will
take place and the overall production of crops will be enhanced.
2.2 Formation of Phosphate-Solubilizing-Bacteria PSB

According to Chabot, et al. (1998), the production of crops can be increased by making the
plants absorb the proper amount of phosphorous, which stimulate the extent to which the plants
grow. It has also been found that when the occurrence of rhizobacteria takes place across the
plant roots, then they also stimulate the absorption of phosphorus in plants. It is required that
there should be the dense colonization of PSB in order to sustain the absorption of phosphorus is
the plants at the maximum level. Therefore, the broader the rhizosphere the more effective the
absorption of phosphorus will take place. Moreover, it is also necessary that there exists
appropriate level of minerals and nutrients to ensure the proper benefits of the colonization of
rhizobacteria or PGPR. The current study uses the sample of rich and poor soil consisting of the
texture of silty-clay and silty loam under the presence of rhizobia PSM leguminosarum to
examine the growth of maize. The temperature was 25 centigrade under the greenhouse where
six seeds were disseminated to analyze their growth rate by adding the fertilizer N-NH 4NO3, KKCl and P-superphosphate. The results from the conjugated strains of RlLux + indicated that the
plant growth, i.e. maize growth was not affected by the fertilization of soil with phosphorus in
poor soil as well as the colonization of bacteria PSM was also not affected by the procedure.
Therefore, the study postulates that plant growth can be enhanced by the solubility of
phosphorus, but the procedure is effected only in the presence of rich soil.
Vasquez, et al. (2000) conducted a study to validate the presence of microbes which help in the
process to make phosphorus accessible in soluble state in the rhizosphere. There exists a large
quantity of inorganic phosphorus in sea and these phosphoric ions contain a negative charge
makes tight bonds with minerals like aluminium, calcium or iron and make such compounds
which are considered to be soluble by certain microorganisms; mainly fungi. The conversion of
10

insoluble phosphorus to soluble phosphorus takes place by the production of some organic acids
by the fungi. These fungi or bacteria are surrounded by the rhizoshpere which stimulates the
nitrogen fixation process and increase the phosphorus intake of the plants resulting in the
enhanced growth and production of plants or crops. In the current study, an experiment
consisting of sample soil cores, which was found to be enriched with calcium, aluminium and
iron was examined and two species of mangrove plants were harvested and the microorganisms
from the soil were studied. The insoluble phosphorus was present higher in quantity than soluble
phosphorus. The results from the culture study indicated the presence of phosphorus in mangrove
roots and Bacillus licheniformis was found to be accountable for the absorption of phosphorus.
The solubility of phosphorus was found to be enhanced when the fungal colony was exposed to
A. Niger indicating the direct correlated effect between them. Furthermore, all types of bacteria
produced some organic acids to solubilize the insoluble phosphorus and these organic acids were
identified as acetic acids, lactic and isobutyric acid which helped in the immobilization of
phosphorus.
The study conducted by Oliveira, et al. (2008) aims to examine the microorganisms in order to
evaluate their activity related to the conversion of insoluble phosphorus. It has been inferred that
microorganisms play a vital role in the development of plants by providing them soluble and
absorbable minerals for their growth. The study conducted experiment on the soil samples which
were extracted from the rhizosphere of maize and four samples were gathered which possessed
noticeable difference in phosphorous nutrient. The colonies of microorganisms around the
rhizosphere were examined which consisted of 45 in number and were selected on the basis of
their efficiency to convert phosphorus and the process took place under the culture which
consisted of phytic acid i.e. C6H18O24P6, soybean lecithin, tricalcium phosphate and aluminium
11

