Production of Indole acetic acid By Rhizobium, Azotobacter, and Pseudomonas spp. Isolated from soil

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTFOR THE AWARD OF MASTER OF

SCIENCE (BIOTECHNOLOGY)

BHAGWAN MAHAVIR COLLEGE OF M.Sc BIOTECHNOLOGY

(AFFILIATED TO VEER NARMAD SOUTH GUJARAT UNIVERSITY UNIVERSITY)

SURAT

2012

ACKNOWLEDGEMENT

I thank the almighty whose blessings have enabled me to accomplish my dissertation work

successfully.

I am very much thankful to all my professors and my co-guidance Mr. Murtuza Hazoori in

our institute who made us work hard, taught us how to manage everything skillfully and made us

into confident individuals.

It is my pride and privilege to express my sincere thanks and deep sense of gratitude to my

Project guidance Miss.Priya Bande, Department of Biotechnology and Environmental Sciences,

MITCON, pune for her valuable advice, splendid supervision and constant patience through

which this work was able to take the shape in which it has been presented. It was her valuable

discussions and endless endeavors through which I have gained a lot. Her constant

encouragement and confidence-imbibing attitude has always been a moral support for me.

My sincere thanks to Miss. Neha Vora and Mr. Chandrashekharkulkarni, Head Department

of Biotechnology and Environmental Sciences, MITCON, pune for his immense concern

throughout the project work.

I also wish to thank all my friends, for providing the mandatory scholastic inputs during my

course venture.

Finally, I wish to extend a warm thanks to everybody involved directly or indirectly with my

work.

The whole credit of my achievements goes to my parents and my brothers who were always

there for me in my difficulties. It was their unshakable faith in me that has always helped me to

proceed further

INDEX

Abstract

List of figure

List of table

Introduction

Materials and method

Results and Discussion

Conclusion

Aknowlegement

References

Appendix

LIST OF FIGURE

Figure No. CONTENT PAGE NO.

1 Fig no.1 Root nodules of Chick pea plant

2 Fig no.2: Rhizobium colonies on YEMA

Medium

3 Fig no.3: Gram’s staning of Rhizobium

4 Fig no.4: Detection of IAA Production by

Rhizobium

5 Fig no.5:Azotobacter colonies on

Ashbhy’s Medium

6 Fig no.6: Gram’s staning of Azotobacter.

7 Fig no. 7:Detection of IAA Production by

Azotobacter

8 Fig no.8: Pesudomonas colonies on

nutrient agar medium

9 Fig no.9: Gram’s staning of

Pesudomonas

10 Fig no.10: Detection of IAA Production

by Pesudomonas

11 Fig No.11: Thin layer chromatography

for IAA

LIST OF GRAPH AND TABLE:

Graph No. CONTENT Page No.

1 Morphological Characters of isolates

( Rhizobium, Azotobacter, and Pseudomonas)

2 Biochemical tests of isolates ( Rhizobium,

Azotobacter, and Pseudomonas)

3 IAA assay for preparation of standard graph

4 Effect of Tryptophan on IAA productionby Rhizobium

5 Effect of carbon sources on IAA productionby Rhizobium

6 Effect of PH on IAA production by Rhizobium

7 Effect of Tryptophan on IAA productionBy Azotobacter

8 Effect of Carbon source on IAA productionby Azotobacter

9 Effect of PH on IAA productionby Azotobacter

10 Effect of Tryptophan on IAA productionBy Pseudomonas

11 Effect of Carbon source on IAA productionBy Pseudomonas

12 Effect of PH on IAA productionBy Pseudomonas

1. INTRODUCTION 1.1 What is indole?

Indole is an aromatic heterocyclic organic compound. It has a bicyclic structure, consisting

of a six-membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring.

Indole is a popular component of fragrances and the precursor to many pharmaceuticals.

Compounds that contain an indole ring are called indoles. The indolic amino acid tryptophan

is the precursor of the neurotransmitter serotonin.

The name indole is a portmanteau of the words indigo and ole um , since indole was first

isolated by treatment of the indigo dye with oleum.

1.2 General properties and occurrence

Indole is a solid at room temperature. Indole can be produced by bacteria as a degradation

product of the amino acid tryptophan. It occurs naturally in human feces and has an intense

fecal odor. At very low concentrations, however, it has a flowery smell, and is a constituent

of many flower scents (such as orange blossoms) and perfumes. It also occurs in coal tar. The

corresponding substituent is called indolyl.

Indole undergoes electrophilic substitution, mainly at position 3. Substituted indoles are

structural elements of the tryptophan-derived tryptamine alkaloids like the neurotransmitter

serotonin, and melatonin. Other indolic compounds include the plant hormone Auxin

(indolyl-3-acetic acid, IAA), the anti-inflammatory drug indomethacin, the betablocker

pindolol, and the naturally occurring hallucinogen dimethyltryptamine (N,N-DMT).

1.2 History

Indole chemistry began to develop with the study of the dye indigo. Indigo can be converted

to isatin and then to oxindole. Then, in 1866, Adolf von Baeyer reduced oxindole to indole

using zinc dust In 1869, he proposed a formula for indole.

Certain indole derivatives were important dyestuffs until the end of the 19th century. In the

1930s, interest in indole intensified when it became known that the indole nucleus is present

in many important alkaloids, as well as in tryptophan and auxins, and it remains an active

area of research today.

