ISOLATION, IDENTIFICATION AND OPTIMIZATION OF PHOSPHATE SOLUBILIZING MICROORGANISMS BY

PHYSICAL AND CHEMICAL MUTAGENS, CARBON AND NITROGEN SOURCES ON THE PRODUCTION OF

PHOSPHATASE AND LIPASE

THESIS SUBMITTED TO

BHARATHIDASAN UNIVERSITY

FOR THE AWARD OF THE DEGREE OF

DOCTOR OF PHILOSOPHY

IN

BOTANY

By

J. RAJESH KUMAR, M.Sc., M.Phil.,

Research Supervisor Dr. M.H. Muhammad Ilyas M.Sc., Ph.D.,

Associate Professor of Botany

P.G. AND RESEARCH DEPARTMENT OF BOTANY JAMAL MOHAMED COLLEGE (AUTONOMOUS)

(ACCREDITED WITH ‘A’ GRADE BY NAAC- CGPA 3.6 OUT OF 4.0)

TIRUCHIRAPPALLI- 620 020 TAMIL NADU, INDIA

July 2011

Date:

CERTIFICATE

This is to certify that the thesis entitled “Isolation, identification

and optimization of phosphate solubilizing microorganisms by

physical and chemical mutagens, carbon and nitrogen sources

on the production of phosphatase and lipase”, submitted to

Bharathidasan University, Tiruchirappalli - 620 024, for the award of the degree

of Doctor of Philosophy in Botany, is an authentic record of original work

carried out by Mr. J. RAJESHKUMAR under my guidance and supervision

during the study period at the PG and Research Department of Botany, Jamal

Mohamed College, Tiruchirappalli- 620 020, Tamil Nadu, India.

I further certify that no part of this thesis has been submitted any where

else for the award of any degree, diploma, associateship, fellowship or other

similar titles to any candidate.

(M.H. MUHAMMAD ILYAS)

Dr. M.H. MUHAMMAD ILYAS Associate Professor

Jamal Mohamed College (Autonomous)

(Nationally reaccredited with ‘A’ grade by NAAC in 2009)

PG & Research Department of Botany

Date:

DECLARATION

I do hereby declare that this work entitled “Isolation, identification and

optimization of phosphate solubilizing microorganisms by

physical and chemical mutagens, carbon and nitrogen sources

on the production of phosphatase and lipase”, has been originally

carried out by me under the guidance of Dr. M.H. Muhammad Ilyas, Associate

Professor, PG and Research Department of Botany, Jamal Mohamed College,

Tiruchirappalli- 620 020, and this work has not been submitted elsewhere for

any other degree.

(J. RAJESHKUMAR)

ACKNOWLEDGEMENTS

I have pleasure in writing this page in my dissertation to express my happiness

which I feel to acknowledge all those who helped me to complete this work

successfully.

It gives me great pleasure expressing my deep sense of indebtedness and

gratitude to my research guide Dr. M.H. Muhammad Ilyas, M.Sc., Ph.D., Associate

Professor, PG and Research Department of Botany, Jamal Mohamed College

(Autonomous), Tiruchirapalli – 620 020 for all the encouragement and valuable

guidance received by me at every stage of the work. The useful discussion I had with

him, both inside and outside the college, were always a source of inspiration to me.

I sincerely thank Hajee Dr. M. Sheik Mohamed, M.Com., M.Phil., Ph.D,

PGDCA, FICWA, FMSPI, PGDFM., Dip. In M.A. former Principal, Jamal

Mohamed College, Tiruchirapalli – 620 020 for having given me this wonderful

opportunity to do my Ph.D work at Jamal Mohamed College , Tiruchirapalli – 620

020.

My earnest thanks and deep of gratitude to Hajee Dr. R. Kadher

Mohiadeen, M.Com., M.Phil., Ph.D. Principal, Jamal Mohamed College,

Tiruchirapalli – 620 020 for his encouragement.

I am very grateful to Dr.S.Ahemed John, M.Sc., M.Phil., Ph.D., Associate

Professor and Head, PG and Research Department of Botany, Jamal Mohamed

College (Autonomous), Tiruchirapalli– 620 020 for their constant encouragement and

support.

I sincerely thank both Teaching and Non teaching staff members of PG and

Research Department of Botany, Jamal Mohamed College (Autonomous),

Tiruchirapalli – 620 020 for their generous help and Co-operation.

My special heartfelt thanks are due to Dr. N.Sengottain, M.Sc., M.Phil.,

Ph.D. Associate Professer and Head, Department of Botany and Microbiology,

Urumu Dhanalakhsmi college, Tiruchirapalli for having provided me with all the

facilities and encouragement throughout the course of this work.

I extent my sincere gratitude to Mr. P. Malaiyarasa Pandian, M.Sc.,

M.B.A., M.Phil. Lecturer, Department of Microbiology, Hindusthan College of Arts

and Science, Chennai, and Sangeetha Menan for his keen interest and valuable

suggestions in the progress of my work.

I extend my thanks to Mr. A. Shanmugasundaram, Lecturer, Department of

Microbiology, Urumu Dhanalakshmi College, for their appreciable help during the

course my research.

I express my thanks to Dr. Subramaniyam V. Mantha, Ph.D. R&D Director,

Green Signal BioPharma Private Ltd., Chennai for his encouragement and valuable

suggestions. I express my profound sense of gratitude to Dr. Srinivasan, Ph.D for his

valuable enthusiastic support and encouragement.

I also thank Mr.Shafi for his computer typing and designing.

Finally, no words of gratitude will be sufficient to pay sincere thanks to my

Mother whose inspiration and dedication helped me in finishing the project work

J. RAJESHKUMAR

CONTENTS

Page No.

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 11

3. MATERIALS AND METHODS 66

4. RESULTS 82

5. DISCUSSION 138

6. SUMMARY AND CONCLUSION 159

7. REFERENCES

LIST OF TABLES

Table No.

Title

1 Rhizosphere Microbial Density.

2 Ratio between Rhizosphere microbes and PSB

3 Cultural, Morphological and biochemical characteristics of isolated bacteria

4 Efficacy of phosphate solubilization of UV treated Aspergillus niger

5 Efficacy of phosphate solubilization of UV treated Aspergillus fumigatus

6 Efficacy of phosphate solubilization of UV treated Penicillium sp

7 Efficacy of phosphate solubilization of UV treated Pseudomonas strains

8 Efficacy of phosphate solubilization of Sodium azide treated Aspergillus niger

9 Efficacy of phosphate solubilization of Sodium azide treated Aspergillus fumigatus

10 Efficacy of phosphate solubilization of Sodium azide treated Penicillium sp

11 Efficacy of phosphate solubilization of Sodium azide treated Pseudomonas strains

12 Efficacy of phosphate solubilization of Ethyl Methane Sulphonate (EMS) Aspergillus niger

13 Efficacy of phosphate solubilization of Ethyl Methane Sulphonate (EMS) Aspergillus fumigatus

14 Efficacy of phosphate solubilization of Ethyl Methane Sulphonate (EMS) treated Penicillium sp

15 Efficacy of phosphate solubilization of Ethyl Methane Sulphonate (EMS) treated Pseudomonas strains

16 Efficacy of phosphate solubilization of Aspergillus niger (ANems120) grown on different Carbon sources

17 Efficacy of phosphate solubilization of Aspergillus niger (ANems120) grown on different Nitrogen sources

Table No.

Title

18 Efficacy of phosphate solubilization of Aspergillus niger (ANsa120) grown on different Carbon sources

19 Efficacy of phosphate solubilization of Aspergillus niger (ANsa120) grown on different Nitrogen sources

20 Efficacy of phosphate solubilization of Penicillium sp.(PEsa150) grown on different Carbon sources

21 Efficacy of phosphate solubilization of Penicillium sp.(PEsa150) grown on different Nitrogen sources

22 Efficacy of phosphatase activity of UV treated Aspergillus niger

23 Efficacy of phosphatase activity of fungal strains UV treated Aspergillus fumigatus

24 Efficacy of phosphatase activity of Penicillium sp.

25 Efficacy of phosphatase activity of UV treated Pseudomonas sp.

26 Efficacy of phosphatase activity of Sodium azide treated Aspergillus niger strains

27 Efficacy of phosphatase activity of Sodium azide treated Aspergillus fumigatus strains

28 Efficacy of phosphatase activity of Sodium azide treated Penicillium sp.

29 Efficacy of phosphatase activity of Sodium azide treated Pseudomonas sp.

30 Efficacy of phosphatase activity of Ethyl Methane Sulphonate (EMS) Aspergillus niger

31 Efficacy of phosphatase activity of Ethyl Methane Sulphonate (EMS) treated Aspergillus fumigatus

32 Efficacy of phosphatase activity of Ethyl Methane Sulphonate (EMS) treated Penicillium sp.

33 Efficacy of phosphatase activity Ethyl Methane Sulphonate (EMS) treated Pseudomonas sp.

34 Efficacy of phosphatase activity of Aspergillus niger (ANems120) grown on different Carbon sources

35 Efficacy of phosphatase activity of Aspergillus niger (ANems120) grown on different Nitrogen sources

36 Efficacy of phosphatase activity of Aspergillus niger (ANsa120)grown on different Carbon sources

Table No.

Title

37 Efficacy of phosphatase activity of Aspergillus niger (ANsa120) grown on different Nitrogen sources

38 Efficacy of phosphatase activity of Penicillium sp.(PEsa150) grown on different Carbon sources

39 Efficacy of phosphatase activity of Penicillium sp.(PEsa150) grown on different Nitrogen sources

40 Efficacy of Lipase activity of UV treated Aspergillus niger

41 Efficacy of Lipase activity of UV treated Aspergillus fumigatus

42 Efficacy of Lipase activity of UV treated Penicillium sp

43 Efficacy of lipase activity of UV treated Pseudomonas sp.

44 Efficacy of lipase activity of Sodium azide treated Aspergillus niger

45 Efficacy of lipase activity of Sodium azide treated Aspergillus fumigatus

46 Efficacy of lipase activity of Sodium azide treated Penicillium sp.

47 Efficacy of lipase activity of Sodium azide treated Pseudomonas sp.

48 Efficacy of lipase activity of Ethyl Methane Sulphonate (EMS) treated Aspergillus niger

49 Efficacy of lipase activity of Ethyl Methane Sulphonate (EMS) treated Aspergillus fumigatus

50 Efficacy of lipase activity of Ethyl Methane Sulphonate (EMS) treated Penicillium sp

51 Efficacy of lipase activity of Ethyl Methane Sulphonate (EMS) treated Pseudomonas sp.

52 Efficacy of lipase activity of fungal strains grown on different Carbon sources

53 Efficacy of lipase activity of Bacterial strains grown on different Carbon sources

54 Efficacy of lipase activity of fungal strains grown on different Nitrogen sources

55 Efficacy of lipase activity of Bacterial strains grown on different Nitrogen sources

56 Screening for IAA by Wild and Mutated Bacterial and Fungal strains

57 Quantification study of IAA by Wild and Mutated Bacterial and Fungal strains

LIST OF FIGURES

Figure No

Title

1 Phosphate solubilization efficacy by wild strains

2 Phosphate solubilization efficacy by Fungal strains treated with chemical mutagen

3 Efficacy of phosphatase activity by wild strains

4 Efficacy of phosphatase activity by Fungal strains treated with chemical mutagen

5 Efficacy of Lipase activity by wild strains

6 Efficacy of Lipase activity by Chemical treated Fungal Strains

LIST OF PLATES

Plate No

Title

1 Biochemical Characterization of Bacterial culture

2 Growth of Fungal colonies on Sabouraud agar plates

3 Phosphate solubilization of Bacterial and Fungal cultures in Pikovskaya agar

4 Phosphate solubilization and phosphatase production by fungal cultures in Pikovskaya broth

5 Indole Acetic Acid production by bacterial and fungal cultures in Czepekdox broth and thin layer chromatogram for IAA

1

Introduction

Microorganisms play a very important role in almost every sector. One

can find applications (uses) of microorganisms in agriculture, forestry, food,

industry, medicine, and environment. The scope and significance of

microbiology has enlarged manifold, particularly when importance of

environment was realized globally and the word environment was used in a

much wider sense in terms of totality to include almost everything, every bit of

nature.

Besides being important in biogeochemical cycling of nutrients, microbes

play vital role in maintenance of soil fertility and in crop protection. Microbes

are being exploited in two important ways as biofertilizers, and creating new

nitrogen-fixing organisms. Most of the chemical reactions that take place in the

soil, leading to increased availability of several major and micronutrients often

have active contribution of microbes. The nitrogen-fixing bacteria, blue green

algae, and phosphate solubilizing bacteria are well known to enhance availability

of major nutritional elements like nitrogen and phosphorus to plants whereas the

decomposer bacteria are instrumental for recycling, and thereby increasing, the

availability of carbon and several micronutrients from plant residues to soil.

Phosphate Solubilization

Phosphorus has been called “the key of life” because it is directly

involved in most of the life processes. Next to nitrogen it is invariably classified

as one of the macronutrients and it is a key element in frequency of use as

fertilizer. It serves as a primary energy source for microbial oxidation. It is a

2

constituent substance in life processes. Soil cannot give high yields if it is

deficient in phosphate.

Soil contains both organic and inorganic forms of phosphorus. The

organic forms of the element in soil are compounds of phytins, phospholipids

and nucleic acids that come mainly by the way of decaying vegetation.

Plants take phosphate in the form of soluble orthophosphate ions but due

to the presence of Ca, Mg, K, Na, Al and Fe ions in soil, the soluble

orthophosphate is converted to insoluble form. Because of this process plants

utilize very little amount of phosphate, even though phosphorus containing

fertilizers are added (Vivekkumar et al., 1999).

This unmanaged excess of phosphate application is known to cause

environmental, economic problems and leads to pollution due to soil erosion and

runoff water containing large amounts of soluble phosphate (Brady, 1990). The

runoff from phosphate loaded soil is accepted as the main factor in

eutrophication of natural water reservoirs (Del Campillo et al., 1999).

Traditional phosphate fertilizer production is based on chemical processing of

insoluble mineral phosphate high-grade ore, which includes an energy intensive

treatment with sulfuric acid at high temperature. This process is environmentally

undesirable, not least because of the release of contaminants into the main

product, gas streams and byproducts. These contaminants must be dealt with as

potential air and water environmental pollutants (Vassilev and Vassileva, 2003).

Furthermore, use of phosphate fertilizers has become a costly affair and there is

a need for alternative sources.

3

Some microorganisms are known to be involved in the solubilization of

insoluble phosphates (Alexander, 1977). These phosphate-solubilizing

microorganisms render insoluble phosphate into soluble form through the

process of acidification, chelation and exchange reactions. This process not only

compensates for higher cost of manufacturing fertilizers in industry but also

mobilizes the fertilizers added to soil (Rodriguez and Reynaldo, 1999).

Therefore, many researchers have tried to increase the plant-available phosphate

fraction by means of Phosphate solubilizing microorganisms (PSMs) such as

Achromobacter sp, Agrobacterium sp, Alcaligens sp, Bacillus cereus,

B. polymyxa, B. megaterium, B. subtilis, Pseudomonas striata and Xanthomonas

sp and Fungi like Aspergillus niger, A. flavus, A. fumigatus, Penicillium sp and

Rhizopus sp.

Soil microorganisms are not only associated in the cycling of phosphorus

but also participate in solubilization of inorganic phosphorus and in the

mineralization of organic phosphorus (Agnihothori, 1970; Ostwal and Bhide,

1972).

Mechanism of Phosphate Solubilization

Different mechanisms have been suggested for the solubilization of

inorganic phosphorus by phosphate solubilizers. It is generally accepted that the

major mechanism of mineral phosphate solubilization is the action of organic

acids synthesized by soil microorganisms (Halder et al., 1990). Production of

organic acids results in acidification of the microbial cell and its surroundings.

Consequently, inorganic phosphate may be released from a mineral phosphate

by proton substitution for Calcium ion (Goldstein, 1994). The production of

organic acids by phosphate solubilizing bacteria has been well documented.

Among them, gluconic acid seems to be the most frequent agent of mineral

4

phosphate solubilization. It is reported as the principal organic acid produced by

phosphate solubilizing bacteria such as Pseudomonas sp., Erwinia herbicola,

Pseudomonas cepacia and Burkholderia cepacia. Another organic acid

identified in strains with phosphate-solubilizing ability is 2-ketogluconic acid,

which is present in Rhizobium leguminosarum, Rhizobium meliloti, Bacillus

firmus, Strains of Bacillus liqueniformis and Bacillus amyloliquefaciens were

found to produce mixtures of lactic, isovaleric, isobutyric, and acetic acids.

Other organic acids, such as glycolic, oxalic, malonic, and succinic acid, have

also been identified among phosphate solubilizers. (Rodriguez and Fraga,

1999).

There is also experimental evidence that supports the role of organic acids

in mineral phosphate solubilization. Halder et al. (1990) showed that the organic

acids isolated from a culture of Rhizobium leguminosarum solubilized an amount

of P nearly equivalent to the amount that was solubilized by the whole culture.

Besides this, treatment of the culture filtrates from several Rhizobium strains

with pepsin or removal of proteins by acetone precipitation did not affect

phosphate release capacity, showing that this was not an enzymatic process.

However, neutralization with NaOH destroyed the solubilization activity.

Alternative possibilities, other than organic acids for mineral phosphate

solubilization have been proposed based on the lack of a linear correlation

between pH and the amount of solubilized phosphate. In addition, no significant

amounts of organic acid production could be detected from a phosphate

solubilizing fungus, Penicillium sp. Studies have shown that the release of

hydrogen ions to the outer surface in exchange for cation uptake or with the help

of hydrogen ion translocation ATPase could constitute alternative ways for

solubilization of mineral phosphates.

5

Other mechanisms have been considered, such as the production of

chelating substances by microorganisms as well as the production of inorganic

acids, such as sulfuric, nitric, and carbonic acid. However, the effectiveness of

these processes has been questioned and their contribution to phosphate release

in soil appears to be negligible. The phosphatase enzyme plays a key role in

transforming organic forms of Phosphate in to plant available inorganic form.

They provide to be active in all three components of the rhizosphere soil,

microbes and plant root. There are two types of phosphatase, according to their

pH optima.

Both acid and alkaline phosphatase have been found as external and

internal enzyme in algae and bacteria. Alkaline phosphatase are metalo proteins.

The organic function of soil phosphorus has received relatively little attention

because of its complex nature, although this fraction may account for 15-20% of

phosphate on soil. In order to become available these phosphate compounds

must be hydrolyzed by phosphatase. This enzyme may therefore be very

important in phosphorus nutrition of plants (Tarafdar et al., 1992). Chelating

substances and inorganic acids such as sulfuric, nitric, and carbonic acid are

considered as other mechanisms for phosphate solubilization. However the

effectiveness and their contribution to P release in soils seems to be less than

organic acid production.

Lipase

Lipases are the enzymes that catalyze the hydrolysis of triglycerides to

glycerol and fatty acids. Microbial lipases are relatively stable and are capable of

catalyzing a variety of reactions. They are potential important for diverse

industrial applications.

6

Degreasing is an essential step in the production of glove and clothing

leather. Degreasing helps to obtain soft and pliable leather for garment

manufacture. Degreasing is carried out by emulsification with detergents or by

solvent extraction. It is well known that organic solvents like kerosene, petrol,

perchloroethylene and trichloroethylene are highly unsafe, hazardous to the

workers and heavily pollute the environment. Lipase which are projected as

alternatives for solvents and detergents, catalyse the breakdown of fats and can

be obtained from animal, microbial and plant sources. (Kamini et al., 1999).

Lipase enzymes play a vital role in many fields such as food, dairy

Leather, paper, pharmaceutical, detergent, textile and cosmetic industries. Lipase

are secreted by microorganisms like bacteria, yeasts, molds and a few protozoa.

The production of lipase by microorganisms depends largely on the species,

strains and culture conditions. Microbial lipases are diverse in their enzymatic

properties, substrate specificity and are usually more thermo stable than animal

or plant lipases (Vanitha, 2002).

Microbial lipases represent the major commercial source of this enzyme.

In recent years, research on lipases, mainly of microbial origin, has increased

because of their great commercial potential and also due to the potential of this

enzyme to both hydrolyze fats and synthesize specific esters with desirable

flavor properties for industrial application. Lipases are of considerable

commercial and industrial potential. Furthermore there is an increasing interest

in the development of new application for these enzymes in products and

processes (Paul and Carles, 1992).

7

Indole Acetic Acid

Plant growth promoting rhizobacteria are considered to promote plant

growth directly or indirectly. They can exhibit a variety of characteristics

responsible for influencing plant growth. Plant hormones and other regulatory

chemicals are now used in a variety of applications for commercial reasons to

control some aspects of plant development. Auxins were the first plant hormones

to be discovered. The principle auxin in plants is indole-3-acetic acid (Yurekli et

al., 2003).

Diverse soil microorganisms including bacteria, fungi and algae are

capable of producing physiologically active quantities of auxins, which may

exert pronounced effects on plant growth and establishment. Many of them can

produce auxins in axenic cultures. Most of the species use tryptophan to produce

indole-3-acetic acid (IAA), mainly through the indole-3-pyruvic acid and

tryptamine pathways (Tudzynski and Sharon. 2002).

For many years it was assumed that tryptophan (Trp) was the only

precursor of IAA. However, work with tryptophan-auxotrophic mutants and

isotope labeling has established that IAA biosynthesis can occur via a

tryptophan- independent route also (Normanly, 1997; Venis and Napier, 1991),

although in the presence of Trp, microbes release greater quantities of IAA and

related compounds. There is firm evidence that indole-3-acetic acid (IAA)

(Arshad and Frankenberger, 1991; Sarwar and Frankenberger, 1994; Barea and

Brown, 1974; Brown, 1972; Brown and Burlingham, 1968; Lee et al., 1970;

Scott, 1972), gibbe-rellins and cytokinins (Sarwar and Frankenberger, 1994;

Barea and Brown, 1974), all produced by plants and essential to their growth and

development, are produced also by various bacteria which live in association

with plants. There is also evidence that the growth hormones produced by the

8

bacteria can in some instances increase growth rates and improve yields of the

host plants (Arshad and Frankenberger, 1991; Sarwar and Frankenberger, 1994;

Barea and Brown, 1974). It is possible that bacteria capable of phosphate

solubilization may improve plant productivity both by hormonal stimulation and

by supplying phosphate.

It is generally agreed that indole-3-acetic acid (IAA) is the major and

most abundant auxin in plants and plays a key role in the regulation of plant

growth and development (Moore, 1989, Luthen et al., 1999 and Davies, 1995). It

is presumed that plant growth promoting rhizobacteria producing plant growth

regulators play a critical role in plant growth promotion. To assess this

hypothesis, local isolates of phosphate solubilizing microbes were screened for

their intrinsic ability to produce IAA in the presence and absence of L-

tryptophan (Shahab et al., 2009).

Mutation

UV irradiation and Ethyl Methane Sulphonate (EMS) have long been

used as a model DNA damaging agent to study not only UV specific repair

processes but also general repair and DNA damage tolerance pathways. Several

UV sensitive mutants have been found to be defective in both nucleotide

excision repair and in DNA damage tolerance pathways. The mutants can

provide valuable insights into DNA repair process and mechanism of

mutagenesis (Srinorakutara et al., 2008).

Mutation alters the genotype of microorganisms, when it expresses that

leads to alter the character or death of microorganisms. The Ultra Violet

radiation forms thymine dimer in gene sequence. But the photolyase enzymes

present in living system break the thymine dimer and correct it. The increasing

9

exposure time to UV radiation may form the thymine dimer in gene sequence

that code photolyase enzyme. In this situation the thymine dimer can not break

by the enzyme of living system. The chemical agents such as Sodium Azide,

Ethyl Methane Sulphonate may alter the gene sequence by altering the base

pairs. The azide ion alters the structure of cytosine such that it forms hydrogen

bonds with adenine, rather than guanine. This produces a C -> T transition. Ethyl

Methane Sulphonate is a strong mutagenic agent. It alkylates N7 of Guanine and

severely alters the base pairing.

In the recent past, a variety of studies are conducted to improve the

productivity of enzymes and microbial conversion process using random

mutagenesis, typically which involve production of microbial enzymes, plant

hormones and phosphate solubilization.

The potential mechanism for phosphate solubilization might be

acidification either by proton extrusion associated with ammonium assimilation

(De Freitas et al., 1997 and Reyes et al., 1999) or by organic acid production

(Cunningham and Kuiack, 1992). Acid phosphatases and phytases secreted by

these microorganisms also have an important role in phosphate solubilization

(Richardson et al., 2000). Reyes et al. (1999) isolated the mutants of Penicillium

rugulosum which showed high phosphate solubilizing activity compared to wild

type strain. A significant increase in soluble phosphate level was observed in

case of UV induced mutants of A. tubingensis compared with the wild type.

There might be a possibility of alteration at genetic level in case of mutants

(Achal et al., 2007).

In the present research work, the bacterial and fungal strains were tried to

improve for their phosphate solubilizing capacity by physical and chemical

10

treatments. Their ability in the production of phosphatase, lipase and Indole

Acetic Acid were analysed. The effect of different carbon and nitrogen sources

on the production of phosphatase, lipase and on phosphate solubilization were

studied.

11

Review of Literature

In recent years, attention has been paid to the microbial products, which

have significant agricultural value. Microorganisms produce secondary

metabolites and some of which have phosphate solubilizing capabilities. Some

of the microorganisms are isolated from the soil and their phosphate

solubilization capabilities were improved by using physical and chemical

mutagenesis.

Phosphate solubilization

Soil generally contains adequate amount of organic and inorganic

phosphorus. But most of these remain unavailable to plants. The efficiency of

water soluble phosphorus is usually low and it’s recovery does not exceed 20%

(Darmwal et al., 1989). But by the help of phosphate solubilizers, phosphate

availability in soil and the utilization of phosphate by plants are increased.

(Gaur and Sacher, 1980). Microbial solubilization of insoluble phosphate,

especially low grade and it’s use in agriculture is receiving greater attention.

This process not only compensates for higher cost of manufacturing fertilizers in

industry but also mobilizes the fertilizers added to the soil.

Isolation and identification

Johnston (1954) isolated fungal cultures from rhizosphere soil and

identified as Aspergillus niger, A. terreus and Penicillum digitatum based on

their spore structure and colony morphology. A number of reports are available

related to phosphate solubilization in different soils. Sen and Paul (1957)

reported that the solubilization of calcium phosphate and iron phosphate in

liquid media by four Bacillus sp, which were isolated from the glands of Cassia

occidentalis. Casida (1959) isolated phosphate solubilizing fungi Aspergillus

12

niger, A. flavus, A. nidulans, A. terreus and Penicillium liliacinum from

rhizosphere soil. Chhonokar and Subba Rao (1967) isolated fungal cultures from

legume root nodules and identified as Aspergillus flavus, A. niger and A.

nidulans based on their spore structure and colony morphology.

Ahamed and Jha (1968) isolated Phosphate Solubilizing Bacteria, fungi

and actinomyces from soil samples of Bihar on carrot extract agar. From their

studies, it is identified that bacteria were more in number than other types of

microbes whereas, the fungi performed better solubilization than other types of

microbes. Bhurat and Sen (1968) examined the leaf surface of wheat, pea, barley

and gram for the presence of Phosphate Solubilizing Bacteria and isolated nearly

15 isolates of various species.

Sethi and Subba Rao (1968) screened 48 isolates of fungi from Delhi and

Ludhiana soils for their potentialities to solubilize tri calcium phosphate and

calcium phosphate in culture media. The results indicated that about seventeen

species belonging to genera like Aspergillus, Penicillum, Cladosporium,

Fusarium, and Pacecilomyces were found to be significantly effective. Metha

and Bhide (1970) selected 42 isolates with solubilization capacity of Tri Calcium

Phosphate in culture out of 149 soil fungal cultures. Most efficient solubilizers

were of Aspergillus sp., Penicillum sp., Pythium sp., Curvularia sp.,

Chaetomium sp. and Humicola sp.

Agnihothori (1970) evaluated phosphate solubilizing potential of several

fungi isolated from forest tree seedbeds. Aspergillus niger, A. flavus, Fusarium

oxysporum, Sclerotium roltsii, Clindracladium sp. and Penicillium sp. were

significant to solubilize phosphate. Gaur et al. (1973) isolated Aspergillus

carbonum, Aspergillus flavus, Aspergillus fumigatous, and Aspergillus wentii

13

from Mussourie, Jhamarkotra and Maton Rock Phosphates and studied the

solubilization capacity. Rao et al. (1982) isolated phosphate dissolving

actinomycetes from soil and this organism was identified as Streptomyces

species from its slide culture.

Venkateswaralu et al. (1984) isolated phosphate-solubilizing

actinomycetes from orchid soils using modified Pikovskaya medium. Singh et

al. (1984) identified phosphate solubilizing fungal cultures viz., Aspergillus

awamori and A. niger based on their colony color, colony morphology and

spore structure. Darmwal et al. (1989) isolated phosphate solubilizing fungal

cultures from different samples of rhizosphere of wheat, gram, garden soils and

composed materials. Thakker et al. (1993) isolated phosphate solubilizing gram

positive and gram negative bacteria from different crops and compost samples

and identified the pure culture on the basis of cell morphology, staining reaction,

biochemical and physiological characteristics as outlined in 8th edition of

Bergey’s Manual of Determinative and Systemic Bacteriology.

Phosphate solubilization assay

Arora and Gaur (1979) inoculated the bacterial and fungal culture into

Pikovskaya broth and after incubation the water-soluble phosphate in the

supernatant was estimated by the King’s B method improved by Sherman with

Klett-Sommerson colorimeter using red filter. Thakker et al. (1993) inoculated

the broth culture containing phosphate-solubilizing bacteria, adjusted with

optical density 1.0 into pikovskaya broth. The phosphate solubilization was

assayed interval of 3 days by chlorostannous reduced molybdophosphoric acid

method.

14

Varshanarishan et al. (1995) cut 80mm mycelial disc (=75 x 106 spores

per ml) from four days old culture, grown on Czapek’s dox agar by sterilized

cork borer under aseptic condition into 50ml sterile pikovskaya broth. After

incubation, the water soluble phosphorus in the culture filtrate was estimated

every 24 hours up to 7days by chlorostannous reduced molybdophosphoric acid

method. Seshadri et al. (2000) inoculated a single colony of Azospirillum

halopraeferans from nutrient agar to sterile broth containing water insoluble

phosphorus. After incubation, the cultures were harvested on alternate days,

centrifuged at 10, 000 rpm for 15 minutes and then subjected for phosphate

estimation by paramolybdate blue method.

Metabolic mechanism of Phosphate solubilization

The potential mechanism for phosphate solubilization might be

acidification either by proton extrusion associated with ammonium assimilation

(De Freitas et al., 1997; Reyes et al., 1999) or by organic acid production

(Cunningham and Kuiack, 1992). Acid phosphatase and phytases secreted by

microorganisms also have an important role in phosphate solubilization

(Richardson et al., 2000).

Katznelson and Bose (1959) isolated rhizosphere bacteria from wheat,

that oxidized Glucose and alanine and they observed over one third of cultures

tested were capable of dissolving insoluble phosphate in the form of calcium

phosphate. Goswami and Sen (1962) isolated three bacterial strains and

examined for their Tri Calcium Phosphate solubilizing activity in Pikovskaya

broth.

Kucey (1984) Phosphate-solubilizing, total bacterial and fungal

populations were determined by serial dilution and plate counting. He reported

15

that the fungi were superior than bacteria in solubilizing both freshly precipitated

calcium phosphate and Idaho Rock Phosphate. Fungi also retained this ability

over many sub culturing transfers. A high percentage of the bacterial isolates lost

their solubilizing ability when sub cultured. A significant correlation was found

between an organism's ability to solubilize freshly precipitated calcium

phosphate in agar plates and Idaho Rock Phosphate in solution culture.

Cerezine et al. (1988) grew the fungus Aspergillus niger in a stationary

culture containing modified citrate medium supplemented with fluoroapatite.

Solubilization of insoluble phosphate was increased with fungi growth, reaching

maximum after 11 days of the inoculation. Soluble phosphate levels were

correlated with pH of the culture medium but not with titrable acidity values,

probably due to metabolic activity of fungus resulting from consumption of

sugar in the culture medium, They also studied the effect of carbon and nitrogen

source on phosphate solubilization.

Halder et al. (1991) also studied the phosphate solubilization efficacy of

Bradirhizobium strains by using different amount of phosphates of both

hydroxyapitite and tri-calcium phosphate in liquid cultures. Phosphate

solubilization was related to pH decrease caused by the strains. Nitrogen in the

form of ammonium effected high solubilization of phosphate in the medium.

However yeast Extract of nitrate was also remarkably effective as a nitrogen

source to support PO4 solubilization, Glucose was best carbon source for the

process. Two fungal isolates, Penicillium bilaji and Penicillium fuscum were

found to solubilize different amounts of Rock Phosphate in liquid culture and the

relationship between inorganic P solubilization and showed pH drop by each

isolate. The also reported the role of Nitrogen in P solubilization metabolism of

P. bilaji. (Asea et al., 1988).

16

Cunningham and Kuiack (1992) isolated Penicillium bilaii to study the

solubilization of mineral phosphates and enhance plant uptake of phosphate.

Using agar media with calcium phosphate and the pH indicator alizarin red S,

the influence of the medium composition on phosphate solubility and medium

acidification was recorded. Three gram positive and four gram negative

Phosphate Solubilizing Bacteria were isolated from rhizosphere of different

crops and compost samples. Among the bacteria, Enterobacter aerogens, was

found to be most effective tri Calcium Phosphate (TCP) & Rock Phosphate

solubilizer. In presence of tri Calcium Phosphate and Rock Phosphate, the effect

of carbon, nitrogen sources and pH of the medium on phosphate solubilization

were also studied. (Thakkar et al. 1993).

Singal et al. (1994) Aspergillus japonicus and A. fetidus were found to

solubilize five types of Indian Rock Phosphates at pH 8 and 9. Solubilization

was higher in the presence of pyrite than in controls lacking either pyrite or

fungal inoculum. Both the Aspergillus were found to be good pyrite solubilizers

and could grow over a wide pH range. Solubilization of Rock Phosphates was

the result of organic acid release and pyrite oxidation. Aspergillus aculeatus

isolated from rhizosphere of gram, Solubilized Phosphate from TCP in

Pikovskaya Medium. The phosphate solubilizing activity was highest after 48h

of fungal growth in the presence of Glucose and ammonium sulphate as the best

carbon and nitrogen sources. They also reported the growth of the

microorganisms and the pH of the growth medium showed no correlation with

phosphate solubilizing activity (Varsanarsian et al.1995).

Two species like Penicillium aurantiogriseum and Pseudomonas sp.

having high abilities in solubilizing inorganic phosphates (hydroxylapatite and

17

brushite) were used to examine solubilization mechanisms. No direct contact

between microorganisms and Calcium phosphates (Ca-Ps) were necessary for

effective solubilization (Illmer and Schinner, 1995).

Vassilev et al. (1996) successfully cultivated phosphate solubilizing

Aspergillus niger on sugar beet-waste material supplemented with Rock

Phosphate. Nine Streptosporangium isolates of Eudilius eugeniae (earthworm)

casts were found to be acid tolerant, Rock Phosphate solubilizers which could

grow on synthetic Glucose or carboxyl methyl-cellulose nitrogen free or

ammonium chloride enriched media, as the sole carbon source. These isolates

could be exploited in the industrial production of microbial phosphate fertilizers

which would enhance organic residues and plant nutrients recycling in acid soils

of Nigeria. (Mba, 1996).

Two soil types, loamy sand and sandy soils, were treated with atrazine,

pyrethrin and a mixture of metobromuron and metolachor for eight weeks in the

laboratory to determine the effect of the chemicals on soil microbial populations

and their mineralization activities (Taiwo and Oso, 1997).

Aspergillus niger, citric acid producing strain grown on olive cake-

based medium, was able to solubilize Rock Phosphate. Solubilization of

insoluble phosphate increased during the solid-state fermentation process.

Various combinations of olive cake and Rock Phosphate, previously treated or

untreated by the fungus, were introduced into a calcareous, phosphorus

deficient soil to improve the growth of Trifolium repens in a greenhouse

experiment. Greater growth and phosphate uptake of Mycorrhizal and Non-

Mycorrhizal plants were achieved when microbe treated olive cake and Rock

18

Phosphate were applied to soil compared with all other treatments. (Vassileva

et al., 1997).

The interactive effect of phosphate-solubilizing bacteria and Arbuscular

Mycorrhizal fungi on plant use of low bioavailable soil phosphate sources was

evaluated using soil microorganisms which integrated isotopic dilution

techniques. The microbial inoculum consisted of the Arbuscular Mycorrhizal

fungus Glomus intraradices and two phosphate-solubilizing rhizobacterial

isolates Enterobacter sp. and Bacillus subtilis. The Mycorrhizosphere

interactions between bacterial and fungal plant associates was identified and

it’s contribution to the biogeochemical phosphorus cycling was also

investigated. (Toro et al., 1997).

The effect of immobilization technique on phosphate-solubilization by

Enterobacter sp., was reported. Various amounts of the immobilized

bioparticles were applied in a repeated batch fermentation process in order to

solubilize Venezuelan Rock Phosphate. Phosphate solubilization was

significantly higher compared to a free-cell single-batch control. Whole and

destroyed immobilized bioparticles were introduced into a soil enriched with

Rock Phosphate to improve the growth of onion plants. (Vassileve et al 1998).

Spores of Aspergillus niger were encapsulated in Agar, Calcium Alginate

and Potassium Carrageenan and the encapsulated-fungus were tested for it’s

phosphate solubilizing capability using the culture medium supplemented with

different concentrations of Rock Phosphate. The highest average soluble

phosphate concentration was obtained with agar-cell beads as compared with

other encapsulated systems (Vassileve et al. 1998).

19

Kim et al. (1998) examined the phosphate solubilization capability of

Enterobacter agglomerans by organic energy source in unsterilized soil. After

the enrichment of organic energy source, they examined the phosphatase activity

and available phosphate concentration. Pany et al. (1998) collected surface soil

samples, treated with different phosphate sources and analyzed for microbial

population with respect to total bacteria and Phosphate Solubilizing Bacteria.

Anusuya and Jayarajan (1998) identified Trichoderma viride fungal culture

isolated from rhizosphere soil and compare the phosphate solubilizing capacity

of the fungus against Bacillus sp.

Pseudomonas cepacia is known as a Rock Phosphate solubilizer in

bioreactors and in soils. BarYosef et al. (1999) determine the production rates of

gluconic acid and 2-ketogluconic acid by the bacteria in the presence of clay

minerals which prevail in soils, and the resulting rate and extent of

orthophosphate release into the suspension solutions. An isolate of A. niger was

found to be very effective in solubilizing Rock Phosphate. The influence of

nutritional requirements such as carbon sources, nitrogen sources and different

concentrations of KH2PO4 on Rock Phosphate solubilization was extensively

studied. Moreover, the effect of heavy metals, such as Mn2+, Co2+, Zn2+, Cu2+ as

metal chloride and Al3+ as aluminium sulphate, on the solubilization rate of Rock

Phosphate was illustrated. A biotic effect of organic acids such as citric and

oxalic acids, as major metabolites of A. niger, on the solubilization of Rock

Phosphate, was studied. The obtained results revealed that there is a relationship

between the rate of solubilization of Rock Phosphate and the extracellular

exudates including organic acids, alkaline and acid phosphatase.

Penicillium radicum, a phosphate-solubilizing fungus isolated from the

rhizosphere of wheat roots and its ability to solubilize inorganic phosphate was

20

studied in vitro. The fungus was grown in liquid medium cultures containing

either ammonium or nitrate as the sole source of nitrogen. The titratable acidity,

pH and concentrations of organic acids and soluble phosphate were determined

periodically during incubation. Phosphate solubilization was generally higher

when ammonium rather than nitrate was the sole source of nitrogen. Soluble

phosphate concentrations in the culture medium were directly proportional to the

titrable acidity and organic acid concentration and inversely related to pH.

(Whitelaw et al., 1999).

Sudhansupal (1999) isolated 23 Phosphate Solubilizing Bacterial cultures.

Of the 23 isolates the acid tolerant strain was identified as Bacillus sp on the

basis of morphological and biochemical tests as described in Bacteriological

manual. Artidave and Patel (1999) isolated Phosphate Solubilizing Bacteria

from soil. They identified the bacteria up to genus level by positive test of the

organism for pigment production on King’s B medium and by cultural,

morphological and biochemical characteristics. Based on the above results and

oxidase positive reaction in King’s B medium, they identified the isolated

culture was Pseudomonades.

Phosphate-solubilizing bacteria and solubilization of Mussoorie

phosphate rock were examined in simulated fish ponds enriched with compost

and exogenous introduction of phosphate-solubilizing bacteria. They found that

the combined effects of the Phosphate-solubilizing bacterial population of both

exogenous and compost origin with short generation time. The relationship

between the phosphorus level of water and Phosphate-solubilizing bacteria

population was expressed in exponential equations (Sahu and Jana, 2000).

Vazquez et al. (2000) isolated thirteen Phosphate Solubilizing Bacterial strains

from rhizosphere of mangroves using culture media containing tri basic calcium

21

phosphate and they analyzed the bacterial culture for the production of organic

acids.

Shekhar et al. (2000) isolated Phosphate Solubilizing Bacterial strains

from the rhizosphere of chickpea and alkaline soils. These bacterial strains were

screened for the phosphate solubilizing ability in the presence of 10 percentage

salt, pH 12 and temperature 45oC. Four strains of Phosphate Solubilizing

Bacteria were isolated from the rhizosphere of chickpea and alkaline soils. All

four strains demonstrated diverse levels of phosphate solubilization activity

under in vitro conditions in the presence of various carbon and nitrogen sources.

