EXTRACTION AND CHARACTERIZATION OF

SIDEROPHORES FROM PSEUDOMONAS SP.

Mentor:
Dr. Dweipayan Goswami

Aayushi Purohit (16-BT-043)

Bhavya Kansara (16-BT-015)
Mahima Patel (16-BT-035)
Shailee Patel (16-BT-037)
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 CONTENTS:

•AIMS
•OBJECTIVES
•RESULT AND DISCUSSIONS
•CONCLUSION

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 AIM

Extraction and characterization of Siderophore from

Pseudomonas sp.

OBJECTIVES

 Confirmation and evaluation of siderophore using Thin Layer Chromatography.

 Use of several solvent systems for TLC.
 Calculate the Rf values for the obtained bands.
 Anti-Fungal assays for Aspergillus niger.
 Compatibility test between Pseudomonas aeruginosa and Staphylococcus aureus.
 Biochemical tests to confirm the group of our extract.
 PGPR activity test on chick pea.
 Carry out HPTLC and analyze the bands obtained.

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 Results and Discussion

1) TLC solvent systems. Their RF Value and Photos under UV light (make sure we
include all three objectives mentioned above)
1. Isopropyl alcohol: water (9:1)
2. Isopropyl alcohol: water (1:1)
3. Isopropyl alcohol: butanol: water (3:1:1)
4. Butanol: Acetic acid: water (3:1:1)
5. Butanol: Acetic acid: water (12:3:5)
6. Isopropyl alcohol: butanol: ammonia: water (9:3:3:1)
7. Isopropyl alcohol: butanol: ammonia: water (10:6:3:1)
8. Ethanol: water (7:3)

After trial and error method the solvent system of Isopropyl alcohol: butanol: ammonia:
water (10:6:3:1) was standardized and the Rf values were calculated of the same.

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 Fig 1: TLC plate as seen Fig 2: TLC plate dipped
under UV light. Solvent in CAS Dye
system-7
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 Table 1: Distance travelled by solute

Sr. No. 1x Bands (cm) 2x Bands (cm)

1 2.6 2.6

2 2.8 2.8

3 3.2 3.4

4 3.7 3.9

Solvent system = 4.1cm

Table 2: Rf Values

Sr. No. 1x Bands (cm) 2x Bands (cm)

1 0.63 0.63
2 0.68 0.68
3 0.78 0.82
4 0.90 0.95
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 Fig 3: Antifungal Test of Fig 4:Compatibility Test between
Pseudomonas sp. with Pseudomonas sp. and
Aspergillus niger Staphylococcus aureus

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Table 3: Biochemical tests for groups present in Siderophore

GROUP IDEAL OBSERVED INFERENCE

RESULT RESULT I
N
a) Absorbance Peak positive S
Hydroxamat peak obtained E
e between at… R
420-450 nm T

P
b) Absorbance Peak negative I
Catecholate peak at obtained C
490nm at…… T
U
c) Pink colour Pink colour Positive R
Carboxylate disappears disappears E
on addition on addition S
of test of test
sample sample H
E
R
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E
 6) Plant growth promoting activity (PGPR) activity:

A B
A

Fig 5: PGPR activity of Pseudomonas sp. Chickpea. A) Control (D/W) B) Treated with
Pseudomonas. Growth till 4 days.

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 A B

C D

Fig 6: Progressive growth of seeds under controlled conditions. A) Day-4 B) Day-7

C) Day-10 D) Day-15

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Fig 7: A) Length comparison between plant grown under control and treated condition.
B) Measurements of the plants

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Table 4: Morphological measurements of plants

Root Shoot Total Fresh No. of No. of

Type Dry Weight
Length Length Length Weight Leaves Branches

52 94 146 0.483 0.124 25 3

Control
82 104 186 0.514 0.099 16 2

57 88 145 0.398 0.121 16 2

98 174 272 0.786 0.133 39 4

Treated
118 178 296 0.744 0.13 38 4

119 155 274 0.624 0.121 36 3

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7) HPTLC of the extract

Fig 8: HPTLC of extract along with phytohormones- IAA, IBA &

Tryptophan. A) HPTLC Plate B) HPTLC Plate as observed under UV light.

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Fig 9: HPTLC scan. Observation of different peaks indicating the
separation different hormones, siderophores and pigments.
(Dhavalkumar Patel et al, 2018)

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*

HPTLC REFERENCE : A resourceful methodology to profile indolic auxins

produced by rhizo-fungi using spectrophotometry and HPTLC.
Dhavalkumar Patel, Anoshi Patel, Disha Vora, Sudeshna Menon,
Sebastian Vadakan, Dhaval Acharya and Dweipayan Goswami

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