phosphate. The microbes consisting of bacteria were isolated by identifying the presence on
nucleotide in their rDNA whereas fungi were isolated by examining their internal transcribed
spacer that was present in their rDNA. The conversion of phosphorus was found to be highest in
bacterium mainly knows as Bacillus species and Burkholderia species and was their rate of
immobilization of phosphorus was measured which was found at the rate of 67% out of the total
amount of phosphorus from the compound of tricalcium phosphate. It has been observed that the
solubility of microorganisms and the immobilization of phosphorus highly depend on the nature
of the phosphorus compound with which the soil is enriched and on the microorganisms that are
involved in the process of phosphorus fixation.
According to Chen, et al. (2006), the production from plants can be increased by the soil
microbes that play a vital role in the conversion of phosphorus from insoluble to soluble form.
The microbes inoculants in the form of the PSM are responsible to increase the absorption of
phosphorus in the plants. The study was conducted to examine the stains in order to isolate and
identify the bacteria that help in phosphorus solubilization. The sample from the non-cropped
site was taken from the upper 3cm and was diluted up to the pH 7 with the addition of tricalcium
phosphate and the mixture was analyzed to find the bacterial strains. The results indicated the
presence of phosphorus solubilizing bacteria consisting of Serratia marcescens and Bacillus
megaterium and Arthrobacter. Furthermore, Arthrobacter proved the maximized solubilization of
phosphorus among the all three types of bacteria. These bacteria contained the culture strains of
enzymes that helped in the solubilization of P mainly known as citric, lactic and propionic acids.
Some isolates also showed small traces of various enzymes. The results also indicated that the
greater the pH of the solution was found the smaller the number of acids was produced by the
microorganisms. This study also prevailed the presence of Rhodococcus and Delftia which were
12

found for the very first time in the isolation of phosphorus solubilization and proved their part by
secreting enzymes and converting the phosphorus. Therefore, the importance of PSM bacteria in
order to increase the phosphorus intake in plants and reduce the pollution by decreasing the use
of phosphate fertilizers has taken the boundless importance in the modern era.
The study conducted by Rashid, et al. (2004) emphasized on the importance of microbes in terms
of bacteria and fungi that play their important part in the conversion and decomposition of soil
nutrients. These microbes are generally present in the rhizoshpere where the solubilization
process takes place. The experiment took place by the studying the sample of rhizosphere from
rice in the petri plates which indicated the presence of microorganisms in broth medium. The
tricalcium phosphate was poured on the slides or petri plates and the change in pH was observed
and the drop in pH indicated the production of organic acids. The organic acids consisting of
acetic, gluconic, fumeri and succinic acids were found by conducting the HPLC tests. The
bacterial strains generated a large amount of organic acids as compared to the fungal strains.
Aspergillus in fungi and Bacillus in bacteria were the highest producers of organic acids amongst
the other microbes. The overall result indicated that the fungi were able to immobilize
phosphorus at the rate of 0.1%-0.4% which were found to be greater in phosphorus
immobilization than bacteria. The statistical techniques showed the positive direct relation
among the secretion of organic acids and the solubility of phosphorus whereas negative relation
was found among the pH and the phosphorus solubility.
Das, et al. (2003) conducted a study by using the two types of herbicides in order to examine the
effects of herbicides on the development and mechanism of PSM. The study postulated that the
microorganisms possess the tendency to breakdown number of organic compounds in order to
provide nutrients to the host and it is more likely to find the mechanisms stays the same when
13

they are encountered with herbicides mainly consisting of oxadiazon and oxyfluorfen. On the
contrary, some herbicides have proven their negative effects on the activities of microorganisms
resulting in the harmful effects on plants. The experiment took place by using the nutrients
enriched soil in which rice seeds were sown and the rhizosphere sample was collected from
there. These soil samples were collected after removing the roots of the seedlings for 60 days.
The diluted mixture of soil and distilled water was mixed with tricalcium phosphate, which
indicated the presence of microbes colonies. After that, sodium bicarbonate was added in order
to examine the rhizosphere. The results indicated that the presence of herbicides has fastened the
activities of PSM and the phenomena was rationalized with the indication that the
microorganisms used these herbicides to get energy and hence promoted the decomposition of
insoluble phosphorus nutrients in the soil. Furthermore, the number of microorganisms was
increased day by day with the presence of herbicides in the soil and the crop was also effected
with this activity by reaching at the stage of flowering by the 30 th day. Therefore, the overall
study indicated that the presence of herbicides stimulated the PSM activity and growth, which
resulted in the greatest amount of phosphorus solubilization and hence, the maximum amount of
phosphorus became available for the plants resulting in enhanced growth and development of
crops.
Hameeda, et al. (2008) argued that the soil may be enriched with minerals and contain higher
amount of phosphorus, but their availability is not considered to be adequate for the plant uptake
in order to utilize it for the growth and development and it happens due to the availability of
other minerals available in the soil specifically calcium which reacts with the phosphorus and the
fixation process takes place in which the insoluble form of phosphorus is generated. The
fertilizers that are applied to the soil usually convert into the insoluble form of phosphorus
14