Acid production may have contributed to phosphate solubilization, but was not

the only reason for phosphate release into the medium. The strains showed

varied levels of phosphate solubilization when the effects of different sources of

nitrogen were examined during growth (Nautiyal et al., 2000).

Fenice et al. (2000) studied the relationship between gluconic acid

production and phosphate solubilization by the encapsulated fugal strain

Penicillium variabile. They also reported that the degree of phosphate

solubilization was not influenced by decreasing the Glucose concentration in the

cultivation medium.

Vassileve et al. (2001) examined phosphate solubilization and organic

acid production by using immobilized microbial cells. The immobilized cells

showed higher degree of phosphate solubilization and organic acid production

than free cells. Kang et al. (2002) screened for phosphate solubilizing fungal

strains in the field soil at Taegu, South Korea. Such strains were identified as

Fomitopsis sp. Phosphate solubilizing ability of Fomitopsis sp. was studied on

four different insoluble phosphates, viz. Tri Calcium Phosphate, Rock

22

Phosphate, Aluminium phosphate and Hydroxyapatite. Tri Calcium Phosphate

was found to be solubilized maximally, while Hydroxyapatite could not be

solubilized by the isolated fungal strain. Further, the effect of salinity under in

vitro conditions on the solubilization activity of Rock Phosphate was also

observed.

Three isolates of Aspergillus tubingensis and two isolates of Aspergillus

niger isolated from rhizospheric soils were tested on solubilization of different

Rock Phosphates. All the isolates of Aspergillus were capable of solubilizing all

the natural Rock Phosphates. A. tubingensis showed maximum percent of

solubilization in all the Rock Phosphates tested in this study when compared to

other isolates. (Reddy et al. 2002). Pseudomonas corrugata, a soil isolate,

initially obtained from a temperate location in Sikkim, was examined for its Tri

Calcium Phosphate solubilizing ability along a wide temperature range, from

psychrophillic to mesophillic. The study has implications for developing carrier-

based microbial inoculants for improved growth of plants in the mountains

(Pandey et al. 2002).

A bacterial strain isolated from rhizospheric soil of grasses growing

spontaneously in Spanish soil, actively solubilized phosphates in vitro when

calcium phosphate was used as a phosphorus source. This strain was Gram-

negative, strictly aerobic, rod-shaped and motile. The strain produced catalase,

but not oxidase. Cellulose, Casein, Starch, Gelatin, Aesculin and Urea were not

hydrolyzed. Growth was observed with many carbohydrates as the carbon

source. (Peix et al. 2003).

The Mineral Phosphate Solubilization (MPS) was studied in ten

Aspergillus niger strains. MPS activity was measured in solid (Pikovskaya's

23

medium) as well as liquid media using different phosphate sources, carbon

sources and nitrogen sources All the strains showed a zone of clearance of

Tricalcium Phosphate in Pikovskaya's medium in plates and solubilized

Dicalcium and Tricalcium Phosphates in broth efficiently. Among the carbon

sources Aspergillus niger preferred mannitol for higher phosphate solubilization.

Nitrogen in the form of nitrate was very effective in solubilizing inorganic

phosphates. Xylose and urea were the poorest sources of carbon and nitrogen for

all the strains of Aspergillus. Phosphate release was associated with reduction in

pH (Seshadri et al., 2004).

A phosphate-solubilizing microorganism isolated from rhizospheric soil

and temporarily identified as Burkholderia glathei produced gluconate and

acetate using Glucose as a carbon source and its metabolic activity caused the

pH of the liquid medium to decrease as low as 4.4. Whole-cell fatty acids

methyl ester profile and 16S rDNA sequence analysis were employed to isolate

and identify the bacterial groups that actively solubilized phosphates in vitro

from rhizosphere soil of various crops of Korea. Out of several hundred colonies

that grew on Pikovskaya’s medium 13 best isolates were selected based on the

solubilization of insoluble phosphates in liquid culture and further characterized

and identified (Chung et al. 2005).

The fungal strains were isolated from agriculture soil, having potential to

solubilize insoluble inorganic phosphates were characterized. Two fungal

isolates were tested for their Tricalcium Phosphate solubilization efficiency in

both solid and liquid medium. Isolates were identified as Aspergillus sp. and

Penicillium sp. Phosphate solubilization activity in liquid broth culture in

presence of various carbon and nitrogen sources, relation between pH and

phosphate solubilization were recorded (Pradhan and Sukla, 2005).

24

The phosphate-solubilizing bacterium was isolated from soil in Pingdong

and Taiwan and identified as B. cepacia based on its 16S ribosomal DNA

(rDNA) sequence. The supernatant was filtered through a 0.22 µm Millipore

filter and assessed for bacterial population, pH, organic acids, and phosphate

solubilizing capability (Lin et al., 2006). The phosphate solubilizing fungi were

isolated to assess the solubilization ability of phosphate fractions in various soil.

The highest numbers of isolates with high solubilization capacity were detected

in pasture soil, followed by tropical rain forest and forest patch soils. Pasture soil

presented both the largest contents of insoluble phosphates and the largest

number of fungal isolates with phosphate-solubilizing ability. The range and size

of phosphate fractions influenced the number of fungi and their ability to

solubilize hardly soluble phosphates (Barroso et al., 2006).

The possible action of Phosphate Solubilizing Bacteria on the

leguminous–rhizobia symbiosis was studied in the region where the available

phosphorus distribution is not uniform. Sinorhizobium meliloti,

Bradyrhizobium japonicum and two phosphorus-solubilizing strains of

Pseudomonas putida were used for growth promotion treatments. (Rosas et al.,

2006). Fankem et al. (2006) collected seven rhizosphere soil samples from oil

palm tree of Cameroon. The collected soil samples were air dried, crushed to

pass through 2mm sieves and the Phosphate Solubilizing Bacteria were isolated

by using the soil samples. For the estimation of phosphate solubilization Reyas

basal medium having Calcium, Aluminum and Iron phosphates was used. At the

end of incubation time, it appeared that, phosphate solubilization resulted from a

combined effect of pH decrease of the media and organic acids production.

Furthermore, each of the tested isolates was able to produce at least one of the

most important organic acids such as Citrate, Malate and Tartrate. Among the

25

ten isolates tested, three were identified as Pseudomonas fluorescens and would

be considered as potential biofertilizers.

To develop environment friendly biofertilizer, solubilizing insoluble

phosphates, salt- and pH-tolerant, insoluble inorganic phosphate solubilizing

bacterium was isolated from soybean rhizosphere. On the basis of its

physiological and biochemical characteristics, this bacterium was identified as

Pantoea agglomerans. Glucose medium consisting of ammonium nitrate was the

optimal and best medium for phosphate solubilization (Son et al., 2006). Two

phosphate and potassium solubilizing strains were isolated from the soil of

Tianmu Mountain, Zhejiang Province (China) and they were phenotypically and

phylogenetically characterized (Hu et al., 2006).

The isolates from different composts such as farm waste compost, rice

straw compost, Gliricidia vermicompost, and macrofauna, showed Rock

Phosphate solubilization in buffered medium in plate culture. When tested in

Rock Phosphate broth medium, all the isolates, Enterobacter cloacae, Serratia

marcescens, Serratia sp, Pseudomonas sp., and Pseudomonas sp. showed

gluconic acid production and solubilized Rock Phosphate. In the presence of

different carbon sources, cellulose degrading and Phosphate solubilizing strains

showed a drop in pH and solubilized Rock Phosphate. Significantly, these

bacteria isolated from composts and macrofauna solubilized Rock Phosphate in

the presence of various pure carbon substrates and crop residues and their

importance in soil/rhizosphere conditions are discussed. (Hameeda et al., 2006).

A field experiment has been conducted with four systemic herbicides at

their recommended field rates to investigate their effects on growth and

activities of aerobic non-symbiotic N2-fixing bacteria and Phosphate

Solubilizing Microorganisms in relation to availability of nitrogen and

26

phosphorus in the rhizosphere soils as well as yield of the rice crop. Application

of herbicides, in general, highly stimulated the population and activities of the

target microorganisms, which resulted in a greater amount of atmospheric

nitrogen fixation and phosphate solubilization in the rhizosphere soils of the test

crop. (Das and Debnath, 2006).

Chen et al. (2006) isolated, screened and characterized 36 strains of

Phosphate Solubilizing Bacteria (PSB) from Central Taiwan. Mineral

Phosphate Solubilizing (MPS) activities of all isolates were tested on tri

calcium phosphate medium by analyzing the soluble phosphate content after

72 h of incubation at 30 °C. Biosolubilization of Rock Phosphate using a

Penicillium sp., an Aspergillus sp., Pleurotus ostreatus, Bradyrhizobium

elkanii and their fungal–rhizobial biofilms was investigated. The study

identified an effective method of fungal–rhizobial biofilm mediated

solubilization of Rock Phophate. (Jayasinghearachchi and Seneviratne, 2006).

The phosphate solubilizing fungi Aspergillius spp., Penicillium spp. and

Fusarium spp. and bacteria B.subtilis, and B.megatherium were collected from

saline affected area of Amravati district. The strains were used to reduce the

salinity of soil by utilizing their organic acid metabolic activity. (Rajankar et

al., 2007). Vassilev et al. (2007) utilized dry olive waste, as a substrate for

phytase production and Rock Phosphate solubilization by Citric Acid

producing Aspergillus niger. Corn steep liquor, Yeast Extract and Ammonium

Nitrate were used as nitrogen source for phosphate solubilization. Both

enzyme production and phosphate solubilization depended on water medium

content, type of nitrogen source, inoculum size and the presence and initial

concentration of Phosphate in the medium.

27

Perez et al. (2007) conducted a survey of Phosphate-solubilizing bacteria

naturally colonizing a limonitic crust in the south-east region of Venezuela. A

total of 130 heterotrophic bacterial isolates showing different degrees of mineral

Tri Calcium Phosphate (Ca3 (PO4)2) solubilizing activities were isolated and

their phosphate solubilizing efficacy were detected with different phosphate

source. The 10 best Ca3 (PO4)2 solubilizers were characterized by partial

sequencing analysis of their respective 16S rRNA genes.

Eupenicillium parvum, a phosphate-solubilizing microorganism was

isolated from the tea rhizosphere. The fungus developed a phosphate

solubilization zone on modified Pikovskaya agar, supplemented with tri calcium

phosphate. Quantitative estimation of phosphate solubilization in Pikovskaya

broth showed high solubilization of Tri Calcium Phosphate and Aluminium

phosphate. The fungus also solubilized north Carolina Rock Phosphate and

Mussoorie Rock Phosphate, and exhibited high levels of tolerance against

desiccation, acidity, salinity, Aluminium, and Iron. Solubilization of inorganic

phosphates by the fungus was also observed under high stress levels of

Aluminium, Iron, and desiccation, though the significant decline in phosphate

solubilization was marked in the presence of Aluminium than Iron (Vyas et al.

2007).

Strain Paecilomyces marquandii was isolated from soil deficient in

phosphate on Pikovskaya’s medium. To study the effect of different carbon

sources on phosphate solubilization, Pikovskaya medium was modified where tri

calcium phosphate was replaced with other phosphate source and Glucose was

replaced with 9 individual carbon compounds viz. Fructose, Galactose, Glycerol,

Lactose, Maltose, Mannose, Sorbitol, Starch and Sucrose. The nitrogen sources

were evaluated similarly by replacing Ammonium Sulphate with six different

nitrogen sources viz. Ammonium Chloride, Asparagine, Calcium Nitrate,

28

Potassium Nitrate, Sodium Nitrate and Urea and their effect on phosphate

solubilization were reported. (Ahuja et al. 2007).

Narula et al. (2007) isolated Phosphate Solubilizing Bacteria from the

rhizosphere soil of eternal rays and reference strains of A. chroococcum,

Pantoea, Pseudomonas were compared for phosphate solublizing ability over a

period of 18 days. Without much effect on pH, cumulative effect of these entire

factors might have had an indirect effect on phosphate solubilization.

Shahab and Ahmed (2008) studied that the phosphate solubilization

efficiency of ten soil bacteria for various parameters like carbon sources such as

Glucose, Fructose, Sucrose and Lactose, variable concentration of Sodium

Chloride and Glucose. Glucose was the most favorable carbon source for

solubilization while lactose is the least favorable carbon source. Acinetobacter

lwoffi, Pseudomonas aeruginosa and Bacillus thuringiensis were found to be the

most promising isolates.

Kang et al. (2008) stated that Aspergillus sp, a soil isolate had excellent

potential to solubilize Rock Phosphate in vitro. The process was influenced by

the presence of various concentrations of local loess (red soil). The simultaneous

occurrence, in our experiment, of high levels of solubilized phosphate and

synthesized citric acid, together with the lowest reached pH values, confirmed

the role of citric acid in the phosphate solubilization mechanism. When the soil

was present, phosphate release was better correlated than citrate synthesis with

Hydrogen ion concentration. Changes in soluble phosphate concentration did not

follow a sigmoid pattern. The ability of organism to release phosphatase was

also studied.

29

The mechanisms involved in the weathering processes of insoluble Rock

Phosphate of Moroccan phosphate mine by phosphate solublizing strains

isolated from Moroccan phosphate mine indicated that the isolates produce

siderophore but not organic acids. (Hamdali et al. 2008). Thermo-tolerant

phosphate-solubilizing microbes including bacteria, actinomycetes, and fungi

were isolated from different compost plants and biofertilizers to prepare the

multi-functional biofertilizer. Most of the isolates possessed amylase, Carboxy

Methyl Cellulase, Chitinase, Pectinase, Protease, Lipase, and Nitrogenase

activities. All isolates could solubilize calcium phosphate and Israel Rock

Phosphate. Adding these microbes can shorten the period of maturity, improve

the quality, increase the soluble phosphorus content, and enhance the

populations of phosphate-solubilizing and proteolytic microbes in biofertilizers

(Chang and Yang. 2009).

Park et al. (2009) investigated the ability of Pseudomonas fluorescens to

solubilize insoluble phosphate via two possible mechanisms: proton excretion by

ammonium assimilation and organic acid production. There were no clear

differences in pH and phosphate solubilization between Glucose-ammonium and

Glucose-nitrate media. phosphate solubilization was significantly promoted with

Glucose compared to Fructose. Regardless of nitrogen sources used,

Pseudomonas fluorescens solubilized little insoluble phosphate with Fructose.

High performance liquid chromatography analysis showed that Pseudomonas

fluorescens produced mainly gluconic and tartaric acids with small amounts of

2-ketogluconic, formic and acetic acids. During the culture, the pH was reduced

with increase in gluconic acid concentration and was inversely correlated with

soluble phosphate concentration.

Khan et al. (2009) described the occurrence, mechanisms and role of

Phosphorus solubilizing bacteria in crop production. Plants acquire phosphorus

30

from soil solution as phosphate anion. It is the least mobile element in plant and

soil contrary to other macronutrients. It precipitates in soil as orthophosphate or

is absorbed by Ferric and Aluminum oxides through legend exchange.

Phosphorus Solubilizing Bacteria play role in phosphorus nutrition by enhancing

its availability to plants through release from inorganic and organic soil

phosphate pools by solubilization and mineralization. Principal mechanism in

soil for mineral phosphate solubilization is lowering of soil pH by microbial

production of organic acids and mineralization of organic Phosphates by acid

phosphatase. Use of phosphorus solubilizing bacteria as inoculants increases

phosphate uptake. These bacteria also increase prospects of using phosphatic

rocks in crop production. Greater efficiency of Phosphate Solubilizing Bacteria

has been shown through co-inoculation with other beneficial bacteria and

mycorrhiza.

Srividya et al. (2009) isolated and characterized the fungal strains from

agriculture soil, having potential to solubilize insoluble inorganic phosphates on

Pikovskya’s medium with Tricalcium Phosphate. Aspergillus niger and

Penicillium sp. showed high phosphate solubilisation efficiency on Pikovskya’s

medium with Tricalcium Phosphate in liquid broth in 5 days of growth. A. niger,

showed maximum phosphate solubilization efficiency on Pikovskya’s agar solid

and liquid medium in 5 days of growth. Aspergillus sp showed diverse levels of

phosphate solubilization activity in both solid and liquid broth culture in

presence of various carbon and nitrogen sources and different media. Phosphate

Solubilizing Microorganisms convert insoluble phosphates into soluble forms

generally through the process of acidification, chelation and exchange reactions.

Joseph and Jisha (2009) isolated Phosphate Solubilizing Bacteria

possessing the ability to solubilize insoluble inorganic phosphates from

rhizosphere soil. Eighty-one potential PSBs thus obtained were quantitatively

31

screened for phosphate solubilization. Of these, four bacteria such as

Acetobacter liquefaciens, Acetobacter sp., Pseudomonas gladioli and one

unidentified strain found to be efficient phosphate solubilizers. They were

selected for further evaluation, and found that they solubilize Tricalcium

Phosphate in buffered as well as non-buffered media. The efficiency of

phosphate solubilization was decreased in buffered media compared to non-

buffered media. The buffering capacity of the medium reduced the effectiveness

of Phosphate Solubilizing Bacterias in releasing Phosphate from Tricalcium

Phosphates.

Bacterial solubilization of insoluble inorganic phosphate has been studied

as a means of providing available phosphorus for crop production. Bacterial

abilities to solubilize Calcium Phosphate and Rock Phosphate have been

identified to be related with their abilities to produce Gluconic acid and

Ketogluconic acid. However, there is no information regarding the relationship

between bacterial ability to solubilize Aluminum phosphate and their ability to

produce organic acids. Bacterial ability to solubilize Calcium and Aluminum

phosphates were determined as the concentration of soluble phosphate in the

filtrate of bacterial cultivation media, while bacterial ability to produce organic

acids were assessed from the accumulated organic acids in its. Organic acids

related with the solubilization of calcium phosphate differ from the ones related

with the solubilization of Aluminum Phosphate. Moreover, there is similarity in

the production of organic acids related to the solubilization of Aluminum

Phosphates and Iron phosphate (Prijambada et al., 2009).

Saha and Biswas (2009) isolated, purified and characterized Phosphate

Solubilizing Bacteria from different fertility gradient with regards to N, P and K

status of soil through their insoluble mineral phosphate-source utilization

patterns. Four insoluble phosphate sources; purulia Rock Phosphate, Mussourie

32

Rock Phosphate, crystalline iron and Aluminum Phosphate were charged in

basic Pikovskaia solid medium. Growth pattern of the isolates on those

phosphate sources was recorded. Different communities utilized different

Phosphate sources in different magnitudes.

Kumar et al. (2010) isolated six Phosphate Solubilizing Bacteria from

paddy fields of Eastern Uttar Pradesh, India and identified as members of

Enterobacter and Exiguobacterium genera. Of the six isolates, Enterobacter sp.

exhibited high level of phosphate solubilization in liquid medium.

Exiguobacterium sp. showed increased phosphate solubilization efficiency under

alkaline pH.

Ekin. (2010) evaluated the effect of application of Phosphate Solubilizing

Bacteria, Bacillus Sp, with and without varying amounts of phosphorus fertilizer

on growth and yield of sunflower under field conditions. The application

Phosphate Solubilizing Bacteria was able to mobilize phosphate efficiently in

the sunflower and improved seed quality and oil yield. It also enhanced the head

diameter, seed weight, kernel ratio and oil content and increases in oil yield.

However, when phosphate solubilizing bacteria was used in conjunction with

phosphatic fertilizers, a much greater effect was observed.

Kapri and Tewari. (2010) isolated fourteen strains of Trichoderma sp

from the forest tree rhizospheres of Pinus, Deodar, Bamboo, Guava and Oak on

Trichoderma selective medium. The isolates were tested for their in-vitro P-

solubilizing potential using National Botanical Research Institute Phosphate

(NBRIP) broth containing Tricalcium Phosphate as the sole phosphate source,

and compared with a standard culture of T. harzianum. All the cultures were

found to solubilize Tricalcium Phosphate but with varying potential. Extra-

cellular acid and alkaline phosphatases of the fungus were induced only in the

33

presence of insoluble phosphorus source. The study explores high Phosphate

solubilizing potential of Trichoderma sp., which can be exploited for the

solubilization of fixed phosphates present in the soil, thereby enhancing soil

fertility and plant growth.

Chakraborty et al. (2010) collected four hundred isolates from soil

samples from forest, river basin, agricultural fields and rhizosphere of plantation

crops of North Bengal. The isolates were screened for phosphate solubilizing

activity on Pikovskaya’s agar medium. Among the screened isolates, ninety

showed phosphate solubilizing activity. Out of these, ten isolates belonging to

Aspergillus niger, A. melleus and A. clavatus were selected for further in vitro

evaluation of phosphate solubilization using Tricalcium Phosphate and Rock

Phosphate. The study revealed that the isolates could solubilize Tricalcium

Phosphate better than Rock Phosphate. Selected isolates were mass multiplied

using farm-yard manure and were tested in vivo for their growth promoting

activity in soybean. While all the isolates promoted growth, A. niger was found

to be most effective.

The importance of rhizospheric microbial phosphate solubilization has

now been well documented. However, the performance of these microbes is

greatly affected by various environmental stresses such as salt stress, pH stress,

temperature stress etc. In this study, two stress tolerant phosphate solubilizing

rhizobacteria Arthrobacter sp. and Bacillus sp. have been isolated from tomato

rhizosphere and characterized with various morphological and biochemical tests.

Phosphate Solubilizing Bacteria were screened on the basis of their phosphate

solubilization and strains with high phosphate solubilizing ability were then

tested against wide range of temperature, pH, and salt stresses. Their ability to

solubilize other insoluble phosphates, such as ferric phosphate and aluminum

phosphate was also studied. In addition to phosphate solubilizing ability these

34

strains also demonstrated various plant growth promoting and biocontrol

activities including Indole Acetic Acid production. These two strains have the

potential to be used as plant growth promoting rhizobacteria (Samiran et al.,

2010).

The ability to solubilize insoluble inorganic phosphate compounds by

Gluconacetobacter diazotrophicus was studied using different culture

approaches. Qualitative plate assays using Tricalcium Phosphate as the sole

phosphate source showed that G. diazotrophicus produced solubilization only

when aldoses were used as the carbon source. In batch cultures with

hydroxyapatite as the phosphate source and Glucose as the carbon source, more

than 98% of insoluble phosphate was solubilized. Continuous cultures of G.

diazotrophicus showed significant activities under carbon or phosphate

limitation. It was suggest that G. diazotrophicus is an excellent candidate to be

used as biofertilizer because in addition to the already described plant growth-

promoting abilities of this organism, it shows a significant mineral phosphate

solubilization capacity (Crespo et al., 2011).

Mutation

Mutations are heritable changes in genetic material. The mutation can

cause Macroalterations or Microalterations. Sometimes it causes a silent

mutation that does not result in a changed amino acid sequence, i.e., the new

codon just happens to code for the same amino acid. Macroalterations are large

changes, such as duplications, deletions, inversions or rearrangements of a large

number of bases. Microalterations involve single base pairs. Transitions are

Purine changes to an alternate Purine, Pyrimidine changes to an alternate

Pyrimidine. Transversions is a position with a Pyrimidine changes to have a

Purine; or, Purine to Pyrimidine.

35

Morphological Mutants have altered shape. Lethal Mutants die as a result

of having the mutation. Conditional Mutants are normal under one condition

(permissive), but abnormal under another (restrictive). Biochemical Mutants

cause defects in biochemical pathways for a substance, which is then deficient.

Mutation mechanism

A mutagen or mutagenic agent is a substance that increases the mutation

rate more than the naturally occurring rate. These are substances, conditions and

forms of energy that significantly increase the frequency of mutations. Examples

of forms of energy are Ultraviolet light, X-rays, Cosmic energy, Gamma

radiation, Alpha particles, Beta particles and Neutrons. Examples of substances

that are mutagens are Nitrous acid, Hydroxylamine, Ethyl Ethane Sulfonate, 5-

bromouracil, Celery, Benzo (a) Pyrene, Acridine dyes.

Ultraviolet Radiation causes Pyrimidine dimers. Gamma and X-Rays:

These can act directly on DNA. Alkylating agents are chemicals that donate

alkly groups to other molecules. Ethyl Methane Sulfonate (EMS) is an example.

Base analogs are similar to the actual correct base and so get incorporated into

the DNA as would its natural counterpart. The problem is that, if they are more

prone to tautomeric shifts than the natural base, the frequency for mutation goes

up, substantially. The compound 5-bromouracil is an example of an analog to

thymine. It undergoes a tautomeric shift to base pair with guanine instead of

adenine, causing a transition. Deaminating agents cause the loss of the amino

group. Deaminating agents would increase the frequency of Cytosine

deamination, greatly. Nitrous acid is a deaminating agent. Intercalation agents

are compounds that can slide between the nitrogenous bases in a DNA molecule.

This tends to cause a greater likelihood for slippage during replication, resulting

36

in an increase in frameshift mutations. Hydroxylating agents add an OH group to

a position on the DNA base cytosine, causing a G:C to A:T transition.

37

The mechanisms of action of mineral phosphate solubilization, the effect

of carbon, nitrogen and phosphate sources on phosphate solubilization (MPS)

were studied in the wild-type Mps+ Penicillium rugulosum strain and in

negative (Mps−) and superpositive (Mps++) mutants derived from it. Mps+

phenotype was strongly associated with the production of gluconic or citric

acids. (Reyes et al. 1999)

Ray et al. (1999) isolated Corynebacterium species from spoiled coconut,

was found capable of producing extracellular Lipase. The Lipase activity of the

strain was tried to improve by mutagenic technique using a chemical mutagen N-

38

methyl, N-nitro, N-nitroso guanidine. They also investigated the superior carbon

and nitrogen sources for Lipase production by the mutant strain.

A pot experiment was conducted in the green house to investigate the

establishment of phosphate solubilizing strains of Azotobacter chroococcum,

including soil isolates and their N-methyl-Nnitro-N-nitrosoguanidine mutants,

in the rhizosphere. The solubilization capacity and IAA production ability of

mutants were also studied (Kumar et al., 2001).

The high mineral phosphate solublizing wild strain Penicillium

rugulosum and two of its mutants were used to investigate phosphate

solubilization in liquid cultures contains two Venezuelan phosphate rocks and

Utah variscite. Solubilization of the various P sources by the wild-type and the

first mutant resulted mostly from the action of organic acids. The organic acids

are likely involved in the solubilization of the other phosphate rock (Reyes et al.,

2001).

The enhancement of Lipase production from Aspergillus niger was

attempted by ultraviolet (UV) and nitrous acid mutagenesis, and the mutants

were selected on media containing bile salts. Nitrous acid mutants exhibited

increased efficiency for Lipase production when compared with UV mutants in

submerged fermentation. The hyper producing UV and nitrous acid mutants

were further subjected to a second step of mutagenesis to devise an economical

and ecofriendly technique for Lipase production by the effective use of

hydrocarbons (Mala et al., 2001).

An indigenous strain of Aspergillus niger isolated from an oil mill waste

was used to analyze Lipase activity. The mutant strains were isolated by

39

exposing the parent using ultra-violet and N-methyl, N-nitro, N-nitroso

guanidine. The effect of different carbon and nitrogen sources were also studied

on the mutant which produce high Lipase activity (Ellaiah et al. 2002).

Pseudomonas fluorescens strains and their cold tolerant mutants were

examined for their Tri Calcium Phosphate solubilizing activity in broth media at

10°Cand 25°C. Invariably, all the cold tolerant mutants were found more

efficient than their respective wild type counterparts for phosphate solubilization

activity at 10°C as compared to 25°C (Das et al. 2003).

Top of Form

Bottom of Form

A study for screening and selection of cold tolerant mutants of Pseudomonas

fluorescens strains based on phosphate solubilization ability and subsequent

effect on plant growth promotion under in vitro and in situ conditions was

conducted. Of all the mutants tested, two were selected, as there was a 21-fold

increase in one mutant and a 10-fold decrease in another mutant over their

respective wild types. (Katiyar and Goel. 2003).

Chakraborty et al. (2003) made a attempt to isolate hyper motile mutant

of Bradyrhizobium spp. through UV mutagenisis. A 30W germicidal lamp

exiting most light rays at 256 nm was used as UV source for the purpose of

inducing mutations. A 10 ml suspension of washed cells from an exponential

phase in phosphate buffer was taken in a sterilized petri dish and exposed to

UV rays in a dark from a distance of 20 cm with continuous shaking. Samples

of 0.1 ml were withdrawn from the plate at an interval of 10 sec and inoculated

into Yeast Extract Mannitol broth to examine the growth of suffering cells.

Bapiraju et al. (2004) investigated the Lipase activity by Rhizopus species

isolated from coconut oil mill waste. They improved the Lipase activity of the

40

strain by natural selection and random mutagenesis by UV and N-Methyl, N-

Nitro, N-Nitroso Guanidine.

Mutants of Aspergillus niger NCIM 1207, isolated by subjecting conidia

to UV-irradiation, were tested for the production of Lipase. Mutants UV-10 and

ANCR-1 showed seven fold and five fold enhanced productivity of enzyme,

respectively, over the wild strain in shake flask culture when grown in SOB

medium containing 1% olive oil. Maximum Lipase activity was obtained in the

culture broth when UV-10 was grown in medium supplemented with 0.5%

Triton X-100. A higher concentration of oil in the medium did not help Lipase

production in the case of mutant UV-10. Similarly no increase in enzyme levels

was observed when mutant UV-10 was grown in medium supplemented with

Glucose. However, the addition of Glucose in the medium resulted in increased

levels of Lipase production by wild strain, Aspergillus niger NCIM 1207

(Mahadik et al., 2004).

Azospirillum sp was isolated from soil sample paddy field at

Thirumangalam, Madurai. The isolated Azospirillum sp. was treated with UV

rays Acridine Orange and Ethidium Bromide. The mutants were tested for the

production of auxin under trp (-) & trp (+) condition compared with wild strains.

The mutants produced more auxin than the wild type. The results revealed that

the Auxin production by mutant was independent of Tryptophan (Kulanthaivel et

al., 2006).

Twenty-three bacterial isolates were screened for their mineral

phosphate–solubilizing ability on Pikovskaya and National Botanical Research

Institute’s Phosphate (NBRIP) agar. The majority of the isolates exhibited a

strong ability to solubilize Hydroxyapatite in both solid and liquid media. The

41

solubilization in liquid medium corresponded with a decrease in the pH of the

medium. Serratia marcescens GPS-5, known for its biocontrol of late leaf spot

in groundnut, emerged as the best solubilizer. S. marcescens GPS-5 was

subjected to Ethyl Methane Sulfonate mutagenesis, and a total of 1700 mutants,

resulting after 45 minutes of exposure, were screened on buffered NBRIP

medium for alterations in mineral phosphate solubilizing ability compared with

that of the wild type (Tripura et al., 2007).

Phenotypic mutants of Aspergillus tubingensis were obtained by UV

irradiation and phosphate solubilization ability of these mutants were studied and

compared with the wild type strain. These mutants also showed maximum Acid

Phosphatase and Phytase activity. These results suggest that phosphate

solubilization by these isolates is due to lowering of pH of the culture filtrate and

also the activity of Acid Phosphatase and Phytase (Achal et al. 2007).

The ability of several Pseudomonas spp. to solubilize Ca3 (PO4)2 was

compared. While all Pseudomonas sp were found to facilitate a decrease in pH

and solubilize inorganic phosphate by the production of extracellular organic

acids, strains varied by producing either gluconic or 2-ketogluconic acid. It was

further investigated that the genetic mechanisms involved in inorganic phosphate

solubilization by Pseudomonas spp., a transposon mutant library of P.

fluorescens was screened for mutants with reduced Ca3 (PO4)2 solubilization

ability. Mutations in the gcd and pqqE genes greatly reduced the solubilization

ability, whereas mutations in the pqqB gene only moderately reduced this ability.

The combination of biochemical analysis and genomic comparisons revealed

that alterations in the pqq biosynthetic genes, and the presence/absence of the

gluconate dehydrogenase (gad) gene, fundamentally affect phosphate

solublization in strains of P. fluorescens (Miller et al., 2010).

42

43

Phosphatase

The effect of phosphate on the production of phosphatases by Aspergillus

awamori var. kawachii was studied. The concentration of phosphatase,

produced, was distinguished between high and low phosphate medium. The

phosphatase fractions produced in the low phosphate medium were different

from those of the high phosphate medium (Ohta et al., 1968).

A simple method of assaying soil phosphatase activity was described. It

involves colorimetric estimation of the p-nitrophenol released by phosphatase

activity when soil is incubated with buffered (pH 6·5) Sodium p-nitrophenyl

phosphate solution and toluene at 37° C for 1 hr. The method is rapid and

precise, and it has significant advantages over methods previously proposed for

assay of soil phosphatase activity (Tabatabai and Bremner. 1969).

The occurrence and distribution of a special group of bacteria, capable

of dissolving insoluble phosphates, were studied in marine environments,

especially in sediments. The phosphatase activity was also investigated. There

was a positive correlation between the total phosphate content and the

phosphatase activity. The phosphatase activity was recorded in all samples,

irrespective of salinity variations (Ayyakkannu and Chandramohan, 1971).

A group of yeasts isolated from burned children and 14 reference strains

were tested for phosphatase activity by using phenolphthalein phosphate

substrates. Phosphatase activity was widely distributed among various species

and strains representing seven genera. The greater enzyme activity of the yeast

cell was not related to more rapid or greater cell growth or decrease in the pH of

culture media. Extracellular constitutive heat-labile acid phosphatase was found

in broth filtrates of some strains (Smith et al., 1973).

44

The bacteria Lysobacter enzymogenes was studied for it’s extracellular

phosphatase during the stationary phase of growth. The cells also produced a

cell-associated alkaline phosphatase. This enzyme is found in the particulate

fraction of cell extracts and may be membrane bound. The production of both

phosphatases, especially the extracellular enzyme, is reduced by inorganic

phosphate (Richard and Tigerstrom, 1984).

The thermostability properties of three Acid Phosphatases viz, Aspergillus

fumigatus Phytase, Aspergillus niger Phytase, and A. niger Acid Phosphatase

(pH 2.5), were investigated by measuring enzymatic activity. The Phytases of

A. fumigatus and A. niger were both denatured at temperatures between 50 and

70°C. A. niger pH 2.5 acid phosphatase displayed considerably higher

thermostability, denaturation, conformational changes, and irreversible

inactivation were observed only at temperatures of 80°C (Wyss et al., 1998).

A Phosphatase overproducing Citrobacter sp. was grown in an air-lift

reactor in steady state continuous culture under limitation of Carbon, Phosphorus

or Nitrogen. Substantial biofilm formation and the highest Phosphatase activity,

were observed under Lactose limitation (Allan et al., 2002). The fungus

Rhizopus delemar produced extracellular and cellular acid phosphatase during

the growth in starch-supplemented medium in the presence or absence of copper

ions. The levels of both acid phosphatase activities were observed. The pH

optimum of the enzyme was determined to be in the range of 3.5–4.5 using p-

nitrophenyl phosphate as a substrate (Tsekova and Galabova. 2003).

The effect of several carbon sources on the production of Alkaline

Phosphatase by the thermotolerant Aspergillus caespitosus was analysed. The

45

pH of the medium likely affects the process of enzyme secretion according to the

carbon source used. (Guimaraes et al., 2003). Sarapatka et al. (2004) studied

Acid Phosphatase activity on the root systems of cereal varieties and also in

nutrient medium on which crops were planted under conditions of changing pH

and phosphorus supply. After 10 days of cultivation the plant roots were

harvested, homogenized and the acid phosphatase activity was measured. The

correlation of increasing pH and available phosphorus level in the nutrient

medium with acid phosphatase activity in the root system of various species and

cereal cultivars was recorded. The Phosphatase Activity of Ectomycorrhizal

roots and nutrient status of Pine seedlings inoculated with Mycorrhizal fungi

were studied under different physical factors, namely relative humidity, light

intensity and concentration of acid phosphatase activity (Jha et al. 2006).

Padmapriya and Anand (2010) studied nine filamentous cyanobacterial

isolates for the screening for amylase, protease, beta lactamae and phosphatase

enzymes. They stated that the extracellular activity of acid phosphatase enzyme

in the non-heterocystous forms, Phormidium minnesotensis and P. boryanum,

showed a decrease as the growth increased, reaching negligible levels on the 20th

day. In the heterocystous forms, both the acid phosphatase extracellular and

intracellular activities were absent up to a period of 8 days. In the case of

alkaline phosphatase intracellular activities, a gradual decrease in the enzyme

content from the first interval is observed in almost all the forms.

Phosphatase and Phosphate Solubilization

Bacterial strains were obtained from a soil sample and culture collection

center and they were used for enzyme production in a low-concentration

phosphate medium. Various concentrations of Sodium Phosphate or Calcium

were added in experiments to study the regulation of phosphatase synthesis.

46

Alkaline Phosphatase activity was measured spectrophotometrically by

monitoring the release of para nitro phenol from para nitro phenyl phosphate

(PNPP) at 400 nm (Prada et al. 1996).

The use of Phosphate Solubilizing Bacteria as inoculants simultaneously

increases phosphate uptake by the plant and crop yield. Strains from the genera

Pseudomonas, Bacillus and Rhizobium are among the most powerful phosphate

solubilizers. The principal mechanism for mineral phosphate solubilization is the

production of organic acids, and Acid Phosphatases play a major role in the

mineralization of organic phosphorous in soil. Several Phosphatase encoding

genes have been cloned and characterized and a few genes involved in Mineral

Phosphate Solubilization have been isolated. Therefore, genetic manipulation of

Phosphate Solubilizing Bacteria to improve their ability to improve plant growth

may include cloning genes involved in both mineral and organic phosphate

solubilization, followed by their expression in selected rhizobacterial strains

(Rodriguez and Fraga.1999).

Seawater and sediment samples were collected and were screened for

phosphate solubilization by streaking them on to Hydroxyapatite medium11. The

phosphate solubilization was expressed as positive and negative depending on

the halo formation. The cultures which showed halo formation around their

colonies were considered to be the phosphate solubilizers. These solubilizers of

inorganic phosphate were further screened for Phosphatase production (De

Souza et al., 2000).

The strains, viz., phosphobacteria, yeast and mildew were compared for

their Phosphorus Dissolving Mechanism and phosphorus solubilizing abilities.

Acids, Acid Phosphatase, and Alkaline Phosphatase which have synergic effects

47

on phosphate dissolution were studied. The variation in activities of Acid

Phosphatase and Alkaline Phosphatase of Phosphate Solubilizing Microbes were

also examined (ChuanQing and WeiYi.2005).

Ponmurugan and Gopi (2006) conducted an experiment to enumerate the

population density of phosphobacteria in the rhizosphere soils of various crops

using Ketznelson and Bose medium following dilution plate technique. Efforts

have been made to isolate phosphobacteria from these soils and isolated strains

were inoculated in specific media containing specific substrates to produce

growth regulating substances such as Indole Acetic Acid and Gibberellic Acid

and Phosphatase enzyme.

Aspergillus niger was studied for its Acid Phosphatase and Invertase

activity in defined media supplemented with various sucrose concentrations.

Ontogenic changes in extracellular, cytoplasmic, and wall-bound enzyme

activities of A. niger were studied. Growth in terms of fresh weight showed

inverse correlation with pH. At higher pH values, both enzyme activities were

higher in the medium supplemented with low sucrose concentration. It was

observed that the more the fresh weight of fungi decreased, the greater was the

enzyme activity observed (Pawar and Thaker, 2009).

Lipase

Lipases are produced by a number of microorganisms, including bacteria,

fungi, and yeasts. In recent years, research on microbial Lipases has increased

because of their practical applications in industry, as in the hydrolysis of fats,

production of fatty acids and food additives, synthesis of esters and peptides,

resolution of racemic mixtures, or additives in detergents.

48

Constein and Thomas (1902) reported that the difference in the sources of

Lipase used by different workers, the diversity of the data report on this subject

seems to partly due to the fact that the many efforts thus far made to purify the

enzyme have not met with definite success.

Juichiro et al. (1963) reported that the Lipase is one of the enzymes

whose chemical and enzymological properties have not yet been fully elucidated

despite the vast literature accumulated in the past. Conflicting reports exists

concerning its requirement for co-factors, specific to substrates, mechanism of

reactions and other properties started their experiment with an extensive

screening test of moulds which led to selection of several strains of Rhizopus and

Aspergillus niger as most potent Lipase formers. Successful isolation of the

enzyme could be made from the bran-koji culture of Aspergillus niger, and the

enzyme was finally obtained in a crystalline form.

Wen Hsiung et al. (1972) isolated a thermophilic fungus which can

produce a thermostable Lipase (glycerol ester hydrolyses) from compost soil and

identified this as Humicola lanuginose. The culture conditions and some

properties of crude Lipase preparation were described in the previous report. In

an attempt to obtain more comprehensive information on the thermostable

Lipase, a lightly purified enzyme preparation is an essential pre-requisite of the

purified Lipase of Humicola lanuginose.

Wen Hsiung et al. (1973) reported on substrate specificity and mode of

action of the Lipase of thermophilic fungus Humicola lanuginose s-38. It was

made clear that mode of action of Humicola Lipase on triolein and on

phosphatidyls ethanolamine was identical. The Humicola Lipase had no activity

of lipoprotein Lipase. Determination of the lipolytic activity after a certain time

49

of incubation, the maximal lipolytic activity and a time integrated lipolytic

activity are compared as estimations for potential hydrolytic capacity of

microorganisms. (Jonsson and Snygg. 1974).

Sugiura et al. (1975) reported that long chain fatty acids generally had an

effect on Lipase production by Candida paralipolytica in both the cells and the

broth. A study on the influence of olive oil in Lipase production by Rhizopus

species, Penicillium roquefortii and Mucor species showed that the cellular

activity increased in some cases and decreased in others. Nobuhiro and Omar

(1977) isolated many microorganisms which are known to produce different

types of Lipases. Almost all microbial Lipases which have been reported can be

regarded as acid Lipases or neutral Lipases when they are classified by their

optimum pH values.