Auxins were the first plant hormones discovered. Charles Darwin was among the first

scientists to dabble in plant hormone research. In his book "The Power of Movement in

Plants".

It was in 1885 that Salkowski discovered indole-3-acetic acid (IAA) in fermentation media

(Salkowski, 1885). The isolation of the same product from plant tissues would not be found

in plant tissues for almost 50 years. IAA is the major auxin involved in many of the

physiological processes in plants (Arteca, 1996).

1.3 Indole Acetic Acid

IDENTIFICATION OF IAA

IUPAC NAME 2-(1H-indol-3-yl)acetic acid

Molecular formula C10H9NO2

Molar mass 175.184

Physical state Off white crystal

Melting point 168-170 ( 441-443K)

Solubility in water Very slightly in water

PH < 7.0

Vapor density 5

Stability Stable under ordinary condition ( light

sensitive )

Other name Indole 3-Acetic acid. Indolylacetic acid , 1-H

Indole 3-acetic acid,Indole acetic

acid,Heteroauxin,IAA

Indole-3-acetic acid, also known as IAA, is a heterocyclic compound that is a phytohormone

called auxin. This colourless solid is native plant compound, potent and the most important

auxin. The molecule is derived from indole, containing a carboxymethyl group (acetic acid).

Structure of Indole 3-Acetic Acid

The phytohormone auxins play a central role in plant growth and development as a regulator

of numerous biological processes, from cell division, elongation and differentiation to tropic

responses, fruit development and senescence. Auxins are employed to induce rooting, callus

formation, flowering, parthenocarpy and so on. They can also prevent abscission of leaves,

flowers and fruits. The action and interaction of some growth regulators like auxins regulate

most of the physiological activities and growth in plants. Naturally occurring substances with

indole nucleus possessing growth-promoting activity are referred to as auxins, chemically it

is Indole acetic acid. (IAA) Not only plants but also microorganisms can synthesize auxins

and cytokinins. The ability to synthesize phytohormone is widely distributed among plant-

associated bacteria. 80% of the bacteria isolated from plant rhizosphere produce IAA. Many

bacteria are known to produce IAA.

1.4 Biosynthesis and biological activity of IAA

IAA is chemically similar to the amino acid tryptophan which is generally accepted to be the

molecule from which IAA is derived. Three mechanisms have been suggested to explain this

conversion:

Tryptophan is converted to indolepyruvic acid through a transamination reaction.

Indolepyruvic acid is then converted to indoleacetaldehyde by a decarboxylation reaction.

The final step involves oxidation of indoleacetaldehyde resulting in indoleacetic acid.

Tryptophan undergoes decarboxylation resulting in tryptamine. Tryptamine is then oxidized

and deaminated to produce indoleacetaldehyde. This molecule is further oxidized to produce

indoleacetic acid.

Biochemical pathway of IAA

As recently as 1991, this 3rd mechanism has evolved. IAA can be produced via a tryptophan-

independent mechanism. This mechanism is poorly understood, but has been proven using

trp(-) mutants. Other experiments have shown that, in some plants, this mechanism is

actually the preferred mechanism of IAA biosynthesis.

The enzymes responsible for the biosynthesis of IAA are most active in young tissues such as

shoot apical meristems and growing leaves and fruits. The same tissues are the locations

where the highest concentrations of IAA are found. One way plants can control the amount

of IAA present in tissues at a particular time is by controlling the biosynthesis of the

hormone. Another control mechanism involves the production of conjugates which

are,simple terms, molecules which resemble the hormone but are inactive.

1.4.1 Synthesis

Chemically, it can be synthesized by the reaction of indole with glycolic acid in the

presence of base at 250 °C.

Many methods for its synthesis have been developed since its original synthesis from

indole-3-acetonitrile.

1.5 Functions of Indole Acetic acid ( Auxin )

Stimulates cell elongation

Stimulates cell division in the cambium and, in combination with cytokinins in tissue

culture

Stimulates differentiation of phloem and xylem

Stimulates root initiation on stem cuttings and lateral root development in tissue culture

Mediates the tropistic response of bending in response to gravity and light

The auxin supply from the apical bud suppresses growth of lateral buds

Delays leaf senescence

Can inhibit or promote (via ethylene stimulation) leaf and fruit abscission

Can induce fruit setting and growth in some plants

Involved in assimilate movement toward auxin possibly by an effect on phloem transport

Delays fruit ripening

Promotes flowering in Bromeliads

Stimulates growth of flower parts

Promotes (via ethylene production) femaleness in dioecious flowers

Stimulates the production of ethylene at high concentrations

1.6 Indole Acetic Acid Producing Bacteria

1.6.1 Rhizobium spp.

These are gram negative soil bacteria. The root nodulating bacteria invented by hellriegal and

wilfarth (1988) was first isolated and named radicola by beijerinck in 1988. The generic name

was changed later to Rhizobium. Rhizobium is one of the important nitrogen-fixing bacteria.

They form a symbiotic association with leguminous plants to form nodules in the roots of host

plant. These nodules are the sites of nitrogen fixation. Active nodules contain a red pigment

called leghaemoglobin.

1.6.1.1 Classification

Family : Rhizobiaceae.

Genus : Azorhizobium for stem nodulation (Sesbania rostrata)

Bradyrhizobium (soyabean, lupin, cowpea miscellany i.e. cowpea, green gram, red gram,

chickpea, groundnut)

Rhizobium (pea, lentil, bean, lathyrus, berseem, lotus).