following the fixation process and very small amount of phosphorus is absorbed into the plants
which prove to be quite inadequate for the proper growth of plants. Therefore, the necessity of
PSM is originated because these PSM are able to solubilize the insoluble form oh phosphorus
making it available for the plants. The study conducted an experiment in glasshouse by sowing
the maize seeds which were provided with nutrients for ten days and peat consisted of
microorganisms i.e. marcescens and Pseudomonas were also inserted and at the end, rhizosphere
was collected. The results indicated that the presence of PSB produced organic acids, i.e.
gluconic acid, which indicated the decomposition process of insoluble phosphorus and the
observed growth in the length of shoot, root enhancement and decreased vulnerability of crops
was found. The study did not provide any result for the increase in the PSB during the process
indicating that the number of PSB included in the peat remained same throughout the process.
The study concluded that the use of marcescens and Pseudomonas during the incubation of seeds
will enhance the phosphorus solubilization process and will increase the crop yield.
The comprehensive work of Perez, E. (2007) has made an attempt to discover the activity of PSB
in the soil that is found to be enriched with iron. The PSB bacteria decompose the inorganic
phosphorus to organic phosphorus so that it can be absorbed by the plants and in return, these
bacteria absorb sugar, basically C-compounds, from the plants. These bacteria produce organic
acids as by-product which indicates that the process of immobilization has been taken place. The
soil was collected from the area which contained 58% to 60% ferrous oxides and the amount of
phosphorus indicated to be very low. The soil samples were sterilized and were diluted with the
media known as NBRIP and tricalcium-sulphate. The appearance of halozone took place. On the
contrary, no traces of iron phosphate were found in the experiment. The isolates from the
halozone were identified to have the bacterial colonies of Burkholderia, Ralstonia species and
15

Pantoea. The identification of bacterial colonies took place by analyzing their rDNA with the
sequencing analysis. The occurrence of the very small amount of gluconic acid was found which
proved the immobilization of phosphorus at a lower pace. The study suggested that when the soil
is enriched with minerals like Fe then fixation process takes place and the activity of PSB proves
to be less effective than the soil where Fe is found in lesser proportion. Furthermore, the bacteria
strains which were used in this experiment were proven to work effectively under the presence of
aluminium. Therefore, it has been highly advised that the soil should be examined properly and
only such bacterium should be used for the immobilization process that are found to be efficient
in the conversion of phosphorus.
The significance of phosphorus in the plant growth has been discussed earlier and it has been
described that the absorption of phosphorus takes place in the soil in the soluble form. The
absorbed phosphorus reacts with the minerals available in the soil, i.e. aluminium, calcium and
ferrous which converts the phosphorus into the insoluble form by the fixation process. In order to
make phosphorus available to the plants the phosphobacteria play a vital role in which they
decompose the inorganic form of phosphorus while releasing organic acids as a by-product. The
experiment conducted by Ponmurugan and Gopi (2006) aimed to estimate the availability of
PSB. The rhizospheres were collected from the cultivation of brinjal, groundnut, cotton and
maize took place. The rhizoshpere sample was diluted using the Bose media and after that
halozone were found indicating the bacterial colonies. These colonies were examined by under
the PVK medium and tricalcium phosphate. The results of the study indicated that the number of
phosphobacteria different on account for each type of plant. The number of PSB available was
highest in the groundnut sample and the second highest was found to be the cotton sample. The
result proved that the immobilization process took place in all the crops indicated by the
16