Watanabe et al. (1977) identified the alkaline Lipase producing

microorganisms, their culturing conditions and some properties of the crude

enzymes obtained. Oso (1978) carried out a study on the ability of Tararomyces

emersionstolk to produce extracellular Lipase in stationary liquid medium under

various conditions. The best temperatures for Lipase synthesis and activity were

40-45°C, and Lipase production was tested at all the temperature (37-55°).

Akhtar and Nadeem (1983) proposed the effect of triglycerides on the

growth of Mucor hiemalis and the production of Lipase and mycelial lipids on

addition of 1% triglycerides to the fermentation medium. The added

triglycerides seemed to be utilized through the formation of free fatty acids. The

lipids produced per gram mycelia were high initially (260mg/g dry weight at 48

hrs), and showed reduced production later. With increase in growth the

50

maximum mycelia lipids per 100ml of culture medium was obtained from

176mg/100ml.

Recent investigations have shown that enzymes have potential in large

scale processing of lipid material, particularly in the areas of fat splitting,

synthesis reversal of hydrolysis and interiesterification. (Posorske. 1984).

Warner et al. (1984) set out to determine the kinetics of lipolysis of olive oil,

coconut oil and tallow and the effects of temperature concentration of Lipase and

pH on commercially available Lipase from Candida rugosa. The effects of

calcium and sodium ions in olive oil lipolysis at room temperature were

determined in the present study.

Dow and Fhee (1986) developed simple and rapid calorimetric method to

determine the Lipase activity for fat splitting. A further simplification of the

method of Lowry and finally for determination of free fatty acids by eliminating

the solvent evaporation and centrifugation steps for Lipase assay. Macrae and

Hammond (1985) reported that Lipase produced by thermophilic fungi have

drawn great deal of attention lately due to their possible application in the food,

pharmaceutical and detergent industries. With newer applications in light, more

active Lipases are being sought from microorganisms.

Nahas (1988) initiated to investigate the effect of different growth media

and conditions on Lipase production by Rhizopus oligosporus, after studying the

factors influencing the growth and production of extracellular Lipase by

Rhizopus oligosporus. The yields of Lipase was maximum at 25°C and pH 6.5

after 3 days shaking culture which enhanced growth but decreased Lipase

production.

51

Johri et al. (1990) studied extracellular Lipase of Sporotrichum

thermophile. It was found that 15%-20% of the enzyme remained cell bound. It

was therefore of interest to examine Lipase secretion by protoplasts derived from

the mycelium. Since immobilization of protoplasts can improve stability and

reusability, entrapment of protoplasts of Sporotrichum thermophile in alginate

was attempted.

Martin (1990) presented his study on the Lipase catalyzed alcoholysis

sunflower oil in order to optimize conditions for the synthesis. Gerald and

Tsuneo (1991) presented that it is possible to obtain nearly theoretical yields of

monoglycerides (approximately 90% wt) using Pseudomonas sp. Lipase and a

yield of approximately 80% wt using Mucor miehei Lipase under appropriate

conditions.

Valerie et al. (1992) isolated strains of Penicillium cyclopium,

Aspergillius niger Aspergillis flavus and Rhizopus arrhizus secrete extracellular

enzymes exhibited good synthetic activities in organic solvent. A loop fixed-bed

reactor was designed for the continuous synthesis of esters.

Lipase biosynthesis occurred in medium without lipids, but for improved

production an inducer was needed. The source and concentration of an inducer

had no significant effect. Starch as an additional carbon source stimulated Lipase

biosynthesis when used in small amounts. Addition of NH4NO3 as a nitrogen

source, KH2PO4 as a phosphate source as well as Mg ions to the medium with

initial pH 5.0 gave the best yield (Pokorny et al., 1994).

There are other reports on the application of microbial Lipase to the

hydrolysis of racemic esters transesterification and to racemization in situ to

52

yield optically pure enantiomers for the manufacture of chiral symptoms. In

addition to racemization in situ Lipases are also capable of catalysing synthetic

reactions, which lead to the production of life-saving drugs (Lee, 1995).

Parmar et al. (1996) reported that because of their excellent capability for

specific region selective reaction in a variety of organic solvents with broad

substrate recognition, Lipases have emerged as an important biocatalyst in

biomedical application. Hilda and Gobinith (1997) reported that oil pollution is a

serious obstacle to photosynthesis, a fundamental life process in plant kingdom,

which affects the food chain and productivity of the sea. Oil is also absorbed by

fish eventually to reach and endanger human life. The lipolytic activity of

physiologically diverse microorganism can be used to degrade oil spills in

marine environment. The fungus Acremonium alternate exhibited varied

efficiency in hydrolyzing the various oils. It degraded olive and ground nut oils

to a maximum efficiency.

Gombert et al. (1999) studied growth and enzyme production by a

Brazilian strain of Penicillium restrictum. Solid waste from the babassu oil

industry was used as the basic nutrient source and was supplemented with

peptone, olive oil or starch at different C:N ratios. Lipase activity was very

sensitive to the kind and the level of supplementation, and decreased as protease

level and pH in the media increased.

Different carbon sources affecting growth and Lipase production in

Candida rugosa were studied by using batch cultures on defined medium.

Carbohydrates and acids non-related to fats and lipids or fatty acids were used

as a carbon sources to induce Lipase production. The highest yields of enzyme

were obtained with lipids or fatty acids as carbon sources (Dalmau et al., 2000).

53

A response surface approach has been used to study the production of

extracellular Lipase by Candida cylindracea. Medium constituents were

optimized for the production of Lipase by central composite design. A 25 design

matrix was used in the experiments with only the carbon source being varied.

The Glucose and olive oil were used as the carbon source All the other medium

constituents namely yeast extract, malt extract, peptone and tween 80 were

simultaneously optimized (Muralidhar et al., 2001).

Coca et al. (2001) used strains from bacteria, fungi and yeast for a

quantitative screening of lipase-production using 2% of olive oil as carbon

source. The production and kinetic characteristics of extracellular lipases

expressed by the best producers such as Aspergillus niger and A. fumigatus were

studied. The more ideal carbon source for lipase synthesis was selected. The

optimum pH and temperature for extract enzymatic activities were also

investigated.

The Lipase production abilty of Rhizopus arrhizus NCIM 877, 878, 879

and Aspergillus niger NCIM 1207 were studied in synthetic oil based medium

under submerged conditions. Rhizopus strains showed major intracellular

activity while A. niger NCIM 1207 produced mainly extracellular activity. The

pH and temperature optima for Lipase production and activity and the efficacy

of Lipase production by submerged and solid state fermentation in A. niger

NCIM 1207 were also studied (Mahadik et al., 2002).

The n-alkane degrading Pseudomonas pseudomallei was investigated for

its extracellular Lipase activity. The stability of the immobilized Lipase in

54

organic solvents, retention of activity, pH and thermo stability of Lipase

produced were studied (Kanwar and Goswami, 2002).

Arya et al. (2003) stated that commercial porcine pancreas Lipase was

immobilized covalently onto alkyl amine glass-beads affixed on the inner wall of

a plastic beaker by an adhesive. The immobilized enzyme retained 10.8% of the

initial activity of free enzyme with a conjugation yield of 52-mg/g supports. The

optimum pH and incubation temperature were decreased, while time for linearity

and λm for triolein of enzyme were increased after immobilization.

Achamma et al. (2003) isolated a bacterium, producing extracellular

Lipases was isolated from coconut oil cake and identified as Bacillus mycoides

by morphological and biochemical characteristics. The incubation period for

maximum growth and Lipase production, optimum pH and temperature for the

production of Lipase were optimized. Shake condition induced more Lipase

production. They also studied the best carbon, nitrogen and mineral source for

Lipase production.

The effect of different lipid sources added to basal medium was verified

to improve Lipase production. Maximum growth and Lipase activity was studied

in the presence of different surfactants. The best temperature for Lipase

production was also recorded (Silva et al., 2005).

A thermophilic isolate Bacillus coagulans produced an extracellular

alkaline Lipase, the production of which was substantially enhanced when the

type of carbon source, nitrogen source and the initial pH of culture medium were

consecutively optimized (Satyendrakumar et al., 2005).

55

The non-conventional yeast Yarrowia lipolytica produces an extracellular

Lipase encoded by the LIP2 gene. Mutant strains with enhanced productivity

were previously obtained either by chemical mutagenesis or genetic engineering.

One of these mutants, named LgX64.81 to select new overproducing strains was

used for amplification of the LIP2 gene. A process for Lipase production in

bioreactors and compared Lipase production levels in batch and fed-batch

cultures were also developed (Fickers et al., 2005).

Rodriques et al. (2006) reported that Rhizopus homothalicus was

cultivated for Lipase production in Solid State Fermentation (SSF) using

sugarcane bagasse as a support and impregnated with a liquid medium. The

production of an extra cellular Lipase from Rhizopus homothallicus was

improved by modification of media nutrients. These results are promising

because these strain producing high Lipase concentrations in an in expensive and

simple medium, which facilitates its recovery and purification. Lipase extraction

from the fermented solid was also studied. Efficient recovery of the enzyme was

used.

The microbial production of extra-cellular Lipase on olive-mill

wastewater by Geotrichum candidum, Rhizopus arrhizus, Rhizopus oryzae,

Aspergillus oryzae, Aspergillus niger, Candida cylindracea and Penicillium

citrinum were investigated. All strains were able to grow on the undiluted olive-

mill wastewater, producing extra-cellular Lipase activity. The efficacy of Lipase

production by the best strain Candida cylindracea on different and nitrogen and

oil source were recorded (Annibale et al., 2006).

Two Lipase-producing yeast strains named Saccharomyces cerevisiae and

Williopsis californica were isolated from Olive oil. The ratio of the aqueous to

56

the organic phase which influence the maximum enzyme activity was

determined. The microbiological analysis carried out on commercial extra virgin

olive oil, produced in four different geographic areas, demonstrated that the

presence of Lipase-producing yeast varied from zero to 56% of the total yeasts

detected, according to the source of oil samples (Ciafardini et al., 2006).

Lipase production in Aspergillus niger was tested by Falony et al. (2006)

using both submerged fermentation and solid-state fermentation on a mineral

culture medium and wheat bran, respectively. The optimization of the culture

medium was carried out for both fermentation. The maximum lipase activity was

obtained during the submerged fermentation in a medium containing Glucose

and olive oil. The lipase activity was reached maximum in solid-state

fermentation process with a medium containing ammonium sulphate and urea.

The optimum pH and temperature for enzymatic activity were also investigated.

The bacteria that could grow on media containing olive mill waste water

(OMW) were isolated and their Lipase production capacities were investigated.

The strain possessing high Lipase activity among 17 strains grown on tributyrin

agar medium was identified as Bacillus sp. The effect of initial pH on the Lipase

activity was investigated in tributyrin medium and the liquid medium

composition was improved by replacing tributyrin, with different carbon sources

(Sevagi et al., 2007).

The strain for Lipase production was Penicillium expansum, isolated from

the waste of a rap oil manufactory in China. The strain was used to produce

Lipase by submerged fermentation. The enzyme so produced was purified from

the fermentation broth by DEAE-Sepharose chromatography (Lianghua et al.,

2007).

57

An extracellular Lipases producing thermophilic Bacillus sp. was

isolated. The strain was used to produce Lipase by submerged fermentation. The

Lipases so produced were purified and characterized by novel techniques.

(Nawani and Kaur, 2007).

Damaso et al. (2008) aimed to produce lipases by solid-state fermentation

using, as substrate, agroindustrial residue supplemented with by-products from

corn oil refining process or olive oil. For a group of ten fungi strains selected in

the first steps, the lipase activity obtained by solid-state fermentation varied from

dry substrate. Among the evaluated strains, the Aspergillus niger mutant was

selected by presenting the best enzymatic production. For the fermentation tests,

two substrates were also investigated. Wheat bran and corn cob, both

supplemented with olive oil. Additionally, three industrial by-products from

corn oil refining (soapstock, stearin and fatty acids) were evaluated as substitutes

to the olive oil in the function of lipases production inducer.

Amara and Salem (2009) studied that two strains of Pseudomonas

aeruginosa for lipase production from Castor oil aiming to control the different

castor oil wastes and technical lipase production. Both strains are able to grow

on media consists of Castor oil and Yeast extract at 37°C. The lipase enzyme

activity at 37, 60 and 75°C in presence of p-nitrophenyl palmitate were

investigated. Lipase produced from the two strains revealed mesophilic and

thermophilic activities. They recommended for using P. aeruginosa lipases for

castor oil waste control and for the production of mesophilic and thermophilic

lipases under mesophilic conditions.

Rani and Panneerselvam (2009) selected and tested certain organisms

for lipase activity. Among the organism tested, Aspergillus fumigatus, A. terreus,

58

Penicillium chrysogenum, P. funiculosum and Fusarium moniliforme were

reported as a high lipase producer. Various environmental parameters like pH,

temperature and nutritional parameters such as carbon and nitrogen sources were

also investigated to optimize the production of lipase.

Pogaku et al. (2010) isolated a bacterial strain from an oil contaminated

soil and was identified as Staphylococcus sp. It was screened for lipase activity

on tributyrin agar and spirit blue agar medium. Maximum lipase production was

observed at 48 h of growth. Peptone was found to be as an ideal nitrogen source

for lipase production. Addition of any nitrogen source other than peptone to the

medium resulted in a significant reduction of enzyme production. Lower lipase

production was noted when an inorganic nitrogen source was used as the sole

nitrogen source. Starch was used as a major carbon source for optimum

production of lipase. Of the natural oils, olive oil was able to induce more lipase

rather than the oils like groundnut, coconut, castor oils. The pH and temperature

effect on lipase production were also investigated.

Out of 64 fungal isolates obtained, 43 exhibited lipase activity. It was

found that maximum lipase activity was obtained in liquid medium by Fusarium

oxysporum. Various physicochemical parameters such as pH, temperature,

incubation period, volume ratio, agitation and effect of different carbon and

nitrogen sources were studied in order to determine the optimum conditions for

lipase production. The lipase present in the broth was partially purified with

ammonium sulphate, ethanol and acetone (Rifaat et al., 2010).

Chouhan and Dawande (2010) studied that the production of extracellular

lipase from P. aeruginosa on Rhodamine B agar plates containing olive oil as

ingredient, analysed spectrophotometrically using olive oil as well as Tween-20

59

as substrate, purified and precipitated by 30% saturated ammonium sulphate,

showed the maximum lipase activity as compared to the crude enzyme.

Salihu et al. (2011) screened ten microorganisms for their potential to

produce lipase in palm oil mill effluent based medium. Among the 10 organisms,

the most promising strain was Candida cylindracea which showed appreciable

activity both on agar plates and liquid cultures. Medium supplementation by

NH4Cl and olive oil led to high enzyme activity. However, supplementation

with organic nitrogen sources resulted in better enzyme activity. Addition of

malt extract, peptone and olive oil into the medium greatly influenced the lipase

production. Among the oils that were tested, olive oil was found to be the best

for the expression of extracellular lipase activity in an optimized palm oil mill

effluent supplemented medium.

The culture and desired growth conditions for the production of

extracellular lipase from Aspergillus niger was extensively investigated by

Hosseinpour et al. (2011). Enzyme production was carried out in a submerged

culture using major nutrients from soya flour as main constituent of the media.

The optimum weight percentage of soya flour, Glucose and olive oil

concentrations on lipase production were defined. Combination of nitrogen

sources such as yeast extract and peptone were found suitable nitrogen sources.

Effect of pH, Temperature and Calcium ions on lipase activity were also

investigated.

Kakde and Chavan (2011) screened the lipolytic activity abnormal

safflower seeds. Dominant fungi were isolated from abnormal oilseeds on Potato

Dextrose Agar. Total twenty fungi were isolated. Out of that lipase enzyme

activity of ten dominant fungi other than Aspergillus sp. was studied by using

60

different nutritional sources like carbon, nitrogen, phosphorus, sulphur,

antibiotic and vitamin sources. It was found that carbon sources like Fructose

and sucrose induces lipase activity while starch, lactose and carboxyl methyl

cellulose inhibits lipase activity. Nitrogen sources like casein and peptone which

are organic forms stimulated maximum lipase enzyme production of storage

fungi. Sulphur sources like calcium sulphate and ferrus sulphate reduced the

lipase enzyme production by storage fungi while, phosphorus source like

disodium hydrogen ortho-phosphate, ammonium phosphate and potassium di-

hydrogen ortho-phosphate stimulated lipase enzyme production. Antibiotic like

ampicillin, norfloxacin and tetracycline reduced the lipase production of storage

fungi. Lipase activity of storage fungi was reduced in presence of vitamin source

like riboflavin while, folic acid and vitamin C stimulated the lipase enzyme

production.

Indole Acetic Acid

Pathogenic strains of Xanthomonas campestris pv. glycines, and

nonpathogenic strains were compared for their ability to produce indole

compounds, including the plant hormone Indole Acetic Acid (IAA) in liquid

media with or without supplementation with L-Tryptophan. Several additional

strains of plant-pathogenic Xanthomonads and Pseudomonads were also tested

for Indole Acetic Acid production to determine whether in vitro production of

Indoles present in culture filtrates were identified by thin-layer chromatography,

high-performance liquid chromatography, UV spectroscopy, mass spectroscopy,

and gas chromatography-mass spectrometry and were quantitated by high-

performance liquid chromatography. All strains examined, produced Indole

Acetic Acid when liquid media were supplemented with L-Tryptophan (Fett et

al. 1987).

61

Bric et al. (1991) developed a new assay that differentiates between

Indole Acetic Acid (IAA) producing and nonproducing bacteria on a colony

plate lift. Medium supplemented with 5 mM L-Tryptophan is inoculated with

isolates of interest, overlaid with a nitrocellulose membrane, and then incubated

until bacterial colonies reach 1 to 2 mm in diameter. The membrane is removed

to a filter paper saturated with Salkowski reagent and incubated until distinct red

haloes form around the colonies. The colorimetric reaction to Indole Acetic Acid

is limited to a region immediately surrounding each colony, is specific to isolates

producing Indole Acetic Acid, occurs within 1 h after the membrane is placed in

the reagent, and is sensitive to as little as 50 p mol of Indole Acetic Acid in a 2-

mm spot. This assay, used for quantifying epiphytic and endophytic populations

of Indole Acetic Acid producing isolates of Pseudomonas syringae subsp.

savastanoi and for detecting Indole Acetic Acid producing colonies of other

Pseudomonads and Erwinia herbicola. The assay provides a rapid and

convenient method to screen large numbers of bacteria.

Auxin production by 131 strains of Pseudomonas syringae subsp.

savastanoi was investigated with the aim of looking for correlations among this

characteristic and the origin of the strains, the types of symptoms, and the host

plant. Most of the P. syringae subsp. savastanoi strains, except those isolated

from ash, produced auxin and harbored Indole Acetic Acid genes. Among ash

strains, which were pathogenic only on ash were found to produce auxin and to

harbor Indole Acetic Acid genes (Garden et al., 1992).

Pseudomonas sp., a potent phosphorus solublizer, also produced

significant levels of siderophore and Indole Acetic Acid. Siderotyping indicated

it was P. aeruginosa siderovar 1. Cadmium, Nickel, and Chromium resistant

mutants were developed and characterized for their PGPR properties. Mutants

62

were stable under non-selective pressure. However, they were able to promote

root and shoot elongation in soybeans at a significant level in the presence of

metals unfamiliar to the wild type (Gupta et al., 2002).

Patten and Glick. (2002) isolated the growth-promoting bacterium

Pseudomonas putida and an Indole Acetic Acid deficient mutant constructed by

insertional mutagenesis. The canola seedling primary roots from seeds treated

with wild-type P. putida were on average 35 to 50% longer than the roots from

seeds treated with the Indole Acetic Acid deficient mutant and the roots from

uninoculated seeds. In addition, exposing mung bean cuttings to the wild-type

strain stimulated the formation of many, very small, adventitious roots.

Formation of fewer roots was stimulated by treatment with the Indole Acetic

Acid deficient mutant. These results suggest that bacterial Indole Acetic Acid

plays a major role in the development of the host plant root system.

Yurekli et al. (2003) determined the physical and chemical conditions

necessary for optimal biosynthesis of Indole Acetic Acid by Lentinus sajor-caju.

Glucose was determined to be superior to sucrose as carbon and energy source.

The synthesis of Indole Acetic Acid in a nitrogen-free medium or in a medium

with low external phosphate was substantially reduced. Light exposed and non-

agitated cultures grown in dark had also reduced levels of Indole Acetic Acid

compared to agitated cultures grown in dark. The highest Indole Acetic Acid

level was determined in cultures grown in Glucose containing medium (pH 7.5)

on a rotary shaker (150 rpm) at 30 °C in dark. The biological activity of Indole

Acetic Acid obtained from the extra-cellular culture of Lentinus sajor-caju was

determined using oat coleptile growth test.

63

Maor et al. (2004) studied the effects of tryptophan, Indole-3-acetic Acid,

and Indole-3-acetamide on Indole-3-acetic Acid biosynthesis in fungal axenic

cultures and on in planta indole-3-acetic acid production by the fungus. Indole-3-

acetic Acid biosynthesis was strictly dependent on external tryptophan and was

enhanced by tryptophan and indole-3-acetamide.

The bacteria Sphingomonas sp, Microbacterium sp, Mycobacterium sp,

Bacillus sp, Rhizobium sp. Rhodococcus sp, Cellulomonas sp, Pseudomonas sp,

and Micrococcus luteus isolated from the roots of greenhouse tropical orchids

were shown to produce Indole-3-acetic Acid (IAA) and to excrete it into the

culture liquid. The presence and activity of Indole-3-acetic Acid were

demonstrated colorimetrically, by thin-layer chromatography, and by biotests.

The associated bacteria varied in their ability to excrete indole compounds (1–

28μg/ml nutrient broth). Addition of tryptophan to the growth medium enhanced

the production of phytohormone (Tsavkelova et al., 2005).

Bhuvaneswari et al. (2006) extracted Indole-3-acetic acid (IAA) and

estimated from three species of Camarosporium and Colleatotrichum falcatum.

Auxin generally occurs bound with an amino acid or sugar. Over six different

precursor molecules for auxins have been reported. Auxin production was

tryptophan dependant. No IAA or any other indoles were detected in the

absence of tryptophan, while in the presence of tryptophan IAA production was

high. IAA production was more in culture filtrate when compared to the

mycelium of the fungi.

Rhizobium isolates from root region of Sesbania procumbens and stem

nodules of S. rostrata and S. procumbens were shown to produce indole-3-acetic

acid (IAA) in culture supplemented with L-Tryptophan. Production of IAA was

64

maximal after 72 h of incubation when the bacteria reached stationary phase of

growth. The cultural requirements were optimized for maximum IAA

production. The effect of carbon and nitrogen sources revealed that Glucose and

potassium nitrate were best promoters for IAA production over controls. The

IAA from this isolate was extracted, purified and identified by thin layer

chromatography. (Sridevi and Mallaiah. 2007).

The microbial community Streptomyces, Bacillus, Erwinia and

Pseudomonas genera were isolated from Paphiopedilum appletonianum and

Pseudomonas, Bacillus and Flavobacterium genera were isolated from

Pholidota articulate, produced indole-3-acetic acid (IAA). Variations in its

biosynthesis among the strains of the same genus were also observed. The

highest auxin level was detected during the stationary growth phase (Tsavkelova

et al., 2007).

The bacterial strains including Mesorhizobium, Azotobacter, and

phosphate-solubilizing bacteria were isolated and tested for siderophore,

ammonia, Indole Acetic Acid production and phosphate solubilization in vitro.

Among the isolates, M. ciceri, A. chrococcum and Bacillus were positive for

IAA, The diameter of the P solubilization zone, phosphate released in liquid

medium and concomitant drop in pH were also recorded (Wani et al., 2007).

Phytohormone-like acting compounds have been suggested to be involved

in the phytostimulatory action exerted by the plant-beneficial rhizobacterium

Bacillus amyloliquefaciens. Analyses with culture filtrates of Bacillus

amyloliquefaciens demonstrated the presence of indole-3-acetic acid (IAA),

corroborating it as one of the pivotal plant-growth-promoting substances

65

produced by this bacterium. In the presence of 5 mM tryptophan, a fivefold

increase in IAA secretion was registered. (Idris et al., 2007).

Forty three strains were isolated from knots induced by Pseudomonas

savastanoi in different olive cultivars. All the selected bacteria were shown to

produce variable amounts of the plant growth hormone indole-3-acetic acid

(IAA). Their gene sequences were analysed by 16s rDNA method (Ouzari et al.,

2008).

The effect of phosphate-solubilizing fungi such as strains of Aspergillus

awamori, and Penicillium citrinum isolated from rhizosphere of various crops,

was observed on the growth and seed production of chickpea plants in pot

experiments. All phosphate-solubilizing fungi isolates were biocompatible and

produced growth-promoting hormone, Indole Acetic Acid (IAA), varying in

concentration (Mittal et al., 2008).

About on hundred rhizobial bacterial strains were isolated, identified and

tested for Indole-3-acetic Acid in Luria Broth medium supplemented with

tryptophan. They used modified nitrocellulose membrane method as a qualitative

test for the ability of IAA production by the isolates (Etesami et al., 2009).

Indigenous soil samples were tested for Phosphate solubilization.

Efficient phosphate solubilizngbacteria were isolated. Effect of four different

media on phosphate solubilization were determined. Auxin production by these

bacteria were determined via bioassay and high performance liquid

chromatography by the bacteria in liquid culture. Indole Acetic Acid and indole

butyric acid were produced by these bacteria in varying concentration with and

without the addition of tryptophan. These bacteria showed stimulatory effects on

66

the growth of root and shoot elongation of Mung beans (Vigna radiata). Three

promising isolates were investigated to establish the effect on plant growth

(Shahab et al., 2009).

The filamentous cyanobacterium Arthrospira platensis strain was isolated

from a rice field. The ability of this strain to synthesize the bioactive compound

Indole Acetic Acid was demonstrated. IAA was extracted from the culture of

Arthrospira platensis strain and its identity was confirmed by thin-layer

chromatography as well as by high-performance liquid chromatography. The

IAA precursor L-tryptophan was required for IAA biosynthesis (Mehboob et al.,

2010).

A total of 216 bacterial strains were isolated from rice rhizospheric soils

in Northern Thailand. The bacterial strains were initially tested for solubilization

of inorganic phosphate, Indole Acetic Acid production, selected strains were

then tested for optimized conditions for Indole Acetic Acid production. It was

found that all strains had solubilized inorganic phosphate. The best Indole Acetic

Acid producer was identified by biochemical testing and DNA sequence

analysis as Klebsiella. In addition to being the best Indole Acetic Acid

producer, this strain was a high phosphate solubilizer. The production of Indole

Acetic Acid was further confirmed by extraction of crude Indole Acetic Acid

from this isolate and subsequent Thin Layer Chromatography analysis

(Chaiharn and Lumyong, 2011).

67

Materials and Methods

Glass wares cleaning

All the glass wares were soaked in Potassium dichromate solution (100 g

of Potassium dichromate in 1 liter of distilled water followed by slow addition of

500 mL concentrated Sulfuric acid) for about 12 hrs and rinsed in tap water.

Finally, they were washed in distilled water, dried and used.

Sterilization

The glass wares were sterilized at 180ºC for one hour in hot air oven. All

the media were autoclaved at 15 lbs pressure for 15 min.

Chemicals

All the chemicals and media used in this work were purchased from Hi-

media, Ranbaxy and Sigma limited and distilled water was used throughout the

study.

pH adjustment

The pH of the medium was adjusted with 0.1N NaOH or 0.1N HCl.

Chemicals used

1. Sodium Azide (Sigma) Chemical Mutagen.

2. Ethyl Methane Sulphonate (Sigma) Chemical Mutagen.

3. Ammonium Molybdate (Ranbaxy) for Chloromolybdic acid reagent

preparation.

4. Stannous Chloride (Ranbaxy) for Chlorostannous acid reagent

preparation.

5. Hydrochloric acid (Ranbaxy) Solution preparation and pH adjustment.

68

6. Phosphate Buffer (pH 7.0)

Dissolve 1.20g of sodium dihydrogen phosphate and 0.885g of disidium

hydrogen phosphate in 1 liter volume distilled water.

7. Chloromolybdic acid reagent for Phosphate Estimation

Ammonium molybdate (15g) was dissolved in 400ml of warm distilled

water. The solution was cooled and 300ml of 10.0N Hydrochloric acid

was added slowly. This was diluted to 1000ml after cooling.

8. Chlorostannous acid reagent for Phosphate Estimation

Stannous chloride (25g) was dissolved in 50ml of concentrated

Hydrochloric acid and was diluted to 500ml using recently boiled

distilled water. This solution was diluted with 1.2N HCl to about one

liter.

9. Sulphuric acid (Ranbaxy) (7N) for Phosphate estimation

196 ml concentrated sulphuric acid in 1000 ml distilled water.

10. Staining solutions Kit (Himedia) contains solutions of Gram Crytal Violet, Gram Iodine andGram Safranin for Gram reaction of bacteria.

11. Kovac reagent (Himedia) for Biochemical identification of bacteria.

12. Barrit’s reagent (Himedia) for Biochemical identification of bacteria.

13. Lacto Phenol Cotton Blue (Himedia) Spore staining of fungal cultures.

14. Potassium di-hydrogen orthophosphate (Ranbaxy)for Standard graph

preparation for Phosphate estimation.

15. Paranitro phenyl phosphate (PNP-P) (Sigma) (1%) : Substrate for Acid

Phosphatase assay.

Dissolve 1 g of paranitro phenyl phosphate in 100 ml of distilled water.

16. Para nitro phenol (PNP) (sigma): Preparation of standard graph for Acid

Phosphatase assay

Dissolve 1.5 g of paranitro phenol in 100 ml of distilled water.

17. Magnesium chloride (Ranbaxy) (1mM) for Acid Phosphatase assay

Dissolve 20.331 mg of Magnesium Chloride in 100 ml of distilled water.

69

18. Sodium Hydroxide (Ranbaxy) (0.2M) for Acid Phosphatase assay

Dissolve 8 g of pellets Sodium hydroxide in 1000 ml of distilled water.

19. Sodium Acetate buffer (Ranbaxy) (0.1 M, pH 5.8) for Acid Phosphatase

assay

a) Dissolve 0.6g of Sodium acetate in 100 ml of Distilled water.

b) Dissolve 0.6 ml of acetic acid in 100 ml of distilled water.

From ‘a’ take 90 ml and from ‘b’ take 10 ml make the final volume of

100 ml.

20. Gum Arabic (Ranboxy) for Lipase Assay

3g Gum Arabic in 100 ml distilled water.

21. Olive Oil (Himedia) for Lipase Assay.

22. O.1 M Phosphate buffer (pH 7.0) for Lipase Assay.

23. Alcoholic Phenolphthalein indicator (Himedia) for Lipase Assay.

24. Sodium Hydroxide (Ranbaxy) (0.02N)

Diluted from 1.0N Sodium hydroxide stock solution. (20ml stock made

up to 1000ml with distilled water.

25. Sodium Hydroxide (Ranbaxy) (1.0N)

40 g Sodium Hydroxide pellets dissolved in distilled water and made up

to 1000ml with distilled water.

26. Silica gel G (Ranbaxy) for Thin Layer Chromatograpy.

27. Salkowski reagent for Indole Acetic Acid estimation

2.03 g Ferric chloride dissolved in 500 ml water and 300 ml concentrated

sulphuric acid.

28. Ethyl acetate, Chloroform and Formic acid (Ranbaxy) solvent system for

Indole acetic Acid identification by Thinlayer Chromatograpy.

29. Glucose, Sucrose, Lactose and Mannitol (Ranboxy) act as carbon sources.

30. Ammonium sulphate, Potassium Nitrate, Sodium Nitrate and Urea

(Ranboxy) act as nitrogen sources.

70

Media used

Nutrient agar medium (Hi-media) for isolation and culture maintenance.

Sabouraud agar medium (Hi-media) for isolation and culture

maintenance.

Starch agar medium (Hi-media) for biochemical identification of bacteria.

Casein agar medium (Hi-media) for biochemical identification of

bacteria.

Certimide agar (Hi-media) for biochemical identification of bacteria.

MR-VP broth (Hi-media) for biochemical identification of bacteria.

Christensen citrate agar medium (Hi-media) for biochemical

identification of bacteria.

TSI agar medium (Hi-media) for biochemical identification of bacteria.

SIM agar medium (Hi-media) for biochemical identification of bacteria.

Urea agar base (Hi-media) for biochemical identification of bacteria.

Gelatin agar (Hi-media) for biochemical identification of bacteria.

Trypticase Nitrate broth (Hi-media) for biochemical identification of

bacteria.

Asparagine- Proline broth (Hi-media) for biochemical identification of

bacteria.

Pikovskaya medium (Hi-media) for isolation of Phosphate solubilizing

bacteria.

Pikovskaya broth (Hi-media) for Phosphate estimation and Acid

Phosphatase assay.

Czapek’s dox broth (Hi-media) Lipase and Indole Acetic Acid assay.

Carbohydrate fermentation medium contains Glucose or Sucrose or

Lactose or mannitol as a carbon source for biochemical identification of

bacteria.

71

Soil sampling

Different types of rhizosphere soil were collected from the rice fields in

and around Mannachanalur area of Trichy ditrict, Tamilnadu for the isolation of

bacterial and fungal cultures.

Isolation of phosphate solubilizing Microorganism

(Manjugupta and Bhriguvansji, 1997).

These soils were mixed and subjected to serial dilution technique using

the stock soil suspension which contains 10 g of soil sample in 100 ml of sterile

distilled water. From the serial diluted suspension, 1 mL of suspension from

10-3 to 10-6 dilution was taken for pour plate technique in Nutrient agar,

Sabouraud agar and Pikovskaya agar medium. The Nutrient agar plates were

observed after 2 days of incubation for enumeration of bacterial colonies, the

Sabouraud agar plates were observed after 3days of incubation for enumeration

of fungal colonies. After 2-3 days of incubation, the plates were observed for

phosphate solubilizing bacterial and fungal cultures from Pikovskaya agar

medium. The bacterial cultures were then transferred to nutrient agar slants and

fungal cultures to the Sabouraud agar slants for further studies.

Identification of Bacteria (Cowan, 1974) and Fungi (Josephgilman, 2001)

The bacterial culture was identified up to genus level by cultural,

morphological and biochemical characteristics. From the 24 hrs broth culture of

the isolated bacteria, 1ml of the culture was inoculated into Kings B agar,

Cetrimide agar, Asparagine –Proline broth, MR-VP broth and carbohydrate

fermentation tubes. One loop ful of culture was inoculated into Christensan

citrate agar, Urea agar, Gelatin agar, Starch agar, SIM medium (Stab) along with

Oxidase and Catalase test.

72

After 24 – 48 hrs incubation, the results were observed in the Kings B

agar and Cetrimide agar for colony morphology and pigmentation of the

bacteria. Asparagine –Proline broth was observed for its fluorescents activity in

UV transilluminator and other tests for the biochemical characterization purpose.

The bacterial culture was identified based on the observed results using

bacteriological manual.

The fungal cultures were identified based on the colony morphology and

spore structure. The spore structure was identified microscopically by Lacto

phenol cotton blue method.

The above isolated bacterial and fungal cultures were tested for its

Phosphate solubilization property and Phosphatase, Lipase and Indole Acetic

Acid producton cabablity in liquid broth.

Phosphate solubilization in liquid broth

Inoculation (Sudhansupal, 1999; Varshanarishan et al., 1995)

The pure bacterial culture from nutrient agar slant was inoculated into

nutrient broth. After 18 hrs of incubation, 0.5 mL of nutrient broth culture (1

O.D) was inoculated into 100 mL Pikovskaya broth containing 1g of Tricalcium

Phosphate. The flask was incubated at 37 ºC. The Pure fungal cultures from

Sabouraud agar slants were transferred to Sabouraud agar plates. After four

days of incubation at 27 ºC, two 8 mm disc were cut from the plates by sterile

borer from all three fungal plates and were inoculated into 100 ml Pikovskaya

broth separately. The flasks were incubated at 27 ºC. Uninoculated Pikovskaya

broth served as control in each case. Each experiment was done in triplicate set.

73

Phosphate estimation (Artidave and Patel, 1999; Seshadri, 1995)

Growth medium was withdrawn aseptically at three days interval from

each flask and centrifuged at 10, 000 rpm for 20 min. The supernatant was

analyzed for phosphate content by Chlorostannous Reduced Molybdophosphoric

Blue Color Method. The clear 10 mL of supernatant was collected in a 50 mL

volumetric flask and 10 mL of chloromolybdic acid reagent was added along the

sides to the flask. The content was diluted to 40 mL and 5 drops of

chlorostannous acid reagent was added. After thorough mixing the volume was

made up to 50 mL quickly. The optical density was measured at 600 nm and the

value was obtained with the help of the standard curve. Pigments produced by

some of the cultures interfere with color development which was overcome by

the addition of 1g of activated charcoal to each flask. The solution was filtered

and the filtrate was taken for further analysis.

Preparation of standard graph

Potassium di-hydrogen orthophosphate (0.2195g) was dissolved in

400 mL distilled water and 25 mL of 7N H2SO4 was added. This solution was

made up to 1000 mL with distilled water, which served as stock phosphate

solution. From that 20 mL solution was taken and diluted to 500 mL, which was

secondary phosphate solution. From the secondary solution 0.5 mL, 1 mL, 2.5

ml, 5 mL, 7.5ml, 10.0ml, 12.5ml, 15.0ml, 20.0ml and 25ml solutions was taken

and Phosphate estimation was carried out and Optical Density (O.D) value was

obtained as described above. The O.D values (X-axis) and phosphate in ppm

(Y-axis) were plotted on a graph.

Random UV mutational studies for Bacteria (Kumar and Dhruv, 1990)

The 24 hrs old parent culture was used for the preparation of bacterial

suspension, which was diluted with phosphate buffer (pH 7.0) contains 108

74

cells/ml. The above culture was inoculated over the surface of nutrient agar

plates. Then the plates were exposed to UV light (2600 Ao) at a distance of 15cm

in different time intervals and one plate was kept as growth control without

exposing to UV light for control. After the above process the plates were

covered with black paper to avoid light induced repair mechanism and were

incubated at 37ºC for 2 days.

Then the cultures were tested for phosphate solubilization as described

above.

Random UV mutational studies for Fungi (Ellaiah et al., 2002)

The growth of 72 hrs old fungal cultures were scrapped off in 5ml sterile

distilled water and diluted with 45 ml of sterile distilled water containing Tween

80 (1:4000). Sterile glass beads were added and were shaked on a rotary shaker

for 30 min to break the hyphal mycelium. The suspension was filtered by using

sterile cotton cloth to remove the mycelium. The spore suspension was prepared

in phosphate buffer (pH 7.0) containing 106 spores per ml. Five ml quantities of

the spore suspension was pipette aseptically into sterile petri dishes of 90 mm

diameter having a flat bottom. Then the plates were exposed to UV light (2600

Ao) at a distance of 15cm from the center of the Germicidal lamp (UV light

source) in different time intervals.

The exposure times were 10, 20, 30, 40, 50, 60, 70 and 80 min. Each UV

exposed spore suspension was stored in dark overnight to avoid photo

reactivation, then was serially diluted in phosphate buffer and plated on

Sabouraud agar medium. The plates were incubated for 5 days at 27°C and the

number of colonies in each plate were counted. Each colony was assumed to be

formed from a single spore.

75

Then the cultures were tested for phosphate solubilization as described

earlier.

The unexposed fungal suspension was inoculated into the Sabouraud

agar medium that served as a growth (positive) control.

Random Chemical mutational study (Bapiraju et al., 2004)

Sodium Azide and Ethyl Methane Sulphonate (EMS) were the chemical

mutagens used in the present study for the strain improvement of phosphate

solubilizers. The spore suspension of fungal cultures was prepared by using

phosphate buffer pH 7.0 as described earlier. To 9 ml of spore suspension, 1 ml

of sterile solution of Sodium Azide (250 μg ml-1 in phosphate buffer) and Ethyl

Methane Sulphonate (EMS) (150 μg ml-1 in phosphate buffer) was added. The

24 hrs old bacterial culture was used for the preparation of bacterial suspension,

which was diluted with phosphate buffer (pH 7.0) contains 108 cells/ml. To a 9

ml of bacterial suspension, 1 ml of sterile solution of Sodium Azide (250 μg ml-1

in phosphate buffer) and Ethyl Methane Sulphonate (EMS) (150 μg ml-1 in

phosphate buffer) was added. The above culture was inoculated and Samples

were withdrawn from the reaction mixture at an interval of 30, 60, 90, 120 and

150 min. and centrifuged for 10 min. at 5000 rpm. The cells were washed three

times with sterile distilled water and again re-suspended in 10 ml sterile buffer.

The samples were serially diluted in the same buffer and plated over Nutrient

agar and Sabouraud agar medium for bacteria and fungi respectively.

Then the cultures were tested for phosphate solubilization as described

earlier.

76

The unexposed fungal suspension was inoculated into the Sabouraud

agar medium that served as a growth (positive) control.

Effect of different carbon sources

To study the effect of different carbon sources on phosphate

solubilization, the carbon source of Pikovskaya broth i. e. Glucose was replaced

by Lactose, Sucrose and Mannitol. The mutated cultures which were superior in

Phosphate solubilization were inoculated, incubated and Phosphate

solubilization assay was carried out as described earlier.

Effect of Different Nitrogen Sources

To study the effect of different nitrogen sources on phosphate

solubilization, the nitrogen source of Pikovskaya broth i.e. Ammonium Sulphate

was replaced by Potassium Nitrate, Sodium Nitrate and Urea. The mutated

cultures which were superior in Phosphate solubilization were inoculated,

incubated and Phosphate solubilization assay was carried out as described

earlier.

