Describe by dr.Subham Preetam

1. INHIBITION OF PATHOGENIC BACTERIAL BIOFILM BY
BIOSURFACTANT PRODUCED BY LYSINIBACILLUS
FUSIFORMIS S9
ARUN KUMAR PRADHAN, NILOTPALA PRADHAN, LALA BEHARI SUKLA, PRASANNA
KUMAR PANDA & BARDA KANTA MISHRA
BIOPROCESS AND BIOSYSTEMS ENGINEERING
VOLUME 37,NUMBER 2,IF-2.3
Centre for Biotechnology
Siksha ‘O’ Anusandhan
( Deemed to be University )
Your name : Subham Preetam
Reg. no : 1861621001
Semester : 3

2. ABSTRACT
• A biosurfectant producing microbe isolated from a river
bank was identified as Lysinibacillus fusiformis S9.
• It was identified with help of biochemical tests and 16s
rRNA gene phylogenetic analysis.
• The biosurfectant S9BS produced was purified and
characterized as glycolipid.

3. INTRODUCTION
• Biosurfactants are amphiphilic surface active molecules produced
by microorganisms, such as bacteria, fungi and yeast.
• As they are biodegradable and nontoxic to humans.
• Biosurfactants mostly applicable in biologically applicable
industries.
• Biosurfactants modulate the attachment of some bacteria on a
particular interface surface while inhibiting the attachment of others,
by forming a conditioning layer on it. This helps to detachment of
biofilm formation by perticular microorganisms.

4. OBJECTIVES
• The biosurfactant showed remarkable inhibition of biofilm
formation by pathogenic bacteria like E.coli and Streptococcus
mutants .
• At concentration of 40 µml-1
the biosurfactant did not show any
bacterial activity but restricted the biofilm formation completely .
• The biosurfactant inhibited bacterial attachment and biofilm
formation equally well on hydrophobic and hydrophilic surface like
catheter tubing and glass.
• This property is significant in many biochemical applications where
the molecule should help in preventing biofouling of surfaces
without being toxic to biotic system.

5. METHODS
THIN LAYER CHROMATOGRAPHY (TLC)
• Biosurfactant solution in chloroform was spotted on a TLC plate and eluted with
chloroform : methanol : water (65 : 15 : 2) in solvent chamber .
FOURIER – TRANSFORM INFRARED SPECTROGRAPHY (FTIR)
• The functional group present in the biosurfactant were determined using FTIR.
NUCLEAR MAGNETIC RESONANCE (NMR)
ANALYSIS
• TLC purified biosurfactant was subjected to analysis with NMR . Denatured
chloroform was used as solvent for NMR sample preparation .

6. RESULTS AND DISCUSSION
Screening of biosurfactant producing bacteria
• The formation of dark blue was due to a reaction between the CTAB –
methylene blue complex and the glycolipid biosurfactant(fig. 1a)
• A transparent zone was found around the bacterial colony on blood
agar plate indicating the hemolytic property of S9 bacteria due to
production of biosurfactant (Fig. 1b).
• Cell free supernatant drop showed spreading over the parafilm surface
in drop collapse method-I . Here water droplets did not show any such
spreading (Fig. 1c) .
• Dispersion of oil drop was observed on the surface of S9 cell
supernatant in drop collapse method-II (Fig. 1d).

7. Fig-1 Screening for biosurfactant producing bacteria Lysinibacillus fusiformis
S9: (A) daywise CTAB plate result showinh deep blue halozones and positive
control SDS, (B) Blood agar plate result showing transparent zone, (C) Drop
collapse test – I on parafilm surface and (D) Drop collapse test - II in test tube

8. IDENTIFICATION OF BIOSURFACTANT PRODUCING
BACTERIA
• Biochemical characteristics of S9 strain matched with the genus L.
fusiformis as shown .The 16S rRNA gene sequencing (Fig. 2a) and
phylogenetic tree analysis (Fig. 2b) confirmed it as L. fusiformis. Hence the
strain was classified as L. fusiformis S9.
• The bacteria L. Fusiformis s9 used produced a glycolipid type of
biosurfactant .
• This bacterium belongs to order Bacillales and family Bacillaceae .