presence of phytohormones. The statistical analysis showed the positive relation between the
capacity of solubilizing phosphorus and the formation of phytohormones. The study concluded
that with the help of phosphobacteria the growth of the plants can be increased due to the
provision of adequate amount of phosphorus to the plants.
The study conducted by Babana and Antoun (2006) suggested that the deficiency of phosphorus
has become a crucial factor with respect to the plant growth, and whereas the cost of using
phosphorus-fertilizers has been increased which makes the agricultural process expensive and
less profitable. However, the use of bacteria to help plants to absorb the required amount of
phosphorus has achieved the attention of researchers and several studies are being conducted on
this aspect to improve the availability of soluble phosphorus to the soil. The silty-clay soil was
used in the experiment where the plots for the experiment were built. These plots were applied
with the phosphate fertilizers in order to enrich them with the availability of phosphorus. These
plots were inoculated with the PSM including Pseudomonas and Penicillium. The seeds of wheat
were sown in the plots and the after their growth of 3 months the plants growth and development
was analyzed. The study provided the evidence by indicating that the use of Pseudomonas is
beneficial for the growth of crops because this bacteria helped in the immobilization of
phosphorus and higher quantity of phosphor-enzymes were found. Moreover, the growth of the
plant was found to be at a greater pace than the plants in which Penicillium was used.
In the last decades, researchers have emphasized on the utilization of phosphorus-solubilizingmicrobes in order to enhance the crop yield and for the reduction of cost by decreasing the
quantity of phosphorus-fertilizers. The microbes which are unambiguously found in the
rhizosphere are responsible in the development of plants and decomposing inorganic phosphorus
to organic phosphorus. The experiment conducted by Afzal and Bano (2008) strived to explain
17

the mechanism of PSB. The seeds of wheat were sown in the soil characterized as sandy loam
and were applied with the phosphorus-fertilizers in order to enrich the soil with phosphorus. The
preparation for the insertion of PSB took place by using the PVK medium. When plants were in
the growing stage, the samples of soil were gathered for the analysis of phosphorus absorption.
The results of the study indicated that the growth of plants was stimulated due to the adequate
absorption of phosphorus. The plant development indicated the better absorption of water and
nutrient by measuring the length of root and shoot. When the PSB containing Rhizobium was
doubled in the soil, then the growth of the plant again found to be at a greater pace than the
single dosage of PSB. Therefore, the study postulated and favored the previous studies with
respect to the positive correlation of phosphorus-fertilizers, PSB Rhizobium and the overall yield
of the crop.
2.3 Phosphorus Solubilizing Mechanism

According to Holford (1997), the phosphorus present in the soil is considered to be insoluble for
the plants and the process of phosphorus conversion takes place when the phosphate ions are
released from the rocks during rain and erosions in the form of inorganic phosphates and are
absorbed in the soil. The soil consists of soil microorganisms, also known as soil microbes,
which ensure the nutrient and phosphorus availability to the plants by converting inorganic
phosphorus to organic phosphorus by means of immobilization or the fixation process for
phosphorus which helps in the absorption of phosphorus in the plants. The process of
immobilization takes place when microbes i.e. Mycorrhizal fungi or PSB that is able to absorb
the inorganic form of phosphorus or phosphorus ions and release organic acids that contain
groups of chemical out of which it also consists of certain hydroxyl and carboxyl which react
with the inorganic phosphorus causing it to form organic phosphorus which is eventually soluble
18

