BIOFILM INHIBITORY ACTIVITY OF BIOSURFACTANTS ISOLATED FROM THE

MARINE BACTERIA

ByPandi selvi, K.

Reg.No:09MBT12

Under the guidance of Dr. S. SATHEESH

LecturerInternational Centre for Nanobiotechnology

Manonmaniam Sundaranar UniversityRajakkamangalam – 629 502

Kanyakumari DistrictTamil Nadu, India

Synopsis

• Introduction• Objectives• Materials and Methods• Results and Discussion• Summary• Acknowledgement• References

INTRODUCTION

• Biofilm is an assemblage of microbial cells that is irreversibly associated with a surface and enclosed in a matrix of primaroly polysaccharide material.

• Biofilm form when bacteria adhere to surfaces and begin to excrete a glue-like substance that protects and anchors them to material such as metals such as metals, plastic, soil, medical implants and tissue.

Cont…

• In industry, biofilms can cause corrosion of pipes, reduce heat transfer in cooling systems and plug water filters.

• The major difficulties in controlling surface-attached bacteria is their enhanced resistance to antimicrobial agents.

• Surface active compounds (surfactants) have been empolyed to prevent bacterial adhesion to surfaces.

CONTROL OF BIOFILM

• A biofilm control programme is detection and assessment of various biofilm components like thickness of slime layer, algal and bacterial species involved, extent of extracellular polymeric substances (EPS) and inorganic components.

• The new techniques for biofilm control like ultrasound, electrical fields, hydrolysis of EPS and methods altering biofilm adhsion.

BIOSURFACTANT

• Surfactants both chemical and biological are amphiphilic compounds which can reduce surface and interfacial tensions by accumulating at the interface of immiscible fluids.

• It play an essential role in the swarming motility of microorganisms and participate in cellular & physiological processes of signalling and differentiation as well as in the biofilm formation.

Cont…

• There are several advantages over the chemical surfactants such as lower toxicity, higher biodegradability, better environmental compatibility, higher foaming , high selectivity and specific activity at extreme temperatures, pH and salinity and the ability to synthsized from renewable feedstock.

OBJECTIVES

• to isolate and characterize the biosurfactant from seaweeds and gastropods,

• to check the antibacterial activity,

• to analyse the impact of biosurfactant on bacterial adhesion,

• to characterize the biosurfactant by TLC, HPLC & NMR.

MATERIALS AND METHODS

• Isolation of surface associated bacteria (SAB),

• Biochemical characterization of isolated bacterial strains,

• Gram’s staining• Motility test• Indole production test• Methyl-red test• Voges-proskauer test• Citrate utilization test • Starch hydrolysis• Urea hydrolysis• Fermentation of carbohydrate• Catalase test• Gelatin hydrolysis test

Cont…

• Isolation of biosurfactant (Oil displacement test),

• Antibacterial activity of biosurfactant against biofilm bacteria,

• Influence of biosurfactants on the adhesion of biofilm bacteria to surface,

• Characterization of biosurfactant by TLC, HPLC & NMR.

Results and Discussion

+ - Positive

• - Negative

S1 - Seaweeds

S2 - Seaweeds

S3 - Seaweeds

S4 - Seaweeds

G1 - Gastropods

Biosurfactant screening methods

Antimicrobial activity of biosurfactant isolates from the srain S1, S2 against target bacteria

Antimicrobial activity of biosurfactant isolated from the strain

S 3, S4 & G1.

Adhesion assay using biosurfactant of the strain S1 & S 3 (number of bacterial cells adhered on glass coupons)

Thin layer chromatogram of the biosurfactant isolated from the strain S1, S2, S3, S4 & G1.

HP LC spectrum of biosurfactant isolated from the strain S1

H P LC spectrum of biosurfactant isolated from the strain S 3

H P LC spectrum of biosurfactant isolated from the strain S4

1 H NMR spectrum of the biosurfactant isolated from the strain S 3

13 C NMR spectrum of the biosurfactant isolated from the strain S3

DISCUSSION

• In conclusion, result of the present study revealed that bacteria associated with marine invertebrates and seaweeds has the potential to produce biosurfactants.

• Further studies on this aspect may improve our knowledge on the application of these biosurfactants in biomedical and environmental field.

SUMMARY

• The present study was carried out to assess the biofilm inhibitory activity of bacteria associated with seaweeds and gastropods.

• Biosurfactant was tested for antimicrobial activity against five bacterial strains (E.coli, Bacillus sp, Klebsiella sp, V. harveyi, V. parahaemolyticus).

• The biosurfactant was characterized by TLC, HPLC & NMR.

• Further studies on biosurfactants, provides tremendous applications in industrial fields as detergents in biomedical and therapeutical fields.

ACKNOWLEDGEMENT• I owe to almighty God and my beloved mother for their blessings and help, without which I might never

have been successful.

• It is my privilege to express my deep sense of gratitude and sincere thank to my guide Dr. S. Satheesh, Lecturer, International Centre for Nanobiotechnology, Rajakkamangalam for providing necessary facilities to carry out my project in this institution.

• It is my privilege to express my deep sense of gratitude and sincere thank to my co-guide Dr. S. G. Prakash Vincent, Associate Professor, for his invaluable contribution, encouragement, optimistic approach in any problem and systematic guidance to make this out a successful project.

• I take this opportunity to express my hearty thanks to Dr.L.Uma, Head Department of Marine Biotechnology, Bharathidasan University, Trichy, Dr.S.Mary Josephine Punitha, Assistant Professor, Dr.G. Immanuel, Assistant Professor, Dr. T. Citarasu, Assistant Professor for their encouragement and support throughout this study.

• I would like to express my hearty thanks to my beloved friends and scholars who helped me directly and indirectly for the completion of my project work.

• I wish to express my sense of appreciation to my sisters who patiently endured and gave encouragement and support throughout my studies.

• Once again I thank to god for making everything possible with the help of our guides, family members, friends and everyone who helped me. (K.Pandi selvi)

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