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1. Isolation and Purification of Bioactive
Compound Isolated Marine Streptomyces
albus from Tiruchendur Coast
Under the Guidance of
Prof. S. Umamaheswari
Presented by
A L Nirmal Shankar
Reg .No: 20204012502113

2. Applications of Marine
Sediments
• Secondary metabolites are natural products
produced by living organisms.
• Play key roles in defense & cell to cell
communication.
• Evolving with nature together with optimized
activities of the targets.
• Natural products play an important role in drug
discovery & used for treatment of diseases.
• >60% of clinically used antibiotics are originally
from Actinobacteria, especially Streptomyces.
INTRODUCTION

3.  Streptomyces- Largest Genus of Actinobacteria
 Gram positive with High GC content
 Aerobic Bacteria
 Filamentous forming mycelial structure like fungi.
 Branching strands: 0.5 -1.0 µm in dm
 Largest Genus producing bioactive compounds
 (antibacterial, antifungal, antiparasitic & anti cancer activity)
 Noted for the production of broad-spectrum antibiotics
Streptomyces sp.

4. OBJECTIVES
04 Partial purification
using Ammonium
Sulphate Precipitation.
06
.
FTIR
05
Column
Chromatography
03 Molecular characterization
of Actinomycetes sp. using
16s r RNA sequencing.
02
Primary Antibacterial
screening
01 Isolation of
Streptomyces sp. From
Tiruchendur Sea Coast.

5. WORK PLAN
Collection of marine soil
sample from Tiruchendur
coast
01
05 Partial purification of secondary
metabolities using precipitation.
03
Identification of Streptomyces
sp. using biochemical
characterization and 16s r RNA
sequencing.
06
Purification of bioactive
compound through column
chromatography & FTIR
04
Antibacterial activity of
various culture using agar
well diffusion.
02
Isolation of Streptomyces sp.
from marine sediment
sample.

6. Identification
Isolation
Streptomyces sp. isolated from the marine sample by Serial dilution method.
Microbiological Characterization
 Gram’s Staining
 Culture Morphology
Biochemical Characterization
 IMVIC
 Motility, Oxidase
 Starch Hydrolysis
 Urease Test
 Catalase Test Ref: Rahman et al., 2010
Functional Characterization of Bioactive Compound
Antibacterial Assay (Agar Well Diffusion Method) Ref: Balachandran et al.,2012
METHODOLOGY

7. Molecular Characterization
16s r RNA Sequencing
The 16S rRNA gene sequencing involves the following steps: Extraction of DNA, PCR amplification,
Purification and sequencing and Sequence similarities and Phylogenetic analysis.
Wilson et al., 1990
Antibiotic Sensitivity Test
Antibiotic sensitivity and resistance of Streptomyces sp. (MBT-02) were
assessed by disc diffusion method.
Peela S et al., 2006
Ammonium Sulfate Precipitation Method and Dialysis
Precipitation of the isolated Streptomyces sp. (MLT-02) bacterial culture with
Ammonium sulfate was done to purify the proteins present in the cultures.
Functional Characterization
Using Column chromatography and Mass Spectral Analysis functional bioactive compounds -
Characterized. Weisburg et al., 1991

8. RESULTS
Marine actinomycetes
actinomycetes
Isolation of Actinomycetessp.
Pure culture of Actinomycetessp.
Gram-positive

9. S. No Biochemical Tests Results
1.
Indole Test
+
2.
Methyl Red Test
+
3.
Vogues-Prausker Test
_
4.
Starch Hydrolysis Test
+
5.
Carbohydrate Fermentation Test +
6.
Oxidase Test +
7.
Catalase Test +
8.
Spore-staining Test -
9.
Urease Test +
10.
Motility Test _
11.
Gelatinase Test +
12.
Citrate Utilization Test +

10. Antibiotic SensitivityTest for Streptomycessp.

11. Antibacterial Activity of Streptomyces sp. against Test Pathogens
1.3
1.1
0.7
0.9
1.5
0.8
1
0.7
E. coli S. aureus P. aureginosa B. subtilis
Antibacterial activity of Streptomyces sp. against
Test pethogens
Sample(S1) Streptomycin
Antibacterial activity of Streptomyces sp.
against E.coli

12. Molecular Analysis by 16s rRNA sequencing

13. Purified bioactive compound producing
Streptomyces sp. cultured for mass production
01
Collection of the crude extract
04
Ammonium sulphate precipitation
(80% saturation)
02
Centrifugation (15,000 rpm, 15mins)
03
AmmoniumSulphatePrecipitation

14. ColumnChromatography
Absorbance Protein Concentration (mg/mL)
0.1808 0.8476
0.2023 0.9126
0.1602 0.6217
0.1832 0.8588
0.1307 0.6127
0.1349 0.6324

15. Antibacterial Assay
Test Bacterial
Strains
Zone of Inhibition (mm)
Fr-1 Fr-2 Fr-3 Fr-4 Fr-5 Fr-6
Escherichia coli 03 04 _ _ _ 04
Pseudomonas
montailliei
_
07
_ _ 03 _
Bascillus subtilis _ 06 _ _ _ _
Staphylococcs
aureus
01
11
02 _ _ _
Klebsiella
pneumoniae
05
08
_ 07 _ 10
Vibrio harveyi - 02 _ 04 03 _
5 4
8 7
11
6
Zone
of
inhibition(mm)
Antimicrobial activity of Column
Fraction-II

16. FTIRanalysis for the Purified compound (F-II)

17. CONCLUSION
 The Actinomycetes culture were isolated from Marine soil sample in Tiruchendur
Coast. The isolated culture was preliminarily characterized by Gram’s staining and
Biochemical tests.
 The isolated marine actinomycetes was identified as Streptomyces albus through 16S
rRNA sequencing.
 The Streptomyces albus culture was purified by Ammonium sulfate precipitation for
purifying the protein content - 75% saturation.
 The 75% saturated precipitate was further subjected to dialysis to remove the salt and
the precipitated culture was used for the Column Chromatography.

18.  The bioactive compounds produced by the isolated strain were purified by
Column Chromatography.
 The crude sample was then fractionized using six purified fractions among which
the 2nd fraction exhibited the highest antibacterial activity when compared with
the other fractions.
 The potential fraction (F-II) obtained by column chromatography was subjected
to FTIR.

19.  Isolation and characterization of Marine Streptomyces (Mobeen shaik, G,Giria
sankar, M.Iswarya) 2017
 Purification and structure elucidation -(Kothagorla venkata raghava rao, palla
mani ) 2017
 Antimicrobial potential - (Mysoon Al-Ansari, Noorah Alkubaisi) 2019
 Extraction and Seperation - (R.Balachandar, N.Karmegam, R.Subbaiya) 2018
REFERENCES