Acid phosphatase Production

Inoculation (Sudhansupal, 1999; Varshanarishan et al., 1995)

The pure bacterial culture from nutrient agar slant was inoculated on

nutrient broth. After 18 hours incubation, 0.5 ml of nutrient broth culture (1

O.D) was inoculated in 100 ml Pikovskaya broth containing 1g of Tricalcium

phosphate. The flask was incubated at 37oC. The pure fungal cultures from

Sabouraud agar slants were transferred to Sabouraud agar plates. After four

days incubation at 27oC, two 8 mm disc were cut from the plates of all three

fungal plates and were inoculated into 100 ml Pikovskaya broth. The flasks

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were incubated at 27oC. Uninoculated Pikovskaya broth served as control in

each case. Each experiment was done in triplicate set.

Acid phosphatase assay (Eileen ingham et al., 1979)

The enzyme Acid phosphatase was assayed using para- nitrophenyl

phosphate (PNP-P) as substrate. The reaction mixture contained 2.5 ml (0.1M)

sodium acetate buffer (pH 5.8), I ml (1mM) Magnisium chloride, 0.5 ml 1%

PNP-P and 0.5ml of a suitable dilution of enzyme preparation. One ml of the

reaction mixture was transferred to 2ml of 0.2M Sodium Hydroxide before and

after 15 min incubation at 370C to stop the reaction. The Sodium Hydroxide

solution added before incubation act as a control sample for each analysis. The

amount of Para- nitro phenol (PNP) liberated was measured by recording the

absorbance at 420 nm using an appropriate calibration curve. Activity is

expressed as μmol PNP liberated min-1 .The blank was run in a similar manner

using distilled water.

Preparation of Standard curve

Dissolve 1.0 g of Para- nitro phenol in water, and dilute the solution to

1000 ml. This is standard Para nitro phenol solution. Store the solution in a

refrigerator. To prepare the standard graph, dilute 1 ml of the standard Para-

nitro phenol solution to 100 ml in a volumetric flask and mix the solution

thoroughly. Then pipette 0, 1, 2, 3, 4 and 5-ml aliquots (equaling to 0, 10, 20, 30,

40 and 50 μg of Para- nitro phenol) of this diluted standard solution into 5 ml

volumetric flask and made up to 5 ml by addition of water, and proceed as

described above. The O.D value (Y axis) and corresponding Para nitro phenol

concentration (X axis) were plotted in a graph.

78

Random mutational studies

Physical and chemical mutations were carried out as described earlier.

The bacterial and fungal cultures, obtained after Physical and chemical mutation

were tested for Acid Phosphatase as described earlier.

Effect of Different Carbon Sources

To study the effect of different carbon sources on Phosphate

solubilization, the carbon source of Pikovskaya broth i. e. Glucose was replaced

by Latose, Sucrose and Mannitol. Then the mutated cultures which were

superior in Acid Phosphatase were inoculated, incubated and Acid Phosphatase

assay was carried out as described earlier.

Effect of Different Nitrogen Sources

To study the effect of different nitrogen sources on Phosphate

solubilization, the nitrogen source of Pikovskaya broth i.e. Ammonium Sulphate

was replaced by Potassium Nitrate, Sodium Nitrate and Urea. The mutated

cultures which were superior in Acid Phosphatase actvty in Pikovskaya broth

were inoculated, incubated and Acid Phosphatase assay was carried out as

described earlier.

Lipase Production

Inoculation (Sudhansupal, 1999; Varshanarishan et al., 1995)

The pure bacterial culture from nutrient agar slant was inoculated on

nutrient broth. After 18 hours incubation, 0.5 ml of nutrient broth culture (1

O.D) was inoculated in 100 ml Czapek’s dox broth containing 1% olive oil as

inductor and also 10% olive oil in 100ml Czapek’s dox broth instead of Sucrose.

The pure fungal culture from Sabouraud agar slant were transferred to

Sabouraud agar plates. After four days incubation at 27°C, two 8mm disc were

cut from all three fungal plates and inoculated into 100ml Czapek’s dox broth

79

containing 1% olive oil as inductor and also 10% olive oil in 100ml Czapek’s

dox broth instead of Sucrose. The flasks were incubated at 27° C. Uninoculated

Czapek’s dox broth served as a control in each case. Each experiment was done

in triplicate set.

Lipase assay (Ray et al., 1999)

Growth medium was withdrawn aseptically at three days interval from

each flask and centrifuged at 3000rpm for 15 minutes and the supernatant was

collected and used for lipase activity.

The enzyme solution prepared by mixing the supernatant solution with

1ml of olive oil and 3% gum Arabic in 250ml conical flask and the contents

were emulsified using magnetic stirrer for 5 minutes at top speed.

After emulsification, 10ml of enzyme solution was taken in a flask

followed by 20ml of double distilled water and 5ml of 0.1M phosphate buffer

(pH 7). This mixture was kept on rotary shaker for 30 minutes at 120rpm.

Simultaneously the same protocol was followed except for the enzyme solution

and this was treated as a control. After 30 minutes a drop of 1% alcoholic

phenolphthalein solution was added and titrated against 0.02 N Sodium

Hydroxide (NaOH) till the appearance of pale pink colour.

Lipase activity

One unit of lipase activity was defined as the amount of enzyme required

to release one µ mol of free fatty acid in one min under standard assay

conditions.

Volume of Normality of NaOH consumed × NaOH Lipase activity = ––––––––––––––––––––––––––––––––––––––––––– × 1000 Time of incubation × Volume of enzyme solution expressed as Unit/ Substrate

80

Random mutational studies

Physical and chemical mutation was carried out as described earlier. The

bacterial and fungal cultures, obtained after Physical and chemical mutation

were tested for Lipase as described above.

Effect of different Carbon Sources

To study the effect of different carbon sources on lipase production, the

carbon source of Czapek‘s dox broth i.e. Sucrose was replaced by Lactose,

Glucose and Mannitol. The mutated cultures which were superior in Lipase

activity were inoculated, incubated and Lipase assay was carried out as

described earlier. When Sucrose, Lactose, Glucose and Mannitol as a carbon

source in Czapek’s dox broth, 1% olive oil was used as a inducer.

Effect of different Nitrogen Sources

To study the effect of different nitrogen sources on lipase production, the

Nitrogen source of Czapek’s dox broth i.e. Sodium Nitrate was replaced by

Potassium Nitrate, Ammonium Sulphate and Urea. The mutated cultures which

were superior in Lipase activity were inoculated, incubated and Lipase assay was

carried out as described earlier. When Sodium Nitrate Potassium Nitrate,

Ammonium Sulphate and Urea as a nitrogen source in Czapek’s dox broth, 1%

olive oil was used as a inducer.

Indole Acetic Acid (IAA)

Indole Acetic Acid Production (Maria Guineth et al., 2000)

The isolated bacterial and fungal cultures were subjected to Indole Acetic

Acid production by inoculating the cultures in Czapek’s dox broth.

81

The pure bacterial culture from nutrient agar slants was inoculated on

nutrient broth. After 18 hrs incubation, 0.5 mL of nutrient broth culture (1 O.D)

was inoculated in 100 mL Czapek’s dox broth and incubated at 280C for 3 days.

The pure fungal cultures from sabouraud agar slants were transferred to

Sabouraud agar plates. After 5 days incubation at 270C two 8mm discs were cut

from the fungal plates & were inoculated separately into 100ml Czapek’s dox

broth. The flask were incubated at 300C for 4 days. (Mittal et al., 2008)

Screening of Indole Acetic Acid producer (Maria Guineth et al., 2000)

Culture broth was withdrawn aseptically after three days incubation for

bacteria and five days incubation for fungi from each flask and centrifuged at

3000 rpm for 15 minutes and the supernatant was collected and used for IAA

assay by Thin layer chromatography. The centrifuged sample of each broth were

brought to pH 3.0 and extracted three times with ethyl acetate. The organic

phase was concentrated to dryness and the diluted with 0.5 ml methanol.

Application of this solution on silica gel G plate (20cm X 5cm) and

chromatogram was developed with Chloroform –ethyl acetate-formic acid

(5:3:2) solvent system and then developed with Salkowiski reagent.

Quantitative estimation of Indole Acetic Acid (Tsavkelova et al., 2007)

The quantitative estimation of Indole Acetic Acid in broth cultures were

determined by a colorimetric assay technique using Salkwoski reagent. The two

part of the supernatant, one part of the Salkowski’s reagent was added. The O.D

was determined at 530nm in a spectrophotometer. A standard curve was

prepared from serial dilutions of IAA stock solution.

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Preparation of standard graph for Indole Acetic Acid

1. 100mg of Indole Acetic Acid was weighed and dissolved in 100ml of

distilled water by slight heating. From this stock solution, Indole Acetic

Acid standard solutions were as prepared in different concentrations.

2. 8ml of the Indole Acetic Acid standard solution was taken with 2ml

Salkowski reagent. It was incubated at room temperature in dark for 30

minutes. Blank was prepared by taking 8ml of distilled water with 2ml of

Salkowski reagent.

3. Optical density was read at 530nm and plotted a graph for the

concentration of Indole Acetic Acid against optical density.

Estimation of Indole Acetic Acid

1. 8ml of culture filtrate was taken and 2ml of Salkowski reagent was added

to it. It was incubated at room temperature in dark for 30 minutes for the

development of pink colour.

2. The optical density was read at 530nm.

3. The results were read from standard graph and expressed µg/L

Random mutational studies

Physical and chemical mutation was carried out as described earlier. The

bacterial and fungal cultures, obtained after Physical and chemical mutation

were tested for IAA qualitatively and quantitatively as described above.

83

Results

ISOLATION AND IDENTIFICATION

The Nutrient agar plates were observed after 2days incubation for

bacterial enumeration and the Sabouraud agar plates were observed after 3days

incubation for fungal enumeration. It was found that more number of bacterial

and fungal colonies in 103 and 104 dilution, 255 colony forming units of bacteria

and 25 colony forming units of fungi in 105 dilution, 29 colony forming units of

bacteria and 3 colony forming units of fungi in 105 dilution and in 107 dilution, it

was 3 colony forming units of bacteria and less than 1 colony forming units

fungi after incubation. Finally, it was calculated the initial mother suspension

contains 3 x 107 colony forming units of bacteria per mL and 3 x 106 colony

forming units of fungi per mL. The results were presented in Table 1.

The same serial diluted suspension (103 to 107 dilutions) were inoculated

into Pikovskaya agar the plates were observed after 3 days of incubation for

phosphate solubilizing microorganisms (PSB). The plates were inoculated with

the suspensions such as 103 and 104 dilution, showed more colonies and were too

numerous to count for both bacteria and fungi. In 105 dilution, 16 bacterial and 7

fungal colonies and in 106 dilution, 2 bacterial and 1fungal colonies were found

to be phosphate solubilizers. Based on the results, the between rhizosphere

microbes and phosphate solubilizers were calculated which was 15:1 for bacteria

and 3.5:1 for fungi. The results were presented in Table 2.

The Phosphate solubilizing bacterial cultures were found to be single type

based on colony morphology in Kings B agar and cetrimide agar and gram

reaction by gram staining. Pale white, translucent colonies Circular, convex, and

smooth colonies and Green mucoid colonies were observed in Kings B agar and

84

Cetrimide agar respectively. The gram staining showed gram negative reaction.

Further, it was observed as Indole, Urease, Methyl Red, Vogus Proskauer and

Hydrogen sulphide production were negative and Citrate, Gelatin hydrolysis,

Nitrate reduction, Starch hydrolysis, Catalase and Oxisdase were positive. The

isolated bacteria was motile, produced green color pigment in Cetrimide agar

and Asparagine –proline broth which fluorescents in UV Trans illuminator.

Carbohydrate fermentation tubes showed no gas production and acid production

found on top of the tube.

The isolated bacterial cultures were identified as Pseudomonas sp. by

cultural, morphological and biochemical characteristics. The results were

presented in Table 3.

Plate-1 showes the colonies of Pseudomonas sp. on Nutrient and

Cetrimide agar Asparagine –Proline broth tube in UV Trans illuminator and

Oxidase test.

Among the fungal cultures isolated, three fungal colonies were shown

clear zone around them in Pikovskaya agar medium were selected for further

studies. The fungal cultures were identified as Aspergillus niger, Aspergillus

fumigatus and Penicillium sp. based on the below observations in macroscopic

and microscopic methods.

Aspergillus niger showed black surface pigmentation with a dense felt of

conidiophores. Conidiophores are long, smooth walled and have round shaped

terminal vesicle which support a biseriate of phialides on the vesicle. Over the

phialides are the round conidia forming radial chains.

85

Aspergillus fumigatus showed green surface pigmentation with a dense

felt of conidiophores. Conidiophores are colorless, rough walled and have round

shaped terminal vesicle which support a biseriate of phialides on the vesicle.

Over the phialides are the round conidia forming radial chains.

Penicillium sp. showed green color colonies consisting of a dense felt of

conidiophores. Microscopically, conodiophores show branching, and phialides

produced in groups from branched metulae, giving brush-like appearance.

Conidia are globuse, greenish and smooth.

Plate-2 shows the growth of fungal colonies on Sabouraud agar plates

and Plate-3 shows the Phosphate solubilization of fungal colonies in Pikovskaya

agar.

Random UV Mutagenesis

The bacterial colonies gradually decreased with increasing exposure

time. There was no growth on plates which were exposed to UV light more than

8 minutes. Four bacterial strains resistant for 7 min UV exposure were selected,

designated as PSUV 1, PSUV 2, PSUV 3 and PSUV 4 and transferred to nutrient

broth and agar slants for further study.

The fungal cultures were selected from the plates exposed to 30, 40, 50

and 60 min UV exposure time and they were given the code numbers as

ANuv30, ANuv40, ANuv50 and ANuv60 for Aspergillus niger, Aspergillus

fumigatus mutants were AFuv30, AFuv40, AFuv50 and AFuv60 whereas the

Penicillium species mutants were given the code numbers PEuv30, PEuv40,

PEuv50 and PEuv60. There was no growth observed the plates inoculated with

fungal cultures at 70 and 80 miniutes UV exposure time.

86

Random Chemical Mutagenesis

The Aspergillus niger, Aspergillus fumigatus mutants cultures were

selected from the plates exposed to 30, 60, 90 and 120 min exposure time where

as Penicillium species mutants cultures from 60, 90, 120 and 150 min exposure

time of Sodium azide.

The Sodium Azide treated Aspergillus niger mutants were given the code

numbers ANsa30, ANsa60, ANsa90 and ANsa120, Aspergillus fumigatus

mutants were AFsa30, AFsa60, AFsa90 and AFsa120 whereas the Penicillium

species mutants were given the code numbers PEsa60, PEsa90, PEsa120 and

PEsa150.

The Aspergillus niger, Aspergillus fumigatus mutants cultures were

selected from the plates exposed to 30, 60, 90 and 120 min exposure time where

as Penicillium species mutants cultures from 60, 90, 120 and 150 min exposure

time of Ethyl Methane Sulphonate.

The Ethyl Methane Sulphonate treated Aspergillus niger mutants were

given the code numbers ANems30, ANems60, ANems90 and ANems120,

Aspergillus fumigatus mutants were AFems30, AFems60, AFems90 and

AFems120 whereas the Penicillium species mutants were given the code

numbers PEems60, PEems90, PEems120 and PEems150.

Then the cultures were tested for Phosphate estimation, Acid

Phosphatase, Lipase and Indole Acetic acid production.

87

Phosphate Solubilization

Phosphate Solubilization Capacity of UV Treated Aspergillus niger

The results of Phosphate Solubilization capacity of UV treated

Aspergillus niger were presented in Table 4.

The phosphate solubilization capacity of UV treated Aspergillus niger

(ANuv60) on day three recorded maximum efficacy in solubilization (3.05ppm

of Phosphate) followed by ANuv50 strain (2.85 ppm of Phosphate), ANuv30

(2.65 ppm of Phosphate) and ANuv40 (2.50 ppm of Phosphate). The wild

Aspergillus niger which was used as a standard control solubilized 2.20ppm of

Phosphate in the third day of incubation.

The phosphate solubilization capacity of UV treated Aspergillus niger

(ANuv60) on day six recorded maximum efficacy in solubilization (4.65ppm of

Phosphate) followed by ANuv50 strain (4.35 ppm of Phosphate), ANuv30 (4.05

ppm of Phosphate) and ANuv40 (3.85 ppm of Phosphate). The wild Aspergillus

niger which was used as a standard control solubilized 3.65 ppm of Phosphate in

the sixth day of incubation.

The phosphate solubilization capacity of UV treated Aspergillus niger

(ANuv60) on day nine recorded maximum efficacy in solubilization (5.85ppm of

Phosphate) followed by ANuv50 strain (5.50 ppm of Phosphate), ANuv30 (5.00

ppm of Phosphate) and ANuv40 (4.75 ppm of Phosphate). The wild Aspergillus

niger which was used as a standard control solubilized 4.60 ppm of Phosphate in

the ninth day of incubation.

The phosphate solubilization efficacy of ANuv60 strain increased 38.64%

on third day, 27.40% phosphate solubilization on sixth day and 27.17%

88

phosphate solubilization on ninth day of incubation using wild type as a standard

(100%).

Phosphate Solubilization Capacity of UV Treated Aspergillus fumigatus

The results of Phosphate Solubilization capacity of UV treated

Aspergillus fumigatus were presented in Table 5.

The phosphate solubilization capacity of UV treated Aspergillus

fumigatus (AFuv50) on day three recorded maximum efficacy in solubilization

(3.55ppm of Phosphate) followed by AFuv60 strain (3.35 ppm of Phosphate),

AFuv30 (3.25 ppm of Phosphate) and AFsa40 (3.15 ppm of Phosphate). The

wild Aspergillus fumigatus which was used as a standard control solubilized 2.95

ppm of Phosphate in the third day of incubation.

The phosphate solubilization capacity of UV treated Aspergillus

fumigatus (AFuv50) on day six recorded maximum efficacy in solubilization

(3.95ppm of Phosphate) followed by AFuv60 strain (3.85 ppm of Phosphate),

AFuv30 (3.65 ppm of Phosphate) and AFsa40 (3.65 ppm of Phosphate). The

wild Aspergillus fumigatus which was used as a standard control solubilized 3.25

ppm of Phosphate in the sixth day of incubation.

The phosphate solubilization capacity of UV treated Aspergillus

fumigatus (AFuv50) on day nine recorded maximum efficacy in solubilization

(4.55ppm of Phosphate) followed by AFuv60 strain (4.45 ppm of Phosphate),

AFuv40 (4.15 ppm of Phosphate) and AFsa30 (3.85 ppm of Phosphate). The

wild Aspergillus fumigatus which was used as a standard control solubilized 3.65

ppm of Phosphate in the ninth day of incubation.

89

The phosphate solubilization efficacy of AFuv50 strain increased 20.34%

on third day, 21.54% phosphate solubilization on sixth day and 24.66%

phosphate solubilization on ninth day of incubation using wild type as a standard

(100%).

Phosphate Solubilization Capacity of UV Treated Penicillium sp

The results of Phosphate Solubilization capacity of UV treated

Penicillium sp were presented in Table 6.

The phosphate solubilization capacity of UV treated Penicillium sp

(PEuv50) on day three recorded maximum efficacy in solubilization (3.75 ppm

of Phosphate) followed by PEuv40 strain (3.65 ppm of Phosphate), PEuv30

(3.45 ppm of Phosphate) and PEuv60 (3.40 ppm of Phosphate). The wild

Penicillium sp which was used as a standard control solubilized 3.30 ppm of

Phosphate in the third day of incubation.

The phosphate solubilization capacity of UV treated Penicillium sp

(PEuv50) on day six recorded maximum efficacy in solubilization (4.40 ppm of

Phosphate) followed by PEuv40 strain (4.25 ppm of Phosphate), PEuv60 (4.20

ppm of Phosphate) and PEuv30 (4.10 ppm of Phosphate). The wild Penicillium

sp which was used as a standard control solubilized 3.85 ppm of Phosphate in

the sixth day of incubation.

The phosphate solubilization capacity of UV treated Penicillium sp

(PEuv50) on day nine recorded maximum efficacy in solubilization (4.75 ppm of

Phosphate) followed by PEuv40 strain (4.45 ppm of Phosphate), PEuv60 (4.35

ppm of Phosphate) and PEuv30 (4.25 ppm of Phosphate). The wild Penicillium

90

sp which was used as a standard control solubilized 4.00 ppm of Phosphate in

the sixth day of incubation.

The phosphate solubilization efficacy of PEuv50 strain increased 13.64%

on third day, 14.29% phosphate solubilization on sixth day and 18.75%

phosphate solubilization on ninth day of incubation using wild type as a standard

(100%).

Phosphate Solubilization Capacity of UV Treated Pseudomonas sp.

The results of Phosphate Solubilization capacity of UV treated

Pseudomonas sp were presented in Table 7.

The phosphate solubilization capacity of UV treated Pseudomonas sp

(PSuv3) on day three recorded maximum efficacy in solubilization (4.15 ppm of

Phosphate) followed by PSuv4 strain (4.05 ppm of Phosphate), PSuv2 (3.95 ppm

of Phosphate) and PSuv1 (3.85 ppm of Phosphate). The wild Pseudomonas sp

which was used as a standard control solubilized 3.80 ppm of Phosphate in the

third day of incubation.

The phosphate solubilization capacity of UV treated Pseudomonas sp

(PSuv4) on day six recorded maximum efficacy in solubilization (4.35 ppm of

Phosphate) followed by PSuv3 strain (4.25 ppm of Phosphate), PSuv2 (4.10 ppm

of Phosphate) and PSuv1 (3.95 ppm of Phosphate). The wild Pseudomonas sp

which was used as a standard control solubilized 4.15 ppm of Phosphate in the

sixth day of incubation.

The phosphate solubilization capacity of UV treated Pseudomonas sp

(PSuv4) on day nine recorded maximum efficacy in solubilization (4.65 ppm of

91

Phosphate) followed by PSuv3 strain (4.45 ppm of Phosphate), PSuv2 (4.30 ppm

of Phosphate) and PSuv1 (4.15 ppm of Phosphate). The wild Pseudomonas sp

which was used as a standard control solubilized 4.75 ppm of Phosphate in the

ninth day of incubation.

All mutants of Pseudomonas strains treated with UV on day nine showed

decreased phosphate solubilization activity when compared to wild type.

Phosphate Solubilization Capacity of Sodium Azide Treated Aspergillus

niger

The results of Phosphate Solubilization capacity of sodium azide treated

Aspergillus niger were presented in Table 8.

The phosphate solubilization capacity of sodium azide treated Aspergillus

niger (ANsa120) on day three recorded maximum efficacy in solubilization

(3.45ppm of Phosphate) followed by ANsa90 strain (2.95 ppm of Phosphate),

ANsa60 (2.50 ppm of Phosphate) and ANsa30 (2.40 ppm of Phosphate). The

wild Aspergillus niger which was used as a standard control solubilized 2.20ppm

of Phosphate in the third day of incubation.

The phosphate solubilization of sodium azide treated Aspergillus niger

(ANsa120) on day six recorded maximum efficacy in solubilization (4.95 ppm of

Phosphate) followed by ANsa90 strain (4.65 ppm of Phosphate) ANsa60 (4.30

ppm of Phosphate) and ANsa30 (4.10 ppm of Phosphate). The wild Aspergillus

niger which was used as a standard control solubilized 3.65 ppm of Phosphate in

the sixth day of incubation.

92

The phosphate solubilization of sodium azide treated Aspergillus niger

(ANsa120) on day nine recorded maximum efficacy in solubilization (6.15 ppm

of Phosphate) followed by ANsa90 strain (5.80 ppm of Phosphate) ANsa60

(5.55 ppm of Phosphate) and ANsa30 (5.05 ppm of Phosphate). The wild

Aspergillus niger which was used as a standard control solubilized 4.60ppm of

Phosphate in the ninth day of incubation.

The phosphate solubilization efficacy of ANsa120 strain increased 56.8%

on third day, 35.6% phosphate solubilization on sixth day and 33.6% phosphate

solubilization on ninth day of incubation using wild type as a standard (100%).

Here the percentage of phosphate solubilization is decreased with the increasing

the day of incubation.

Phosphate Solubilization Capacity of Sodium Azide Treated Aspergillus

fumigatus

The results of Phosphate Solubilization capacity of sodium azide treated

Aspergillus fumigatus were presented in Table 9.

The phosphate solubilization capacity of sodium azide treated Aspergillus

fumigatus (AFsa120) on day three recorded maximum efficacy in solubilization

(4.05ppm of Phosphate) followed by AFsa90 strain (3.45 ppm of Phosphate),

AFsa60 (3.25 ppm of Phosphate) and AFsa30 (3.10 ppm of Phosphate). The

wild Aspergillus fumigatus which was used as a standard control solubilized 2.95

ppm of Phosphate in the third day of incubation.

The phosphate solubilization capacity of sodium azide treated Aspergillus

fumigatus (AFsa120) on day six recorded maximum efficacy in solubilization

(4.65ppm of Phosphate) followed by AFsa90 strain (4.00 ppm of Phosphate),

93

AFsa60 (3.75 ppm of Phosphate) and AFsa30 (3.25 ppm of Phosphate). The

wild Aspergillus fumigatus which was as a standard control solubilized 3.45 ppm

of Phosphate in the sixth day of incubation.

The phosphate solubilization capacity of sodium azide treated Aspergillus

fumigatus (AFsa120) on day nine recorded maximum efficacy in solubilization

(5.50ppm of Phosphate) followed by AFsa90 strain (5.30 ppm of Phosphate),

AFsa60 (4.25 ppm of Phosphate) and AFsa30 (3.95 ppm of Phosphate). The

wild Aspergillus fumigatus which was used as a standard control solubilized 3.65

ppm of Phosphate in the ninth day of incubation.

The phosphate solubilization efficacy of AFsa120 strain increased 37.0%

on third day, 43.0% phosphate solubilization on sixth day and 50.6% phosphate

solubilization on ninth day of incubation using wild type as a standard (100%).

Here the percentage of phosphate solubilization is increased with the increasing

the day of incubation.

Phosphate Solubilization Capacity of Sodium Azide Treated Penicillium sp

The results of Phosphate Solubilization capacity of sodium azide treated

Penicillium sp were presented in Table 10.

The phosphate solubilization capacity of sodium azide treated Penicillium

sp (PEsa150) on day three recorded maximum efficacy in solubilization (3.80

ppm of Phosphate) followed by PEsa120 strain (3.70 ppm of Phosphate), PEsa90

(3.60 ppm of Phosphate) and PEsa60 (3.50 ppm of Phosphate). The wild

Penicillium sp which was used as a standard control solubilized 3.30 ppm of

Phosphate in the third day of incubation.

94

The phosphate solubilization capacity of sodium azide treated Penicillium

sp (PEsa150) on day six recorded maximum efficacy in solubilization (4.85 ppm

of Phosphate) followed by PEsa120 strain (4.65 ppm of Phosphate), PEsa90

(4.50 ppm of Phosphate) and PEsa60 (4.15 ppm of Phosphate). The wild

Penicillium sp which was used as a standard control solubilized 3.85 ppm of

Phosphate in the sixth day of incubation.

The phosphate solubilization capacity of sodium azide treated Penicillium

sp (PEsa150) on day nine recorded maximum efficacy in solubilization (5.95

ppm of Phosphate) followed by PEsa120 strain (5.65 ppm of Phosphate), PEsa90

(5.05 ppm of Phosphate) and PEsa60 (4.35 ppm of Phosphate). The wild

Penicillium sp which was used as a standard control solubilized 4.00 ppm of

Phosphate in the ninth day of incubation.

The phosphate solubilization efficacy of PEsa150 strain increased 15.1%

on third day, 25.9% phosphate solubilization on sixth day and 48.7% phosphate

solubilization on ninth day of incubation using wild type as a standard (100%).

Phosphate Solubilization Capacity of Sodium Azide Treated Pseudomonas sp

The results of Phosphate Solubilization capacity of sodium azide treated

Pseudomonas sp were presented in Table 11.

The phosphate solubilization of sodium azide treated Pseudomonas sp.

(PSsa120) recorded maximum efficacy in solubilization (3.45 ppm of Phosphate)

followed by PSsa90 strain (3.35 ppm of Phosphate), PSsa60 strain (3.15 ppm of

Phosphate) and PSsa30 strain (2.85 ppm of Phosphate). The wild Pseudomonas

sp which was used as a standard control solubilized 3.80 ppm of Phosphate in

the third day of incubation.

95

The phosphate solubilization of sodium azide treated Pseudomonas sp.

(PSsa120) recorded maximum efficacy in solubilization (4.25 ppm of Phosphate)

followed by PSsa90 strain (4.05 ppm of Phosphate), PSsa60 strain (3.95 ppm of

Phosphate) and PSsa30 strain (3.65 ppm of Phosphate). The wild Pseudomonas

sp. which was used as a standard control solubilized 4.15 ppm of Phosphate in

the sixth day of incubation.

The phosphate solubilization of sodium azide treated Pseudomonas sp

(PSsa120) recorded maximum efficacy in solubilization (4.65 ppm of Phosphate)

followed by PSsa3 strain (4.50 ppm of Phosphate), PSsa60 strain (4.45 ppm of

Phosphate) and PSsa30 strain (4.15 ppm of Phosphate).. The wild Pseudomonas

sp which was used as a standard control solubilized 4.75 ppm of Phosphate in

the ninth day of incubation.

All mutants of Pseudomonas strains treated with sodium azide on day

three, six and nine showed decreased phosphate solubilization activity when

compared to wild type.

Phosphate Solubilization Capacity of Ethyl Methane Sulphonate Treated

Aspergillus niger

The results of Phosphate Solubilization capacity of Ethyl Methane

Sulphonate treated Aspergillus niger were presented in Table 12.

The phosphate solubilization capacity of Ethyl Methane Sulphonate

treated Aspergillus niger (ANems120) on day three recorded maximum efficacy

in solubilization (2.85ppm of Phosphate) followed by ANems90 strain (2.65

ppm of Phosphate), ANems60 (2.50 ppm of Phosphate) and ANems30 (2.30

96

ppm of Phosphate). The wild Aspergillus niger which was used as a standard

control solubilized 2.20ppm of Phosphate in the third day of incubation.

The phosphate solubilization capacity of Ethyl Methane Sulphonate

treated Aspergillus niger (ANems120) on day sixth recorded maximum efficacy

in solubilization (4.75ppm of Phosphate) followed by ANems90 strain (4.45

ppm of Phosphate), ANems60 (4.25 ppm of Phosphate) and ANems30 (3.90

ppm of Phosphate). The wild Aspergillus niger which was used as a standard

control solubilized 3.65ppm of Phosphate in the sixth day of incubation.

The phosphate solubilization capacity of Ethyl Methane Sulphonate

treated Aspergillus niger (ANems120) on day ninth recorded maximum efficacy

in solubilization (6.25ppm of Phosphate) followed by ANems90 strain (5.65

ppm of Phosphate), ANems60 (5.40 ppm of Phosphate) and ANems30 (4.75

ppm of Phosphate). The wild Aspergillus niger which was used as a standard

control solubilized 4.60 ppm of Phosphate in the ninth day of incubation.

The phosphate solubilization efficacy of ANems120 strain increased

29.5% on third day, 30.1% phosphate solubilization on sixth day and 35.8%

phosphate solubilization on ninth day of incubation using wild type as a standard

(100%).

Phosphate Solubilization Capacity of Ethyl Methane Sulphonate Treated

Aspergillus fumigatus

The results of Phosphate Solubilization capacity of Ethyl Methane

Sulphonate treated Aspergillus fumigatus were presented in Table 13.

97

The phosphate solubilization capacity of Ethyl Methane Sulphonate

treated Aspergillus fumigatus (AFems120) on day three recorded maximum

efficacy in solubilization (3.95ppm of Phosphate) followed by AFems90 strain

(3.75 ppm of Phosphate), AFems60 (3.55 ppm of Phosphate) and AFems30

(3.25 ppm of Phosphate). The wild Aspergillus fumigatus which was used as a

standard control solubilized 2.95 ppm of Phosphate in the third day of

incubation.

The phosphate solubilization capacity of Ethyl Methane Sulphonate

treated Aspergillus fumigatus (AFems120) on day six recorded maximum

efficacy in solubilization (4.45ppm of Phosphate) followed by AFems90 strain

(4.15 ppm of Phosphate), AFems60 (3.80 ppm of Phosphate) and AFems30

(3.45 ppm of Phosphate). The wild Aspergillus fumigatus which was used as a

standard control solubilized 3.25 ppm of Phosphate in the sixth day of

incubation.

The phosphate solubilization capacity of Ethyl Methane Sulphonate

treated Aspergillus fumigatus (AFems120) on day nine recorded maximum

efficacy in solubilization (5.15ppm of Phosphate) followed by AFems90 strain

(4.65 ppm of Phosphate), AFems60 (4.15 ppm of Phosphate) and AFems30

(3.85 ppm of Phosphate). The wild Aspergillus fumigatus which was used as a

standard control solubilized 3.65 ppm of Phosphate in the ninth day of

incubation.

The phosphate solubilization efficacy of AFems120 strain increased

33.8% on third day, 36.9% phosphate solubilization on sixth day and 41.0%

phosphate solubilization on ninth day of incubation using wild type as a standard

(100%).

98

Phosphate Solubilization Capacity of Ethyl Methane Sulphonate Treated

Penicillium sp

The results of Phosphate Solubilization capacity of Ethyl Methane

Sulphonate treated Penicillium sp were presented in Table 14.

The phosphate solubilization capacity of Ethyl Methane Sulphonate

treated Penicillium sp (PEems150) on day three recorded maximum efficacy in

solubilization (4.80 ppm of Phosphate) followed by PEems120 strain (4.55 ppm

of Phosphate), PEems90 (4.05 ppm of Phosphate) and PEems60 (3.65 ppm of

Phosphate). The wild Penicillium sp which was used as a standard control

solubilized 3.30 ppm of Phosphate in the third day of incubation.

The phosphate solubilization capacity of Ethyl Methane Sulphonate

treated Penicillium sp (PEems150) on day six recorded maximum efficacy in

solubilization (5.70 ppm of Phosphate) followed by PEems120 strain (5.10 ppm

of Phosphate), PEsa90 (4.90 ppm of Phosphate) and PEems60 (4.70 ppm of

Phosphate). The wild Penicillium sp which was used as a standard control

solubilized 3.85 ppm of Phosphate in the sixth day of incubation.

The phosphate solubilization capacity of Ethyl Methane Sulphonate

treated Penicillium sp (PEems150) on day six recorded maximum efficacy in

solubilization (5.95 ppm of Phosphate) followed by PEems120 strain (5.45 ppm

of Phosphate), PEems90 (5.30 ppm of Phosphate) and PEems60 (5.15 ppm of

Phosphate). The wild Penicillium sp which was used as a standard control

solubilized 4.00 ppm of Phosphate in the ninth day of incubation.

The phosphate solubilization efficacy of PEems150 strain increased

45.4% on third day, 48.0% phosphate solubilization on sixth day and 48.7%

99

phosphate solubilization on ninth day of incubation using wild type as a standard

(100%).

Phosphate Solubilization Capacity of Ethyl Methane Sulphonate Treated

Pseudomonas sp

The results of Phosphate Solubilization capacity of Ethyl Methane

Sulphonate treated Pseudomonas sp were presented in Table 15.

The phosphate solubilization of Ethyl Methane Sulphonate treated

Pseudomonas sp (PSems150) on day three recorded maximum efficacy in

solubilization (2.45 ppm of Phosphate) followed by PSems120 strain (2.40 ppm

of Phosphate), PSems90 (2.35 ppm of Phosphate) and PSems60 (2.30 ppm of

Phosphate). The wild Pseudomonas sp which was used as a standard control

solubilized 3.80 ppm of Phosphate in the third day of incubation.

The phosphate solubilization of Ethyl Methane Sulphonate treated

Pseudomonas sp (PSems150) on day six recorded maximum efficacy in

solubilization (3.25ppm of Phosphate) followed by PSems120 strain (3.05 ppm

of Phosphate) PSems90 (2.90 ppm of Phosphate) and PSems60 (2.80 ppm of

Phosphate). The wild Pseudomonas sp which was used as a standard control

solubilized 4.15 ppm of Phosphate in the sixth day of incubation.

The phosphate solubilization of Ethyl Methane Sulphonate treated

Pseudomonas sp (PSems150) on day nine recorded maximum efficacy in

solubilization (4.25ppm of Phosphate) followed by PSems120 strain (4.15 ppm

of Phosphate) PSems90 (3.55 ppm of Phosphate) and PSems60 (3.30 ppm of

Phosphate). The wild Pseudomonas sp which was used as a standard control

solubilized 4.75 ppm of Phosphate in the ninth day of incubation.

100

All mutants of Pseudomonas strains treated with Ethyl Methane

Sulphonate on day three, six and nine showed decreased phosphate

solubilization activity when compared to wild type.

Effect of Different Carbon and Nitrogen Sources on the Efficacy of

Phosphate Solubilization

The superior mutated cultures of Phosphate solubilization in Pikovskaya

broth were identified as Ethyl Methane Sulphonate treated Aspergillus niger

(ANems120), Sodium Azide Treated Aspergillus niger (ANsa120) and Sodium

Azide Treated Penicillium sp (PEsa150) and their efficacy of Phosphate

solubilization on different Carbon and Nitrogen Sources were studied.

Effect of Different Carbon Sources on the Efficacy of Phosphate

Solubilization of Ethyl Methane Sulphonate Treated Aspergillus niger

(ANems120)

The results of different carbon sources on the efficacy of phosphate

solubilization of Ethyl Methane Sulphonate treated Aspergillus niger

(ANems120) were presented in Table 16

The efficacy of phosphate solubilization of ANems120 was maximum in

the medium containing Glucose (2.85 ppm of Phosphate) followed by Sucrose

(2.40 ppm of Phosphate), Lactose (2.20 ppm of Phosphate) and Mannitol (2.05

ppm of Phosphate) in the third day of incubation.

The efficacy of phosphate solubilization of ANems120 was maximum in

the medium containing Glucose (4.75 ppm of Phosphate) followed by Sucrose

101

(3.75 ppm of Phosphate), Lactose (3.60 ppm of Phosphate) and Mannitol (3.05

ppm of Phosphate) in the sixth day of incubation.

The efficacy of phosphate solubilization of ANems120 was maximum in

the medium containing Glucose (6.25 ppm of Phosphate) followed by Sucrose

(4.85 ppm of Phosphate), Lactose (4.60 ppm of Phosphate) and Mannitol (4.35

ppm of Phosphate) in the ninth day of incubation.

Effect of Different Nitrogen Sources on the Efficacy of Phosphate

Solubilization of Ethyl Methane Sulphonate Treated Aspergillus niger

(ANems120)

The results of different nitrogen sources on the efficacy of phosphate

solubilization of Ethyl Methane Sulphonate treated Aspergillus niger

(ANems120) were presented in Table 17.

The efficacy of phosphate solubilization of ANems120 was maximum in

the medium containing Ammonium sulphate (2.85 ppm of Phosphate) followed

by Urea (2.40 ppm of Phosphate), Potassium nitrate (2.35 ppm of Phosphate)

and Sodium nitrate (1.90 ppm of Phosphate) in the third day of incubation.

The efficacy of phosphate solubilization of ANems120 was maximum in

the medium containing Ammonium sulphate (4.75 ppm of Phosphate) followed

by Urea (3.70 ppm of Phosphate), Potassium nitrate (3.70 ppm of Phosphate)

and Sodium nitrate (2.65 ppm of Phosphate) in the sixth day of incubation.

The efficacy of phosphate solubilization of ANems120 was maximum in

the medium containing Ammonium sulphate (6.25 ppm of Phosphate) followed

by Urea (4.75 ppm of Phosphate), Potassium nitrate (4.70 ppm of

102

Phosphate)and Sodium nitrate (3.20 ppm of Phosphate) in the ninth day of

incubation.

Effect of Different Carbon Sources on the Efficacy of Phosphate

Solubilization of Sodium Azide Treated Aspergillus niger (ANsa120).

The results of different carbon sources on the efficacy of phosphate

solubilization of sodium azide treated Aspergillus niger (ANsa120) were

presented in Table 18

The efficacy of phosphate solubilization of ANsa120 was maximum in

the medium containing Sucrose (3.55 ppm of Phosphate) followed by Glucose

(3.45 ppm of Phosphate), Lactose (3.05 ppm of Phosphate) and Mannitol (1.85

ppm of Phosphate) in the third day of incubation.

The efficacy of phosphate solubilization of ANsa120 was maximum in

the medium containing Glucose (4.95 ppm of Phosphate) followed by Sucrose

(4.53 ppm of Phosphate), Lactose (3.55 ppm of Phosphate) and Mannitol (2.15

ppm of Phosphate) in the sixth day of incubation.

The efficacy of phosphate solubilization of ANsa120 was maximum in

the medium containing Glucose (6.15 ppm of Phosphate) followed by Sucrose

(4.95 ppm of Phosphate), Lactose (3.95 ppm of Phosphate) and Mannitol (2.90

ppm of Phosphate) in the ninth day of incubation.

103

Effect of Different Nitrogen Sources on the Efficacy of Phosphate

Solubilization of Sodium Azide Treated Aspergillus niger (ANsa120).

The results of different nitrogen sources on the efficacy of phosphate

solubilization of sodium azide treated Aspergillus niger (ANsa120) were

presented in Table 19.

The efficacy of phosphate solubilization of ANsa120 was maximum in

the medium containing Ammonium sulphate (3.45 ppm of Phosphate) followed

by Urea (2.45 ppm of Phosphate), Potassium nitrate (2.40 ppm of Phosphate)

and Sodium nitrate (1.75 ppm of Phosphate) in the third day of incubation.