for the plants. The study reveals the process of phosphorus fixation and describes its importance
which proves to be viable for the proper growth and development of plants and its deficiency
proves harmful effects on the plants by making them weak and producing abnormal leaf color
indicating the over processing of photosynthesis.
Silcock, et al. (1976) conducted a study in which the extent to which different types of soils
absorb the phosphorus by the fixation process in examined. The study took place when the
phosphorus deficiency was found to cause reduced plant growth in the Mulga soil, which was
found to be enriched with minerals consisting of nitrogen, phosphorus and aluminium. The
experiment took place by analyzing the Mulga soil sample from 15cm zone and soil was tested
with the two types of native grass i.e. Mulga oat and silky umbrella and two types of exotic grass
i.e. Wool grass and buffal grass. The continuous recording of growth and absorption of minerals
took place through the growth process of the grass. The result of the experiment indicated that
the grass that was encountered with the proper fertilization of phosphorus and enriched soil
possessed the astonishing growth in all types of grasses rather than silky umbrella grass. The
types of grass where the proper absorption of phosphorus was found proved to be vulnerable to
weather changes with a prolonged life cycle. The study infers that the seeds of silky umbrella
grass consisted of low level of phosphorus than all the other species of grass which resulted in
the declined growth and slow rate of plant maturity in the overall experiment.
The study conducted by Sheng, et al. (2010) has strived to examine the nitrogen fixation process
and the effects of the availability of microorganisms in the presence of phosphorus in the soil. In
this study, the essentiality for the provision of phosphorus to the plants has been discussed and it
has been inferred that when the existing level of phosphorus diminishes from the ideal required
level of phosphorus from the soil then the crop or plants encounter with the deficiency of
19

phosphorus and face adverse effects. The phosphorus may be available in the soil, but plants fail
to absorb it because of its close bonding with minerals calcium, aluminium or iron. The activity
of PSM takes place in order to convert insoluble phosphorus to soluble phosphorus so that plant
can absorb and utilize the adequate amount of phosphorous essential for their growth. The
experiment in this study took place with the four samples of same soil rice straw was sown and
Pseudomonas was added and the process was stimulated by adding glucose in order for the
formation of soil. The result of the study indicated that the soil was enriched after the phosphorus
fixation process, but this effect was not too significant whereas the significant growth in the plant
was observed. The study encompassed that modification of the soil contents in terms of
microorganisms can enhance the process of phosphorus fixation by motivating the
microorganisms that were already present in the soil even before the assimilation of biofertilizers.
Alam, et al. (2002) conducted a study to infer the immobilization of phosphorus by identifying
the activities of PSM gathered from the maize rhizoshpere. The yield of the crops can be
maximized by ensuring that the plant uptake the required amount of phosphorus in order to grow
and develop properly, whereas the lack of phosphorus availability to the plants causes adverse
effects on the crop yield. Therefore, to meet the phosphorus requirement for the plants, fertilizers
containing soluble forms of phosphorus are applied to the soil, which precipitate easily, and
convert a soluble form to the insoluble form of phosphorus. Hence, PSM is required to convert
the insoluble phosphorus so that it can be absorbed by the plants. Under certain circumstances,
fungi are found to be more active in the phosphorus conversion than bacteria. The experiment
took place by examining the PSM on PKV agar after the distillation and afterwards tricalcium
phosphate was poured on the culture. The pH and solubility of phosphorus were continuously
20

analyzed for seven days. The results of the study indicated the presence of holozone colony
which proved the presence of the isolates of PSM out of which fungi proved to be most
solubilizing in nature for phosphorus. The continuous drop in pH also proved the production of
organic acids by the PSM consisting mainly of oxalic, acetic and citric acids. The results for the
immobilization of phosphorus proved that the bacteria were actively responsible for the
immobilization and the result was rationalized on the basis that fungi colonies are larger in size
than bacteria colonies which may result in higher immobilization by fungi. The study also
suggested that the production of organic acids play important role in the immobilization
mechanism.
Igual, et al. (2001) adopted a less time taking procedure to study the solubilizing activities of
bacteria to convert phosphorus in order to increase plant yield. Phosphorus being the essential
nutrient in the plant growth is not fully available to the plants even if its higher quantities exist in
the soil. The soil may be acidic or alkaline in nature, but the minerals originated in these soil
causes the fixation of phosphorus reduce the solubility process of fertilizers. The bacteria found
in the rhizosphere act as active agents to decompose the phosphorus so that it can be absorbed by
the plants. The experiment took place by taking the sample from rhizosphere and PKV medium
was poured on it and distilled which caused the development of Halozone indicating the PSB
colonies. The mixed culture method was adopted in the study to examine the bacteria that proved
to be beneficial for the crops. Therefore, Pseudomonas and bacillum which are usually found in
the outermost layer of rhizosphere take active part in the process and produce isobutyric acid,
lactic acid, acetic acid and oxalic acid as a by-product. The process declared the immobilization
of phosphorus due to the bacterial activity. The study concluded that with the identification of
21

