ANTIMICROBIAL POTENTIAL OF BIOSURFACTANT PRODUCING

ORGANISMS ISOLATED FROM OIL SPILLED SOIL

BYSHABARI DAWN

ROLL-BUR ENVS NO-2012/13REGISTRATION NO-016884 OF 2011-12

SUPERVISED BYDR SHAMPA DUTTA

DR SRIMANTA GUPTA

DEPARTMENT OF ENVIRONMENTAL SCIENCETHE UNIVERSITY OF BURDWAN

A surfactant is an organic chemical when added to a liquid changes the property of that liquid at a surface or interface. A surfactant produced by microorganism is called biosurfactant. The word surfactant stands for “Surface active agent”

INTRODUCTION

• Surfactants widely used in lubricants, paints, corrosion inhibitor, textiles, detergent, emulsifying agent in house hold, soap-shampoo, foaming agent, food industries, cosmetics, wetting agent, and soil reclamation.• There are many chemical surfactants available in market (SDS,CTAB) but

green revolution fulfilled our aspirations by introducing modern eco-friendly practice so uses of biosurfactant is an approach to support green practice.• Most of the chemical surfactants are hazardous and persistent in nature,

where biosurfactant produce from microorganism is very environment friendly and can be produced from easily available substrate which is also cost effective in compare to chemical surfactant.

INTRODUCTION...

INTRODUCTION...Surfactant classes

AIMS AND OBJECTIVES• The purpose of exploring native biosurfactant producing strains

isolated from oil spilled soil for hydrocarbon recovery.

• Characterization of the native biosurfactant producing strains.

• Studied their certain antibacterial and antifungal activity.

Materials and Methods

LOCATION OF THE EXPERIMENTAL SITE

Petroleum contaminated soil samples were collected from different petroleum stations.

Materials and Methods…

Lakurdi Golapbag Golapbag

Collection of soil sample

Isolation ( Nutrient agar )

Enrichment ( Minimal salt medium, Bushnell-Haas medium)

Biochemical characterization

Assay of biosurfactant producer (Oil spreading assay, Blood agar hemolytic assay)

Antibacterial and Antifungal study

Materials and Methods…

• Isolation of Bacterial strains 24h Oil spilled soil sample + nutrient agar medium Bacterial colony 30°C

• Enrichment of Bacterial strains 24hIsolated microorganisms + MSM agar medium Bacterial colony

30°C

24 h

Selected microorganisms + Bushnell Haas agar medium Bacterial colony

30°C

Materials and Methods…

Biochemical Characterization

1.Gram staining

2.Catalase test

3.Indole production

4.Methyl red

5.Voges Proskaure

6.Citrate utilization

Materials and Methods…

Assay of Biosurfactant production

Oil spreading assay

72 h culture

Centrifugation of bacterial cell culture

Supernatant injected on oil surface

Observation of dispersion zone

Blood agar hemolysis test

Overnight bacterial culture

Inoculate into blood agar medium

Observation of change of color or clear zone

Antimicrobial assay

• Antibacterial assay against E. coli, Pseudomonas, Salmonella and Bacillus

• Antifungal assay against Colletotrichum sp., Rhizopus sp., Alternaria sp. and Penicillium sp.

Materials and Methods…

Results and Discussion

Colony Morphology on Nutrient Agar Medium

Isolated sample Colour Elevation Shape Surface Characteristics

S1 Yellow Convex Irregular Smooth

S2 Muddy Convex Filamentous Smooth

S3 White Convex Irregular Smooth

S4 Yellowish Convex Irregular Smooth

S5 Whitish Convex Round Smooth

S6 whitish Convex Irregular Smooth

S7 Fluorescent yellow Convex Round Smooth

S8 yellow Convex Irregular Smooth

Results and Discussion…

Bacterial Growth on MSM medium Sample

NoPetroleum

oilSunflower oil Mustard oil Coconut oil

S1 ++ + + +

S2 + + + -

S7 + - + -

Bacterial Colony on Bushnell Haas medium Sample No Petroleum

oilSunflower oil Mustard oil Coconut oil

S1 - - - -S2 - - - -S7 + + + +

Results and Discussion…

Bacterial Growth on MSM medium

Results and Discussion…

Petroleum oilSunflower oil Mustard oil Coconut oil

Biochemical CharacteristicsResults and Discussion…

Catalase test Voges-Proskaure test Citrate utilization test

Oil spreading assay

Oil spreading assay of biosurfactant on mustard oil containing plates (0-10 sec)

Oil spreading assay of biosurfactant on petroleum oil containing plates

Blood Haemolysis test

Antimicrobial activity of isolated strainSample Diameter (cm) of the inhibition zone

E.coli Salmonella sp Bacillus sp Pseudomonas spS1 - - - -S2 2.2 - - -S7 - - - -

Antibacterial activity against E.coli. By S2 strain

Antifungal activity against Alternaria sp Antifungal activity against Penicillum sp

 

Antifungal activitySample Diameter (cm) of the inhibition zone

Rhizopus.sp Alternaria.sp Colletorichium.sp Penicillum.sp

S1 - - - -S2 - - - -S7 - 0.2 - 0.7

Conclusion

Conclusion…

• The present experiment was conducted to isolate biosurfactant producing strains from crude oil contaminated soil and only three strain (S1, S2 and S7) able to utilize oil as carbon source in MSM and Bushnell Haas medium.

• Production of biosurfactant is greatly affected by substrate and available carbon source they are utilizing that’s why we observed different growth in respect to 4 different oil ( Petroleum, mustard, coconut and sunflower oil).

• In case of antimicrobial test only S2 showed antibiotic activity against E.coli and S7 showed mild antifungal activity against two fungal species ( Alternaria sp and Penicillium sp)

• These three strain can be further studied for the knowledge of exact amount of the surfactant they are producing and their types and the types of soil microorganism who are best in this production what can be used in oil removal from soil for fresh oil recovery.

AcknowledgementI wish to express my deep sense of gratitude and thanks to Dr. Srimanta Gupta and Dr. Shampa Dutta, Lecturer, Department of Environment Science, The University of Burdwan for his and her constant inspiration keep interest help and guidance at every step of this work.

I am most grateful to Prof. S. Mondal Founder Head of The Department Environmental Science. The University of Burdwan for his valuable suggestion during the period of my dissertation work.

I am most grateful to Dr. Apuraba Ratan Ghosh of Department of Environment Science. The University of Burdwan for his continuous enthusiasm and advice throughout the period of my dissertation work.

I also express my thanks to Sri Gobindo Baidya and Sri Sarkar Nag for their continuous help by supplying essential requirements and books at the time of project preparation.

I express my heartiest thanks to my class mates for their continuous help in the proceeding of my project.

Lastly I express my deep regards and love to my parents and all of my well-wisher for their affectionate blessing and encouragement.

Thank You