The efficacy of phosphate solubilization of ANsa120 was maximum in

the medium containing Ammonium sulphate (4.95 ppm of Phosphate) followed

by Urea (3.85 ppm of Phosphate), Potassium nitrate (3.70 ppm of Phosphate)

and Sodium nitrate (2.30 ppm of Phosphate) in the sixth day of incubation.

The efficacy of phosphate solubilization of ANsa120 was maximum in

the medium containing Ammonium sulphate (6.15 ppm of Phosphate) followed

by Urea (4.70 ppm of Phosphate), Potassium nitrate (4.65 ppm of

Phosphate)and Sodium nitrate (2.75 ppm of Phosphate) in the ninth day of

incubation.

Effect of Different Carbon Sources on the Efficacy of Phosphate

Solubilization of Sodium Azide Treated Penicillium sp (PEsa150).

The results of different carbon sources on the efficacy of phosphate

solubilization of sodium azide treated Penicillium sp (PEsa150) were presented

in Table 20

104

The efficacy of phosphate solubilization of PEsa150 was maximum in the

medium containing Glucose (3.80 ppm of Phosphate) followed by Sucrose (3.35

ppm of Phosphate), Lactose (3.15 ppm of Phosphate) and Mannitol (3.05 ppm of

Phosphate) in the third day of incubation.

The efficacy of phosphate solubilization of PEsa150 was maximum in the

medium containing Glucose (4.85 ppm of Phosphate) followed by Sucrose (3.90

ppm of Phosphate), Lactose (3.80 ppm of Phosphate) and Mannitol (3.75 ppm of

Phosphate) in the sixth day of incubation.

The efficacy of phosphate solubilization of PEsa150 was maximum in the

medium containing Glucose (5.95 ppm of Phosphate) followed by Sucrose (4.60

ppm of Phosphate), Lactose (4.35 ppm of Phosphate) and Mannitol (4.25 ppm of

Phosphate) in the ninth day of incubation.

Effect of Different Nitrogen Sources on the Efficacy of Phosphate

Solubilization of Sodium Azide Treated Penicillium sp (PEsa150)

The results of different nitrogen sources on the efficacy of phosphate

solubilization of sodium azide treated Penicillium sp (PEsa150) were presented

in Table 21

The efficacy of phosphate solubilization of PEsa150 was maximum in the

medium containing Ammonium sulphate (3.80 ppm of Phosphate) followed by

Urea (3.70 ppm of Phosphate), Potassium nitrate (3.35 ppm of Phosphate) and

Sodium nitrate (3.30 ppm of Phosphate) in the third day of incubation.

The efficacy of phosphate solubilization of PEsa150 was maximum in the

medium containing Ammonium sulphate (4.85 ppm of Phosphate) followed by

105

Urea (3.95 ppm of Phosphate), Potassium nitrate (3.90 ppm of Phosphate) and

Sodium nitrate (3.75 ppm of Phosphate) in the sixth day of incubation.

The efficacy of phosphate solubilization of PEsa150 was maximum in the

medium containing Ammonium sulphate (5.95 ppm of Phosphate) followed by

Urea (4.70 ppm of Phosphate), Potassium nitrate (4.60 ppm of Phosphate)and

Sodium nitrate (4.05 ppm of Phosphate) in the ninth day of incubation.

Figure- 1 showes the comparsion of efficacy of Phosphatate solublization

by wild strains and Figure-2 showes the comparsion of efficacy of Phosphatate

solublization by chemical treated fungal strains.

Phosphatase Activity

Efficacy of UV Treated Aspergillus niger on Phosphatase Activity

The results of Phosphatase activity of UV treated Aspergillus niger were

presented in Table 22.

The maximum efficacy of Phosphatase activity of UV treated Aspergillus

niger (ANuv60) on day three recorded (0.165 µmol min-1) followed by ANuv50

strain (0.134 µmol min-1), ANuv30 (0.121 µmol min-1) and ANuv40 (0.103

µmol min-1). The Phosphatase activity of Wild Aspergillus niger which was used

as a standard control produced 0.097µmol min-1 in the third day of incubation.

The maximum efficacy of Phosphatase activity of UV treated Aspergillus

niger (ANuv60) on day six recorded (0.275µmol min-1) followed by ANuv50

strain (0.260 µmol min-1), ANuv30 (0.223 µmol min-1) and ANuv40 (0.218

µmol min-1). The Phosphatase activity of Wild Aspergillus niger which was used

as a standard control produced 0.192 µmol min-1 in the sixth day of incubation.

106

The maximum efficacy of Phosphatase activity of UV treated Aspergillus

niger (ANuv60) on day nine recorded (0.424µmol min-1) followed by ANuv50

strain (0.399 µmol min-1), ANuv30 (0.345 µmol min-1) and ANuv40 (0.341

µmol min-1). The Phosphatase activity of Wild Aspergillus niger which was used

as a standard control produced 0.303 µmol min-1 in the ninth day of incubation.

The Phosphatase activity of ANuv60 strain increased 70.10% on third

day, 43.23% Phosphatase activity on sixth day and 39.93% Phosphatase

activity on ninth day of incubation using wild type as a standard (100%).

Efficacy of UV Treated Aspergillus fumigatus on Phosphatase Activity

The results of Phosphatase activity of UV treated Aspergillus fumigatus

were presented in Table 23.

The maximum efficacy of Phosphatase activity of UV treated Aspergillus

fumigatus (AFuv50) on day three recorded (0.195 µmol min-1) followed by

ANuv60 strain (0.173 µmol min-1), ANuv30 (0.171 µmol min-1) and ANuv40

(0.160 µmol min-1). The Phosphatase activity of Wild Aspergillus fumigatus

which was used as a standard control produced 0.156µmol min-1 in the third

day of incubation.

The maximum efficacy of Phosphatase activity of UV treated Aspergillus

fumigatus (AFuv50) on day six recorded (0.227 µmol min-1) followed by

AFuv60 strain (0.224 µmol min-1), AFuv40 (0.205 µmol min-1) and AFuv30

(0.203 µmol min-1). The Phosphatase activity of Wild Aspergillus fumigatus

which was used as a standard control produced 0.178µmol min-1 in the sixth

day of incubation.

107

The maximum efficacy of Phosphatase activity of UV treated Aspergillus

fumigatus (AFuv50) on day nine recorded (0.269 µmol min-1) followed by

ANuv60 strain (0.265 µmol min-1), AFuv40 (0.232 µmol min-1) and AFuv30

(0.221 µmol min-1). The Phosphatase activity of Wild Aspergillus fumigatus

which was used as a standard control produced 0.197µmol min-1 in the ninth

day of incubation.

The Phosphatase activity of AFuv50 strain increased 25.00% on third

day, 27.53% Phosphatase activity on sixth day and 36.55% Phosphatase activity

on ninth day of incubation using wild type as a standard (100%).

Efficacy of UV Treated Penicillium sp on Phosphatase Activity

The results of Phosphatase activity of UV treated Penicillium sp were

presented in Table 24.

The maximum efficacy of Phosphatase activity of UV treated Penicillium

sp (PEuv50) on day three recorded (0.209 µmol min-1) followed by PEuv40

strain (0.195 µmol min-1), PEuv30 (0.180 µmol min-1) and PEuv60 (0.178 µmol

min-1). The Phosphatase activity of Wild Penicillium sp which was used as a

standard control produced 0.169µmol min-1 in the third day of incubation.

The maximum efficacy of Phosphatase activity of UV treated Penicillium

sp (PEuv40) on day six recorded (0.245 µmol min-1) followed by PEuv50 strain

(0.237 µmol min-1), PEuv60 (0.234 µmol min-1) and PEuv30 (0.228 µmol min-

1). The Phosphatase activity of Wild Penicillium sp which was used as a

standard control produced 0.213 µmol min-1 in the sixth day of incubation.

108

The maximum efficacy of Phosphatase activity of UV treated Penicillium

sp (PEuv50) on day nine recorded (0.267 µmol min-1) followed by PEuv40

strain (0.264 µmol min-1), PEuv60 (0.257 µmol min-1) and PEuv30 (0.239 µmol

min-1). The Phosphatase activity of Wild Penicillium sp which was used as a

standard control produced 0.236 µmol min-1 in the ninth day of incubation.

The Phosphatase activity of PEuv50 strain increased 23.67% on third day,

11.27% Phosphatase activity on sixth day and 13.14% Phosphatase activity on

ninth day of incubation using wild type as a standard (100%).

Efficacy of UV Treated Pseudomonas sp on Phosphatase Activity

The results of Phosphatase activity of UV treated Pseudomonas sp were

presented in Table 25.

The maximum efficacy of Phosphatase activity of UV treated

Pseudomonas sp (PSuv3) on day three recorded (0.227 µmol min-1) followed by

PSuv2 strain (0.215 µmol min-1), PSuv4 (0.210 µmol min-1) and PSuv1 (0.205

µmol min-1). The Phosphatase activity of Wild Pseudomonas sp which was

used as a standard control produced 0.200µmol min-1 in the third day of

incubation.

The maximum efficacy of Phosphatase activity of UV treated

Pseudomonas sp (PSuv3) on day six recorded (0.233 µmol min-1) followed by

PSuv2 strain (0.227 µmol min-1), PSuv4 (0.226 µmol min-1) and PSuv1 (0.225

µmol min-1). The Phosphatase activity of Wild Pseudomonas sp which was

used as a standard control produced 0.234µmol min-1 in the six day of

incubation.

109

The maximum efficacy of Phosphatase activity of UV treated

Pseudomonas sp (PSuv4) on day nine recorded (0.286 µmol min-1) followed by

PSuv3 strain (0.275 µmol min-1), PSuv2 (0.236 µmol min-1) and PSuv1 (0.231

µmol min-1). The Phosphatase activity of Wild Pseudomonas sp which was

used as a standard control produced 0.287µmol min-1 in the ninth day of

incubation.

All mutants of Pseudomonas strains treated with UV on day six and nine

showed decreased Phosphatase activity activity when compared to wild type.

Efficacy of Sodium Azide Treated Aspergillus niger on Phosphatase Activity

The results of Phosphatase activity of sodium azide treated Aspergillus

niger were presented in Table 26.

The maximum efficacy of Phosphatase activity of sodium azide treated

Aspergillus niger (ANsa120) on day three recorded (0.184 µmol min-1) followed

by ANsa90 strain (0.152 µmol min-1), ANsa60 (0.110 µmol min-1) and ANsa30

(0.107 µmol min-1). The Phosphatase activity of Wild Aspergillus niger which

was used as a standard control produced 0.097µmol min-1 in the third day of

incubation.

The maximum efficacy of Phosphatase activity of sodium azide treated

Aspergillus niger (ANsa120) on day six recorded (0.340 µmol min-1) followed

by ANsa90 strain (0.268 µmol min-1), ANsa60 (0.234 µmol min-1) and ANsa30

(0.218 µmol min-1). The Phosphatase activity of Wild Aspergillus niger which

was used as a standard control produced 0.192µmol min-1 in the sixth day of

incubation.

110

The maximum efficacy of Phosphatase activity of sodium azide treated

Aspergillus niger (ANsa120) on day nine recorded (0.474 µmol min-1) followed

by ANsa90 strain (0.421 µmol min-1), ANsa60 (0.401 µmol min-1) and ANsa30

(0.335 µmol min-1). The Phosphatase activity of Wild Aspergillus niger which

was used as a standard control produced 0.303µmol min-1 in the ninth day of

incubation.

The Phosphatase activity of ANsa120 strain increased 89.69% on third

day, 77.08% Phosphatase activity on sixth day and 56.43% Phosphatase activity

on ninth day of incubation using wild type as a standard (100%).

Efficacy of Sodium Azide Treated Aspergillus fumigatus on Phosphatase

Activity

The results of Phosphatase activity of sodium azide treated Aspergillus

niger were presented in Table 27.

The maximum efficacy of Phosphatase activity of sodium azide treated

Aspergillus fumigatus (AFsa120) on day three recorded (0.232 µmol min-1)

followed by AFsa90 strain (0.185 µmol min-1), AFsa60 (0.174 µmol min-1) and

AFsa30 (0.165 µmol min-1). The Phosphatase activity of Wild Aspergillus

fumigatus which was used as a standard control produced 0.156µmol min-1 in

the third day of incubation.

The maximum efficacy of Phosphatase activity of sodium azide treated

Aspergillus fumigatus (AFsa120) on day six recorded (0.274 µmol min-1)

followed by AFsa90 strain (0.229 µmol min-1), AFsa60 (0.203 µmol min-1) and

AFsa30 (0.189 µmol min-1). The Phosphatase activity of Wild Aspergillus

111

fumigatus which was used as a standard control produced 0.178µmol min-1 in

the sixth day of incubation.

The maximum efficacy of Phosphatase activity of sodium azide treated

Aspergillus fumigatus (AFsa120) on day nine recorded (0.365 µmol min-1)

followed by AFsa90 strain (0.351 µmol min-1), AFsa60 (0.241 µmol min-1) and

AFsa30 (0.213 µmol min-1). The Phosphatase activity of Wild Aspergillus

fumigatus which was used as a standard control produced 0.197µmol min-1 in

the ninth day of incubation.

The Phosphatase activity of AFsa120 strain increased 48.71% on third

day, 53.93% Phosphatase activity on sixth day and 85.27% Phosphatase activity

on ninth day of incubation using wild type as a standard (100%).

Efficacy of Sodium Azide Treated Penicillium sp on Phosphatase Activity

The results of Phosphatase activity of sodium azide treated Penicillium sp

were presented in Table 28.

The maximum efficacy of Phosphatase activity of sodium azide treated

Penicillium sp (PEsa150) on day three recorded (0.210 µmol min-1) followed by

PEsa120 strain (0.200 µmol min-1), PEsa90 (0.191 µmol min-1) and PEsa60

(0.181 µmol min-1). The Phosphatase activity of Wild Penicillium sp which was

used as a standard control produced 0.169 µmol min-1 in the third day of

incubation.

The maximum efficacy of Phosphatase activity of sodium azide treated

Penicillium sp (PEsa150) on day six recorded (0.276 µmol min-1) followed by

PEsa120 strain (0.261 µmol min-1), PEsa90 (0.249 µmol min-1) and PEsa60

112

(0.233 µmol min-1). The Phosphatase activity of Wild Penicillium sp used as a

standard control produced 0.213 µmol min-1 in the sixth day of incubation.

The maximum efficacy of Phosphatase activity of sodium azide treated

Penicillium sp (PEsa150) on day nine recorded (0.420 µmol min-1) followed by

PEsa120 strain (0.400 µmol min-1), PEsa90 (0.349 µmol min-1) and PEsa60

(0.261 µmol min-1). The Phosphatase activity of Wild Penicillium sp which was

used as a standard control produced 0.236 µmol min-1 in the ninth day of

incubation.

The Phosphatase activity of PEsa150 strain increased 24.26% on third

day, 29.57% Phosphatase activity on sixth day and 77.96% Phosphatase activity

on ninth day of incubation using wild type as a standard (100%).

Efficacy of Sodium Azide Treated Pseudomonas sp on Phosphatase Activity

The results of Phosphatase activity of sodium azide treated Pseudomonas

sp were presented in Table 29.

The maximum efficacy of Phosphatase activity of sodium azide treated

Pseudomonas sp (PSsa120) on day three recorded (0.185 µmol min-1) followed

by PSsa90 strain (0.180 µmol min-1), PSsa60 (0.171 µmol min-1) and PSsa30

(0.169 µmol min-1). The Phosphatase activity of Wild Pseudomonas sp which

was used as a standard control produced 0.200µmol min-1 in the third day of

incubation.

The maximum efficacy of Phosphatase activity of sodium azide treated

Pseudomonas sp (PSsa120) on day six recorded (0.241 µmol min-1) followed by

PSsa90 strain (0.232 µmol min-1), PSsa60 (0.220 µmol min-1) and PSsa30

113

(0.199 µmol min-1). The Phosphatase activity of Wild Pseudomonas sp which

was used as a standard control produced 0.234µmol min-1 in the six day of

incubation.

The maximum efficacy of Phosphatase activity of sodium azide treated

Pseudomonas sp (PSsa120) on day nine recorded (0.275 µmol min-1) followed

by PSsa90 strain (0.260 µmol min-1), PSsa60 (0.256 µmol min-1) and PSsa30

(0.224 µmol min-1). The Phosphatase activity of Wild Pseudomonas sp which

was used as a standard control produced 0.287µmol min-1 in the ninth day of

incubation.

All mutants of Pseudomonas strains treated with sodium azide on day

three, six and nine showed decreased Phosphatase activity activity when

compared to wild type.

Efficacy of Ethyl Methane Sulphonate Treated Aspergillus niger on

Phosphatase Activity

The results of Phosphatase activity of Ethyl Methane Sulphonate treated

Aspergillus niger were presented in Table 30.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Aspergillus niger (ANems120) on day three recorded (0.131

µmol min-1) followed by ANems90 strain (0.125 µmol min-1), ANems60 (0.104

µmol min-1) and ANems30 (0.099 µmol min-1). The Phosphatase activity of

Wild Aspergillus niger which was used as a standard control produced

0.097µmol min-1 in the third day of incubation.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Aspergillus niger (ANems120) on day six recorded (0.273

114

µmol min-1) followed by ANems90 strain (0.253 µmol min-1), ANems60 (0.222

µmol min-1) and ANems30 (0.210 µmol min-1). The Phosphatase activity of

Wild Aspergillus niger which was used as a standard control produced

0.192µmol min-1 in the sixth day of incubation.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Aspergillus niger (ANems120) on day nine recorded (0.452

µmol min-1) followed by ANems90 strain (0.412 µmol min-1), ANems60 (0.365

µmol min-1) and ANems30 (0.316 µmol min-1). The Phosphatase activity of

Wild Aspergillus niger which was used as a standard control produced 0.303

µmol min-1 in the ninth day of incubation.

The Phosphatase activity of ANems120 strain increased 35.05% on third

day, 42.18% Phosphatase activity on sixth day and 49.17% Phosphatase activity

on ninth day of incubation using wild type as a standard (100%).

Efficacy of Ethyl Methane Sulphonate Treated Aspergillus fumigatus on

Phosphatase Activity

The results of Phosphatase activity of Ethyl Methane Sulphonate treated

Aspergillus fumigatus were presented in Table 31.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Aspergillus fumigatus (AFems120) on day three recorded

(0.219 µmol min-1) followed by AFems90 strain (0.206 µmol min-1), AFems60

(0.189 µmol min-1) and AFems30 (0.175 µmol min-1). The Phosphatase activity

of Wild Aspergillus fumigatus which was used as a standard control produced

0.156 µmol min-1 in the third day of incubation.

115

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Aspergillus fumigatus (AFems120) on day six recorded

(0.258 µmol min-1) followed by AFems90 strain (0.236 µmol min-1), AFems60

(0.212 µmol min-1) and AFems30 (0.197 µmol min-1). The Phosphatase activity

of Wild Aspergillus fumigatus which was used as a standard control produced

0.178 µmol min-1 in the sixth day of incubation.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Aspergillus fumigatus (AFems120) on day nine recorded

(0.318 µmol min-1) followed by AFems90 strain (0.261 µmol min-1), AFems60

(0.231 µmol min-1) and AFems30 (0.214 µmol min-1). The Phosphatase activity

of Wild Aspergillus fumigatus which was used as a standard control produced

0.197 µmol min-1 in the ninth day of incubation.

The Phosphatase activity of AFems120 strain increased 40.38% on third

day, 44.94% Phosphatase activity on sixth day and 61.42% Phosphatase activity

on ninth day of incubation using wild type as a standard (100%).

Efficacy of Ethyl Methane Sulphonate Treated Penicillium sp on

Phosphatase Activity

The results of Phosphatase activity of Ethyl Methane Sulphonate treated

Penicillium sp were presented in Table 32.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Penicillium sp (PEems150) on day three recorded (0.278

µmol min-1) followed by PEems120 strain (0.245 µmol min-1), PEems90 (0.227

µmol min-1) and PEems60 (0.197 µmol min-1). The Phosphatase activity of Wild

116

Penicillium sp which was used as a standard control produced 0.169 µmol min-1

in the third day of incubation.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Penicillium sp (PEems150) on day six recorded (0.381 µmol

min-1) followed by PEems120 strain (0.324 µmol min-1), PEems90 (0.325 µmol

min-1) and PEems60 (0.275 µmol min-1). The Phosphatase activity of Wild

Penicillium sp which was used as a standard control produced 0.213 µmol min-1

in the sixth day of incubation.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Penicillium sp (PEems150) on day nine recorded (0.425

µmol min-1) followed by PEems120 strain (0.371 µmol min-1), PEems60 (0.370

µmol min-1) and PEems90 (0.369 µmol min-1). The Phosphatase activity of Wild

Penicillium sp which was used as a standard control produced 0.236 µmol min-1

in the ninth day of incubation.

The Phosphatase activity of PEems150 strain increased 64.49% on third

day, 78.87% Phosphatase activity on sixth day and 80.00% Phosphatase activity

on ninth day of incubation using wild type as a standard (100%).

Efficacy of Ethyl Methane Sulphonate Treated Pseudomonas sp on

Phosphatase Activity

The results of Phosphatase activity of Ethyl Methane Sulphonate treated

Pseudomonas sp were presented in Table 33.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Pseudomonas sp (PSsa150) on day three recorded (0.113

µmol min-1) followed by PSsa120 strain (0.100 µmol min-1), PSsa90 (0.095

117

µmol min-1) and PSsa60 (0.091 µmol min-1). The Phosphatase activity of Wild

Pseudomonas sp which was used as a standard control produced 0.200µmol

min-1 in the third day of incubation.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Pseudomonas sp (PSsa150) on day six recorded (0.175 µmol

min-1) followed by PSsa120 strain (0.167 µmol min-1), PSsa90 (0.161 µmol

min-1) and PSsa60 (0.150 µmol min-1). The Phosphatase activity of Wild

Pseudomonas sp which was used as a standard control produced 0.234µmol

min-1 in the six day of incubation.

The maximum efficacy of Phosphatase activity of Ethyl Methane

Sulphonate treated Pseudomonas sp (PSsa150) on day nine recorded (0.229

µmol min-1) followed by PSsa120 strain (0.220 µmol min-1), PSsa90 (0.191

µmol min-1) and PSsa60 (0.180 µmol min-1). The Phosphatase activity of Wild

Pseudomonas sp which was used as a standard control produced 0.287µmol

min-1 in the nine day of incubation.

All mutants of Pseudomonas strains treated with Ethyl Methane

Sulphonate on day three, six and nine showed decreased Phosphatase activity

activity when compared to wild type.

Effect of Different Carbon and Nitrogen Sources on the Efficacy of Acid

Phosphatase Production.

The superior mutated cultures of Phosphatase in Pikovskaya broth were

identified as Ethyl Methane Sulphonate treated Aspergillus niger (ANems120),

Sodium Azide Treated Aspergillus niger (ANsa120) and Sodium Azide Treated

118

Penicillium sp (PEsa150) and their efficacy of Phosphatase on different Carbon

and Nitrogen Sources were studied.

Effect of Different Carbon Sources on the Efficacy of Phosphatase Activity

of Ethyl Methane Sulphonate Treated (ANems120).

The results of different carbon sources on the efficacy of Phosphatase

activity of Ethyl Methane Sulphonate treated Aspergillus niger (ANems120)

were presented in Table 34.

The efficacy of Phosphatase activity of ANems120 was maximum in the

medium containing Glucose (0.131 µmol min-1) followed by Sucrose (0.107

µmol min-1), Lactose (0.094 µmol min-1) and Mannitol (0.083 µmol min-1) in the

third day of incubation.

The efficacy of Phosphatase activity of ANems120 was maximum in the

medium containing Glucose (0.273 µmol min-1) followed by Sucrose (0.206

µmol min-1), Lactose (0.182 µmol min-1) and Mannitol (0.167 µmol min-1) in the

sixth day of incubation.

The efficacy of Phosphatase activity of ANems120 was maximum in the

medium containing Glucose (0.452 µmol min-1) followed by Sucrose (0.279

µmol min-1), Lactose (0.257 µmol min-1) and Mannitol (0.229 µmol min-1) in the

ninth day of incubation.

119

Effect of Different Nitrogen Sources on the Efficacy of Phosphatase Activity

of Ethyl Methane Sulphonate Treated Aspergillus niger (ANems120).

The results of different nitrogen sources on the efficacy of Phosphatase

activity of Ethyl Methane Sulphonate treated Aspergillus niger (ANems120)

were presented in Table 35.

The efficacy of Phosphatase activity of ANems120 was maximum in the

medium containing Ammonium sulphate (0.131 µmol min-1) followed by Urea

(0.109 µmol min-1), Potassium nitrate (0.102 µmol min-1) and Sodium nitrate

(0.074 µmol min-1) in the third day of incubation.

The efficacy of Phosphatase activity of ANems120 was maximum in the

medium containing Ammonium sulphate (0.273 µmol min-1) followed by Urea

(0.203 µmol min-1), Potassium nitrate (0.200 µmol min-1) and Sodium nitrate

(0.122 µmol min-1) in the sixth day of incubation.

The efficacy of Phosphatase activity of ANems120 was maximum in the

medium containing Ammonium sulphate (0.452 µmol min-1) followed by Urea

(0.275 µmol min-1), Potassium nitrate (0.271 µmol min-1) and Sodium nitrate

(0.173 µmol min-1) in the ninth day of incubation.

Effect of Different Carbon Sources on the Efficacy of Phosphatase Activity

of Sodium Azide Treated Aspergillus niger (ANsa120).

The results of different carbon sources on the efficacy of Phosphatase

activity of sodium azide treated Aspergillus niger (ANsa120) were presented in

Table 36.

120

The efficacy of Phosphatase activity of ANsa120 was maximum in the

medium containing Sucrose (0.186 µmol min-1) followed by Glucose (0.184

µmol min-1), Lactose (0.165 µmol min-1) and Mannitol (0.076 µmol min-1) in the

third day of incubation.

The efficacy of Phosphatase activity of ANsa120 was maximum in the

medium containing Glucose (0.340 µmol min-1) followed by Sucrose (0.248

µmol min-1), Lactose (0.187 µmol min-1) and Mannitol (0.094 µmol min-1) in the

sixth day of incubation.

The efficacy of Phosphatase activity of ANsa120 was maximum in the

medium containing Glucose (0.474 µmol min-1) followed by Sucrose (0.330

µmol min-1), Lactose (0.213 µmol min-1) and Mannitol (0.134 µmol min-1) in the

ninth day of incubation.

Effect of Different Nitrogen Sources on the Efficacy of Phosphatase Activity

of Sodium Azide Treated Aspergillus niger (ANsa120).

The results of different nitrogen sources on the efficacy of Phosphatase

activity of sodium azide treated Aspergillus niger (ANsa120) were presented in

Table 37.

The efficacy of Phosphatase activity of ANsa120 was maximum in the

medium containing Ammonium sulphate (0.184 µmol min-1) followed by Urea

(0.112 µmol min-1), Potassium nitrate (0.108 µmol min-1) and Sodium nitrate

(0.008 µmol min-1) in the third day of incubation.

The efficacy of Phosphatase activity of ANsa120 was maximum in the

medium containing Ammonium sulphate (0.340 µmol min-1) followed by Urea

121

(0.216 µmol min-1), Potassium nitrate (0.203 µmol min-1) and Sodium nitrate

(0.103 µmol min-1) in the sixth day of incubation.

The efficacy of Phosphatase activity of ANsa120 was maximum in the

medium containing Ammonium sulphate (0.474 µmol min-1) followed by Urea

(0.273 µmol min-1), Potassium nitrate (0.254 µmol min-1) and Sodium nitrate

(0.125 µmol min-1) in the ninth day of incubation.

Effect of Different Carbon Sources on the Efficacy of Phosphatase Activity

of Sodium Azide Treated Penicillium sp (PEsa150).

The results of different carbon sources on the efficacy of Phosphatase

activity of sodium azide treated Penicillium sp (PEsa150) were presented in

Table 38.

The efficacy of Phosphatase activity of PEsa150 was maximum in the

medium containing Glucose (0.210 µmol min-1) followed by Sucrose (0.171

µmol min-1), Lactose (0.168 µmol min-1) and Mannitol (0.162 µmol min-1) in the

third day of incubation.

The efficacy of Phosphatase activity of PEsa150 was maximum in the

medium containing Sucrose (0.288 µmol min-1) followed by Glucose (0.276

µmol min-1), Lactose (0.214 µmol min-1) and Mannitol (0.204 µmol min-1) in the

sixth day of incubation.

The efficacy of Phosphatase activity of PEsa150 was maximum in the

medium containing Glucose (0.420 µmol min-1) followed by Sucrose (0.342

µmol min-1), Mannitol (0.261 µmol min-1) and Lactose (0.253 µmol min-1) in the

ninth day of incubation.

122

Effect of Different Nitrogen Sources on the Efficacy of Phosphatase Activity

of Sodium Azide Treated Penicillium sp (PEsa150).

The results of different nitrogen sources on the efficacy of Phosphatase

activity of sodium azide treated Penicillium sp (PEsa150) were presented in

Table 39.

The efficacy of Phosphatase activity of PEsa150 was maximum in the

medium containing Ammonium sulphate (0.210 µmol min-1) followed by Urea

(0.205 µmol min-1), Potassium nitrate (0.165 µmol min-1) and Sodium nitrate

(0.163 µmol min-1) in the third day of incubation.

The efficacy of Phosphatase activity of PEsa150 was maximum in the

medium containing Ammonium sulphate (0.276 µmol min-1) followed by Urea

(0.223 µmol min-1), Potassium nitrate (0.218 µmol min-1) and Sodium nitrate

(0.209 µmol min-1) in the sixth day of incubation.

The efficacy of Phosphatase activity of PEsa150 was maximum in the

medium containing Ammonium sulphate (0.420 µmol min-1) followed by Urea

(0.270 µmol min-1), Potassium nitrate (0.261 µmol min-1) and Sodium nitrate

(0.229 µmol min-1) in the ninth day of incubation.

Plate-4 shows the Phosphate solubilization and Phosphatase production

by fungal cultures in Pikovskaya broth.

Figure- 3 showes the comparsion of efficacy of Phosphatase activity by

wild strains and Figure-4 showes the comparsion of efficacy of Phosphatase

activity by chemical treated fungal strains.

123

Lipase Activity

Efficacy of UV Treated Aspergillus niger on Lipase Activity

The results of Lipase activity of UV treated Aspergillus niger were

presented in Table 40.

The maximum efficacy of Lipase activity of UV treated Aspergillus niger

(ANuv50) grown in Sucrose medium recorded (2.15 unit g-1 of substrate)

followed by ANuv40 strain (1.95 unit g-1 of substrate), ANuv60 (1.89 unit g-1 of

substrate) and ANuv30 (1.86 unit g-1 of substrate). The Lipase activity of wild

Aspergillus niger which was used as a standard control produced 1.62 unit g-1 of

substrate after 96 hrs incubation.

The maximum efficacy of Lipase activity of UV treated Aspergillus niger

(ANuv50) grown in olive oil medium recorded (0.90 unit g-1 of substrate)

followed by ANuv40 strain (0.62 unit g-1 of substrate), ANuv60 (0.60 unit g-1 of

substrate) and ANuv30 (0.49 unit g-1 of substrate). The Lipase activity of wild

Aspergillus niger which was used as a standard control produced 0.47 unit g-1 of

substrate after 96 hrs incubation.

The Lipase activity of ANuv50 strain increased 32.72% followed by

ANuv40 (20.37%), ANuv60 (16.66%) and ANuv30 (14.81%) after 96 hrs

incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of ANuv50 strain increased 91.49% followed by ANuv40

(31.91%), ANuv60 (27.66%) and ANuv30 (4.26%) after 96 hrs incubation in

olive medium using wild type as a standard (100%).

124

Efficacy of UV Treated Aspergillus fumigatus on Lipase Activity

The results of Lipase activity of UV treated Aspergillus fumigatus were

presented in Table 41.

The maximum efficacy of Lipase activity of UV treated Aspergillus

fumigatus (AFuv50) grown in Sucrose medium recorded (2.09 unit g-1 of

substrate) followed by AFuv60 strain (1.75 unit g-1 of substrate), AFuv40 (1.39

unit g-1 of substrate) and AFuv30 (1.22 unit g-1 of substrate). The Lipase activity

of wild Aspergillus fumigatus which was used as a standard control produced

1.01 unit g-1 of substrate after 96 hrs incubation.

The maximum efficacy of Lipase activity of UV treated Aspergillus

fumigatus (AFuv50) grown in olive oil medium recorded (0.60 unit g-1 of

substrate) followed by AFuv60 strain (0.56 unit g-1 of substrate), AFuv40 (0.38

unit g-1 of substrate) and AFuv30 (0.33 unit g-1 of substrate). The Lipase activity

of wild Aspergillus fumigatus which was used as a standard control produced

0.27 unit g-1 of substrate after 96 hrs incubation.

The Lipase activity of AFuv50 strain increased 106.93% followed by

AFuv60 (73.26%), AFuv40 (37.62%) and AFuv30 (20.79%) after 96 hrs

incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of AFuv50 strain increased 122.22% followed by AFuv60

(107.40%), AFuv40 (40.74%) and AFuv30 (22.22%) after 96 hrs incubation in

olive medium using wild type as a standard (100%).

Efficacy of UV Treated Penicillium sp on Lipase Activity

The results of Lipase activity of UV treated Penicillium sp were presented

in Table 42.

125

The maximum efficacy of Lipase activity of UV treated Penicillium sp

(PEuv60) grown in Sucrose medium recorded (1.69 unit g-1 of substrate)

followed by PEuv50 strain (1.62 unit g-1 of substrate), PEuv40 (1.43 unit g-1 of

substrate) and PEuv30 (1.28 unit g-1 of substrate). The Lipase activity of wild

Penicillium sp which was used as a standard control produced 1.20 unit g-1 of

substrate after 96 hrs incubation.

The maximum efficacy of Lipase activity of UV treated Penicillium sp

(PEuv60) grown in olive oil medium recorded (0.85 unit g-1 of substrate)

followed by PEuv40 strain (0.73 unit g-1 of substrate), PEuv50 (0.69 unit g-1 of

substrate) and PEuv30 (0.48 unit g-1 of substrate). The Lipase activity of wild

Penicillium sp which was used as a standard control produced 0.46 unit g-1 of

substrate after 96 hrs incubation.

The Lipase activity of PEuv60 strain increased 40.83% followed by

PEuv50 (35.00%), PEuv40 (19.17%) and PEuv30 (6.67%) after 96 hrs

incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of PEuv60 strain increased 84.78% followed by PEuv40

(50.00%), PEuv50 (58.7%) and PEuv30 (4.35%) after 96 hrs incubation in olive

medium using wild type as a standard (100%).

Efficacy of UV Treated Pseudomonas sp on Lipase Activity

The results of Lipase activity of UV treated Pseudomonas sp were

presented in Table 43.

The maximum efficacy of Lipase activity of UV treated Pseudomonas sp

(PSuv3) grown in Sucrose medium recorded (2.49 unit g-1 of substrate) followed

126

by PSuv4 strain (2.29 unit g-1 of substrate), PSuv1 (2.22 unit g-1 of substrate)

and PSuv2 (2.19 unit g-1 of substrate). The Lipase activity of wild Pseudomonas

sp which was used as a standard control produced 2.10 unit g-1 of substrate after

96 hrs incubation.

The maximum efficacy of Lipase activity of UV treated Pseudomonas sp

(PSuv3) grown in olive oil medium recorded (0.38 unit g-1 of substrate) followed

by PSuv4 strain (0.34 unit g-1 of substrate), PSuv2 (0.33 unit g-1 of substrate)

and PSuv1 (0.23 unit g-1 of substrate). The Lipase activity of wild Pseudomonas

sp which was used as a standard control produced 0.17 unit g-1 of substrate after

96 hrs incubation.

The Lipase activity of PSuv3 strain increased 18.57% followed by

PSuv4 (9.04%), PSuv1 (5.71%) and PSuv2 (4.28%) after 96 hrs incubation in

Sucrose medium using wild type as a standard (100%). The Lipase activity of

PSuv3 strain increased 123.53% followed by PSuv4 (100.00%), PSuv2 (94.12%)

and PSuv1 (35.29%) after 96 hrs incubation in olive medium using wild type as

a standard (100%).

Efficacy of Sodium Azide Treated Aspergillus niger on Lipase Activity

The results of Lipase activity of sodium azide treated Aspergillus niger

were presented in Table 44.

The maximum efficacy of Lipase activity of sodium azide treated

Aspergillus niger (ANsa90) grown in Sucrose medium recorded (2.61 unit g-1 of

substrate) followed by ANsa60 strain (2.09 unit g-1 of substrate), ANsa30 (1.90

unit g-1 of substrate) and ANsa120 (1.85 unit g-1 of substrate). The Lipase activity

127

of wild Aspergillus niger which was used as a standard control produced 1.62

unit g-1 of substrate after 96 hrs incubation.

The maximum efficacy of Lipase activity of sodium azide treated

Aspergillus niger (ANsa90) grown in olive oil medium recorded (0.98 unit g-1 of

substrate) followed by ANsa60 strain (0.68 unit g-1 of substrate), ANsa120 (0.63

unit g-1 of substrate) and ANsa30 (0.51 unit g-1 of substrate). The Lipase activity

of wild Aspergillus niger which was used as a standard control produced 0.47

unit g-1 of substrate after 96 hrs incubation.

The Lipase activity of ANsa90 strain increased 61.11% followed by

ANsa60 (29.01%), ANsa30 (17.28%) and ANsa120 (14.19%) after 96 hrs

incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of ANsa90 strain increased 108.61% followed by ANsa60

(44.68%), ANsa120 (34.04%) and ANsa30 (8.51%) after 96 hrs incubation in

olive medium using wild type as a standard (100%).

Efficacy of Sodium Azide Treated Aspergillus fumigatus on Lipase Activity

The results of Lipase activity of sodium azide treated Aspergillus

fumigatus were presented in Table 45.

The maximum efficacy of Lipase activity of sodium azide treated

Aspergillus fumigatus (AFsa120) grown in Sucrose medium recorded (1.60 unit

g-1 of substrate) followed by AFsa30 strain (1.45 unit g-1 of substrate), AFsa60

(1.25 unit g-1 of substrate) and AFsa90 (1.22 unit g-1 of substrate). The Lipase

activity of wild Aspergillus fumigatus which was used as a standard control

produced 1.01 unit g-1 of substrate after 96 hrs incubation.

128

The maximum efficacy of Lipase activity of sodium azide treated

Aspergillus fumigatus (AFsa120) grown in olive oil medium recorded (0.44 unit

g-1 of substrate) followed by AFsa30 strain (0.41 unit g-1 of substrate), AFsa60

(0.38 unit g-1 of substrate) and AFsa90 (0.30 unit g-1 of substrate). The Lipase

activity of wild Aspergillus fumigatus which was used as a standard control

produced 0.27 unit g-1 of substrate after 96 hrs incubation.

The Lipase activity of AFsa120 strain increased 58.42% followed by

AFsa30 (43.56%), AFsa60 (23.76%) and AFsa90 (20.79%) after 96 hrs

incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of AFsa120 strain increased 62.96% followed by AFsa30

(51.85%), AFsa60 (40.74%) and AFsa90 (11.11%) after 96 hrs incubation in

olive medium using wild type as a standard (100%).

Efficacy of Sodium Azide Treated Penicillium sp on Lipase Activity

The results of Lipase activity of sodium azide treated Penicillium sp were

presented in Table 46.

The maximum efficacy of Lipase activity of sodium azide treated

Penicillium sp (PEsa120) grown in Sucrose medium recorded (2.01 unit g-1 of

substrate) followed by PEsa90 strain (1.76 unit g-1 of substrate), PEsa30 (1.56

unit g-1 of substrate) and PEsa60 (1.43 unit g-1 of substrate). The Lipase activity

of wild Penicillium sp which was used as a standard control produced 1.20 unit

g-1 of substrate after 96 hrs incubation.

The maximum efficacy of Lipase activity of sodium azide treated

Penicillium sp (PEsa120) grown in olive oil medium recorded (0.99 unit g-1 of

substrate) followed by PEsa90 strain (0.73 unit g-1 of substrate), PEsa60 (0.69

129

unit g-1 of substrate) and PEsa30 (0.48 unit g-1 of substrate). The Lipase activity

of wild Penicillium sp which was used as a standard control produced 0.46 unit

g-1 of substrate after 96 hrs incubation.

The Lipase activity of PEsa120 strain increased 67.50% followed by

PEsa90 (46.67%), PEsa30 (30.00%) and PEsa60 (19.17%) after 96 hrs

incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of PEsa120 strain increased 115.22% followed by PEsa90

(58.70%), PEsa60 (50.00%) and PEsa30 (4.35%) after 96 hrs incubation in olive

medium using wild type as a standard (100%).

Efficacy of Sodium Azide Treated Pseudomonas sp on Lipase Activity

The results of Lipase activity of sodium azide treated Pseudomonas sp

were presented in Table 47.

The maximum efficacy of Lipase activity of sodium azide treated

Pseudomonas sp (PSsa90) grown in Sucrose medium recorded (3.23 unit g-1 of

substrate) followed by PSsa120 strain (3.04 unit g-1 of substrate), PSsa60 (2.93

unit g-1 of substrate) and PSsa30 (2.28 unit g-1 of substrate). The Lipase activity

of wild Pseudomonas sp which was used as a standard control produced 2.10

unit g-1 of substrate after 96 hrs incubation.

The maximum efficacy of Lipase activity of sodium azide treated

Pseudomonas sp (PSsa90) grown in olive oil medium recorded (0.39 unit g-1 of

substrate) followed by PSsa120 strain (0.39 unit g-1 of substrate), PSsa60 (0.35

unit g-1 of substrate) and PSsa30 (0.28 unit g-1 of substrate). The Lipase activity

of wild Pseudomonas sp which was used as a standard control produced 0.17

unit g-1 of substrate after 96 hrs incubation.

