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Title: Screening of Biosurfactant Producing Bacteria From Oil Spilled Soil and Characterization of Biosurfactant

Author: Mohammed Jobaer

Supervisor: Dr. Md. Nural Anwar

Co-Supervisor: Ferdausi Ali

Research Field: Environmental Microbiology

Batch: 11th

Session: 2010-2011


Biosurfactants are usually organic substances which are surface metabolites that vary in their chemical composition. Biosurfactants include lipopeptide, glycolipid, polymeric biosurfactant etc are synthesized by different microorganisms including bacteria, yeast and fungi. Chemical surfactants are being replaced by the biosurfactant because biosurfactants are less toxic, highly biodegradable, highly selective and better environmental compatible. The present study was carried out to isolate biosurfactant producing bacteria, to optimize the cultural conditions for biosurfactant production and characterize the biosurfactants.
Samples were collected from hill view motor, Panchlaish and rowshan garage, Bahaddarhat, CTG. By following enrichment technique, 32 distinct colonies were isolated.
To screen the biosurfactant producer, two methods were employed as primary screening method. These are blood haemolysis test and oil spreading test. On the basis of better haemolytic activity and better oil spreading capability, five isolates were selected for secondary screening. Secondary screening was done by measuring emulsification index (E24). Isolate FS6 and FS7 showed highest E24 in mckeen medium whereas FS6 showed highest E24 in mineral salt medium.
The selected isolates were then identified by various cultural, morphological and biochemical tests. On the basis of these tests the isolates the isolates JR3, JR7, FS6, FS7 and FS22 were identified as Bacillus firmus (JR3), Acinetobacter haemolyticus (JR7), Pseudomonas gladioli (FS6), Pseudomonas caryophylli (FS7) and Pseudomonas aeruginosa (FS22) respectively.
To optimize cultural condition it was observed that JR3, JR7 showed highest biosurfactant production in 3/4 days of incubation at 30oC and the medium pH was 6. Glucose, ammonium nitrate and C/N ratio (1:10) were found to be optimum for biosurfactant production by JR3 but for JR7 the C/N ration was 1:20. Kerosene was found to be better inducer for biosurfactant production for all isolates.
Optimized incubation period was varied from isolate to isolate. FS6, FS22 (2 days), FS7 (3 days) showed highest biosurfactant production in glucose, ammonium nitrate, C/N ratio of 1:10 at 37oC with medium pH of 6 (FS6) and 8 (FS22, FS7).
Structural characterization of the biosurfactant was carried out by TLC method. From this study it was observed that JR3, JR7 and FS6 produce lipopeptide biuosurfactant and FS6, FS7 and FS22 produce glycolipid biosurfactant.
Activity of biosurfactant was carried out by emulsifying properties and antimicrobial activity. All the biosurfactants produced by the isolate showed higher emulsifying property with kerosene but JR7. JR7 showed higher emulsifying ability with soya bean oil.
Antimicrobial activity was evaluated by disc diffusion method and microtitre plate assay. From both study it was observed that extract from FS22 was to be better antimicrobial agent when tested against different lab pathogen like Bacillus cereus, Staphylococcus aureus, E. coli, Serratia mercescens and Salmonella typhi.
Stability under high temperature was done for the biosurfactant by heating the crude supernatant. After heating, it was observed that all the biosurfactant produced by the isolates were seemed to be thermostable.
Effect of pH on the biosurfactant activity was examined by adjusting different pH of the biosurfactant solution. Biosurfactant produced by JR3 and FS22 showed gradual increasing of the E24 with increasing of pH of the solution but FS6, JR7 and FS7 showed almost stable E24 with different pH.