1.6.1.2 Morphology

Unicellular, cell size less then 2µ wide. Short rod, polymorphic, motile with peritricus

flagella,

Gram negative,

Accumulate poly B-hydroxyl butyrate granules.

1.6.1.3 Physiology

Rhizobium

Nature : Chemohetrotrophic, Symbiotic with legume,

C – Source : Supplied by legume through photosynthates,

Monosaccharide, Disaccharide.

N – Source : Fixed from atmosphere

Respiration : Aerobic

Growth : Fast (Rhizobium), slow (Bradyrhizobium).

Doubling time : Fast growers (2 - 4 hours)

Slow growers (6 - 12 hours)

Growth media : YEM/YEMA(Fred).

1.6.1.4 Recommended for Legumes

Pulses : Chickpea, pea, lentil, cowpea, greengram, blackgram, pigeonpea.

Oil seeds : Soyabean, Groundnut.

Fodders : Berseem (egyption clover), lucern.

Increase in Yield : 10-35%

1.6.1.5 Fast and Slow Growers

Generally Rhizobium spp. fall into two distinct categories based on growth characteristics.

Rhizobium meliloti, R. trifolii, R. phaseoli, R. leguminosarum, and rhizobia isolated from nodules

on sesbania, birdsfoot, chickpea, and others are considered "Fast Growers." When incubated at

28°C on a solid medium such as yeast extract mannitol (YMA), visible colonies develop 4 to 5

days. In contrast, the "Slow Growers", Bradyrhizobium japonicum and others, require 6 to 8 days

to produce visible colonies when incubated under the same conditions. However, within these two

general groups, strains of rhizobia vary in their growth rates in culture; the division into slow- and

fast-growers on the basis of time required to develop visible colonies is not fix

COMMON NAME OF CROPS RHIZOBIUM SPP.

(with cross inoculation group)

FAST GROWING RHIZOBIUM

Lucerne, fenugreek Rhizobium meliloti

Egyptain, clover, R. trifolii

Khesri(lathyrus), lentil, pea, common vetch R. leguminosarum

Bean, kidney bean, French bean R. phaseoli

Chickpea, subabul Rhizobium spp.

SLOW GROWING RHIZOBIUM

White lupines, lupines R. lupines

Soybean Bradyrhizobium japonicum

Sun hemp, green gram, black gram, cowpea, peanut,

moth bean

Rhizobium

Table :- Fast grower and slow grower Rhizobium strains

1.6.2 Azotobacter

Azotobacter is a genus of usually motile, oval or spherical bacteria that form thick-walled cysts

and may produce large quantities of capsular slime. They are aerobic, free-living soil microbes

which play an important role in the nitrogen cycle in nature, binding atmospheric nitrogen, which

is inaccessible to plants, and releasing it in the form of ammonium ions into the soil. Apart from

being a model organism, it is used by humans for the production of biofertilizers, food additives

and some biopolymers. The first representative of the genus, Azotobacter chroococcum, was

discovered and described in 1901 by the Dutch microbiologist and botanist Martinus Beijerinck.

Azotobacter are Gram-negative bacteria. They are found in neutral and alkaline soils, in water

and in association with some plant.

1.6.2.1 Classification:

Domain: Bacteria

Kingdom: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Pseudomonadales

Family: Pseudomonadaceae/Azotobacteraceae

Genus: Azotobacter

Number of species : Seven (A. beijerinckii, A. chroococcum, A.paspali, A.vinelandii,

A.agilis, A.azomonas, A.mactroytogenes).

Azotobacter

1.6.2.2 Morphology :

Cell size : Large ovoid cells, Size ranging from 2.0-7.0 x 1.0-2.5 µ

Cell character : Polymorphic

Gram reaction : Negative

Accumulate poly B-hydroxybutyrate granules

1.6.2.3 Physiology

Nature : Chemoheterotrophic, Free living.

Carbon source : A variety of carbon sources(mono, di & certain polysacharide) organic acids.

Nitrogen source : N2 through fixation, amino acids, N2, NH4+, NO3

-

Respiration : Aerobic

Growth media : Ashby/Jensen’s medium

Doubling time : 3 hours

1.6.3 Pseudomonas

Pseudomonas is a genus of gammaproteobacteria, belonging to the family Pseudomonadaceae

containing 191 validly described species. Like most bacteria genera the pseudomonad last

common ancestor lived hundreds of million years ago, however they were classified by humans

at the end of the 19th century. Because of their widespread occurrence in water and in plant

seeds such as dicots, the pseudomonads were observed early in the history of microbiology. The

generic name Pseudomonas created for these organisms was defined in rather vague terms by

Walter Migula in 1894 and 1900 as a genus of Gram-negative, rod-shaped and polar-flagella

bacteria with some sporulating species.

1.6.3.1Classification

Domain: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Pseudomonadales

Family: Pseudomonadaceae

Genus: Pseudomonas

1.6.3.2 Morphology

Pseudomonas aeruginosa is a gram-negative, rod-shaped, asporogenous, and monoflagellated

bacterium that has an incredible nutritional versatility. It is a rod about 1-5 µm long and 0.5-1.0

µm wide. P. aeruginosa is an obligate respirer, using aerobic respiration (with oxygen) as its

optimal metabolism although can also respire anaerobically on nitrate or other alternative

electron acceptors. P. aeruginosa can catabolize a wide range of organic molecules, including

organic compounds such as benzoate. This, then, makes P. aeruginosa a very ubiquitous

microorganism, for it has been found in environments such as soil, water, humans, animals,

plants, sewage, and hospitals.