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The Lipase activity of PSsa90 strain increased 53.80% followed by

PSsa120 (44.76%), PSsa60 (39.52%) and PSsa30 (8.57%) after 96 hrs

incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of PSsa90 strain increased 129.41% followed by PSsa120

(129.41%), PSsa60 (105.88%) and PSsa30 (64.71%) after 96 hrs incubation in

olive medium using wild type as a standard (100%).

Efficacy of Ethyl Methane Sulphonate Treated Aspergillus niger on Lipase

Activity

The results of Lipase activity of Ethyl Methane Sulphonate treated

Aspergillus niger were presented in Table 48.

The maximum efficacy of Lipase activity of Ethyl Methane Sulphonate

treated Aspergillus niger (ANems150) grown in Sucrose medium recorded (3.85

unit g-1 of substrate) followed by ANems120 strain (3.10 unit g-1 of substrate),

ANems90 (2.09 unit g-1 of substrate) and ANems60 (1.90 unit g-1 of substrate).

The Lipase activity of wild Aspergillus niger which was used as a standard

control produced 1.62 unit g-1 of substrate after 96 hrs incubation.

The maximum efficacy of Lipase activity of Ethyl Methane Sulphonate

treated Aspergillus niger (ANems150) grown in olive oil medium recorded (0.94

unit g-1 of substrate) followed by ANems120 strain (0.87 unit g-1 of substrate),

ANems90 (0.77 unit g-1 of substrate) and ANems60 (0.62 unit g-1 of substrate).

The Lipase activity of wild Aspergillus niger which was used as a standard

control produced 0.47 unit g-1 of substrate after 96 hrs incubation.

The Lipase activity of ANems150 strain increased 137.65% followed by

ANems120 (91.35%), ANems90 (29.01%) and ANems60 (17.28%) after 96 hrs

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incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of ANems150 strain increased (100.00%) followed by

ANems120 (85.11%), ANems90 (63.83%) and ANems60 (31.91%) after 96 hrs

incubation in olive medium using wild type as a standard (100%).

Efficacy of Ethyl Methane Sulphonate Treated Aspergillus fumigatus on

Lipase Activity

The results of Lipase activity of Ethyl Methane Sulphonate treated

Aspergillus fumigatus were presented in Table 49.

The maximum efficacy of Lipase activity of Ethyl Methane Sulphonate

treated Aspergillus fumigatus (AFems120) grown in Sucrose medium recorded

(2.76 unit g-1 of substrate) followed by AFems150 strain (2.60 unit g-1 of

substrate), AFems90 (2.26 unit g-1 of substrate) and AFems60 (1.85 unit g-1 of

substrate). The Lipase activity of wild Aspergillus fumigatus which was used as

a standard control produced 1.01 unit g-1 of substrate after 96 hrs incubation.

The maximum efficacy of Lipase activity of Ethyl Methane Sulphonate

treated Aspergillus fumigatus (AFems120) grown in olive oil medium recorded

(0.45 unit g-1 of substrate) followed by AFems150 strain (0.41 unit g-1 of

substrate), AFems90 (0.40 unit g-1 of substrate) and AFems60 (0.33 unit g-1 of

substrate). The Lipase activity of wild Aspergillus fumigatus which was used as

a standard control produced 0.27 unit g-1 of substrate after 96 hrs incubation.

The Lipase activity of AFems120 strain increased 173.26% followed by

AFems150 (157.43%), AFems90 (123.76%) and AFems60 (83.17%) after 96 hrs

incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of AFems120 strain increased 66.67% followed by AFems150

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(51.85%), AFems90 (48.15%) and AFems60 (22.22%) after 96 hrs incubation in

olive medium using wild type as a standard (100%).

Efficacy of Ethyl Methane Sulphonate Treated Penicillium sp on Lipase

Activity

The results of Lipase activity of Ethyl Methane Sulphonate treated

Penicillium sp were presented in Table 50.

The maximum efficacy of Lipase activity of Ethyl Methane Sulphonate

treated Penicillium sp (PEems150) grown in Sucrose medium recorded (1.97

unit g-1 of substrate) followed by PEems120 strain (1.89 unit g-1 of substrate),

PEems90 (1.79 unit g-1 of substrate) and PEems60 (1.65 unit g-1 of substrate).

The Lipase activity of wild Penicillium sp which was used as a standard control

produced 1.20 unit g-1 of substrate after 96 hrs incubation.

The maximum efficacy of Lipase activity of Ethyl Methane Sulphonate

treated Penicillium sp (PEems150) grown in olive oil medium recorded (0.77

unit g-1 of substrate) followed by PEems120 strain (0.63 unit g-1 of substrate),

PEems90 (0.58 unit g-1 of substrate) and PEems60 (0.50 unit g-1 of substrate).

The Lipase activity of wild Penicillium sp used as a standard control produced

0.46 unit g-1 of substrate after 96 hrs incubation.

The Lipase activity of PEems150 strain increased 64.17% followed by

PEems120 (57.50%), PEems90 (49.17%) and PEems60 (36.67%) after 96 hrs

incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of PEems150 strain increased 67.39% followed by PEems120

(36.96%), PEems90 (26.09%) and PEems60 (8.70%) after 96 hrs incubation in

olive medium using wild type as a standard (100%).

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Efficacy of Ethyl Methane Sulphonate Treated Pseudomonas sp on Lipase

Activity

The results of Lipase activity of Ethyl Methane Sulphonate treated

Pseudomonas sp were presented in Table 51.

The maximum efficacy of Lipase activity of Ethyl Methane Sulphonate

treated Pseudomonas sp (PSems120) grown in Sucrose medium recorded (4.45

unit g-1 of substrate) followed by PSems150 strain (4.12 unit g-1 of substrate),

PSems90 (3.53 unit g-1 of substrate) and PSems60 (2.42 unit g-1 of substrate).

The Lipase activity of wild Pseudomonas sp which was used as a standard

control produced 2.21 unit g-1 of substrate after 96 hrs incubation.

The maximum efficacy of Lipase activity of Ethyl Methane Sulphonate

treated Pseudomonas sp (PSems120) grown in olive oil medium recorded (0.61

unit g-1 of substrate) followed by PSems150 strain (0.52 unit g-1 of substrate),

PSems90 (0.47 unit g-1 of substrate) and PSems60 (0.34 unit g-1 of substrate).

The Lipase activity of wild Pseudomonas sp which was used as a standard

control produced 0.22 unit g-1 of substrate after 96 hrs incubation.

The Lipase activity of PSems120 strain increased 101.36% followed by

PSems150 (86.43%), PSems90 (59.72%) and PSems60 (9.50%) after 96 hrs

incubation in Sucrose medium using wild type as a standard (100%). The

Lipase activity of PSems120 strain increased 177.27% followed by PSems150

(136.36%), PSems90 (113.64%) and PSems60 (54.55%) after 96 hrs incubation

in olive medium using wild type as a standard (100%).

134

Effect of Different Carbon and Nitrogen Sources on the Efficacy of Lipase

Activity.

The superior mutated cultures of Lipase in Czapek’s dox broth were

identified as Ethyl Methane Sulphonate treated Aspergillus niger (ANems120)

and Aspergillus fumigatus (AFems120), Aspergillus niger (ANems150),

Pseudomonas sp. (PSems150) and (PSems120) and their efficacy of lipase

activity were studied by using different Carbon and Nitrogen sources.

Effect of Different Carbon Sources on the Efficacy of Lipase Activity.

The results of different carbon sources on the efficacy of lipase activity of

Ethyl Methane Sulphonate treated Aspergillus niger (ANems120) and

Aspergillus fumigatus (AFems120), Aspergillus niger (ANems150),

Pseudomonas sp. (PSems150) and (PSems120) were presented in Tables 52 &

53.

The efficacy of lipase activity of Aspergillus niger (ANems120) was

maximum in the medium containing Sucrose (3.10 Unit/g of substrate) followed

by Glucose (2.94 Unit/g of substrate), Mannitol (1.85 Unit/g of substrate) and

Lactose (1.65 Unit/g of substrate) after 96 hrs incubation.

The efficacy of lipase activity of Aspergillus fumigatus (AFems120) was

maximum in the medium containing Sucrose (2.76 Unit/g of substrate) followed

by Glucose (2.54 Unit/g of substrate), Mannitol (1.69 Unit/g of substrate) and

Lactose (1.25 Unit/g of substrate) after 96 hrs incubation.

The efficacy of lipase activity of Aspergillus niger (ANems150) was

maximum in the medium containing Sucrose (3.85 Unit/g of substrate) followed

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by Glucose (2.44 Unit/g of substrate), Mannitol (1.98 Unit/g of substrate) and

Lactose (1.34 Unit/g of substrate) after 96 hrs incubation.

The efficacy of lipase activity of Pseudomonas sp. (PSems120) was

maximum in the medium containing Sucrose (4.45 Unit/g of substrate) followed

by Glucose (2.95 Unit/g of substrate), Mannitol (2.74 Unit/g of substrate) and

Lactose (1.80 Unit/g of substrate) after 96 hrs incubation.

The efficacy of lipase activity of Pseudomonas sp. (PSems150) was

maximum in the medium containing Sucrose (4.12 Unit/g of substrate) followed

by Glucose (2.45 Unit/g of substrate), Mannitol (2.05 Unit/g of substrate) and

Lactose (1.65 Unit/g of substrate) after 96 hrs incubation.

Effect of Different Nitrogen Sources on the Efficacy of Lipase Activity

The results of different nitrogen sources on the efficacy of lipase activity

of Ethyl Methane Sulphonate treated Aspergillus niger (ANems120) and

Aspergillus fumigatus (AFems120) Aspergillus niger (ANems150) Pseudomonas

sp. (PSems150) and (PSems120)were presented in Tables 54 & 55.

The efficacy of lipase activity of Aspergillus niger (ANems120) was

maximum in the medium containing Sodium nitrate (3.10 Unit/g of substrate)

followed by Potassium nitrate (2.85 Unit/g of substrate), Ammonium sulphate

(2.01 Unit/g of substrate) and urea (1.55 Unit/g of substrate) after 96 hrs

incubation.

The efficacy of lipase activity of Aspergillus fumigatus (AFems120) was

maximum in the medium containing Sodium nitrate (2.76 Unit/g of substrate)

followed by Potassium nitrate (2.67 Unit/g of substrate), Ammonium sulphate

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(2.13 Unit/g of substrate) and urea (1.54 Unit/g of substrate) after 96 hrs

incubation.

The efficacy of lipase activity of Aspergillus niger (ANems150) was

maximum in the medium containing Sodium nitrate (3.85 Unit/g of substrate)

followed by Potassium nitrate (2.24 Unit/g of substrate), Ammonium sulphate

(1.98 Unit/g of substrate) and urea (1.27 Unit/g of substrate) after 96 hrs

incubation.

The efficacy of lipase activity of Pseudomonas sp. (PSems120) was

maximum in the medium containing Sodium nitrate (4.45 Unit/g of substrate)

followed by Potassium nitrate (2.94 Unit/g of substrate), Ammonium sulphate

(2.71 Unit/g of substrate) and urea (1.45 Unit/g of substrate) after 96 hrs

incubation.

The efficacy of lipase activity of Pseudomonas sp. (PSems150) was

maximum in the medium containing Sodium nitrate (4.12 Unit/g of substrate)

followed by Potassium nitrate (2.99 Unit/g of substrate), Ammonium sulphate

(2.64 Unit/g of substrate) and urea (1.40 Unit/g of substrate) after 96 hrs

incubation.

Figure- 5 showes the comparsion of efficacy of lipase activity by wild

strains and Figure-6 showes the comparsion of efficacy of lipase activity by

chemical treated fungal strains.

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Indole Acetic Acid (IAA)

Screening Of IAA Production by Wild And Mutated Strains of Aspergillus

niger, Aspergillus fumigatus and Penicillium sp.

The results of IAA activity in wild and mutated strains of Aspergillus

niger, Aspergillus fumigatus and Penicillium sp were presented in Table 56 and

Plate-5 shows the IAA production by bacterial and fungal cultures in Czapek’s

dox broth and Thinlayer chromatogram for IAA.

The wild and mutated cultures were analysed for IAA production

qualitatively by Thin layer chromatography method. All the wild strains except

Aspergillus fumigatus were positive for IAA production. Among the mutated

strains EMS treated Aspergillus niger and UV, sodium azide and Ethyl

Methane Sulphonate treated Aspergillus fumigatus were negative for IAA

production where as rest of all mutated strains were positive for IAA production.

Quantitative Estimation of IAA Production by Wild And Mutated Strains

of Aspergillus niger, Penicillium sp. and Pseudomonas sp.

The wild and mutated cultures were analysed for IAA production

quantitatively by Spectrophotometric method. The results were presented in

Table 57.

The efficacy of Indole Acetic Acid production of Aspergillus niger was

maximum in ANsa120 (17.53µg/L) followed by ANsa90 (16.73µg/L), ANsa60

(15.65µg/L), ANuv30 (13.01µg/L), ANsa30 (12.75µg/L), ANuv40 (12.47µg/L)

and ANuv60 (11.85µg/L).

The wild Aspergillus niger produced 12.33 µg/L of Indole Acetic Acid.

138

The efficacy of Indole Acetic Acid production of Pencillium sp. was

maximum in PEsa120 (10.64µg/L) followed by PEsa90 (9.75µg/L), PEsa60

(8.88µg/L), PEsa150 (8.83µg/L), PEuv30 (8.67µg/L), PEems60 (8.35µg/L),

PEuv40 (7.55µg/L), PEems90 (7.45µg/L), PEuv50 (7.07µg/L), PEems120

(6.97µg/L), PEuv60 (6.83µg/L) and PEems150 (6.35µg/L).

The wild Pencillium sp. produced 7.68 µg/L of Indole Acetic Acid.

The efficacy of Indole Acetic Acid production of Pseudomonas sp. was

maximum in PSuv3 (8.60µg/L) followed by PSems150 (8.45µg/L), PSems120

(8.35µg/L), PSsa60 (8.23µg/L), PSsa30 (8.08µg/L), PSuv4 (8.08µg/L), PSsa90

(8.00µg/L), PSems90 (8.00µg/L), PSuv2 (7.55µg/L), PSems60 (7.07µg/L),

PSsa120 (6.75µg/L) and PEems150 (6.35µg/L).

The wild Pseudomonas sp. produced 7.50 µg/L of Indole Acetic Acid.

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Discussion

The Phosphate Solubilizing Microorganisms were isolated from rice

rhizosphere soil of Mannachanalur area, Trichy district, Tamilnadu. by using

Pikovskaya agar. Zone formation around the colony in Pikovskaya agar is the

basic criteria for the selection of Phosphate Solubilizing Microorganisms. The

earlier of reports were available for screening of Phosphate Solubilizing

Microorganisms, using Pikovskaya agar. (Hardy et al., 1998; Puente et al., 2004)

showed that the bacteria isolated from the rhizoplane of desert plants, was very

effective in dissolving Calcium phosphates on solid medium.

Phosphate Solubilization

In the present study, the phosphate solubilization efficacy of isolated

fungal strains was higher when compared to the bacterial strains in general.

These results were in coincidence with the earlier findings of Arora and Gaur

(1979);Kucey (1984). Among the fungal strains isolated in the present study, the

Aspergillus niger was found to be predominant phosphate solubilizer followed

by Penicillium sp., Pseudomonas sp. and Aspergillus fumigatus. This may be

due to the production of more organic acid in culture medium by the Phosphate

Solubilizing Microorganism. The more organic acid produced in the culture

medium decrease the pH which is the key indication for more phosphate

solubilization. The Ammonium Sulphate is a nitrogen source of Pikavskaya’s

medium, the acid production in response to the assimilation of cations such as

ammonium is well known fungal phenomenon. The uptake of ammonium by

fungi in a liquid medium commonly leads to a rapid drop in pH of the medium.

Whitelaw et al. (1999) reported that the soluble phosphate concentrations in the

culture medium was directly proportional to organic acid concentration and

inversely related to pH. The main mechanism for phosphate solubilisation by

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Penicillium radicum was acid production leading to a decrease in pH. Halder et

al. (1991) reported the phosphate solubilization was related to pH decrease

caused by the Bradyrhizobium strains. Cerezine et al. (1988) reported that the

soluble phosphate levels were correlated with pH of the culture medium,

probably due to metabolic activity of Aspergillus niger resulting from

consumption of sugar in the culture medium. Pradhan and Sukla. (2005)

described that the Aspergillus sp. solubilized higher phosphate comparing

Penicillium sp from Tricalcium Phosphate. Drop in pH during growth was more

prominent in absence of Tricalcium Phosphate in the liquid medium. This

indicates that absence of soluble phosphate in media induces the acid production.

Illmer et al. (1995) stated that the production of detectable amount of

organic acids by A. niger is an important factor for phosphate solubilization

mechanism. Nevertheless, organic acids alone are involved, although they are

less effective compared to biotic leaching. Narsian and Patel (2000) explained

that the acidic pH was accompanied with phosphate solubilization Aspergillus

aculeatus in all the experiments and concluded no specific correlation could be

established between maximum phosphate solubilization, growth or pH. Seshadri

et al. (2004) also stated that the release of phosphate by Aspergillus niger in the

liquid culture was associated with reduction in pH of culture medium. Barroso et

al. (2006); Achal et al. (2007) studied the relationship between phosphate

solubilization, extensive growth, acid production, and decrease in pH. They

concluded the phosphate solubilization was related to acid production, pH drop

and Aspergillus thubingensis growth in the culture medium.

Sperber (1957), Das (1963), Sethi and Subba Rao (1968) and Ahmed and

Jha (1968) were reported there is no significant relationship could be established

between the quantity of phosphate solubilized and drop in pH. The results of pH

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drop also support the data of phosphate solubilization by Pseudomonas, Bacillus

and Rhizobium, which is associated with acidification of the culture medium, but

the extent of phosphate solubilization and pH drop are not proportionally co-

related (Rodriguez and Fraga, 1999).

Bolan et al. (1994), Sagoe et al. (1997), Alam et al. (2002), and Kumari

et al. (2008) have reported that Malic, Tartaric, Oxalic, and Citric acids have

high capability to release soluble phosphate from insoluble Tricalcium

Phosphate as well as Rock Phosphate. Kang et al. (2008) suggested that the

solubilizing ability is not related to organic acid produced but to the nature of

organic acid produced by Aspergillus sp. Kapri and Tewari (2010) highlighted

the comparative phosphate solubilizing potential of Trichoderma sp. isolated

from Pinus, Deoder, Bamboo and Oak rhizospheres. It was found that all the

isolates were capable of differentially utilizing Tricalcium Phosphate from liquid

medium. This was indicated by the soluble phosphate concentrations and

increase in acidity of the growth medium.

In the present study, the phosphate solubilization efficacy of the wild

strains was observed in the descending order of Aspergillus niger >Penicillium

sp. > Aspergillus fumigatus. Among the UV irradiated fungal strains, ANuv60

was found as the predominant phosphate solubilizing strain followed by

ANuv50, PEuv60, ANuv30, ANuv40, PEuv50 and AFuv50. In chemical

mutagenesis of phosphate solubilizing strains, ANems120 showed the highest

phosphate solubilization activity followed by ANsa120, PEsa150, PEems150,

ANsa90, ANems90, PEsa120, ANsa60 and AFsa120. Achal et al. (2007)

reported a significant increase in soluble phosphate level was observed in case of

UV induced mutants of Aspergillus strains compared with wild strains. The

present study also revealed that UV induced mutant enhanced the phosphate

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solubilization compared with wild strains. There might be a possibility of

alteration at genetic levels in case of mutants. Tripura et al. (2007) reported that

the Ethyl Methane Sulphonate treated microbial strain Serratia marcescens have

increased phosphate solubilization efficiency compared to the wild strains.

Reyes et al. (2001) studied that phosphate solubilization of two UV induced

Penicillium rugulosam along with wild strain. Among the studied strains, MP++

strain solubilized high amount of phosphate and they stated that the

solubilization depends on the type of phosphate source.

In the present study, UV induced Penicillium sp. showed less

solubilization when compared to UV induced Aspergilus niger and the same

showed high solubilization when compared to wild Penicillium sp. This may be

due to more production of organic acid, Acid Phosphatase and Phytase enzyme

by mutant strains, which was supported by Achal et al. (2007). The mechanisms

of action of mineral phosphate solubilization (MPS) were studied in the wild-

type Mps+ Penicillium rugulosum strain and in negative (Mps−) and

superpositive (Mps++) mutants derived from it. MPS activities were measured in

liquid media using Sucrose as carbon source and nitrite, ammonium plus nitrate,

ammonium and arginine as nitrogen sources. Ammonium significantly decreased

phosphate solubilization, and this activity was higher in the Mps++ mutant than

in the wild-type depending on the N sources used. The Mps+ phenotype was

strongly associated with the production of gluconic or citric acids. The results

also suggest for the MPS− mutant the involvement of the H+ pump mechanism in

the solubilization of small amounts of phosphates. (Reyes et al. 1999). Vassileve

et al. (1998) reported the encapsulated spores of Aspergillus niger solubilized

Rock Phosphate in the culture medium due to the production of organic acids.

143

In the present study, Pseudomonas sp. was found to be the lowest

phosphate solubilizer, when compared to the fungal strains. The UV and

chemical mutated Pseudomonas strains were showed decreased phosphate

solubilizing capacity when compared to wild strains. These results were in

acceptable with the findings of Miller et al. (2010), who stated that mutants with

showed calcium phosphate solubilization ability. Mutations in the gcd and pqqE

genes greatly reduced the solubilization ability, whereas mutations in the pqqB

gene only moderately reduced this ability. The combination of biochemical

analysis and genomic comparisons revealed that alterations in the pqq

biosynthetic genes, and the presence/absence of the gluconate dehydrogenase

(gad) gene, fundamentally affect phosphate solublization in strains of

P. fluorescens. Goldstein and Liu (1987) have shown that mineral phosphate

solubilizing activity by Erwinia herbicola is genetically coded in a gene cluster

on the plasmids of microorganisms possessing this activity. It was also reported

that gene expression and mineral phosphate solubilization of bacteria is affected

by the presence of soluble phosphate due to feed back regulation. Kumar et al.

(2001) suggested that it is possible to select physiologically efficient strains of

A. chroococcum through mutagenesis starting from soil isolates and that

microbial inoculants can be used as an economic input to increase crop

productivity with lower fertilizer levels.

The present study indicates that the solubilization efficacy of the fungal

strain Aspergillus niger treated with Sodium Azide (ANsa120) and Penicillium

sp. treated with Ethyl Methane Sulphonate (PEems150) were about 1.5-fold

higher when compared to wild strain. The chemical mutagenic agents such as

Sodium Azide, Ethyl Methane Sulphonate may alter the gene sequence by

altering the base pairs. The azide ion alters the structure of cytosine such that it

forms hydrogen bonds with adenine, rather than guanine. This produces a

144

cytosine to thymine transition. Ethyl Methane Sulphonate is a strong mutagenic

agent. It alkylates N7 of Guanine and severely alters the base pairing.

The phosphate solubilization efficacy of mutant fungal strains

(ANems120, ANsa120 and PEsa150) were demonstrated in the presence of

various carbon and nitrogen sources. The effect of different carbon sources on

phosphate solubilization efficiency of fungal strains was found to be in the

ascending order Mannitol < Lactose < Sucrose < Glucose. This is in

disagreement with the findings of Seshadri et al. (2004) who reported that the

Mannitol was the best carbon source utilized by Aspergillus niger preferred for

higher phosphate solubilization. The present study revealed that Mannitol was

found to be the poorest carbon source and the Urea as one of the predominant

nitrogen sources for phosphate solubilization by Aspergillus niger. This in

confirmation with the findings of Seshadri et al. (2004) who reported the

nitrogen in the form nitrate was very effective and Urea was the poorest source

of nitrogen by Aspergillus niger. This may due to the genetic alteration in the

fungus due to chemical mutation. Reyes et al. (1999) studied that the Sucrose

appeared to be the best carbon source for phosphate solubilization by UV

induced Penicillium rugulosum. Nautiyal et al. (2000) reported that the Glucose

and Lactose were the best carbon source and Sucrose, Sorbitol were identified as

poor carbon source for phosphate solubilization, ammonium and nitrate source

to be equally effective for phosphate solubilization. This is in contrary with the

earlier report of Halder et al (1991) who stated that Glucose decreased the

medium pH most and caused highest solubilization of phosphate followed by

arabinose, sodium acetate, sodium gluconate and sodium succinate. Mohamed et

al (1985); Abd-Alla (1994) reported that ammonium salts induced phosphate

solubilization whereas the nitrate salts repress the phosphate solubilization

activity.

145

Similarly, in the present study, the addition of various nitrogen sources in

the medium to determine their effect on phosphate solubilization efficiency of

the inoculated strains (ANems120, ANsa120 and PEsa150) gave the following

results in the ascending order Sodium Nitrate < Potassium Nitrate < Urea <

Ammonium Sulphate. Whitelaw et al. (1999) reported that the Penicillium strain

solubilized the insoluble phosphate in the culture medium containing ammonium

or nitrate as sole source of nitrogen. Vassileve et al. (2007) compared the

phosphate solubilization efficacy of Aspergillus niger by using corn steep liquor,

Ammonium Sulphate and yeast extract as nitrogen source. The phosphate

solubilization activity of Xanthomonas campestris was measured in both the

wild type and mutant strains using various carbon and nitrogen sources. Glucose

was found to be the best in both (wild 39.9%; mutant 67.1%) strains followed by

Sucrose (46.8%) in the mutant and molasses (36.0%) in the wild type.

Ammonium Sulphate was the best nitrogen source for both the strains, followed

by and Urea. (Sharan et al., 2008). Son et al. (2006) investigated the effect of

carbon sources on the insoluble phosphate solubilization Pseudomonas species.

It solubilized insoluble phosphate with all carbon sources except Adonitol and

Lactose. They also studied all nitrogen sources except Sodium Nitrite, increased

the level of soluble phosphate production. Among the carbon and nitrogen

sources used, Glucose and Ammonium Nitrate was the best carbon and nitrogen

source respectively for the insoluble phosphate solubilization by Pseudomonas

species. In all cases insoluble phosphate solubilization was accompanied by a

distinct pH decrease.

Cerezine et al. (1988) reported the soluble phosphate levels were

correlated with pH of the culture medium probably due to metabolic activity of

fungus resulting from consumption of sugar in the Aspergillus niger culture

medium. Fructose, Glucose, xylose and Sucrose were the carbohydrates that

146

favored ‘P’ solubilization when compared to Galactose and Maltose. Among the

nitrogen sources tested ammonium salts favored the production of larger

amounts of soluble phosphate than peptone, Urea and Nitrate. The results were

positively correlated with the present study. Phosphate solubilization was

decreased with decreasing Glucose concentration; Solubilization was increased

when the nitrogen source was Nitrate by the soil fungus Aspergillus niger

(Hardy et al. 1998). The present study revealed that the carbon source Glucose

and nitrogen source Ammonium Sulphate were the best for phosphate

solubilization, which confirm results of earlier reports. Goenadi et al. (2000)

have reported that the accumulation of more insoluble phosphate using

Aspergillus niger and Glucose as the carbon source. Illmer and Schinner (1992)

have shown that the accumulation of high insoluble phosphate using Glucose

utilizing Penicillium sp. and Pseudomonas sp. respectively. According to

Srividya et al. (2009) the Aspergillus niger utilized the nitrogen salts having

either ammonium group or nitrate group or both as nitrogen source. Ammonium

Sulphate was found to be best in reducing the medium pH and simultaneous

solubilization of phosphate, out of the entire nitrogen sources used and observed

that Aspergillus niger was able to utilize Ammonium Sulphate most efficiently

to decrease the pH of the medium for phosphate solubilization. Fasim et al.

(2002) have reported that the bacterial isolates from the air environments of

tannery solubilize the Zinc Phosphate only in presence of Glucose. Disimmine,

(1998) reported solubilization of Zinc Phosphate by Pseudomonas fluorescens

only in presence of Glucose and slight solubilization in presence of mannose

while no solubilization was detected in presence of Gluconate, Galactose,

Glycerol, Sorbitol and Fructose.

The present study reported that the poorest carbon source and nitrogen

source were Mannitol and Sodium Nitrate respectively for phosphate

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solubilization. The results of present study were negatively correlated with

Barroso et al. (2006) who stated that solubilization of CaHPO4 was enhanced by

Aspergillus niger when the carbon source was Mannitol, Maltose, Galactose and

Glucose (in that order) and this was positively correlated with the present study

when evaluating with nitrogen sources. The solubilization of CaHPO4 or AlPO4

decreased in the following order: Glycine > NH4Cl > NaNO3 and NH4NO3 >

Urea > (NH4)2SO4, respectively. Ammonical nitrogen (NH4+-N) sources were

the most effective in the production of acids and in lowering of the pH.

Previous reports on phosphorus solubilizing microorganisms like

Ectomycorrhizal fungi by Lapeyrie et al., (1991) who attributed the differences

in phosphate solubilization (when ammonium and nitrate were used) to the use

of different mechanisms for the generation of acidity in the culture. Production

of organic acids is an important mechanism, but is not the only possibility for

phosphate solubilization. The other mechanism involved in solubilization of

insoluble phosphates is extrusion of H+ ions during ammonium ions assimilation

by Parks et al. (1990) for Penicillium like fungus, Illmer and Schinner (1995) for

Pseudomonas sp. and Penicillium aurantiogriseum Illmer et al. (1995) for

Aspergillus niger, Penicillium simplicissimum, Pseudomonas sp. and Penicillium

aurantiogriseum. In the present study, the phosphate solubilization using

ammonium salts as the nitrogen source was substantially higher than with other

nitrogen sources. Thus, the results suggested the second mechanism which

acidification of the medium by proton extrusion during ammonium ions was

involved in phosphate solubilization by these isolates.

Phosphatase

The Phosphatase production capacity of isolated bacterial and fungal

cultures were analysed by inoculating the bacterial and fungal isolates into

148

pikovskaya broth. In the present study, para nitro phenyl phosphate was used as

a substrate for Acid Phosphatase assay. The earlier reports were available for the

utilization of para nitro phenyl phosphate as a substrate for Acid Phosphatase

assay (Prada et al. 1996;Puruchothaman, 1994).

In the present study, the Phosphatase production efficacy of the wild

strains were observed in the descending order of Aspergillus niger>

Pseudomonas sp >Penicillium sp. > Aspergillus fumigatus. The UV irradiated

and chemical treated bacterial strains in the present study showed that decrease

in Phosphatase production, when compared to wild strains. Among the UV

irradiated fungal strains, ANuv60 was found the best Phosphatase producing

strain followed by ANuv50, ANuv30, ANuv40, AFuv50, PEuv50, AFuv60,

PEuv40, PEuv60, PEuv30, AFuv40 and AFuv30.

Among the chemical treated fungal strains, ANsa120 was the

predominant fungal strain followed by ANems120, PEems150, ANsa90,

PEsa150, ANems90, PEsa120, AFsa120, PEems120, PEems60, PEems90,

ANems60, AFsa120, AFsa90, PEsa90, ANsa30, AFems120, ANems30,

AFems90, PEsa60, AFsa60, AFems60, AFems30 and AFsa30. The result of the

present study was in agreement with Relwani et al. (2008) who reported that the

enzyme activities such as Acid Phosphatase and Phytase was increased

significantly in mutants of A. tubingensis when compared to the wild type. Achal

et al. (2007) reported that Acid Phosphatase production by UV induced mutants

of Aspergillus tubingensis, might be the reason for highest phosphate

solubilization by mutant strains. The production of the Acid Phosphatase was

favoured by low pH values.

149

Among all physical and chemical treated fungal strains, the best

Phosphatase producing strains such as ANsa120, ANems120 and PEems150

were demonstrated in the presence of various carbon and nitrogen sources in the

present study. The effect of different carbon sources on Phosphatase production

efficacy of fungal strains were found to be in the ascending order of Mannitol<

Lactose < Sucrose < Glucose. Andrea et al. (1990) reported that Pseudomonas

aeruginosa and Rhizobium meliloti, utilized several choline derivatives as the

sole carbon and nitrogen source and increased the production of Acid

Phosphatase activity. Spicers and McGill (1979) demonstrated an increase in

Phosphatase activity during incubation of soil amended with Glucose and

Ammonium Sulphate.

The effect of different nitrogen sources on Phosphatase production

efficacy of fungal strains were found to be in the ascending order of Sodium

Nitrate < Potassium Nitrate < Urea < Ammonium Sulphate in the present study.

The results were in agreement with Semenova et al. (1986) who stated that the

synthesis of the secreted enzyme depended on the sources of carbon and nitrogen

nutrition. The enzyme yield was highest in a medium with Sucrose as a carbon

source and Ammonium salt as a nitrogen source. The secretion of Acid

Phosphatase is stimulated by an increase in the sugar content and a deficiency of

the nitrogen source in the medium.

Semenova et al. (1986) studied that Acid Phosphatase production from

Saccharomyces cerevisiae in presence of phosphate concentration of 10 mM

which showed repression of the Phosphatase production. Sudha and

Purushothaman (2000) reported that Pseudomonas strain isolated from the

freshwater pond system showed repression. Casida (1959) isolated several

species of Aspergillus sp. capable of producing Acid Phosphatase active against

150

organic phosphate in soils. Smith et al. (1973) reported the addition of gradient

amount of Potassium phosphate to Phosphatase test broth was the least

inhibitory to Phosphatase activity by Candida tropicalis. Sarapatka et al. (2004)

stated that the increasing amount of available phosphate increasing the

Phosphatase activity in cereal roots. Pedregosa et al. (1991) reported that

Aspergillus strains produce non repressible, repressible and phosphate inducible

Phosphotases. It is accepted that culture condition may cause fluctuations and

multiplicity of fungal Phosphatases. Ponmurugan and Gopi (2006) stated that

there was a positive correlation between phosphate solubilizing capacity and

Phosphatase enzyme activity. These earlier reports were in agreement with

present results which showed that the increase in inorganic phosphate

concentration due to phosphate solubilization were not repressed the

Phosphatase production by all strains used in this study. But disagreement with

the earlier reports of Semenova et al. (1986) who studied that Acid Phosphatase

production from Saccharomyces cerevisiae in presence of phosphate

concentration of 10 mM which showed repression of the Phosphatase

production. Ohta et al. (1968) stated that the production of Phosphatase by the

black koji fungus Aspergillus awamori is a repressible enzyme.

The earlier reports explained that there are two systems of regulation in

Phosphatase induction in microorganisms. This may be the reason for repressible

and non repressible nature of Phosphatases by inorganic phosphate. Singleton

and Sainsberry (1988) explained that about the phosphate controlled genes or

pho genes, which is the gene cluster, consist of Pho A encodes Phosphatase, Pho

S encodes inorganic phosphate binding site, PhoE encodes porin and Pho B

encodes the regulator gene as like the operon model. Wagner et al. (1995)

reported that Synechoccus strain system of phosphate irrepressible Acid

Phosphatase in addition to the normal repressible system.

151

Lipase

The Lipase production capacity of isolated bacterial and fungal cultures

were analysed by inoculating the isolates in Czapek’s dox broth enriched with

1% Olive oil as a inducer and the same were inoculated in Czapek’s dox broth

replaced carbon source as 10% Olive oil for Lipase production. The number of

reports were available for the addition of inducer to induce the production of

Lipases. Salihu et al. (2011) utilized Olive oil and Palm oil as an inducer. Olive

oil by Rifaat et al. (2010); Hosseinpour et al. (2011); Cocunut oil by Rani and

Panneerselvam (2009) and different lipid sources namely Olive oil, Coconut oil,

Groundnut oil, Triacetin, Tributyrin and Surfactants namely Tween 20, Tween

40, Triton –X was used for acceleration of Lipase by Pogaku et al. (2010). It was

suggested that the Lipase formed might be derived from carbohydrates in the

medium with small quantities of lipid material as inducer (Minoda and Ota,

1980; Iwai et al., 1973).

In the present study, the Lipase activity of isolated bacterial strains were

higher compared to the fungal strains in the present study. Rani and

Panneerselvam (2009) showed that Aspergillus fumigatus, A. terreus,

Penicillium chrysogenum, P. funiculosum and Fusarium moniliforme were

selected as the highest Lipase producers. In the present study, the Lipase

production efficacy of the wild strains was observed in the descending order of

Pseudomonas sp.>Aspergillus niger >Penicillium sp. > Aspergillus fumigatus.

Juichiro et al. (1963) reported that several strains of Rhizopus and Asphergillus

niger as most potent Lipase formers. Successful isolation of the enzyme could be

made from the bran-koji culture of Aspergillus niger. Ogundero (1981) stated

that A. fumigatus and A. nidulans were able to degrade vegetable oils and

triglycerides.

152

Among the UV irradiated bacterial strains in the present study, PSuv3

was found to be the predominant Lipase producing strain grown in both Sucrose

and 10% Olive oil containing medium followed by PSuv4, PSuv2 and PSuv1.

Among the UV irradiated fungal strains, ANuv50 was found the best Lipase

producing strain grown in both Sucrose containing medium followed by

AFuv50, ANuv40, ANuv60, ANuv30, AFuv60, PEuv60, PEuv50, PEuv40,

AFuv40, PEuv30 and AFuv30.

Lawrence et al. (1967) studied the Lipase activity was considerably

increased by nutritional and physical conditions from Pseudomonad. Suzuki et

al. (1988) used the Olive oil as a carbon source for microbial growth and Lipase

production by Pseudomonas sp. Ray et al. (1999) reported the Lipase activity of

the isolated bacterial strain, Corynebacterium sp. was increased 2.3 fold by

mutagenic technique. This was agreed with our present study, since all mutant

strains tested were found to produce more Lipase activity, when compared to

wild strains. The present study also indicated, the strain Pseudomonas sp.

(PSems120) treated with Ethyl Methane Sulphonate, produced 2-fold higher

Lipase activity when compared to wild strain. PSems120 which was the

predominant Lipase producing strain, among all the strains used in the present

study and the bacterial strains PSems150, PSems90, PSsa90 and PSsa120 were

followed the strain PSems120. ANems150 was the best strain for Lipase

production among the chemical mutated fungal strains followed by ANems120,

AFems120, Ansa90, AFems150, AFems90, ANsa60, PEsa120 and PEems150

grown in Sucrose containing medium. Ellaiah et al. (2002) isolated a fungal

strain, which produced Lipase constitutively was used to produce mutants using

physical and chemical agents and the mutant strain with Lipase productivity of

2-fold higher was obtained. Bapiraju et al. (2004) reported that the the UV and

chemical treated fungal strains of Rhizopus sp. showed 133% to 232% higher

153

than the wild strains. Caob and Zhanga (2000) reported that the Lipase

production of UV and chemical treated Pseuomonas mutant 3.25 fold higher

than the wild strain.

The present study stated that the mutant strain PSems120, ANsa90,

PSsa90, AFuv50 and PEsa120 showed 2 fold and PSems150, PEuv40, PEsa90,

AFsa120 and PSsa90 showed 1.5 fold enhanced productivity of enzyme, over

the wild strain. This was in agreement with findings of Mahadik et al. (2004),

who reported that Mutant strains of Aspergillus niger showed seven to five fold

enhanced productivity of Lipase, over the wild strain. Mala et al. (2001) isolated

UV and nitrous acid derived mutants of A. niger selected on media containing

bile salts. Nitrous acid mutants exhibited increased efficiency of Lipase

production compared with wild strain. Mutation alters the genotype of

microorganisms, when it expresses that leads to alter the character or death of

microorganisms. The ultra-violet radiation forms thymine dimer in gene

sequence. But the photolyase enzymes present in living system break the

thymine dimer and correct it. The increasing exposure time to UV radiation may

forms the thymine dimer in gene sequence that code photolyase enzyme. In this

situation the thymine dimer can not break by the enzyme of living system

(Freifelder, 1990; Radman, 1999). The chemical agents such as Sodium Azide,

Ethyl Methane Sulphonate may alter the gene sequence by altering the base

pairs. The azide ion alters the structure of cytosine such that it forms hydrogen

bonds with adenine, rather than guanine. This produces a cytosine to thymine

transition. Ethyl Methane Sulphonate is a strong mutagenic agent. It alkylates

N7 of Guanine and severely alters the base pairing.

In the present study, all the bacterial strains were showed less Lipase

activity, when compared to fungal strains grown in 10% Olive oil containing

154

medium and moreover all the strains, grown in 10% Olive oil containing

medium were showed poorest Lipase activity. This may due to the inoculated

strains failed to utilize more 10% Olive oil as a carbon source for their growth

and subsequently reduction in Lipase activity. This was in agreement with

Mahadik et al. (2004) who stated, higher concentration of oil in the medium did

not help Lipase production in the case of mutant. On the other hand, Sucrose,

Lactose, Mannitol and Glucose may support the growth initially and the inducer

1% Olive oil induced the inoculated strains for more Lipase production in latter

stages. Many researchers have reported the positive effect of sugars on Lipase

production by Aspergillus niger (Pal et al., 1978), by Rhizopus chinensis

(Nakashima et al., 1988) and by Rhizopus oryzae (Salleh et al., 1993). On the

contrary of present study, Muralidhar et al. (2001) stated that their experimental

results indicate that Olive oil is a better carbon source for Lipase production by

Candida cylindraces compared to Glucose. This is supported by Dalmau et al.,

2000; Gordillo et al., 1998 for Candida rugosa. Optimization of the quantity of

Olive oil in the fermentation medium along with the other nutrients resulting in

the rise in enzyme production also contradicts the fact that large quantities of

Olive oil decrease the Lipase activity by for Candida rugosa (Gordillo et al.,

1995).

Nahas (1988) reported that Carbohydrates were good sources of carbon

for growth but low Lipase production was obtained by Rhizopus oligoporus.