9. Fig. 2 16S-rRNA gene phylogenetic analysis of Lysinibacillus fusiformis S9.
(a) Agarose gel electrophoresis showing genomic DNA band and PCR product with
1.5 kb DNA ladder
(b) Phylogenic tree by MEGA 5.05

10. CHARACTERIZATION OF BIOSURFACTANT
• Lysinibacillus fusiformis strain S9 was capable of producing 160-200 mg l-
1
of crude biosurfactant .
• TLC analysis confirmed the biosurfactant to be a glycolipid using TLC and
selective developing reagents for different functional groups (Table 3).
• The FTIR spectra of partially purified biosurfactant from L. Fusiformis S9 is
shown in (Fig. 3).
• Biosurfactant molecule comprised fatty acids and carbohydrates moieties
making it a glycolipid .
Fig. 3 FTIR spectra of biosurfactant produce
by Lysinibacillus fusiformis S9
Table 3 TLC plate analysis for
detection of composition of
biosurfactant molecule
SI. no Spot developers Spot color Indication
1 Iodine vapour Brown Organics
2 Ninhydrin solution No spot No amino
group
3 Orcinol reagent Brown Carbohydrate
4 Bromothymol blue solution Yellow Lipids
5 UV light (366 nm) Blue Organics

11. PEAKS FUNCTIONAL GROUPS
3670 - 3250cm-1
Hydroxyl group
300 – 2800cm-1
Fattyacid chains
800 - 875cm-1
Glycosidic linkage of carbohydrates
1730cm-1
Alkyl ester of fatty acids
Peaks and functioal groups stretching

12. • The biofilm formed on glass cover slips were visualized under fluorescence
microscope after staining with dye. (Fig.5)
• The minimum concentration of biosurfactant required for complete inhibition
of biofilm was 40µg ml-1
for E. coli and S. mutans.
• Adhesiveness of bacteria to a surface is reported to be due to
exopolysaccharides(EPS) produced on the surface of the bacterial cell.
• The biosurfactant inhibited the development of a biofilm on the hydrophilic
surface.
BIOFILM INHIBITION BY EXTRACTE GLYCOLIPID
Fig. 5 Effect of biosurfactants concentration on biofilm formation by
a E. coli and b S. mutans on glass surface

13. •Catheter tubing was used as surface to develop and monitor the formation of
biofilm in the absence and presence of biosurfactant.
•Catheter tubes stained dark violet when biosurfactants was not added to the
medium, where as they stained faintly when incubated in medium with 40µg ml-1
biosurfactant (Fig. 6).
Fig-6. Effect of biosurfactant on biofilm formation on
cathter tubing stained with crystal violet stain .

14. •Biofilm forming bacteria when streaked on the CRA plate, show black pigmentation
of colonies and media when EPS is produced.
•E. Coli and S. Mutans showed black pigment formation on CRA plates in the
absence of biosurfactants due to biofilm formation (Fig-7) and such black pigment
formation was absent in biosurfactant-supplimented CRA plates.
Fig. 7 Effect of biosurfactant on EPS production by E. coli and S. mutans on
Congo Red Agar plates

15. •S9BS was better than SDS and CTAB in inhibiting biofilm formation by both E.coli
And S. mutans and also better than from chemical surfactants .
•The biosurfactant S9BS produced by the bacteria L. Fusiformis S9 was effective in
inhibiting bacterial attachment and biofilm formation on two different types of
surfaces, such as glass and catheter.
•Glass is highly hydrophilic and catheter is highly hydrophobic in nature .
•The biosurfactant is preventing bacterial attachment and antifouling of both
hydrophilic and hydrophobic surfaces which have enormous biomedical application.

16. CONCLUSION
• A biosurfactant producing microorganism L. Fusiformis S9 produce a glycolipid
type of biosurfactant .
• The presence of carbohydrate and fatty acid moieties were confirmed by FTIR
and NMR analysis .
• At a concentration of 40 µg ml-1
, the biosurfactant effectively inhibited biofilm
formation by E. Coli and S. mutans .
• Biosurfactant S9BS proved to be better than chemical surfactant SDS and CTAB
.

17. Thank You