3. MATERIALS AND METHOD3.1 MATERIALS REQUIRED

3.1.1 GLASSWARE

Sterile Petri dishes, Glass slides, Glass beakers, Cover slips, Media bottles, Conical flasks,

Pipette,Media bottles, Test tubes, Micro pipette, Beake , Measuring cylinder,Cavity Slides

Sterile wire loop ,Sterile centrifuge tube, Stirror ,Glass spreader.

3.1.2 EQUIPMENT

Vortex ( cyclo mixture )

Magnetic stirrer ( REMI )

Colony counter ( DBK-instrument interlink )

Microscop ( Labomed )

Laminar air flow ( Micro filt india )

Incubater ( REMI )

Incubater with shaker ( REMI )

Refrigerater

Autoclave ( Meta instrument mumbai )

Centrifuge ( REMI )

Hot air oven ( Meta instrument mumbai )

Water bath ( NEOLAB )

Weighing machine ( ATCO, CITIZEN )

PH meter ( control dynamics )

Uv-visibal spectrophotometer ( Milton roy company )

3.1.3 MATERIAL AND MEDIA

Sterile distilled water

yeast extract mannitol agar ( YEMA)

Congo red

Nutrient agar

Ashbhy’s agar medium

Alcohol (70%)

3.1.4 METHOD

3.1.4.1 SAMPLE COLLECTION:

The rhizopheric soil is used for isolation of microorganism. Soil Sample was collected from

sugarcane and wheat field near by agricultural college,pune

3.1.4.2 ISOLATION OF MICROORGANISM

A. Rhizobium Spp.

For isolation of Rhizobia from root nodules of leguminous plants following method was used

REQUIREMENTS:

1. Recently rooted out leguminous plant roots with nodules

2. Yeast extract mannitol (YEMA) agar plate with congo red

3. 1:1000 aqueous solution of Hgcl2 soluton (1g Hgcl2 dissoled in 1000 ml of

distill water).

4. Ethanol (95%)

5. Sterile petri dishes, forceps, scalpel and slides

PROCEDURE:

o Healthy root nodules were selected, washed with tap water and surface sterilized

by 1:1000 HgCl2,

o washed with sterile distilled water several times.

o These nodules were crushed in a drop of sterile water, inoculated on sterile yeast

extract mannitol agar with Congo red Plates were incubated at room temperature

for 48 hrs.

Root nodules of chick pea plant

o Typical rhizobial colonies on YEMA were opaque, white and mucoid. Colony

characters of a well-isolated colony were studied. Gram staining and motility was

carried out.

B .Azotobacter spp.

REQUIREMENTS:

1. Fertile soil sample having slight reaction.

2. 100 ml Ashby’s mannitol broth in 500-ml flask.

3. Ashby’s mannitol agar medium.

4. Sterile distilled water tubes ( 10 ml ).

5. Spatula

PROCEDUER:

o Inoculate the Flask of Ashby’s mannitol broth with 2g of soil

o Incubate the flask at 25 0c ( room temp.) , until a pellical forms on the surface. This may

require 1-2 weeks of incubation.

o Do not disturb the pellicle once it has been formed.

o Steak a loopful of pellical over the Ashby’s mannitol agar plate, and incubate the plate at

25 0c ( room temp.) for 5-7 days.

o Observe plates for a typical mucoid , dew drops colony’s of Azotobacter.

o Perform Gram’s staning and examine under oil immersion objective.

o Record the results and draw conclusion.

C. Pseudomonas Spp.

Requirements:

1. sterile petri dishes

2. Nutrient agar

3. soil sample

4. distille water

5. Ethanol ( 95%)

PROCEDURE:

o 1g of soil sample on 9 ml of Sterile distille water tubes and mix well.

o After few minutes take 1ml of sample and transfer the another tues with 9ml

distille water.

o Prepare a diution like 10-1,10-2,10-4,10-6…..

o And take a 0.1 ml sample on nutrient agar plate and sprading with glass sprader

and incubate the plate at 37 0c for 24 hs.

o Record the results and draw conclusion.

3.1.5 Gram’s Staning

REQUIREMENTS:

1. Yong culture of microorganism

2. Crystal violets

3. Gram’s iodine

4. Alcohol

5. Distill water

6. Saffranin

PROCEDURE:

1. Prepare a heat fixed smear of the culture.

2. Cover the smear with crystal stain for 1 minutes.

3. Add Gram’s iodine to wash off crystal violet stain and cover it with iodine till the smear

turns coffee brown in color ( approximately 1 min.)

4. Rinse the slide in running water.

5. Add decolorizing solution drop wise at upper end of slides held in inclined position till

the violet color fails to from the smear for normal smear 10-15 seconds are enough.