Lipase production was strongly repressed by higher concentration of Glucose. In

present study revealed that fungal culture utilizing Olive oil as a carbon source

exhibited lowest Lipase producing activity. This result was not in agreement

with those of Paul and Carles (1992). Falony et al. (2006) reported that the A.

niger strain showed more Lipase activity among the test strains used. The

production of Lipase was more significant in culture medium added with lipids

155

as the carbon source than in the culture medium without lipids. Previous works

on the physiology of Lipase production showed that the mechanisms regulating

biosynthesis vary widely in different microorganisms. Results obtained with

Calvatia (Christakopoulos et al., 1992), Rhizopus (Salleh et al., 1993),

Aspergillus (Pokorny et al., 1994), and Rhodotorula (Papaparaskevas et al.,

1992), showed that Lipase production seems to be constitutive and independent

of the addition of lipid substrates to the culture medium, although their presence

enhanced the level of Lipase activity produced. On the other hand, (Shimada et

al., 1992; Rapp, 1995), lipid substrates are necessary for Lipase production by

Geotrichium candidum and Fusarium oxysporum respectively and also,

carbohydrates can act as repressors of its biosynthesis.

In the present study, Lipase production efficacy of mutant fungal strains

(ANems120, AFems120, ANems150, PSems120 and PSems150) were

demonstrated in the presence of various carbon and nitrogen sources. The effect

of different carbon sources on Lipase production efficiency of bacterial and

fungal strains was found to be in the ascending order Mannitol < Lactose <

Glucose < Sucrose. In present study Aspergillus niger, and Penicillium sp.,

utilized Sucrose as a carbon source exhibited highest Lipase producing activities,

these results are in agreement with those of Susumu and Yashio (1975). Elliah

et al. (2002) reported that the Aspergillus niger utilized dextrose as carbon

source for highest Lipase activity. The Lipase production was decreased in the

order of Sucrose < Lactose < Mannitol < Dextrose by the mutant strain of

Aspergillus niger. These results were in contrary to the present study results.

Petrovic et al. (1990) reported maximum Lipase production when

Glucose and peptone were incorporated in the production medium using

Penicillium roquefortii. In the present study, Glucose favoured the production

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of Lipase. This is in contrary with Mahadik et al. (2004) who reported that there

was no increase in enzyme levels was observed when mutant UV-10 was grown

in medium supplemented with Glucose. However, the addition of Glucose in the

medium resulted in increased levels of Lipase production by wild strain,

Aspergillus niger. The maximum Lipase activity was found with the

combination of Sucrose with Olive oil inducer and Glucose with Olive oil

inducer in the present study. But the repressive effect of Glucose with fatty oil is

reported by Dalmau et al. (2000) for Candida rugosa, Nahas (1988) for

Rhizopus oligosporus, Baillargeon et al. (1989) for Geotrichium candidum, and

Rapp (1995) for Fusarium oxysporum. but differ from those obtained for C.

rugosa by Chang et al. (1994). Cordova (1998) reported that the carbon sources

such as Glucose, Fructose, Glycerol, Xylose, Sucrose and Lactose are probably

utilized before Lipase production, there were little or no differences with these

substrates as carbon source.

Damaso et al. (2008) reported that the Aspergillus niger was found to be

the best Lipase producer. Soap stock was the best substrate and inducer compare

to Olive oil and they also explained the repressive effect Olive oil on Lipase

production. Coca et al. (2001) stated that the Aspergillus niger and Aspergillus

fumigatus were the best Lipase producer in the medium containing Olive oil as a

carbon source. This is in contrary with our present results. The present study

revealed that Aspergillus fumigatus utilizing Mannitol and Lactose as a carbon

sources and exhibiting lowest Lipase producing activity. In case of Sucrose and

Glucose exhibited highest Lipase producing activity.

Kakde and Chavan. (2011) found that carbon sources like Fructose and

Sucrose induced Lipase activity while Lactose, Starch and Carboxyl Methyl

Cellulose inhibited Lipase activity by Penicillium chrysogenum. Nitrogen

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sources like Nitrate, Nitrite, Amide, Ammonium, and Protein showed its effect

on Lipase enzyme of fungi. Casein and Peptone which are organic forms

stimulated maximum Lipase enzyme production of storage fungi. Both the

species of Penicillium viz. Penicillium notatum and Penicillium chrysogenum

showed maximum extracellular Lipase activity in presence of Casein and

Peptone. Urea which is an amide form and Ammonium Phosphate which is an

ammonium form hampered the extracellular Lipase enzyme production of fungi.

These reports were found to be similar to our present study results. Chavan and

Kakde, (2009) studied that the Lipase enzyme activity of storage fungi under the

influence of carbon and nitrogen sources. They found that carbon sources as like

Fructose and Sucrose induces Lipase activity while Starch, Lactose and

Carboxyl Methyl Cellulose inhibit Lipase activity. Nitrogen sources as like

Nitrate, Nitrite, Amide, Ammonium, and Protein affect in different ways on

Lipase enzyme of fungi. The results of the present study revealed that the

Lactose and Urea was a poor carbon and nitrogen sources respectively in the

tested bacterial strains like PSems120 and PSems150 and fungal strains like

ANems120, AFems120 and ANems150.

The effect of different nitrogen sources on Lipase production efficiency

of bacterial and fungal strains was found to be in the ascending order Urea

<Ammonium Sulphate < Potassium Nitrate < Sodium nitrite in the present

study. This was in accordance with Lima et al. (2003), who stated that the

Lipase activity by Penicillium sp. was higher when the medium containing only

the inorganic nitrogen sources and in the presence of Olive oil, Ammonium

Sulphate produced less mycelial growth subsequently less Lipase activity. But

the Ammonium Sulphate and Potassium Nitrate combination produced higher

Lipase activity in short fermentation time. Other workers (Freire et al., 1997;

Pereira-Meirelles et al., 1997;Samad et al., 1990) employed Candida lipolytica,

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Penicillium restrictum and Rhizopus sp. reported maximum Lipase production,

when organic nitrogen was used as a nitrogen source. Waller and Comeau

(1990) reported that the incorporation of corn steep liquor into the production

medium gave good Lipase activity by Candida sp. Rodriguez et al. (2006)

reported that Ammonium Sulphate, Ammonium Nitrate and Sodium Nitrate

except Urea reduced the production of Lipase activity compared to the initial

medium containing yeast extract. With Urea, the activity was around six times

higher. A similar observation has been reported using Penicillium sp. (Gombert

et al., 1999). On the other hand, Lima et al. (2003) found that Lipase production

in Penicillium sp. was stimulated using Ammonium Sulphate.

Indole Acetic Acid

The phosphate solubilizing wild and mutant strains of bacteria and fungi

were analysed for their Indole Acetic Acid production efficacy qualitatively by

Thin Layer Chromatography. Phosphate Solubilizing Bacteria are capable of

producing physiologically active Auxins that may.have pronounced effects on

plant growth. (Ponmurugan and Gopi. 2006). Shahab et al. (2009) reported that

the phosphate solubilizing microbes from Mung beans excreted the

phytohormones like Auxin and ethylene.

The results of present study revealed that the wild strains of Pseudomonas

sp., Aspergillus niger and Penicillium sp. have the ability to produce the plant

hormone Indole Acetic Acid except Aspergillus fumigatus. Among the mutated

bacterial and fungal strains, all the strains were able to produce Indole Acetic

Acid except Ethyl Methane Sulphonate treated Aspergillus niger and all the

strains of Aspergillus fumigatus. Since the wild strains failed to produce Indole

Acetic Acid, the mutant Aspergillus fumigatus might not produced the Indole

Acetic Acid.

159

Kulanthaivel et al. (2006) reported that Azosprillum sp. were treated with

UV, Acridine orange and Ethidium bromide. The production of Indole Acetic

Acid by most of mutant strains were higher when compared to wild strains. This

is in agreement with the present study results, which stated most of the mutant

produce more amount of Indole Acetic Acid when compared to wild strains.

Garcia et al. (1980) stated that the mutant Azosprillum brasilense excreated

Indole Acetic Acid more when compare to wild strains. Normanly et al. (1983)

confirmed that the orangepericap mutant Arabidopsis thaliana, accumulate

Auxin greater than wild type.

Tien et al. (1979) reported that Azospirillum and phosphobacteria isolated

from the soil of pearl millet produced Indole Acetic Acid, gibberellic acid and

cytokinin like substances. (Brown, 1972), and some of them are capable of

dissolving phosphate (Barea et al., 1976). Production of Indole Acetic Acid

varies greatly among different species and is also influenced by culture

conditions, growth stage and availability of substrate (s) (Brown, 1972; Vijila,

2000).

160

Summary and Conclusion

The Phosphate solubilizing microorganisms were isolated from rice

rhizosphere soil of Mannachanalur area, Trichy district, Tamilnadu. by

using Pikovskaya agar.

The efficacy of Phosphate solubilization assay and Phosphatase activity

assay of isolated bacterial and fungal strains were analysed by

spectrophotometrically using Pikovskaya broth.

The efficacy of Lipase activity by the isolated bacterial and fungal strains

were analysed spectrophotometrically using Czapadox broth.

Screening of Indole Acetic Acid production of isolated bacterial and

fungal strains were analysed by Thin Layer Chromatography and

spectrophotometric estimation using Czapadox broth.

Random mutational studies such as UV and chemical mutation of isolated

bacterial and fungal strains to improve their phosphate solubilization,

Phosphatase activity, Lipase activity and Indole Acetic Acid production

capability were carried out.

The results of present study revealed that the phosphate solubilization

efficacy of isolated fungal strains was higher when compared to the

bacterial strains.

The phosphate solubilization efficacy of the wild strains was observed in

the descending order of Aspergillus niger >Penicillium sp. > Aspergillus

fumigatus.

Among the UV irradiated fungal strains, ANuv60 was found as the

predominant phosphate solubilizing strain.

ANems120 showed the highest phosphate solubilization activity among

the chemical mutated fungal strains.

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The solubilization efficacy of the fungal strain Aspergillus niger treated

with Sodium Azide (ANsa120) and Penicillium sp. treated with Ethyl

Methane Sulphonate (PEems150) were about 1.5-fold higher when

compared to wild strain.

The effect of different carbon sources on phosphate solubilization

efficiency of fungal strains was found to be in the ascending order

Mannitol < Lactose < Sucrose < Glucose.

The effect of different nitrogen sources on phosphate solubilization

efficiency of fungal strains was found to be in the ascending order

Sodium Nitrate < Potassium Nitrate < Urea < Ammonium Sulphate.

Lipase activity of isolated bacterial strain Pseudomonas sp. higher when

compared to the isolated fungal strains.

The Lipase production efficacy of the wild microbial strains were

observed in the descending order of Pseudomonas sp.>Aspergillus niger

>Penicillium sp. > Aspergillus fumigatus.

Among the UV irradiated bacterial strains in the present study, PSuv3

was found to be the predominant Lipase producing strain grown in both

Sucrose and 10%Olive oil containing medium.

The Pseudomonas sp. (PSems120) was the predominant Lipase producing

strain, among all the strains, which produced 2-fold higher Lipase

activity when compared to wild strain.

The effect of different carbon sources on Lipase production efficiency of

bacterial and fungal strains was found to be in the ascending order of

Mannitol < Lactose < Glucose < Sucrose.

The effect of different nitrogen sources on Lipase production efficiency

of bacterial and fungal strains was found to be in the ascending order of

Urea <Ammonium Sulphate < Potassium Nitrate < Sodium nitrite.

162

The Phosphatase production efficacy of the wild strains were observed in

the descending order of Aspergillus niger.> Pseudomonas sp

>Penicillium sp. > Aspergillus fumigatus.

Among the UV irradiated fungal strains, ANuv60 was found to be the

best Phosphatase producing strain.

Among the chemical treated fungal strains, ANsa120 was the

predominant fungal strain for Phosphatase activity.

The effect of different carbon sources on Phosphatase production efficacy

of fungal strains were found to be in the ascending order of Mannitol<

Lactose < Sucrose < Glucose.

The effect of different nitrogen sources on Phosphatase production

efficacy of fungal strains were found to be in the ascending order of

Sodium Nitrate < Potassium Nitrate < Urea < Ammonium Sulphate.

The wild and mutated strains of Pseudomonas sp., Aspergillus niger and

Penicillium sp. have the ability to produce the plant hormone Indole

Acetic Acid except Aspergillus fumigatus.

It can be concluded from the findings of present study that the wild and

mutant bacterial and fungal strains like Pseudomonas sp., Aspergillus niger,

Penicillium sp. and Aspergillus fumigatus can be exploited biotechnologically

for the production of Phosphatase and Lipase.

Table 1. Rhizosphere Microbial Density.

Microorganism

Dilutions Cfu/ml of Mother

suspension 10-3 10-4 10-5 10-6 10-7

Bacteria

(Cfu/ml)* TNTC TNTC 255 29 3 3X10-7

Fungi

(Cfu/ml)* TNTC TNTC 25 3 - 3X10-6

*mean value of duplicates

Table 2. Ratio between Rhizosphere microbes and PSB

Microorganism

Dilutions Ratio (Rhizosphere

microbes verses PSB) 10-3 10-4 10-5 10-6 10-7

Bacteria (Cfu/ml)*

TNTC TNTC 16 2 - 15:1

Fungi (Cfu/ml)*

TNTC TNTC 07 1 - 3.5:1

* mean value of duplicates

Table 3. Cultural, Morphological and biochemical characteristics of isolated bacteria

S.No. Characteristics of the

test organism Results

Cellular characteristics

1 Morphological identification

Straight rods

2 Staining

characteristics Gram Negative

3 Motility Motile

Cultural characteristics

4 Kings B agar

colonies Pale white, translucent colonies Circular, convex, and smooth colonies

5 Cetrimide agar Green color colonies

6 Asparagine–proline broth

Fluorescent pigment production which fluorescents when observing UV Trans illuminator

Biochemical characteristics

Interpretation Result

7 Indole production

Test

The test organism can not break Tryptophan into Amino group and Indole No change in color after addition of Kovac’s Reagent in SIM medium: Negative

Negative

8 Methyl red Test Methyl Red: neutral acetoin is produced and color changes from Red to Yellow

Negative

9 Vogus Proskauer

Test Voges-Proskauer Test: The medium turns light brown

Negative

10 Hydrogen sulphide

production Test

The absence of a black colorin SIM medium indicates that H2S was not produced. No conversion of Ferrous sulfate to ferrous sulfide: H2S Negative

Negative

11 Citrate utilization

result Test for the ability of bacteria to convert citrate into oxaloacetate.

Positive

12 Starch hydrolysis

This test is used to detect the enzyme amylase which digest the starch present in the medium. Clear zone formation around the colonies indicates starch utilization. .

Positive

Biochemical characteristics

Interpretation Result

13 Gelatin

liquefaction

This test is used to detect the enzyme Gelatinase which digest the Gelatin present in the medium. . Medium liquefied

Positive

14 Urease

production

This test is used to detect the enzyme Urease, which breaks down urea into ammonia. Lack of color change represent negative

Negative

15 Catalase Test

This test is used to detect the enzyme catalase. Liberation of bubbles (O2) immediately after the addition of hydrogen peroxide solution

Positive

16 Oxidase test

This test is based on detecting the production of the enzyme cytochrome oxidase. Appearance of Blue colur after immediate addition of culture in oxidase disc

Positive

17 Nitrate reduction

Test

Add one ml of sulfanilic acid to each tube, then add one ml of dimethyl 1-naphthylamine solution Development of a red color indicates nitrate has been reduced

Positive

18

Acid and gas production in carbohydrate fermentation

Medium

Test organism showed Respiration of sugars - Acid seen in upper part of the tube

showed no gas

production and acid

production found on top of the tube

Table 4. Efficacy of phosphate solubilization of UV treated Aspergillus niger

UV irradiated Aspergillus

niger strains

*‘P’ ppm of filtrate (Day) Increased % of phosphate

Solubilization

(Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

ANuv30 2.65±0.07 4.05±0.09 5.00±0.05 20.45 10.96 8.70

ANuv40 2.50±0.09 3.85±0.11 4.75±0.07 13.64 5.48 3.26

ANuv50 2.85±0.05 4.35±0.06 5.50±0.04 29.55 19.18 19.57

ANuv60 3.05±0.06 4.65±0.07 5.85±0.09 38.64 27.40 27.17

Wild type 2.20±0.05 3.65±0.1 4.60±0.04

*Mean value ± Standard Deviation of triplicates Table 5. Efficacy of phosphate solubilization of UV treated Aspergillus

fumigatus

UV irradiated Aspergillus fumigatus

strains

*‘P’ ppm of filtrate (Day) Increased % of phosphate

solubilization (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

AFuv30 3.25±0.07 3.65±0.06 3.85±0.05 10.17 12.31 5.48

AFuv40 3.15±0.12 3.65±0.05 4.15±0.07 6.78 12.31 13.70

AFuv50 3.55±0.09 3.95±0.08 4.55±0.08 20.34 21.54 24.66

AFuv60 3.35±0.05 3.85±0.08 4.45±0.06 13.56 18.46 21.92

Wild type 2.95±0.04 3.25±0.06 3.65±0.08

*Mean value ± Standard Deviation of triplicates

Table 6. Efficacy of phosphate solubilization of UV treated Penicillium Strains

UV irradiated Penicillium

Strains

*‘P’ ppm of filtrate (Day) Increased % of phosphate

solubilization (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

PEuv30 3.45±0.08 4.10±0.05 4.25±0.06 4.55 6.49 6.25

PEuv40 3.65±0.07 4.25±0.07 4.45±0.08 10.61 10.39 11.25

PEuv50 3.75±0.06 4.40±0.08 4.75±0.05 13.64 14.29 18.75

PEuv60 3.40±0.08 4.20±0.1 4.35±0.11 3.03 9.09 8.75

Wild type 3.30±0.05 3.85±0.06 4.00±0.09

*Mean value ± Standard Deviation of triplicates Table 7. Efficacy of phosphate solubilization of UV treated Pseudomonas

strains

UV treated Pseudomonas

strains

*‘P’ ppm of filtrate (Day) Increased % of

phosphate solubilization (Day)

3rd 6th 9th 3rd

(%) 6th (%) 9th (%)

PSuv1 3.85±0.09 3.95±0.07 4.15±0.08 1.32 -4.8 -12.6

PSuv2 3.95±0.08 4.10±0.04 4.30±0.03 3.95 -1.2 -9.4

PSuv3 4.15±0.07 4.25±0.06 4.45±0.05 9.21 2.41 -6.3

PSuv4 4.05±0.09 4.35±0.09 4.65±0.04 6.58 4.82 -2.1

Wild type 3.80±0.06 4.15±0.07 4.75±0.06

*Mean value ± Standard Deviation of triplicates

Table 8. Efficacy of phosphate solubilization of Sodium azide treated Aspergillus niger

Sodium

azide treated

Aspergillus niger

strains

*‘P’ ppm of filtrate (Day) Increased % of phosphate

solubilization (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

ANsa30 2.40±0.06 4.10±0.05 5.05±0.09 9.0 12.3 9.7

ANsa60 2.50±0.05 4.30±0.09 5.55±0.08 13.6 17.8 20.6

ANsa90 2.95±0.09 4.65±0.1 5.80±0.08 34.0 27.4 26.0

ANsa120 3.45±0.06 4.95±0.11 6.15±0.05 56.8 35.6 33.6

Wild type 2.20±0.05 3.65±0.1 4.60±0.04

*Mean value ± Standard Deviation of triplicates Table 9. Efficacy of phosphate solubilization of Sodium azide treated

Aspergillus fumigatus

Sodium azide

treated Aspergillus fumigatus

strains

*‘P’ ppm of filtrate (Day) Increased % of phosphate

solubilization (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

AFsa30 3.10±0.05 3.45±0.06 3.95±0.1 5.0 6.1 8.2

AFsa60 3.25±0.07 3.75±0.06 4.25±0.9 10.1 15.3 16.4

AFsa90 3.45±0.04 4.00±0.04 5.30±0.12 16.9 23.0 45.0

AFsa120 4.05±0.06 4.65±0.09 5.50±0.05 37.0 43.0 50.6

Wild type 2.95±0.04 3.25±0.06 3.65±0.08

*Mean value ± Standard Deviation of triplicates

Table 10. Efficacy of phosphate solubilization of Sodium azide treated Penicillium strains

Sodium

azide treated

Penicillium strains

*‘P’ ppm of filtrate (Day) Increased % of phosphate

solubilization (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

PEsa60 3.50±0.06 4.15±0.05 4.35±0.07 6.0 7.7 8.7

PEsa90 3.60±0.03 4.50±0.05 5.05±0.04 9.0 16.8 26.2

PEsa120 3.70±0.08 4.65±0.08 5.65±0.09 12.1 20.7 41.2

PEsa150 3.80±0.09 4.85±0.04 5.95±0.06 15.1 25.9 48.7

Wild type 3.30±0.05 3.85±0.06 4.00±0.09

*Mean value ± Standard Deviation of triplicates Table 11. Efficacy of phosphate solubilization of Sodium azide treated

Pseudomonas strains

Sodium azide treated Pseudomonas

strains

*‘P’ ppm of filtrate (Day) Increased % of

phosphate solubilization (Day)

3rd 6th 9th 3rd

(%) 6th (%) 9th (%)

PSsa30 2.85±0.05 3.65±0.1 4.15±0.07 25.00 -12.0 -12.6

PSsa60 3.15±0.07 3.95±0.08 4.45±0.09 -17.1 -4.8 -6.3

PSsa90 3.35±0.05 4.05±0.05 4.50±0.04 -11.8 -2.4 -5.2

PSsa120 3.45±0.08 4.25±0.08 4.65±0.06 -9.2 2.41 -2.1

Wild type 3.80±0.06 4.15±0.07 4.75±0.06

*Mean value ± Standard Deviation of triplicates

Table 12. Efficacy of phosphate solubilization of Ethyl Methane Sulphonate (EMS) Aspergillus niger

EMS

treated Aspergillus

niger strains

*‘P’ ppm of filtrate (Day) Increased % of phosphate

solubilization (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

ANems30 2.30±0.07 3.90±0.06 4.75±0.06 4.5 6.8 3.2

ANems60 2.50±0.05 4.25±0.05 5.40±0.04 13.6 16.4 17.3

ANems90 2.65±0.09 4.45±0.07 5.65±0.06 20.4 21.9 22.8

ANems120 2.85±0.09 4.75±0.07 6.25±0.08 29.5 30.1 35.8

Wild type 2.20±0.05 3.65±0.1 4.60±0.04

*Mean value ± Standard Deviation of triplicates

Table 13. Efficacy of phosphate solubilization of Ethyl Methane Sulphonate (EMS) Aspergillus fumigatus

EMS

treated Aspergillus fumigatus

strains

*‘P’ ppm of filtrate (Day) Increased % of phosphate

solubilization (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

AFems30 3.25±0.03 3.45±0.09 3.85±0.05 10.1 6.1 5.4

AFems60 3.55±0.07 3.80±0.06 4.15±0.06 20.3 16.9 13.6

AFems90 3.75±0.05 4.15±0.06 4.65±0.09 27.1 27.6 27.3

AFems120 3.95±0.08 4.45±0.07 5.15±0.08 33.8 36.9 41.0

Wild type 2.95±0.04 3.25±0.06 3.65±0.08

*Mean value ± Standard Deviation of triplicates

Table 14. Efficacy of phosphate solubilization of Ethyl Methane Sulphonate (EMS) treated Penicillium strains

EMS treated

Penicillium strains

*‘P’ ppm of filtrate (Day) Increased % of phosphate

solubilization (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

PEems60 3.65±0.11 4.70±0.07 5.15±0.06 10.6 22.0 28.7

PEems90 4.05±0.07 4.90±0.1 5.30±0.08 22.7 27.2 32.5

PEems120 4.55±0.09 5.10±0.1 5.45±0.05 37.8 32.4 36.2

PEems150 4.80±0.06 5.70±0.09 5.95±0.04 45.4 48.0 48.7

Wild type 3.30±0.05 3.85±0.06 4.00±0.09

*Mean value ± Standard Deviation of triplicates

Table 15. Efficacy of phosphate solubilization of Ethyl Methane Sulphonate (EMS) treated Pseudomonas strains

EMS treated Pseudomonas

Strains

*‘P’ ppm of filtrate (Day) Increased % of

phosphate solubilization (Day)

3rd 6th 9th 3rd

(%) 6th (%) 9th (%)

PSems60 2.30±0.08 2.80±0.04 3.30±0.07 -39.4 -32.5 -30.5

PSems90 2.35±0.06 2.90±0.05 3.55±0.09 -38.1 -30.0 -25.2

PSems120 2.40±0.08 3.05±0.05 4.15±0.06 -36.8 -26.5 -12.6

PSems150 2.45±0.09 3.25±0.08 4.25±0.05 -35.5 -21.6 -10.5

Wild type 3.80±0.06 4.15±0.07 4.75±0.06

*Mean value ± Standard Deviation of triplicates

Table 16. Efficacy of phosphate solubilization of Aspergillus niger (ANems120) grown on different Carbon sources

Carbon sources vs ANems120 Strains

*‘P’ ppm of filtrate (Day)

3rd 6th 9th

Glucose 2.85±0.09 4.75±0.07 6.25±0.08

Sucrose 2.40±0.05 3.75±0.08 4.85±0.06

Mannitol 2.05±0.04 3.05±0.06 4.35±0.09

Lactose 2.20±0.08 3.60±0.06 4.60±0.06

*Mean value ± Standard Deviation of triplicates; Table 17. Efficacy of phosphate solubilization of Aspergillus niger

(ANems120) grown on different Nitrogen sources

Nitrogen sources vs ANems120 strains

*‘P’ ppm of filtrate (Day)

3rd 6th 9th

Ammonium sulphate 2.85±0.09 4.75±0.07 6.25±0.08

Sodium nitrate 1.90±0.05 2.65±0.09 3.20±0.05

Potassium nitrate 2.35±0.07 3.70±0.09 4.70±0.08

Urea 2.40±0.04 3.70±0.04 4.75±0.05

*Mean value ± Standard Deviation of triplicates; Table 18. Efficacy of phosphate solubilization of Aspergillus niger

(ANsa120) grown on different Carbon sources

Carbon sources vs ANsa120 Strains

*‘P’ ppm of filtrate (Day)

3rd 6th 9th

Glucose 3.45±0.06 4.95±0.11 6.15±0.05

Sucrose 3.55±0.09 4.55±0.04 4.95±0.06

Mannitol 1.85±0.1 2.15±0.08 2.90±0.07

Lactose 3.05±0.06 3.55±0.07 3.95±0.09

*Mean value ± Standard Deviation of triplicates;

Table 19. Efficacy of phosphate solubilization of Aspergillus niger (ANsa120) grown on different Nitrogen sources

Nitrogen sources vs AFsa120 Strains

*‘P’ ppm of filtrate (Day)

3rd 6th 9th

Ammonium sulphate 3.45±0.06 4.95±0.11 6.15±0.05

Sodium nitrate 1.75±0.09 2.30±0.1 2.75±0.05

Potassium nitrate 2.40±0.05 3.70±0.06 4.65±0.07

Urea 2.45±0.06 3.85±0.08 4.70±0.05

*Mean value ± Standard Deviation of triplicates; Table 20. Efficacy of phosphate solubilization of Penicillium sp.(PEsa150)

grown on different Carbon sources

Carbon sources PEsa150 *‘P’ ppm of filtrate (Day)

3rd 6th 9th

Glucose 3.80±0.09 4.85±0.04 5.95±0.06

Sucrose 3..35±0.1 3.90±0.06 4.60±0.05

Mannitol 3.05±0.11 3.75±0.05 4.25±0.04

Lactose 3.15±0.06 3.80±0.07 4.35±0.07

*Mean value ± Standard Deviation of triplicates;

Table 21. Efficacy of phosphate solubilization of Penicillium sp.(PEsa150) grown on different Nitrogen sources

Nitrogen sources PEsa150 *‘P’ ppm of filtrate (Day)

3rd 6th 9th

Ammonium sulphate 3.80±0.09 4.85±0.04 5.95±0.06

Sodium nitrate 3.30±0.07 3.75±0.05 4.05±0.06

Potassium nitrate 3.35±0.04 3.90±0.07 4.60±0.05

Urea 3.70±0.06 3.95±0.04 4.70±0.08

*Mean value ± Standard Deviation of triplicates;

Table 22. Efficacy of phosphatase activity of UV treated Aspergillus niger

UV irradiated Aspergillus

niger strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

ANuv30 0.121±0.04 0.223±0.05 0.345±0.04 24.74 16.15 13.86

ANuv40 0.103±0.02 0.218±0.04 0.341±0.03 6.19 13.54 12.54

ANuv50 0.134±0.03 0.260±0.05 0.399±0.05 38.14 35.42 31.68

ANuv60 0.165±0.02 0.275±0.05 0.424±0.06 70.10 43.23 39.93

Wild type 0.097±0.05 0.192±0.03 0.303±0.04

*Mean value ± Standard Deviation of triplicates Table 23. Efficacy of phosphatase activity of fungal strains UV treated

Aspergillus fumigatus

UV irradiated Aspergillus fumigatus

strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

AFuv30 0.171±0.05 0.203±0.06 0.221±0.06 9.62 14.04 13.86

AFuv40 0.160±0.05 0.205±0.05 0.232±0.08 2.56 15.17 12.54

AFuv50 0.195±0.04 0.227±0.07 0.269±0.04 25.00 27.53 31.68

AFuv60 0.173±0.05 0.224±0.04 0.265±0.03 10.90 25.84 39.93

Wild type 0.156±0.04 0.178±0.04 0.197±0.06

*Mean value ± Standard Deviation of triplicates

Table 24. Efficacy of phosphatase activity of Penicillium strains

UV irradiated Penicillium

strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

PEuv30 0.180±0.04 0.228±0.06 0.239±0.06 6.51 7.04 1.27

PEuv40 0.195±0.05 0.245±0.08 0.264±0.04 15.38 15.02 11.86

PEuv50 0.209±0.05 0.237±0.07 0.267±0.06 23.67 11.27 13.14

PEuv60 0.178±0.05 0.234±0.04 0.257±0.05 5.33 9.86 8.90

Wild type 0.169±0.04 0.213±0.06 0.236±0.06

*Mean value ± Standard Deviation of triplicates Table 25. Efficacy of phosphatase activity of UV treated Pseudomonas sp.

UV treated Pseudomonas

strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

PSuv1 0.205±0.04 0.225±0.06 0.231±0.05 2.5 -3.85 -19.51

PSuv2 0.215±0.04 0.227±0.07 0.236±0.05 7.5 -2.95 -17.77

PSuv3 0.227±0.07 0.233±0.04 0.275±0.06 13.5 -0.43 -4.18

PSuv4 0.210±0.03 0.226±0.06 0.286±0.05 5.0 -3.42 -0.35

Wild type 0.200±0.05 0.234±0.05 0.287±0.07

*Mean value ± Standard Deviation of triplicates

Table 26. Efficacy of phosphatase activity of Sodium azide treated Aspergillus niger strains

Sodium azide treated Aspergillus

niger strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

ANsa30 0.107±0.04 0.218±0.04 0.335±0.06 10.30 13.54 10.56

ANsa60 0.110±0.05 0.234±0.03 0.401±0.03 13.40 21.87 32.34

ANsa90 0.152±0.03 0.268±0.03 0.421±0.06 56.70 39.58 38.94

ANsa120 0.184±0.03 0.340±0.03 0.474±0.05 89.69 77.08 56.43

Wild type 0.097±0.05 0.192±0.03 0.303±0.04

*Mean value ± Standard Deviation of triplicates Table 27. Efficacy of phosphatase activity of Sodium azide treated

Aspergillus fumigatus strains Sodium

azide treated Aspergillus fumigatus

strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

AFsa30 0.165±0.04 0.189±0.04 0.213±0.06 5.76 6.18 8.12

AFsa60 0.174±0.03 0.203±0.05 0.241±0.05 11.53 14.04 22.33

AFsa90 0.185±0.06 0.229±0.04 0.351±0.05 18.59 28.65 78.17

AFsa120 0.232±0.04 0.274±0.03 0.365±0.05 48.71 53.93 85.27

Wild type 0.156±0.04 0.178±0.04 0.197±0.06

* Mean value ± Standard Deviation of triplicates

Table 28. Efficacy of phosphatase activity of Sodium azide treated Penicillium strains

Sodium azide treated Penicillium

strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

PEsa60 0.181±0.06 0.233±0.05 0.261±0.04 7.10 9.39 10.59

PEsa90 0.191±0.04 0.249±0.04 0.349±0.04 13.01 16.90 47.88

PEsa120 0.200±0.05 0.261±0.05 0.400±0.06 18.34 22.53 69.49

PEsa150 0.210±0.04 0.276±0.06 0.420±0.08 24.26 29.57 77.96

Wild type 0.169±0.04 0.213±0.06 0.236±0.06

*Mean value ± Standard Deviation of triplicates Table 29. Efficacy of phosphatase activity of Sodium azide treated

Pseudomonas strains

Sodium azide treated Pseudomonas

strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

PSsa30 0.169±0.06 0.199±0.07 0.224±0.03 -15.5 9.39 10.59

PSsa60 0.171±0.04 0.220±0.06 0.256±0.06 -14.5 16.90 47.88

PSsa90 0.180±0.05 0.232±0.07 0.260±0.04 -10.0 22.53 69.49

PSsa120 0.185±0.06 0.241±0.03 0.275±0.04 -7.5 29.57 77.96

Wild type 0.200±0.05 0.234±0.05 0.287±0.07

*Mean value ± Standard Deviation of triplicates

Table 30. Efficacy of phosphatase activity of Ethyl Methane Sulphonate (EMS) Aspergillus niger

EMS treated Aspergillus

niger strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

ANems30 0.099± 0.210± 0.316± 2.06 9.37 4.29

ANems60 0.104±0.03 0.222±0.05 0.365±0.04 7.21 15.62 20.46

ANems90 0.125±0.04 0.253±0.06 0.412±0.03 28.86 31.77 35.97

ANems120 0.131±0.05 0.273±0.05 0.452±0.07 35.05 42.18 49.17

Wild type 0.097±0.05 0.192±0.03 0.303±0.04

*Mean value ± Standard Deviation of triplicates

Table 31. Efficacy of phosphatase activity of Ethyl Methane Sulphonate (EMS) treated Aspergillus fumigatus

EMS treated Aspergillus

niger strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

AFems30 0.175±0.04 0.197±0.06 0.214±0.05 12.17 10.67 8.62

AFems60 0.189±0.05 0.212±0.04 0.231±0.07 21.15 19.10 17.25

AFems90 0.206±0.05 0.236±0.07 0.261±0.05 32.05 32.58 32.48

AFems120 0.219±0.05 0.258±0.04 0.318±0.04 40.38 44.94 61.42

Wild type 0.156±0.04 0.178±0.04 0.197±0.06

*Mean value ± Standard Deviation of triplicates

Table 32. Efficacy of phosphatase activity of Ethyl Methane Sulphonate (EMS) treated Penicillium strains

EMS treated Penicillium

strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

PEems60 0.197±0.05 0.275±0.05 0.370±0.05 16.56 29.10 56.78

PEems90 0.227±0.05 0.325±0.05 0.369±0.05 34.32 52.58 56.35

PEems120 0.245±0.06 0.324±0.03 0.371±0.05 44.97 52.11 57.20

PEems150 0.278±0.04 0.381±0.07 0.425±0.07 64.49 78.87 80.08

Wild type 0.169±0.04 0.213±0.06 0.236±0.06

*Mean value ± Standard Deviation of triplicates

Table 33. Efficacy of phosphatase activity Ethyl Methane Sulphonate (EMS)

treated Pseudomonas strains

EMS treated Pseudomonas

strains

*Phosphatase activity µmol min-1 (Day)

Increased % of phosphatase activity (Day)

3rd 6th 9th 3rd (%) 6th (%) 9th (%)

PSems60 0.091±0.05 0.150±0.07 0.180±0.06 -54.5 29.10 56.78

PSems90 0.095±0.05 0.161±0.05 0.191±0.05 -52.5 52.58 56.35

PSems120 0.100±0.03 0.167±0.04 0.220±0.05 -50.0 52.11 57.20

PSems150 0.113±0.04 0.175±0.08 0.229±0.06 -43.5 78.87 80.08

Wild type 0.200±0.05 0.234±0.05 0.287±0.07

*Mean value ± Standard Deviation of triplicates

Table 34. Efficacy of phosphatase activity of Aspergillus niger (ANems120) grown on different Carbon sources

Carbon sources vs ANems120 Strains

*Phosphatase activity µmol min-1 (Day)

3rd 6th 9th

Glucose 0.131±0.05 0.273±0.05 0.452±0.07

Sucrose 0.107±0.04 0.206±0.05 0.279±0.04

Mannitol 0.083±0.05 0.167±0.03 0.229±0.05

Lactose 0.094±0.04 0.182±0.07 0.257±0.04

*Mean value ± Standard Deviation of triplicates; Table 35. Efficacy of phosphatase activity of Aspergillus niger (ANems120)

grown on different Nitrogen sources

Nitrogen sources vs ANems120 strains

*Phosphatase activity µmol min-1 (Day)

3rd 6th 9th

Ammonium Sulphate 0.131±0.05 0.273±0.05 0.452±0.07

Sodium Nitrate 0.074±0.04 0.122±0.06 0.173±0.08

Potassium Nitrate 0.102±0.04 0.200±0.05 0.271±0.03

Urea 0.109±0.07 0.203±0.05 0.275±0.06

*Mean value ± Standard Deviation of triplicates Table 36. Efficacy of phosphatase activity of Aspergillus niger

(ANsa120)grown on different Carbon sources

Carbon sources vs ANsa120 Strains

*Phosphatase activity µmol min-1 (Day)

3rd 6th 9th

Glucose 0.184±0.03 0.340±0.03 0.474±0.05

Sucrose 0.186±0.05 0.248±0.07 0.330±0.06

Mannitol 0.076±0.07 0.094±0.04 0.134±0.08

Lactose 0.165±0.04 0.187±0.08 0.213±0.06

*Mean value ± Standard Deviation of triplicates;

Table 37. Efficacy of phosphatase activity of Aspergillus niger (ANsa120) grown on different Nitrogen sources

Nitrogen sources vs ANsa120 Strains

*Phosphatase activity µmol min-1 (Day)

3rd 6th 9th

Ammonium Sulphate 0.184±0.03 0.340±0.03 0.474±0.05

Sodium Nitrate 0.08±0.02 0.103±0.06 0.125±0.07

Potassium Nitrate 0.108±0.08 0.203±0.08 0.254±0.04

Urea 0.112±0.07 0.216±0.07 0.273±0.06

*Mean value ± Standard Deviation of triplicates

Table 38. Efficacy of phosphatase activity of Penicillium sp.(PEsa150)

grown on different Carbon sources

Carbon sources PEsa150 *Phosphatase activity µmol min-1 (Day)

3rd 6th 9th

Glucose 0.210±0.04 0.276±0.06 0.420±0.08

Sucrose 0.171±0.08 0.288±0.06 0.342±0.03

Mannitol 0.162±0.04 0.204±0.04 0.261±0.04

Lactose 0.168±0.05 0.214±0.06 0.253±0.04

*Mean value ± Standard Deviation of triplicates Table 39. Efficacy of phosphatase activity of Penicillium sp.(PEsa150)

grown on different Nitrogen sources

Nitrogen sources PEsa150 *Phosphatase activity µmol min-1 (Day)

3rd 6th 9th

Ammonium Sulphate 0.210±0.04 0.276±0.06 0.420±0.08

Sodium Nitrate 0.163±0.05 0.209±0.03 0.229±0.05

Potassium Nitrate 0.165±0.04 0.218±0.04 0.261±0.05

Urea 0.205±0.05 0.223±0.07 0.270±0.03

*Mean value ± Standard Deviation of triplicates

Table 40. Efficacy of Lipase activity of UV treated Aspergillus niger

UV treated Aspergillus niger strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

ANuv30 1.86±0.08 0.49±0.04 14.81 4.26

ANuv40 1.95±0.06 0.62±0.08 20.37 31.91

ANuv50 2.15±0.05 0.90±0.06 32.72 91.49

ANuv60 1.89±0.05 0.60±0.04 16.66 27.66

Wild type 1.62±0.07 0.47±0.04

*Mean value ± Standard Deviation of triplicates

Table 41. Efficacy of Lipase activity of UV treated Aspergillus fumigatus

UV treated Aspergillus fumigatus strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

AFuv30 1.22±0.09 0.33±0.04 20.79 22.22

AFuv40 1.39±0.08 0.38±0.04 37.62 40.74

AFuv50 2.09±0.09 0.60±0.04 106.93 122.22

AFuv60 1.75±0.06 0.56±0.07 73.26 107.40

Wild type 1.01±0.07 0.27±0.04

*Mean value ± Standard Deviation of triplicates

Table 42. Efficacy of Lipase activity of UV treated Penicillium strains

UV treated Pencillum strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

PEuv30 1.28±0.06 0.48±0.05 6.67 4.35

PEuv40 1.43±0.06 0.73±0.04 19.17 58.70

PEuv50 1.62±0.08 0.69±0.06 35.00 50.00

PEuv60 1.69±0.08 0.85±0.04 40.83 84.78

Wild type 1.20±0.10 0.46±0.04

*Mean value ± Standard Deviation of triplicates

Table 43. Efficacy of lipase activity of UV treated Pseudomonas strains

UV treated Pseudomonas strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

PSuv1 2.22±0.01 0.23±0.02 5.71 8.70

PSuv2 2.19±0.09 0.33±0.08 4.28 26.09

PSuv3 2.49±0.07 0.38±0.03 18.57 36.96

PSuv4 2.29±0.02 0.34±0.05 9.04 67.39

Wild type 2.10±0.08 0.17±0.06

*Mean value ± Standard Deviation of triplicates

Table 44. Efficacy of lipase activity of Sodium azide treated Aspergillus niger

Sodium azide treated Aspergillus niger

strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

ANsa30 1.90±0.08 0.51±0.07 17.28 8.51

ANsa60 2.09±0.05 0.68±0.05 29.01 44.68

ANsa90 2.61±0.07 0.98±0.04 61.11 108.61

ANsa120 1.85±0.06 0.63±0.03 14.19 34.04

Wild type 1.62±0.07 0.47±0.04

*Mean value ± Standard Deviation of triplicates±

Table 45. Efficacy of lipase activity of Sodium azide treated Aspergillus

fumigatus

Sodium azide treated Aspergillus fumigatus

strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

AFsa30 1.45±0.05 0.41±0.04 43.56 51.85

AFsa60 1.25±0.05 0.38±0.06 23.76 40.74

AFsa90 1.22±0.07 0.30±0.03 20.79 11.11

AFsa120 1.60±0.07 0.44±0.07 58.42 62.96

Wild type 1.01±0.07 0.27±0.04

*Mean value ± Standard Deviation of triplicates±

Table 46. Efficacy of lipase activity of Sodium azide treated Penicillium strains

