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
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
PAGE NO.
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.
1
CONTENT
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 production
by Rhizobium
Effect of carbon sources on IAA production
by Rhizobium
5
6
Effect of PH on IAA production by Rhizobium
7
Effect of Tryptophan on IAA production
By Azotobacter
Effect of Carbon source on IAA production
by Azotobacter
8
Page No.
9
10
11
12
Effect of PH on IAA production
by Azotobacter
Effect of Tryptophan on IAA production
By Pseudomonas
Effect of Carbon source on IAA production
By Pseudomonas
Effect of PH on IAA production
By 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 oleum, 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 plantassociated 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
Biochemical pathway 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.
As recently as 1991, this 3rd mechanism has evolved. IAA can be produced via a tryptophanindependent 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
Rhizobium
 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
 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, Rhizobium
moth bean
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
:
 Nitrogen source :
 Respiration
N2 through fixation, amino acids, N2, NH4+, NO3:
 Growth media :
 Doubling time
A variety of carbon sources(mono, di & certain polysacharide) organic acids.
Aerobic
Ashby/Jensen’s medium
:
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 METHOD
3.1 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
Root nodules of chick pea plant
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.
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
Fig No.4 Gram’s staning of Rhizobium
BIOCHEMICAL TEST RESULT OF RHIZOBIUM
BIO CHEMICAL TEST
RESULT
Oxidase test
Positive (+)
Catalase test
Positive (+)
Voges– Proskauer’S Test
Negative (-)
H2s Production Test
Negative (-)
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.
Azotobacter colony on Ashby’s Mannitol Agar
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
9
consistancy
Moist
10
Motility
Motile
11
Remark
Nil
GRAM’S STAINING OF AZOTOBACTER
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 (-)
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 (-)
Carbohydrate utilization Test
Simmon’s Citrate utilization Test
PRODUCTION OF IAA BY AZOTOBACTER
Catalase Test
Production of IAA by Azotobacter in
different Tryptophan concentration
Production of IAA by Azotobacter in
different carbon source
Production of IAA by Azotobacter
in different PH
PLATE COLONY OF PSEUDOMONAS Spp.
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
Oxidase test
Carbohydrate utilization Test glucose,
sucrose, xylose
Catalase Test
H2s Reduction Test
TSI Slant
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.
optical density (530nm)
0.07
0.06
0.06
0.046
0.05
0.04
0.04
0.032
0.035
0.024
0.03
Series 1
0.016
0.02
0.01
0.05
0.01
0.004
0
20
40
60
80 100 120 140
Concentration (µg/ml)
160
180
200
Observation table
Assay of IAA
Sr.no IAA
std.soln
IAA
Distille
conc.(µg/ml) water
(ml)
(ml)
Solawaski’s
Optical density
reagent
(530nm)
(ml)
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
25 min in 0.024
5
1.0
100
1.0
4
room
0.032
6
1.2
120
0.8
4
temp.
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.
350
323
300
IAAconc(µg/ml)
250
200
150
150
Series 1
100
50
0
10
0
2
5
Concentration of tryptophane (mg/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.
500
466
450
IAA conc.(µg/ml)
400
320
350
300
250
Series 1
200
150
150
70
100
50
0
lactose
mannitol
sucrose
glucose
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.
185
180
180
IAA conc.(µg/ml)
175
170
165
160
160
155
Series 1
150
150
145
140
135
5
7
9
PH
Observation table
Sr
Different
Supernatant
Solawaski’s
no.
parameter
of broth (ml)
reagent(ml)
1
Without
2
4
0.004
2
4
0.042
O.D
tryptophan
2
2 mg/ml
Incubation
(try.)
3
5 mg/ml
2
4
Two drops of
in room
0.097
orthophospharic temp. for
(try.)
acid
25min
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
100
90
IAA conc.(µg/ml)
80
70
60
50
5 days
40
30
20
10
0
5
10
tryptophan conc.(µg/ml)
15
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.