6. Rinse the smear with water.

3.1.6 BIOCHEMICAL TEST:

3.1.6.1 CARBOHYDRATES FERMENTATION TEST

REQUIREMENTS:

1. Test culture

2. Nutrient sugur broth

PROCEDURE:

1. Inoculate a loopful of culture into the sugar broth and incubate 37 0c for overnight

2. Observe the tube for acid and gas production

3.1.6.2 METHYL RED (M-R) TEST

REQUIREMENTS:

1. Glucose phosphate broth (GPB) , methyl red indicator.

2. Test culture

PROCEDURE:

1. Inoculate GPB with the test culture and incubate the broth at 37 0c for 48-72 hr.

2. After incubation add about 5 drops of methyl red indicator to the medium

3. Observe for development of the red color

3.1.6.3 VOGES-PROSKAUER (V-P) TEST

REQUIREMENTS:

1. Glucose phosphate broth (GPB)

2. 5% alcoholic alfa napthol and 40% KOH solution.

3. Test culture

PROCEDURE:

1. Inoculate the medium (GPB) with culture and incubate the medium at 37 0c for 24-48 hr.

2. After incubation add 0.6 ml of alfa nephthol and 0.2 ml of KOH solution per ml of

culture broth

3. Shake well after addition of each reagent and slope the tube to increase the aeration. Read

results after 15-60 minutes.

3.1.6.4 CITRATE UTILIZATION TEST

REQUIREMENTS:

1. Simmon’s citrate agar slant

2. Test culture

PROCEDURE:

1. Streak heavily on the surface of agar slant and incubate the slant at 370c for 24-48 hr.

2. Record the color change of the slant after incubation

3.1.6.5 INDOL PRODUCTION TEST

REQUIREMENTS:

1. 1% Tryptone broth and Erlich’s or Kovac’s reagent

2. Test culture.

PROCEDURE:

1. Incubate the tryptone broth with of test culture and incubate at 37 0c for 24 hr.

2. After incubation add 3-4 drops of xylene in the medium and shake it vigorously.

3. Allow the two layers to seprate.

3.1.6.6 HYDROGEN SULPHIDE PRODUCTION TEST

REQUIREMENTS:

1. Standard thiosulphate iron agar stab medium

2. Test cultur

PROCEDURE:

1. Stab the medium with the test culture and incubate the medium at 37 0c for 24hr.

2. After incubation look for the black color in the lower portion of the stab agar medium.

3.1.6.7 UREA HYDROLYSIS TEST

REQUIREMENTS:

1. Stuart’s urea broth

2. Test culture.

PROCEDURE:

1. Inoculate a loopful of test culture in urea broth add incubate at 37 oc for 24 hr.

2. Observe for the change in color of the after incubation

3.1.6.8 NIRATE REDUCTION TEST

REQUIREMENTS:

1. Peptone nitrate broth (PNB).

2. Test culture

3. Zinc dust

4. α-napthylamine reagent (reagent A)

5. Sulphanilic acid reagent (reagent B)

PROCEDURE;

1. Inoculate PNB with a loopful of test culture and incubate the medium at 37 0c.

2. Add 0.5 ml of the reagent A and B each to the test medium in this order.

3. Observe the development of color within 30 seconds after adding test reagent.

4. If no color develops add a pinch of Zinc dust mix them well and observe the development

of red color.

3.1.5.9 GELATIN HYDROLYSIS TEST

REQUIREMENTS

1. Two nutrient gelatin agar tube

2. Test culture

3. Refrigerator

PROCEDURE:

1. Inoculate a loopful of test culture into one of the tube and the second tube is left

uninoculated incubate both the tubes at 37 0c for 24-72 hr.

2. After incubation place both the at 5-10 0c either in refrigerator or in ice water bath for

30-60 min.

3. After refrigeration slightly tilt tubes so as to check the liquefaction of gelatin.

3.1.5.9 CATALASE TEST

REQUIREMENTS:

1. Microscopic glass slide

2. 3% H2O2

3. Test culture

PROCEDURE:

1. Place one or two drops of hydrogen peroxide solution on a glass microscopic slide.

2. With a nicrome wire loop pick up cells from the of a well isolated colony of the test.

3. Observe for the production of the gas bubbles of effervescence.

3.1.5.11 OXIDASE TEST

REQUIREMENTS:

1. Nutrient agar plate

2. Filter paper, platinum wire loop

3. Test culture

4. 1% tetramethyl-p-phenylenediamine dihydrochloride solution

PROCEDURE:

1. Grow the test organism under aerobic condition on nutrient agar medium for 18-24 h.

2. Take a filter paper strip and moisten it with 3-4 drops of tetramethyl-p-

phenylenediamine dihydrochloride solution

3. With the help of platinum wire pick up a colony and make a compact smear on moistened

filter paper.

4. Wait for 10-15 seconds and observe for formation of violet color.

3.1.5.12 TRIPAL SUGAR IRON ( TSI ) AGAR TEST

REQUIREMENTS:

1. TSI agar slant

2. Test culture

PROCEDURE:

1. Streak a loopful of test culture on slant and stab the same culture into butt of the slant.

2. Incubate the TSI slant at 37 0c for 24 hr.

3. After incubation observe the medium for presence of acid/gas/H2S in butt as well in the

slant.

3.1.5 PRODUCTION OF IAA FROM BACTERIA:

3.1.5.1 SCREENING OF IAA PRODUCTION:

All the test strains of Rhizobium ,Azotobacter and Pseudomonas spp. were screened for

IAA production . Briefly, test bacterial culture was inoculated in the respective medium

(YEMB /Ashby’s broth/nutrient broth) with tryptophan ( 2 and 5 mg/ml) or without

tryptophan incubated at 28 ± 2 0C for 1week for rhizobium ,15 days for Azotobacter

and 1 week for Pseudomonas spp.