Sodium azide treated Pencillilum strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

PEsa30 1.56±0.06 0.48±0.04 30.00 4.35

PEsa60 1.43±0.04 0.69±0.06 19.17 50.00

PEsa90 1.76±0.08 0.73±0.04 46.67 58.70

PEsa120 2.01±0.07 0.99±0.03 67.50 115.22

Wild type 1.20±0.10 0.46±0.04

*Mean value ± Standard Deviation of triplicates

Table 47. Efficacy of lipase activity of Sodium azide treated Pseudomonas

strains

Sodium azide treated Pencillilum strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

PSsa30 2.28±0.04 0.28±0.11 8.57 64.71

PSsa60 2.93±0.03 0.35±0.02 39.52 105.88

PSsa90 3.23±0.05 0.39±0.08 53.80 129.41

PSsa120 3.04±0.09 0.39±0.04 44.76 129.41

Wild type 2.10±0.06 0.17±0.06

*Mean value ± Standard Deviation of triplicates

Table 48. Efficacy of lipase activity of Ethyl Methane Sulphonate (EMS) treated Aspergillus niger

EMS treated Aspergillus niger

strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

ANems60 1.90±0.09 0.62±0.06 17.28 31.91

ANems90 2.09±0.10 0.77±0.07 29.01 63.83

ANems120 3.10±0.08 0.87±0.05 91.35 85.11

ANems150 3.85±0.07 0.94±0.03 137.65 100.00

Wild type 1.62±0.07 0.47±0.04

*Mean value ± Standard Deviation of triplicates

Table 49. Efficacy of lipase activity of Ethyl Methane Sulphonate (EMS) treated Aspergillus fumigatus

EMS treated Aspergillus fumigatus

strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

AFems60 1.85±0.05 0.33±0.05 83.17 22.22

AFems90 2.26±0.09 0.40±0.04 123.76 48.15

AFems120 2.76±0.08 0.45±0.06 173.26 66.17

AFems150 2.60±0.09 0.41±0.03 157.43 51.85

Wild type 1.01±0.07 0.27±0.04

*Mean value ± Standard Deviation of triplicates

Table 50. Efficacy of lipase activity of Ethyl Methane Sulphonate (EMS) treated Penicillium strains

EMS treated Penicillium strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

PEems60 1.64±0.06 0.50±0.03 36.67 8.70

PEems90 1.79±0.05 0.58±0.03 49.17 26.09

PEems120 1.89±0.04 0.63±0.04 57.50 36.96

PEems150 1.97±0.08 0.77±0.06 64.17 67.39

Wild type 1.20±0.10 0.46±0.04

*Mean value ± Standard Deviation of triplicates

Table 51. Efficacy of lipase activity of Ethyl Methane Sulphonate (EMS) treated Pseudomonas strains

EMS treated Penicillium strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

PSems60 2.42±0.10 0.34±0.06 9.50 54.55

PSems90 3.53±0.05 0.47±0.09 59.72 113.64

PSems120 4.45±0.07 0.61±0.05 101.36 177.27

PSems150 4.12±0.04 0.52±0.05 86.43 136.36

Wild type 2.21±0.09 0.22±0.12

*Mean value ± Standard Deviation of triplicates

Table 52. Efficacy of lipase activity of fungal strains grown on different Carbon sources

Carbon sources vs ANems120

Strain

Lipase activity

after 96 hrs (Unit/g of

substrate)*

Carbon sources vs AFems120

Strain

Lipase activity

after 96 hrs (Unit/g of

substrate)*

Carbon sources vs ANems150

Strain

Lipase activity

after 96 hrs (Unit/g of

substrate)*

Glucose 2.94±0.06 Glucose 2.54±0.05 Glucose 2.44±0.03

Sucrose 3.10±0.08 Sucrose 2.76±0.08 Sucrose 2.61±0.07

Mannitol 1.85±0.07 Mannitol 1.69±0.07 Mannitol 1.98±0.07

Lactose 1.65±0.09 Lactose 1.25±0.07 Lactose 1.34±0.09

*Mean value ± Standard Deviation of triplicates Table 53. Efficacy of lipase activity of Bacterial strains grown on different

Carbon sources

Carbon sources vs PSems120 Strain

Lipase activity after 96 hrs

(Unit/g of substrate)*

Carbon sources vs PSems150 Strain

Lipase activity after 96 hrs

(Unit/g of substrate)*

Glucose 2.95±0.07 Glucose 2.45±0.08

Sucrose 4.12±0.04 Sucrose 4.12±0.04

Mannitol 2.74±0.06 Mannitol 2.05±0.04

Lactose 1.80±0.04 Lactose 1.65±0..05

*Mean value ± Standard Deviation of triplicates

Table 54. Efficacy of lipase activity of fungal strains grown on different Nitrogen sources

Nitrogen sources vs ANems120

Strain

Lipase activity

after 96 hrs

(Unit/g of substrate)*

Nitrogen sources vs AFems120

Strain

Lipase activity

after 96 hrs

(Unit/g of substrate)*

Nitrogen sources vs ANems150

Strain

Lipase activity

after 96 hrs

(Unit/g of substrate)*

Sodium nitrate

3.10±0.08 Sodium nitrate

2.76±0.08 Sodium nitrate

2.61±0.07

Potassium nitrate

2.85±0.05 Potassium nitrate

2.67±0.09 Potassium nitrate

2.24±0.06

Ammonium sulphate

2.01±0.05 Ammonium sulphate

2.13±0.10 Ammonium sulphate

1.98±0.08

Urea 1.55±0.07 Urea 1.54±0.06 Urea 1.27±0.08

*Mean value ± Standard Deviation of triplicates Table 55. Efficacy of lipase activity of Bacterial strains grown on different

Nitrogen sources

Carbon sources vs PSems120 Strain

Lipase activity after 96 hrs

(Unit/g of substrate)*

Carbon sources vs PSems150 Strain

Lipase activity after 96 hrs

(Unit/g of substrate)*

Sodium nitrate 4.12±0.04 Sodium nitrate 4.12±0.04

Potassium nitrate 2.94±0.07 Potassium nitrate 2.99±0.05

Ammonium sulphate 2.71±0.09 Ammonium sulphate 2.13±0.06

Urea 1.45±0.06 Urea 1.54±0.04

*Mean value ± Standard Deviation of triplicates

Table 56. Screening for IAA by Wild and Mutated Bacterial and Fungal strains

Wild and Mutated strains of

Aspergillus niger

IAA activity

Wild and Mutated strains of

Aspergillus fumigatus

IAA activity

Wild and Mutated strains of

Penicillium sp

IAA activity

Wild and Mutated strains of

Pseudomonas sp

IAA activity

Wild type + Wild type - Wild type + Wild type +

ANuv30 + AFuv30 - PEuv30 + PSuv1 +

ANuv40 + AFuv40 - PEuv40 + PSuv2 +

ANuv50 + AFuv50 - PEuv50 + PSuv3 +

ANuv60 + AFuv60 - PEuv60 + PSuv4 +

ANsa30 + AFsa30 - PEsa60 + PSsa30 +

ANsa60 + AFsa60 - PEsa90 + PSsa60 +

ANsa90 + AFsa90 - PEsa120 + PSsa90 +

ANsa120 + AFsa120 - PEsa150 + PSsa120 +

ANems30 - AFems30 - PEems60 + PSems60 +

ANems60 - AFems60 - PEems90 + PSems90 +

ANems90 - AFems90 - PEems120 + PSems120 +

ANems120 - AFems120 - PEems150 + PSems150 +

Key: + activity; - No activity;

Table 57. Quantification study of IAA by Wild and Mutated Bacterial and Fungal strains

Wild and Mutated strains of

Aspergillus niger

*IAA activity (µg/L)

Wild and Mutated strains of

Penicillium sp

*IAA activity (µg/L)

Wild and Mutated strains of

Pseudomonas sp

*IAA activity (µg/L)

Wild type 12.33±0.12 Wild type 7.68±0.14 Wild type 7.50±0.17

ANuv30 13.01±0.15 PEuv30 8.67±0.17 PSuv1 6.50±0.18

ANuv40 12.47±0.15 PEuv40 7.55±0.18 PSuv2 7.55±0.17

ANuv50 12.33±0.17 PEuv50 7.07±0.11 PSuv3 8.60±0.18

ANuv60 11.85±0.13 PEuv60 6.83±0.17 PSuv4 8.08±0.15

ANsa30 12.75±0.10 PEsa60 8.88±0.15 PSsa30 8.08±0.19

ANsa60 15.65±0.19 PEsa90 9.75±0.19 PSsa60 8.23±0.15

ANsa90 16.73±0.13 PEsa120 10.64±0.12 PSsa90 8.00±0.17

ANsa120 17.53±0.15 PEsa150 8.83±0.10 PSsa120 6.75±0.11

PEems60 8.35±0.11 PSems60 7.07±0.18

PEems90 7.45±0.09 PSems90 7.09±0.17

PEems120 6.97±0.18 PSems120 8.35±0.19

PEems150 6.35±0.14 PSems150 8.45±0.14

*Mean value ± Standard Deviation of triplicates

Plate 1

Biochemical Characterization of Bacterial culture

A. Growth on Cetrimide agar B. Growth on Nutrient agar

C. Asparagine -Proline broth culture tube showed fluorescence in UV-trans illuminator (Magnified image –Right)

D. Oxidase test ( Positive – Left Disc and Control – Right disc)

Plate 2 Growth of Fungal colonies on Sabouraud agar plates

Aspergillus niger

Penicillium sp.

Aspergillus fumigatus

Plate 3 Phosphate solubilization of Bacterial and Fungal cultures in Pikovskaya agar

A. Control

B. Phosphate solubilization by Penicillium sp.

C. Phosphate solubilization by Aspergillus niger

D. Phosphate solubilization by

Aspergillus fumigatus

E. Phosphate solubilization by Pseudomonas sp.

Plate 4 Phosphate solubilization and phosphatase production by fungal cultures in

Pikovskaya broth

Phosphate solubilization by Penicillium sp.

A. Wild strain B. PEems150

Phosphate solubilization by Aspergillus niger

C. Wild strain D. ANsa120

Phosphate solubilization by

Aspergillus fumigatus E. AFsa120 F. Wild strain

Plate 5

Indole Acetic Acid production by bacterial and fungal cultures in Czepekdox broth and thin layer chromatogram for IAA

IAA production by Wild Bacterial and Fungal cultures.

A. Aspergillus niger, B. Pseudomonas sp., C. Penicillium sp., D. Aspergillus fumigatus, E. Control

Thin layer chromatogram for IAA

Figure-1: Phosphate solubilization efficacy by wild strains

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

3rd Day 6th Day 9th Day

Incubation period (in Days)

pp

m o

f P

hos

ph

ate

Aspergillus niger Aspergillus fumigatus Pencillium sp

Figure-2: Phosphate solubilization efficacy by Fungal strains treated with chemical mutagen

0

1

2

3

4

5

6

7

ANuv60 ANsa120 ANems 120 AFuv50 AFsa120 AFems 120 PEuv50 PEsa150 PEems150

Incubation period (in Days)

ppm

of

Ph

osph

ate

3rd Day 6th Day 9th Day

Figure-3: Efficacy of phosphatase activityby wild strains

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

3rd Day 6th Day 9th Day

Incubation period (in Days)

µm

ol m

in-1

Aspergillus niger Aspergillus fumigatus Pencillium sp

Figure -4: Efficacy of phosphatase activityby Fungal strains treated with chemical mutagen

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

ANuv60 ANsa120 ANems120 AFuv50 AFsa120 AFems PEuv50 PEsa150 PEems150

Incubation period (in Days)

µm

ol m

in-1

3rd Day 6th Day 9th Day

Figure-5: Efficacy of Lipase activity by wild strains

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Sucrose medium Olive oil medium

un

it g

-1 o

f su

bst

rate

Aspergillus niger Aspergillus fumigatus Pencillium sp

Figure-6: Efficacy of Lipase activity by Chemical treated Fungal Strains

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

ANuv50 ANsa90 ANems150 AFuv50 AFsa120 AFems120 PEuv60 PEsa120 PEems150

un

it g

-1of

su

bst

rate

Sucrose medium Olive oil medium

i

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LIST OF PUBLICATIONS

1. Strain Improvement of Phosphate Solubilizing Fungal Strains (2010).

Journal of Ecobiotechnology, 2(5): 65-70.

2. Strain Improvement of Pseudomonas sp for the production of Lipase

(2010). Journal of Experimental Sciences, 1(11): 01-03.

Journal of Ecobiotechnology 2/5: 65-70, 2010 ISSN 2077-0464 http://journal-ecobiotechnology.com/

Strain Improvement of Phosphate Solubilizing Fungal Strains Department of Botany, Jamal Mohammed College, (Autonomous), Trichy-620020

*Corresponding author, Email: biojrkumar@rediffmail.com, ilyasjmc@yahoo.co.in, Tel.: ( 0431) 2331235, 2331935, Fax : ( 0431 ) 2331135

Phosphate solubilizing fungi Mutagenesis Enhancement Mutant strains

A laboratory study was conducted to isolate, investigate and enhance the phosphate solubilization efficiency of certain isolated fungal strains viz., Aspergillus niger, Aspergillus fumigatus, Penicillium sp. These fungal strains were isolated from the rhizosphere of paddy fields in Tamilnadu, India and screened for phosphate solubilization. The enhancement of phosphate solubilization of these strains was studied through three types of mutagenesis: UV, sodium azide and ethyl methane sulphonate mutagenesis. The mutant ANuv60 exhibited 127% increased efficiency for phosphate solubilization compared to the wild strains. Similarly, the phosphate solubilization by sodium azide mutant (ANsa120) was 133% higher and EMS mutant (ANems120) was 135% higher than the wild strain. The efficient strains were further studied for the effect of carbon and nitrogen source on phosphate solubilization. Significance of the findings is discussed in detail.

SA: Sodium azide, EMS: Ethyl methane sulphonate, UV: Ultra Violet, ANsa: Sodium azide treated Aspergillus niger, AFsa: Sodium azide treated Aspergillus fumigatus, PEsa: Sodium azide treated Penicillium. ANems: Ethyl methane sulphonate treated Aspergillus niger, AFems: Ethyl methane sulphonate treated Aspergillus fumigatus, PEems: Ethyl methane sulphonate treated Penicillium, ANuv: Ultra Violet treated Aspergillus niger, AFuv: Ultra Violet treated Aspergillus fumigatus and PEuv: Ultra Violet treated Penicillium. 1. Introduction

Phosphorus is called �the key of life� as it is directly involved in most life processes. Next to nitrogen it is invariably classified as one at the macronutrients and is an important key element in frequency of use as fertilizer. It serves as a primary energy source for microbial oxidation. It is a constituent substance in life processes. Soil can not give high yields if it is deficient in phosphate. Soil contains both organic and inorganic forms of phosphorus. The organic forms of the element in soil are compounds of phytins, phospholipids and nucleic acids that come mainly by the way of decaying vegetation.

Plants take phosphate in the form of soluble orthophosphate ions but due to the presence of Ca, Mg, K, Na, Al and Fe ions in soil, the soluble orthophosphate is converted to insoluble form. Because of this process plants utilize very little amount of phosphate, even though phosphatic fertilizers are added (Vivek kumar et al., 1999; Rosas et al., 2006; Jayasinghearachchi and Seneviratne, 2006). Soil microorganisms are reported to be initially associated in the cycling of phosphorus. But they also participate in solubilization of inorganic phosphorus and in the mineralization of organic phosphorus (Agnihothri, 1970; Ostwal and Bhide, 1972).

A large fraction of microbial population can dissolve insoluble inorganic phosphorus known to

occur in the soil (Hayman, 1975). Various microorganisms were reported to solubilize different types of insoluble phosphates. Bacteria such as Achromobacter sp, Agrobacterium sp, Fungi like Aspergillus niger, A. flavus, A. fumigatious, Penicillium sp, Rhizopus sp etc are the important phosphate solubilizers present in the soil.

Different mechanisms have been suggested for the solubilization of inorganic phosphorus by phosphate solubilizers. The production of organic acids in the microenvironment around the root or in the culture media is considered to be the important cause of phosphate solubilization (Sperber, 1957; Bardiya and Gaur, 1972). Achal et al. (2007) reported the phenotypic mutants of Aspergillus tubingensis were obtained by UV irradiation and phosphate solubilization ability of these mutants were studied and compared with wild type strains. However, not much work has been carried on phosphate solubilization by mutated Aspergillus and Penicillium species. Hence the present study has been undertaken to study reveals the efficacy of phosphate solubilization by mutated strains isolated from paddy soils.

2. Materials and Methods Microorganisms and isolation

The fungal strains were isolated from rhizosphere soils of Paddy field, Mannachanallur,

Rajeshkumar Jayaraman and M.H.Muhammad Ilyas Journal of Ecobiotechnology 2/5: 65-70, 2010

one of the largest paddy producing taluk located in Tamil Nadu State, India. The soil samples were screened for phosphate solubilizing fungi in Pikovskaya�s agar medium by conventional dilution plate count method. The fungal strains showing halo formation around their colonies were isolated and grown on Sabouraud agar slants at 27ºC for 3 days and kept in the refrigerator at 40C until further use (Ellaiah et al., 2002). Identification

The fungal cultures were identified based on the colony morphology and spore structure (Pradhan and Sukla, 2005) Random mutational studies Mutagenesis by UV

72 hrs old fungal cultures were scraped off from agar slants and suspended in 5ml sterile distilled water and then diluted with 45 ml of sterile distilled water containing Tween 80 (1:4000). Sterile glass beads were added to the suspension and kept on rotary shaker for 30 min to break the hyphal mycelium. The suspension was filtered to remove the mycelium. The spore suspension was prepared in phosphate buffer (pH7.0) containing 106 spores per ml. Five ml quantities of the spore suspension were transferred aseptically into sterile petri dishes and exposed to UV light (2600 Ao) at a distance of 15cm away from the center of the Germicidal lamp for various time intervals (10, 20, 30, 40, 50, 60, 70 and 80 min). The suspension was agitated by gently rotating the plates in between the time intervals. (Ellaiah et al., 2002).

The UV exposed spore suspensions were stored in dark for overnight to avoid photo reactivation. After overnight incubation, irradiated spore suspensions were serially diluted by using phosphate buffer (pH 7.0) and plated on Sabouraud�s Dextrose agar medium. The plates were incubated for 5 days at 27°C. The colonies were selected on the basis of their morphological characters and were given the code numbers ANuv30, ANuv40, ANuv50, ANuv60, AFuv30, AFuv40, AFuv50, AFuv60, PEuv30, PEuv40, PEuv50 and PEuv60. Mutagenesis by Chemical

Chemical mutagenesis was performed using Sodium azide and Ethyl methane sulphonate (EMS) for the strain improvement of phosphate solubilizers. Spore suspensions of fungal strains were prepared by using phosphate buffer pH 7.0 as described earlier (Ellaiah et al., 2002). To 9 ml of each spore suspension, 1 ml of sterile solution of Sodium azide (250 g ml-1 in phosphate buffer) was added. Similar procedure was adopted for Ethyl methane sulphonate (EMS) (150 g ml-1 in

phosphate buffer). The reaction was allowed to proceed. Control tube was also kept without any chemical mutagen. Samples were withdrawn from the reaction mixture at an interval of 30, 60, 90, 120 and 150 min. and centrifuged for 10 min. at 5000 rpm. The cells were washed three times with sterile distilled water and again re-suspended in 10 ml sterile buffer. The samples were serially diluted in the same buffer and plated on Sabouraud�s Dextrose agar medium. The selected sodium azide treated mutants were given the code numbers ANsa30, ANsa60, ANsa90, ANsa120, AFsa30, AFsa60, AFsa90, AFsa120, PEsa60, PEsa90, PEsa120 and PEsa150.

The EMS treated mutants were given the code numbers ANems30, ANems60, ANems90, ANems120,AFems30, AFems60, AFems90, AFems120,PEems60, PEems90, PEems120 and PEems150 (Bapiraju et al., 2004). Submerged fermentation Inoculation

Pure fungal cultures from agar slants were inoculated to Sabouraud�s Dextrose agar plates and incubated for four days at 27oC. After sufficient growth, 8 mm discs (~106 spores/ml) were cut from the plates using sterile cork borer and were inoculated into 100 ml Pikovskaya�s broth (Narsian et al., 1995). The flasks were incubated at 27oC for 9days. Control Pikovskaya�s broth medium was also kept and the experiment was conducted in triplicate set. Phosphate estimation

Broth culture medium was withdrawn aseptically at three days interval from each flask and centrifuged at 10,000 rpm for 20 minutes (Dave and Patel, 1999). The supernatant was estimated for phosphate content by chlorostannous reduced molybdophosphoric blue colour method (Seshadri, 1995). Effect of different carbon sources

Effect of different carbon sources on phosphate solubilization efficiency of the isolated strains was also studied. The carbon source used was maltose, sucrose and mannitol instead of glucose. Efficient phosphate solubilizing fungal strains were inoculated in to broth media with various carbon sources and incubated. After incubation, phosphate solubilization assay was carried out (Narsian and Patel, 2000). Effect of different nitrogen sources

To study the effect of different nitrogen sources on phosphate solubilization efficiency of the fungal strains, the nitrogen source ammonium sulphate was replaced by potassium nitrate, sodium nitrate and urea. Efficient phosphate solubilizing

Rajeshkumar Jayaraman and M.H.Muhammad Ilyas Journal of Ecobiotechnology 2/5: 65-70, 2010

fungal strains were inoculated and incubated. After incubation, phosphate solubilization assay was carried out (Narsian and Patel, 2000).

3. Results and Discussion The efficacy of phosphate solubilization of

fungal strains treated by ultraviolet rays, sodium

azide and ethyl methane sulphonate (EMS) are presented in the Tables 1, 2 and 3 respectively. The influence of different carbon and nitrogen sources on phosphate solubilization of fungal mutants are reported in the Tables 4 and 5.

Table 1. Efficacy of phosphate solubilization of fungal strains treated by UV

* Values are expressed as mean ± standard deviation (>0.09) of triplicates a Standard for calculation of increased %

Table2. Efficacy of phosphate solubilization of fungal strains treated by sodium azide

*Values are expressed as mean ± standard deviation (>0.09) of triplicates a Standard for calculation of increased %

Table 3. Efficacy of phosphate solubilization of fungal strains treated by Ethyl Methyl Sulphonate (EMS)

* Values are expressed as mean ± standard deviation (>0.09) of triplicates

a Standard for calculation of increased %

Rajeshkumar Jayaraman and M.H.Muhammad Ilyas Journal of Ecobiotechnology 2/5: 65-70, 2010

Table 4. Efficacy of phosphate solubilization of fungal strains grown on different carbon sources

*Values are expressed as mean ± standard deviation (>0.09) of triplicates

Table 5. Efficacy of phosphate solubilization of fungal strains grown on different nitrogen sources

* Values are expressed as mean ± standard deviation (>0.09) of triplicates Phosphate solubilization efficacy of the wild

strains was observed in the descending order of Aspergillus niger >.Penicillium Sp > Aspergillus fumigatus. In the first part of the study, among the UV irradiated fungal strains, ANuv60 was found as the predominant phosphate solubilizing strain followed by ANuv50. In the second part of the study involving chemical mutagenesis of phosphate solubilizing strains, ANems120 showed the highest phosphate solubilization followed by ANsa120. Achal et al. (2007) reported a significant increase in soluble phosphate level was observed in case of UV induced mutants of Aspergillus strains compared with wild strains. The present study also revealed that UV induced mutant enhanced the phosphate solubilization compared with wild strains. There might be a possibility of alteration at genetic levels in case of mutants. Tripura et al. (2007) reported that the EMS treated microbial strains have increased phosphate solubilization efficiency compared to the wild strains.

The phosphate solubilization efficacy of mutant fungal strains (ANems120, ANsa120 and PEsa150) were demonstrated in the presence of various carbon and nitrogen sources. The effect of different carbon sources on phosphate solubilization efficiency of fungal strains was found to be in the ascending order mannitol < lactose < sucrose < glucose. Seshadri et al. (2004) reported

that the mannitol was the best carbon source utilized by fungi preferred for higher phosphate solubilization and the nitrogen in the form nitrate was very effective and urea was the poorest source of nitrogen by Aspergillus niger. The present study revealed that mannitol was the poorest carbon source and the urea was one of the predominant nitrogen sources for phosphate solubilization by Aspergillus niger. This may due to the genetic alteration in the fungus due to chemical mutation. Reyes et al. (1999) studied the carbon source sucrose appeared to be the best carbon source for phosphate solubilization by UV induced fungal strain. Nautiyal et al. (2000) reported that the glucose and lactose were the best carbon source and sucrose, sorbitol were identified as poor carbon source for phosphate solubilization, ammonium and nitrate source to be equally effective for phosphate solubilization. This is in contrary with the report of Halder et al (1991) and Abualla (1994).

Similarly, the addition of various nitrogen sources in the medium to determine their effect on phosphate solubilization efficiency of the inoculated strains gave the following results in the ascending order sodium nitrate < potassium nitrate < urea < ammonium sulphate. Vassileve et al. (1998) reported the encapsulated spores of Aspergillus niger solubilized rock phosphate in the culture medium due to the production of organic acids. Whitelaw et

Rajeshkumar Jayaraman and M.H.Muhammad Ilyas Journal of Ecobiotechnology 2/5: 65-70, 2010

al. (1999) reported that the Penicillium strain solubilized the insoluble phosphate in the culture medium containing ammonium or nitrate as sole source of nitrogen. Vassileve et al. (2007) compared the phosphate solubilization efficacy of Aspergillus niger by using corn steep liquor, ammonium sulphate and yeast extract as nitrogen source.

The present study also indicates, the solubilization efficacy of the fungal strain Aspergillus niger treated with sodium azide (ANsa120) and Penicillium sp treated with EMS (PEems150) were about 1.5-fold higher when compared to wild strain. The chemical mutagenic agents such as sodium azide, ethyl methane sulphonate may alter the gene sequence by altering the base pairs. The azide ion alters the structure of cytosine such that it forms hydrogen bonds with adenine, rather than guanine. This produces a cytosine to thymine transition. Ethyl methane sulphonate is a strong mutagenic agent. It alkylates N7 of Guanine and severely alters the base pairing. 4. Conclusion

Many soil microorganisms are able to solubilize the unavailable forms of phosphate through their metabolic activities. The present study concluded that the treatment of physical and chemical mutagenic agents in fungal strains increased the phosphate solubilization efficacy of the fungal strains. This study also strengthened the idea that addition of carbon and nitrogen sources favors the phosphate solubilization to a certain extent. Acknowledgements

The authors greatly acknowledge the management and the principal of Jamal Mohammed College, Trichy, Tamil Nadu, India for providing the facility. The authors also acknowledge the support rendered by Dr. N.Sengottaian, Head, Department of Botany and Microbiology, Urumu Dhanalakshmi College, Trichy, Tamil Nadu, India. We also thank Mr. P. Malaiarasa Pandian and Miss. Sangeetha Menon for helpful discussion.

References Abd-Alla, M.H., 1994. Phosphatases and the

utilization of organic P by Rhizobium leguminosarum biovar viceae. Biology and fertility of soils 18, 294-296.

Achal, V., Savant V.V., Reddy, M.S., 2007. Phosphate solubilization by a wild type strain and UV-induced mutants of Aspergillus thubingensis. Soil Biology & Biochemistry 39, 695�699.

Agnihotri, V.P., 1970. Solubilization of insoluble phosphate by some soil fungi isolated from nursery seed beds. Canadian Journal of Microbiology 16, 877-880.

Dave, A., Patel, H.H., 1999. Inorganic phosphate solubilizing soil Pseudomonads. Indian Journal of Microbiology 39, 161-164.

Bapiraju, K.V.V.S.N., Sujatha, P., Ramana, T., 2004. Mutation induced enhanced biosynthesis of lipase. African Journal of Biotechnology 3, 618-621.

Bardiya, M.C., Gaur, A.C., 1972. Rock phosphate dissolution by bacteria. Indian Journal of Microbiology 12, 269-271.

Elliaiah, P., Prabhakar, T., Ramakrishna, B., Thaer Taleb, A., Adinarayana, K., 2002. Strain improvement of Aspergillus niger for the production of lipase. Indian Journal of Microbiology 42, 151-153.

Halder, A.K., Mishra, A.K., Chakrabartty, P.K., 1991. Solubilization of inorganic phosphates by Bradyrhizobium. Indian Journal of Experimental Biology 29, 28-31.

Hayman, D.S., 1975. Phosphorus cycling by soil microorganisms and plant roots In: soil microbiology. Butterworths, London, pp. 67-91.

Jayasinghearachchi, H.S., Seneviratne, G., 2006. Fungal solubilization of rock phosphate is enhanced by forming fungal�rhizobial biofilms. Soil Biology & Biochemistry 38, 405�408.

Nautiyal, C.S., Bhadauria, S., Kumar, P., Lal, H., Mond, L. R., Verma, D., 2000. Stress induced phosphate solubilization in bacteria isolated from alkaline soils. FEMS Microbiological Letters 182, 291-296.

Narsian, V., Jungu, T., Patel, H.H., 1995. Mineral phosphate solubilization by Aspergillus aculeatus. Indian Journal of Experimental Biology 33, 91-93.

Narsian, V., and Patel, H.H., 2000. Aspergillus aculeatus as a rock phosphate solubilizer. Soil Biology & Biochemistry 32, 559�565.

Pradhan, N., Sukla, L.B., 2005. Solubilization of inorganic phosphate by fungi isolated from agriculture soil. African Journal of Biotechnology 5, 850-854

Ostwal, K.P., Bhide, V.P., 1972. Solubilization of TCP by soil Pseudomonas. Indian Journal of Experimental Biology 10, 153-154.

Reyes, I., Bernier, L., Simard, R.R., Tanguary, P., Antoun, H., 1999. Characteristics of phosphate solubilization by an isolate of a tropical Penicillium rugulosum and two UV induced mutants. FEMS Microbiology Ecology 28, 291-295.

Rosas, S.B., Andres, J.A., Rovera, M., Correa, N.S., 2006. Phosphate-solubilizing Pseudomonas putida

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can influence the rhizobia�legume symbiosis. Soil Biology & Biochemistry 38, 3502�3505.

Seshadri, S., 1995. Phosphate solubilizing fungi from Thanjavur district. South India.,Ph.D., thesis, Bharathidasan University.

Seshadri, S., Ignacimuthu, S., Lakshminarasimhan, C. 2004. Effect of nitrogen and carbon sources on the inorganic phosphate solubilization by different Aspergillus niger strains. Chemical Engineering Communications 191(8): 1043-1052.

Sperber, J.I., 1957. Solubilization of mineral phosphate by soil bacteria., Nature 180, 994-95.

Tripura, C., Sashidhar, B., Podile, A.R., 2007. Ethyl methanesulfonate mutagenesis-enhanced mineral phosphate solubilization by groundnut-associated Serratia marcescens GPS-5. Current Microbiology 54, 79-84.

Vassilev, N., Vassileva, M., Bravo,V., Serrano, M.F., Nikolaeva,I., 2007. Simultaneous phytase production and rock phosphate solubilization by Aspergillus niger grown on dry olive wastes. Industrial Crops and Products 26, 332�336.

Vassileva, M., Azcon, R., Barea, J.M., Vassilev, N., 1998. Application of an encapsulated filamentous fungus in solubilization of inorganic phosphate. Journal of Biotechnology 63, 67-72.

Vivekkumar, Punia, S., Lakshminarayana, K., Narula, N., 1999. Effect of phosphate solubilizing analogue resistant mutants of agriculture Azotobacter chroococcum on sorghum. Indian Journal of Agricultural Science 69, 198-200.

Whitelaw, M.A ., Harden, T.J., Helyar, K.R., 1999. Phosphate solubilization in solution culture by the soil fungus Penicillium radicum. Soil Biology & Biochemistry 31, 655-665.

Regular Article

Strain Improvement of sp for the Production of Lipase

Rajeshkumar Jayaraman and M.H. Muhammad Ilyas*

Department of Botany, Jamal Mohammed College, Trichy-620020

ABSTRACT: sp. capable of extracellular lipase production was isolated from rhizosphere soil of paddy fields in Tamilnadu, India. The isolated strain was mutated using ultra-violet rays and chemical agents like Sodium Azide and EMS. The study indicated that the lipase activity of the mutant strain using chemical mutagen was 2-fold higher than the wild strain.

Key words: ; Lipase; Mutagenesis; UV; Sodium Azide; EMS Abbreviation: SA: Sodium azide, EMS: Ethyl methane sulphonate, UV: Ultra Violet, PSsa: Sodium azide treated sp.PSems: Ethyl methane sulphonate treated sp. PSuv: Ultra Violet treated sp.

Introduction

Lipase enzyme plays a vital role in many food, dairy, leather, paper, pharmaceutical, detergent, textile and cosmetic industries. Lipases are secreted by microorganisms like bacteria, yeasts, molds and a few protozoa. The production of lipase by microorganisms depends largely on the species, strains and culture conditions. Microbial lipases are diverse in their enzymatic properties, substrate specificity and are usually more thermo stable than animal or plant lipases (Vanitha, 2002). Research on lipase production has intensified in recent years due to its potential application in various industrial processes. There are several reports on isolation of lipase producing microorganisms and the effect of nutritional factors on their growth and lipase production (Kamini ., 1997; Gisela ., 1991; Sangilyandi and Gunasekaran, 1996). Jette and Ziomek (1994) determined the lipase activity by a Rhodamine-Triglyceride-Agarose assay. Several species of have been reported to produce this enzyme (Suziki 1988; Narasaki 1968; Sugiura 1977 and Sugiura and Oikawa, 1977). However, no report is available on lipase production by mutated species. Therefore, the present study has been undertaken to study the efficacy of lipase production after inducing mutation in

strains isolated from rhizophere soil of the paddy fields.

Materials and Methods Microorganism

strain isolated from rhizosphere soils of paddy fields in Mannachanallur, one of the largest paddy producing area located in Trichy district of Tamil Nadu, India. The strain was sub-cultured on nutrient agar slant and incubated at 37ºC for 48hrs. This strain was stored at 4ºC and used as the parent (Wild strain) culture.

Mutagenesis by UV

The 24 hr old nutrient broth of the parent culture was diluted with phosphate buffer (pH 7.0) to contain 108 cells/ml. The culture was spread plated on nutrient agar plates. Then the plates were exposed for various time intervals (1 to 10 min) to UV light (2600 Ao) at a distance of 15cm (Kumar and Dhruv, 1990). After the stipulated time interval, each set of plates were covered with black paper to avoid photo reactivation and incubated at 37ºC for 2 days. The unexposed plate containing the wild strain served as positive control. On incubation, the resistant strains were selected based on colony development in nutrient agar plates and given code names as PSuv 1, PSuv 2, PSuv 3 and PSuv 4 .These strains were transferred to nutrient agar slants and further screened for lipase activity.

Mutagenesis by Chemical

Sodium azide and ethyl methane sulphonate (EMS) were the chemical mutagens used for mutagenesis of the parent culture,

sp. The bacterial suspension was diluted in similar way as that for U.V mutagenesis. To nine ml of bacterial suspension, one ml of sterile solution of sodium azide (50 g ml-1 in phosphate buffer) was added. Similar method was adopted for ethyl methane sulphonate (50 g ml-1 in phosphate buffer). The reaction was allowed to proceed. Samples were withdrawn from the reaction mixture at intervals of 30, 60, 90, 120, 150 and 180 min and immediately centrifuged for 10 min at 5000 rpm. The pellet was washed three times with sterile phosphate buffer and resuspended in 10 ml of the same buffer (pH 7.0). The samples were serially diluted using the same buffer and plated on nutrient agar. The plates were incubated at 37ºC for two days. The selected sodium azide treated mutants were designated as PSsa30, PSsa60, PSsa90 and PSsa120, whereas ethyl methane sulphonate treated mutants were designated as PSems60, PSems90, PSems120 and PSems150.

Submerged fermentation

18 hr old culture broth of wild type and mutant strains of sp. was used for this study. 0.5 ml of each strain (O.D

= 1.0) was inoculated into 100ml Czapek-Dox broth containing 1% olive oil as inducer. Sucrose and 10% olive oil as the carbon source. The flasks were incubated at 37°C for 96hrs. Uninoculated Czapekdox broth served as control. Each experiment was done in triplicates.

Lipase assay

Culture broth was withdrawn aseptically at three days interval from each flask and centrifuged at 3000 rpm for 15 minutes and the supernatant was collected and used for lipase activity (Ray .,1999). Lipase activity in culture broth was determined by titrimetry using olive oil as substrate (Vanitha, 2002).

Results and discussion

Lipase activity of sp treated by ultraviolet rays, sodium azide and ethyl methane sulphonate are presented in the Table 1, 2 and 3 respectively. The results indicated that lipase activity was more in sucrose medium than in olive oil medium after 96 hrs of UV Sodium azide and EMS treatments. The enzyme activity was found to be higher in mutant strains than the wild type (Table 1, 2 and 3). Efficacy of lipase activity by the mutant strains was observed in the descending order of EMS mutants > SA mutants > UV mutants. In the first part of the study, among the UV irradiated bacterial strains, PSuv3 was found as the predominant lipase producing strain followed by PSuv4. In the second part of the study involving chemical mutagenesis, PSems120 showed the highest lipase activity followed by PSems150.

The lipase activity considerably increased depending on nutritional conditions. Similar observations were made by Lawrence (1967) in (Suzuki 1988). Ray (1999) reported the lipase activity increased in isolated bacterial strains by chemical mutagens. Ellaiah (2002) reported that isolated fungal strain produced lipase with physical and chemical mutagens and the mutant has higher lipase activity than the wild strain. The increase in the production of lipase by the mutagens both physical and chemical may be due to the alternation of genotype of the micro organisms. Moreover, they may alter the gene sequence

(Freifelder, 1990; Radman, 1999). The azide ion alters the structure of cytosine such that it forms hydrogen bonds with adenine, rather than guanine. This produces a cytosine to thymine transition. Ethyl methane sulphonate is a strong mutagenic agent. It alkylates N7 of

Guanine and severely alters the base pairing. The present study also indicated that the strain of sp treated with EMS produced 2-fold (PSems120) higher amount of lipase compared to wild strain.

Table 1. Lipase activity of sp treated by ultraviolet rays

UV treated sp strains Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of Lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

PSuv1 2.22±0.01 0.23±0.02 5.71 29.31

PSuv2 2.19±0.09 0.33±0.08 4.28 91.95

PSuv3 2.49±0.07 0.38±0.03 18.57 118.39

PSuv4 2.29±0.02 0.34±0.05 9.04 92.52

Wild typea 2.10±0.08 0.17±0.06

*Values are expressed as mean ± standard deviation of triplicatesaStandard for calculation of increased %

Table 2. Lipase activity of sp treated by sodium azide

Sodium azide treated sp strains

Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

PSsa30 2.28±0.04 0.28±0.11 8.57 58.05

PSsa60 2.93±0.03 0.35±0.02 39.52 103.45

PSsa90 3.23±0.05 0.39±0.08 53.80 124.14

PSsa120 3.04±0.09 0.39±0.04 44.76 121.26

Wild typea 2.10±0.06 0.17±0.06

*Values are expressed as mean ± standard deviation of triplicates aStandard for calculation of increased %

Table 3. Lipase activity of sp treated by EMS

EMS treated sp strains Lipase activity after 96 hrs(Unit/g of substrate)*

Increased % of lipase activity

Sucrose medium

Olive oil medium

Sucrose medium

Olive oil medium

PSems60 2.42±0.10 0.34±0.06 9.50 54.55

PSems90 3.53±0.05 0.47±0.09 59.72 113.64

PSems120 4.45±0.07 0.61±0.05 101.36 177.27

PSems150 4.12±0.04 0.52±0.05 86.43 136.36

Wild typea 2.21±0.09 0.22±0.12

*Values are expressed as mean ± standard deviation of triplicates aStandard for calculation of increased %

Lipases are enzymes that catalyze the hydrolysis of triglycerides to glycerol and fatty acids Microbial lipases are relatively stable and are capable of catalyzing a variety of reactions: they are potentially of importance for divorce industrial applications. The present study concluded the addition of chemical agents in bacterial strains may produce the higher quantity of lipase enzyme.

Acknowledgements The authors greatly acknowledge the management and the

principal of Jamal Mohammed College, Trichy, Tamil Nadu, India for providing the facility. The authors also acknowledge the support rendered by Dr. N.Sengottaian, Head, Department of Botany and Microbiology, Urumu Dhanalakshmi College, Trichy, Tamil Nadu, India. We also thank Mr. P. Malaiarasa pandian and Miss. Sangeetha menon for helpful discussion.

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