PSB and proper characterization of soil, it is possible to provide specific bacteria that can make
the phosphorus absorption process more efficient and can cause increased productivity of crops.
The comprehensive work of Sahin et, al. (2004) with the purpose of analyzing the effects of
phosphorus solubilizing bacteria on the growth and development of plants under the given
specific environmental conditions and types of soils. Rhizosphere containing the PSB has been
used for the conversion of phosphorus soluble so that the plants can uptake the adequate amount
of phosphorus and can be utilized. Furthermore, when nitrogen-fixating bacteria and PSB are
inoculated together, then they prove to be more efficient and the crop yield increases at the
higher pace. The experiment was conducted at the experimental farm where the nitrogen fixing
microbes and PSB i.e. Bacillus was added altogether where the seeds of sugar beets and barley
were sown. The soil of the farm was found to be clay loam at the average temperature of 42
centigrade. The result of the study showed that the overall crop yield of sugar was increased by
the inoculation of the bacillus bacteria, but the crop yield for the barley remained the same
throughout the trial of two years. The study concluded that the overall production of sugar beet
can be increased by applying the phosphorus-fertilizers provided with specific bacterial strains.
Moreover, the crop yield can vary depending on the type of plants, soil and environmental
conditions.
When the plants are provided with the appropriate amount of phosphorus then these plants
results by having the deeper roots and become invulnerable to weathering and wind thrusts. The
alkaline soil forms the bond with phosphorus, making the phosphorus insoluble for the plants.
The proper absorption of phosphorus causes the efficient biochemical activity in plants, mainly
consists of photosynthesis in which plants obtain starch, which is considered to be the basic and
important activity in plants. Furthermore, the proper provision of phosphorus will result in the
22

early fruit ripening and flower formation, which as a whole, indicate the effective and efficient
growth. The phosphorus is assimilated from the soil to the plants in the form of phosphate anion
and this absorption is found to be at low paced which increase need to ensure the availability of
phosphorus in the soil. The lack of phosphorus in plants will cause the negative effects on the
growth and development of plants. Moreover, all the types of soil contain insoluble or inorganic
amount of phosphorus where fertilizers can be included in order to increase the amount of
phosphorus available to the plant and this process will convert the soluble form of phosphorus
which is essential for the plants. The study conducted by Mehrvarz & Chaichi (2008) took place
an experiment in the research field. The soil was characterized as clay loam and the soil was
applied with the fertilizer known as triple-supper-phosphate. Pseudomonas was added in the soil
and the barley seeds were sown. These bacteria played vital role in the decomposing of the
inorganic phosphorus of organic phosphorus in order to make it available for the plants. These
bacteria produced by-products in terms of organic acids ensuring the process of immobilization
has been taken place. The soil samples were collected, sterilized and were diluted with the media
known as KVP media and tricalcium-sulphate. The appearance of halozone took place. The
occurrence of very small amounts of gluconic acid was found which proved the immobilization
of phosphorus at a lower pace. The study suggested that when the soil is enriched with
phosphorus and PSB then fixation process takes place. Therefore, it has been concluded that the
application of PSB inoculants should be practiced in order to increase the overall crop yield and
cost effectiveness
23
CHAPTER 3: METHODOLOGY
3.1 Sample
The soil samples were collected from neighboring places consisting of banana soil, graveyard
soil, garden soil and ground soil and the samples were gathered from the site of cultivating areas.
The samples for each soil were taken 6 times from different sites at the depth of 17 inches. These
samples were dried and then all 6 samples were mixed together by making one sample for each
type of soil.
3.2 Preparation of medium