Cultures were centrifuged at 3000 rpm for 30 min.

Two milliliters of the supernatant was mixed with 2 drops of orthophosphoric acid and 4

ml of Solawaski’s reagent (50 ml, 35% perchloric acid, or HCL , 1 ml 0.5 M FeCl3).

3.1.6.2 PREPARATION OF INOCULUM

Inoculum were used in order to obtain maximum production of IAA and best inoculum was used

for further studies. Inoculum was prepared by a loopful organism into 5 ml Normal saline or

nutrient broth and incubated at 28 0c for 48hr. and transfer a 2 ml of old culture into respective

fermentation broth .

3.1.6.3 PREPARSTION OF STANDARD GRAPH

Standard graph of IAA was prepared as mentioned by Different IAA concentrations are prepared

as aqueous solution of IAA ranging from 10 microgram/ml to 100 micrograms/ml. To each 1 ml

of the standard, 2ml of 0.5 M FeCl3 in 35% perchloric acid or HCL i.e. Salkowaski reagent is

added and readings are taken after 25 minutes at 530 nm by UV Visibl

Espectrophotometer( Milton Roy company). Standard graph is prepared by plotting

concentration of IAA in micrograms/ml Vs Optical Density at 530 nm.

3.1.6.4 EFFECT OF CARBON SOURCES ON IAA PRODUCTION

IAA production was detected on different carbon source like glucose, sucrose, lactose, and

mannitol 1% w/v. supplemented with 2 mg/ml of tryptophan. IAA production was studied by

using Salkowaski reagent after 24, 48 and 72 hrs. for Rhizobium, and pseudomonas. Azotobacter

IAA production studied at 5,10 and 15 days after incubation period.Cultures selected for the

study i.e.Rhizobium isolated from chickpea plant and Azotobacter, pseudomonas from wheat soil

in near agicultural field.

3.1.6.5 EFFECT OF PH ON IAA PRODUCTION

To study the extent of IAA produced by the different isolates at different pH, on

YEMB ,Ashby’s broth , nutrient broth with 2 mg/ml of tryptophan is adjusted to different pH as

5, 7, and 9. Media were inoculated with 1% inoculum of O.D.600 1.0 and incubated at 28 0c for

24 hrs. IAA production was studied by using Salkowaski reagent.

3.1.6.6 EXTRACTION OF CRUDE IAA

A proper inoculam of Pseudomonas spp. inoculated in 100 ml of nutrient broth and strains of

Azotobacter was inoculated in 100 ml of Ashbhy’s broth amended with 2 and 5 mg/ml of

tryptophan and incubated at 28 ± 2oC for 1 week on a shaker incubator. Bacterial cells were

separated from the supernatant by centrifugation at 10,000 rpm for 30 min. The supernatant was

acidified to pH 2.5 to 3.0 with 1 N HCl and extracted twice with ethyl acetate at double the

volume of the supernatant. Extracted ethyl acetate fraction was evaporated to dryness in a rotator

evaporator at 40 oC. The extract was dissolved in 0.5 ml of methanol and kept at -20 oC.

3.1.6.7 CONFIRMATION OF IAA BY USING TLC

After incubation period broth was centrifuged at 10000 rpm for 10 minutes. pH of broth

brought to 3.0 using HCL. 4:1 aliquots of liquid portion of centrifuged sample were extracted

three times with ethyl acetate. The organic phase was concentrated to dryness and then diluted

with 0.5 ml methanol. This solution along with the standard IAA was applied on silica gel G

plate and TLC was run by using a solvent system chloroform: Ethyl acetate: Formic acid in 5:3:2

proportion or benzene : n-butanol 17.5 :6.25 ml and developed by using Salkowaski reagent. Red

colour spots were developed. Rf value of the standard and IAA produced by the selected isolates

was calculated.

3.1.6.8 THIN LAYER CHROMATOGRAPHY

COMPONENTS:

1. TLC Plate ( Silica gel G, Distiile water )

2. TLC chamber ( Solvent Benzene: n-butanol )

3. Solvent system or mobile phase :

This comprises of a solvent or solvent mixture recommended for the purpose. The mobile

phase used should be particulate free and of highest purity for proper development of TLC

spots. The solvents recommended are chemically inert with the sample, stationary phase.

4. Spreyer ( Solawaski’s reagent or ehman’s reagent )

5. Hot air oven

PROCEDURE

o The stationary phase is applied onto the plate uniformly and then allowed to dry and

stabilize. But now a days ready made plates are preferred.

o A thin mark is made at the bottom of the plate with a capillary to apply the sample spots.

o Then samples solutions are applied on the spots marked on the line at equal distances.

o The mobile phase is poured into the TLC chamber to a level few centimeters above the

chamber bottom. A filter paper moistened in mobile phase is placed on the inner wall of

the chamber to maintain equal humidity in the entire chamber.

o Then the plate prepared with sample spotting is placed in TLC chamber such that the side

of the plate with sample line is towards the mobile phase. Then the chamber is closed

with a lid.

o The plate is immersed such that sample spots are well above the level of mobile phase but

not immersed in the solvent.

o Allow sufficient time for development of spots. Then the plates are removed and allowed

to dry. The sample spots are visualized in suitable UV light chamber or any other

methods as recommended for the said sample.

The Rf value can be calculated as:

Rf = distance of sopt traveled

Distance of solvent traveld

Solvent traveled

TLC plate

RESULT AND DISSCUTION

PLATE COLONY OF RHIZOBIUM Spp.

Fig No.3 Rhizobium colonies on YEMA with congo red

MORPHOLOGICAL CHARACTERISTIC

Sr. no. COLONY CHARACTER RHIZOBIUM CHARACTER

1 Size Small

2 Shape Circular

3 Colour White

4 Margin Entire

5 Elevation Convex

6 Consistency Mucoid

7 Opasity translucent

8 Motility Motile

9 Remark Nil

GRAM’S STANING OF RHIZOBIUM

After performing Gram’s staning Gram negative short rod bacteria are observed

BIOCHEMICAL TEST RESULT OF RHIZOBIUM

BIO CHEMICAL TEST RESULT

Oxidase test Positive (+)

Catalase test Positive (+)

Voges– Proskauer’S Test Negative (-)

H2s Production Test Negative (-)

Fig No.4 Gram’s staning of Rhizobium

Triple Iron Sugar( TSI ) Slant Positive (+)

Nitrate Reduction Test Negative (-)

Methyl red ( M-R) Test Negative (-)

Citrate Utilization Test Positive (+)

Indole Production Test Positive (+)

Urea Hydrolysis Test Negative (-)

Gelatin Hydrolysis Test Negative (-)

CARBOHYDRATE UTILIZATION TEST

Glucose Negative (-)

Sucrose Negative (-)

Lactose Negative (-)

Xylose Positive (+)

Mannitol Negative (-)

Carbohydrate utilization test Oxidase test

H2S Reduction test Simmon’s Citrate utilization test

Triple Sugar Iron Test Catalase Test

PRODUCTION OF IAA BY RHIZOBIUM Spp.

Production of IAA by Rhizobium in different Tryptophan concentration

Production of IAA by Rhizobium in different carbon source

Production of IAA by Rhizobium in different PH

PLATE COLONY OF AZOTOBACTER Spp.

MORPHOLOGICAL CHARACTER OF AZOTOBACTER

Sr. no. COLONY CHARACTER AZOTOBACETR CHARACTER

1 Size Small

2 Shape Circular

3 surface Smooth

4 Colour White

5 Margin Entire

6 Elevation Convex

7 Consistency Mucoid

8 Opasity translucent

Azotobacter colony on Ashby’s Mannitol Agar

9 consistancy Moist

10 Motility Motile

11 Remark Nil

GRAM’S STAINING OF AZOTOBACTER

BIOCHEMICAL TEST OF AZOTOBACTER

BIO CHEMICAL TEST RESULT

Oxidase test Positive (+)

Catalase test Positive (+)

Voges– Proskauer’S Test Positive (+)

H2s Production Test Negative (-)

Gram’s staining of Azotobacter

Triple Iron Sugar( TSI ) Slant Positive (+)

Nitrate Reduction Test Negative (-)

Methyl red ( M-R) Test Positive (+)

Citrate Utilization Test Positive (+)

Indole Production Test Positive (+)

Urea Hydrolysis Test Negative (-)

Gelatin Hydrolysis Test Negative (-)

CARBOHYDRATE UTILIZATION TEST

Glucose Positive (+)

Sucrose Positive (+)

Lactose Negative (-)

Xylose Positive (+)

Mannitol Negative (-)

PRODUCTION OF IAA BY AZOTOBACTER

Carbohydrate utilization Test Catalase Test

Simmon’s Citrate utilization Test

Production of IAA by Azotobacter in different Tryptophan concentration

Production of IAA by Azotobacter in different carbon source

PLATE COLONY OF PSEUDOMONAS Spp.

Production of IAA by Azotobacter in different PH

Pseudomonas colony on Nutrient agar medium

MORPHOLOGICAL CHARACTER OF PSEUDOMONAS

Sr. no. COLONY CHARACTER AZOTOBACETR CHARACTER

1 Size Small

2 Shape Rod

3 surface Smooth

4 Colour Green

5 Margin Entire

6 Elevation Convex

7 Consistency Mucoid

8 Opasity translucent

9 consistancy Moist

10 Motility Motile

11 Remark Nil

GRAM’S STAINING OF PSEUDOMONAS

BIOCHEMICAL OF PSEUDOMONAS

BIO CHEMICAL TEST RESULT

Oxidase test Positive (+)

Catalase test Positive (+)

Voges– Proskauer’S Test Negative (-)

H2s Production Test Negative (-)

Triple Iron Sugar( TSI ) Slant Negative (-)

Nitrate Reduction Test Negative (-)

Methyl red ( M-R) Test Positive (+)

Citrate Utilization Test Positive (+)

Indole Production Test Positive (+)

Urea Hydrolysis Test Negative (-)

Gelatin Hydrolysis Test Negative (-)

CARBOHYDRATE UTILIZATION TEST

Glucose Positive (+)

Sucrose Positive (+)

Lactose Negative (-)

Xylose Positive (+)

Mannitol Negative (-)

Simmon’s Citrate utilization test Carbohydrate utilization Test glucose,

sucrose, xylose

STANDARD GRAPH OF IAA

Straight-line graph indicates direct proportion between concentrations of IAA and the extent of

red colour developed. Hence production of IAA by the organisms was confirmed.