Pikovaskaya agar or PKV agar was used as a medium to isolate PSM which is specifically used
for the detection of PSB. The pH of the medium was sustained at 7. The components of the PKV
agar and their quantity used in the process is shown in Table 1.
Composition
Glucose
Yeast Extract
(NH4)2SO4
Magnesium Sulphate (MgSO4.7H2O)
Calcium Phosphate [Ca3(PO4)2]
NaCl
KCl
MnSO4.2H2O
FeSO4.7H2O
Agar
Gram/ml
10
0.5
0.5
0.1
5
0.2
0.2
0.002
0.002
10
Table 1 Preparation of Medium
3.3 Isolation of PSM

In order to isolate PSM, two processes were used consisting of serial dilution and spread-platemethod. The heterogeneous soil sample of 1 gram was taken in the test tube with 9 ml distilled
24

water (autoclaved) and was mixed by thorough shake and the diluted solution of 10^- 1 was
formed. 1 ml of this mixture was taken into the other test tube and the mixture was sterilized
with 9 ml of distilled water to form 10^-2. The same process was repeated till the formation of
diluted solution up to 10^-5. Furthermore, 0.1 ml was taken from the every diluted solution on
which the medium known as Pikovaskaya agar (PVK) was spread which consisted of tricalcium
phosphate (insoluble). Halo zones were formed indicating the microorganisms colonies which
were picked. These colonies were purified by using PVK medium in order to study the colonies.
3.4 Morphological Characterization

The isolates of colonies were observed under microscope and in order to characterize the PSB by
examining the shape of the microorganisms, the size of the microorganisms and the elevation of
the microorganisms. Further observations were made in accordance with their color, surface,
texture and the distance or margins between the microorganisms were studied morphologically.
3.5 Gram Staining

The standard procedure was used to study the characteristics of gram staining in the isolate.
During the procedure, first of all the smear of the glass slide was taken by using an inoculating
loop and it was set to get dried. The smear was then fixed by encountering it with flame and heat
caused it to get fixed. After that, crystal violet was added to the time period of thirty seconds.
Afterwards, it was washed with distilled water and was set to get dried. Later on, grams iodine
was added the time period of sixty seconds and was washed by using the ethyl alcohol mixture.
After a while, Saffranin was added to the time period of thirty seconds and was washed by using
the distilled water. Blotting paper was used to get it dry. The slide was then observed under the
microscope. Two types of bacteria colonies were found in which the pink colonies showed the
25

presence of gram negative bacteria, whereas the observation of purple colonies represented the
presence of gram positive bacteria.
CHAPTER 4: RESULTS AND DISCUSSION

4.1 Isolation of PSB
The halozone of phosphate solubilization was found in three soil samples, indicating that the
proficiency of phosphorus solubilization has been generated.
Figure. 1 Halozone in
Banana Soil
These halozones were obtained from the medium of banana

soil sample which showed the dilution of 10^-3. Therefore, the
banana soil was found to be
proficient
for
the
immobilization of phosphorus
indicating the presence of
bacteria that are responsible
Figure. 2 Halozone in
Garden Soil
for the solubilization of phosphorus i.e. PSB. The halozone in banana soil can be seen in Figure
1.
The result obtained from the halozone of the garden soil sample proved its proficiency of the
immobilization of phosphorus by indicating the existence of phosphorus-solubiling-bacteria and
the garden soil demonstrated the dilution of 10^-4. The halozone is shown in figure 2.
The third soil sample consisting of graveyard soil also showed the positive result by having the
ability to immobilize phosphorus, which in turn indicated the presence of PSB and the banana
26