Oxidase test Catalase Test

H2s Reduction Test TSI Slant

20 40 60 80 100 120 140 160 180 2000

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.00400000000000001

0.010.016

0.024

0.0320000000000001

0.0350000000000001

0.0400000000000001

0.046

0.0500000000000001

0.0600000000000001

Series 1

Concentration (µg/ml)

optic

al d

ensit

y (5

30nm

)

Observation table

Assay of IAA

Sr.n

o

IAA

std.soln

(ml)

IAA conc.

(µg/ml)

Distille

water

(ml)

Solawaski’s

reagent

(ml)

25 min in

room

temp.

Optical density

(530nm)

1 0.2 20 1.8 4 0.004

2 0.4 40 1.6 4 0.010

3 0.6 60 1.4 4 0.020

4 0.8 80 1.2 4 0.024

5 1.0 100 1.0 4 0.032

6 1.2 120 0.8 4 0.035

7 1.4 140 0.6 4 0.040

8 1.6 160 0.4 4 0.046

9 1.8 180 0.2 4 0.050

10 2.0 200 0.0 4 0.060

Standard IAA for preparation of standard graph.

IAA production by Rhizobium Spp.

Effect of tryptophane concentration

Most of the organisms produce IAA in presence of tryptophan. In present study it was observed

that as the concentration of tryptophan in the medium increases, the amount of IAA produced

increased and also study the IAA production in 72 hr.and 5 days incubation period. In 72hr the

IAA production is (10µg/ml,150µg/ml,323µg/ml) ) IAA production was found to be decreased

in 5 days incubation.

0 2 50

50

100

150

200

250

300

350

10

150

323

Series 1

Concentration of tryptophane (mg/ml)

IAA

conc

(µg/

ml)

Effect of Carbon source on production of IAA

For finding out the most favourable carbon source giving maximum IAA production mannitol

from YEMB is replaced by different sugars. maximum IAA production in presence of glucose

followed by sucrose, mannitol, and lactose in descending order. A reported 1% glucose as a

preferred carbon source for IAA production. In glucose was a best carbon source for production

of IAA. Glucose as a carbon source production of IAA was 466 µg/ml.

lactose mannitol sucrose glucose0

50

100

150

200

250

300

350

400

450

500

70

150

320

466

Series 1

IAA

con

c.(µ

g/m

l)

Effect of PH on IAA production

To decide the optimum pH for IAA production, the isolates are inoculated in YEMB amended

with 2.0 mg/ml of tryptophan having different pH such as 5, 7, 9. A rhizobium showed little

amount of IAA production at pH 5 whereas maximum production found at pH 7. IAA production

decreased at pH 9.

5 7 9135

140

145

150

155

160

165

170

175

180

185

150

180

160

Series 1

PH

IAA

conc

.(µg/

ml)

Observation table

Sr

no.

Different

parameter

Supernatant

of broth (ml)

Solawaski’s

reagent(ml)

Two drops of

orthophosphari

c acid

Incubation

in room

temp. for

25min

O.D

1 Without

tryptophan

2 4 0.004

2 2 mg/ml

(try.)

2 4 0.042

3 5 mg/ml

(try.)

2 4 0.097

4 Glucose 2 4 0.140

5 Sucrose 2 4 0.096

6 Lactose 2 4 0.024

7 Ph-5 2 4 0.045

8 Ph-9 2 4 0.050

Production of IAA by Azotobacter Spp.

Effect of tryptophan concentration on IAA production

Tryptophan is a induced the IAA production in fermentation broth. In Azotobacter spp. In

Ashby’s broth with different tryptophan concentration showed the different concentration IAA

was produced.The incubation

5 10 150

10

20

30

40

50

60

70

80

90

100

5 days

tryptophan conc.(µg/ml)

IAA

conc

.(µg/

ml)

1.0 Abstract

The phytohormone auxins play a central role in plant growth and development as a regulator

of numerous biological processes, from cell division, elongation and differentiation to tropic

responses, fruit development and senescenc. Chemically auxins is a indole acetic acid A

bacterial(Rhizobium,azotobacter,pseudomonas Spp.) were isolated from rhizospheric soils

was collected from sugarcane and wheat field near agicultural college,pune These isolates

were identify by Gram’s staning,motility and Biochemical test. the production of indole

acetic acid (IAA) in a medium with 0, 1, 2 and 5 mg/ml of tryptophan. A low amount of IAA

production was recorded by Azotobacter,Rhizobium and pseudomonas strains without

tryptophan addition. Azotobacter isolates showed high level (7.3 to 32.8 mg/ml) production

of IAA at 5 mg/ml of tryptophan while at 1 and 2 mg/ml the production was in the range of

1.47 to 11.88 and 5.99 to 24.8 mg/ml, respectively. Production of IAA in fluorescent

Pseudomonas isolates increased with an increase in tryptophan concentration from 1 to5

mg/ml.Azotobacter and Rhizobium were production of high amount IAA in glucose as a

carbon source and PH-7.IAA was detect by solawaski’s reagent and subsequent TLC

analysis. A specific spot from the extracted IAA preparation was found corresponding with

the standard spot of IAA with same Rf value.Among studies on diffrenent isolates like

pseudomonas,Rhizobium, and azotobacter using tryptophan as inducer Azotobacter showed

the highest IAA production ability.