soil sample exhibited is dilution of 10^-4. There were no
Figure. 3 Halazone in
Graveyard Soil
isolates formed in the ground soil sample. The halozone in

graveyard soil are shown in figure 3.
The results indicated the presence of gram negative bacteria
due to its features which were containing the colorless
colonies with the absence of any pigment; the cells were
found to be gram negative with rod-like structure. Moreover, the biochemical reactions indicated
the presence of Pseudomonas fluorescens which were found during the process of gram staining.
On the contrary, the isolates indicating the presence of gram positive bacteria due to its features
containing the white colonies with slimy shape and margins among them was found to be
irregular. These colonies possessed the existence of Bacillus megaterium which was proved by
the biochemical reactions during the gram staining. Therefore, the presence of Pseudomonas and
megaterium indicated that the immobilization mechanism for the conversion of insoluble
phosphorus to the soluble phosphorus took place.
CHAPTER 5: CONCLUSION
The study conducted an experiment by analyzing the four soil samples of banana, graveyard,
garden and ground soil in order to isolate the microorganisms that isolate phosphorus. The results
of the present study indicated that the three soil samples, i.e. Garden, graveyard and banana soil
in which the isolates were formed in the samples from garden soil, graveyard soil and banana
soil. These samples indicated the formulation of halozone which represented the
microorganisms colony lying under the PVK medium. These isolates were studied under the
microscope, which confirmed the existence of bacteria which are responsible to convert
27

phosphorus to soluble forms. The current study has declared the presence of bacteria, i.e.
Pseudomonas by which the immobilization of phosphorus took place. The absorption of
phosphorus is considered to be essential for plant development and has found to be the primary
nutrient for the plant growth and development. Microorganisms like bacillus margarita and
Pseudomonas break down inorganic or insoluble form of phosphorus and convert it into the
soluble or organic form so that phosphorus can be utilized by plants. This immobilization of
phosphorus mechanisms causes the secretion of organic acid, which confirms the activity of PSB
mechanism. The organic acid secreted as by-product from the bacteria cause the decrease in the
pH of soil, which proves the microorganism activity for the conversion of phosphorus.
In agricultural soil, inorganic form of phosphorus has already been existed, but the phosphorus
tends to be insoluble and becomes useless for the plant. The phosphorus added to the soil in the
form of phosphorus-fertilizers can increase the intake of phosphorus to the plants for a while, but
there exists other minerals in the soil like calcium, aluminium, magnesium and iron which start
precipitating with the soluble phosphorus and turns it into the insoluble form or inorganic form.
In this way, phosphorus becomes unavailable to the soil and the plants face adverse effects of by
being deficient in phosphorus. Moreover, the absence of soluble phosphorus from the soil will
cause the increased demand of soluble phosphorus and if more phosphorus-fertilizers are applied
then the cost for the crop yield will be increased. Therefore, PSB are required for the continuous
decomposition of insoluble phosphorus or inorganic phosphorus so that the plants will
continuously get the required amount of phosphorus and the hazardous effects of phosphorus
deficiency can be eliminated and plants can uptake all the essential nutrients properly.
In this way, the bacteria that break down the inorganic form of phosphorus can be used along
with the bio-fertilizers which will ultimately increase the phosphorous efficiency in soil by
28

breaking down the insoluble phosphorus to the soluble phosphorus and that will improve the
plant nutrient intake and will increase the crop yield. Furthermore, when these bacteria or PSM
will be used as bio-fertilizers then not only the availability of phosphorus will be increased, but
the usage of phosphorus fertilizers will be reduced, which will play an important part in reducing
the environmental pollution and the overall agricultural and crop yield will be increased.
Effective provision of phosphorus to the soil will result in the proper growth and development of
the plant with stronger root and shoot structure, increased productivity of fruits and flowering,
reduced vulnerability towards diseases and weathering. Therefore, phosphorus not only proves to
be essential for the plant as a nutrient, but it has also become important to be used in order to
increase the agricultural capacity.
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