Biosurfactant Lab Report

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Area of isolate
For the isolate that can produce biosurfactant, we collected the sample from oil spilled area, from automobile workshop because the nature of the place will determine the type of microorganism.
Sample of the soil was collected from the automobile workshop. The sample of the soil was taken to the laboratory for analysis via sterile polythene bag. At the time of sample collection, the temperature and pH of the soil sample was 30℃ and 7 respectively.
Collection of bacteria from the sample and its isolation
In 50ml of R2B broth, the soil sample of 5 gram inoculated and incubated at 25℃ for 72 hours. After incubation, serial dilution of the medium taken place from 〖10〗^(-1) to 〖10〗^(-6) in sterile
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After solidification of agar, the fresh colonies from the culture were taken and streaked on the blood agar. Incubated at 37℃ for 48-72 hours. After incubation, we were looking for the clear zone around the microbial colonies. This clear zone are the sign of biosurfactant producing microorganisms.
Biochemical analysis of biosurfactant producing microorganisms
The biosurfactant producing microorganisms then characterized by different types of test includes Motility Test, Gram Staining, Indole Test, Methyl Red Test, Citrate Test, Spore Staining, Catalase Test, Voges-Proskauer Test, Casein Hydrolysis, Starch Hydrolysis, Lipid Hydrolysis, Gelatin Liquifaction Test, Gelatin Hydrolysis, Oxidase Test.
Results after these test, we will able to find the closest match of our sample with known bacterial genus and assign the bacterial signature according to Bergey’s manual.
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Incubated at 25℃ for 7 days under the shaking condition. After 7 days, centrifuge it at 5000rpm and at temperature of 4℃ for 20 minutes. Discard the pellet that contains bacterial cells, take supernatant. Make the pH of supernatant of 2 by using "M" "H" _2 "S" "O" _4. Add choloroform and methanol of ratio 2:1 and of equal volume. Mix the mixture and left it for overnight for the process of evaporation. The white coloured sediment appeared that is the biosurfactant.
Drying of biosurfactant
Poured the sediment in the sterile petriplate of known weight. Put the plate in the Hot Air Oven at 100℃ for 30 minutes. After drying, the weight of biosurfactant was calculated with the help of following formula-
Dry wt. of biosurfactant = (wt. of plate after drying – wt. of empty plate)
Preliminary characterization of biosurfactant
It was done by TLC method that is Thin Layer Chromatography method. Silica gel poured on the glass plate. A mark of biosurfactant was placed on the glass plate which is poured by silica gel. The biosurfactant ran on silica plate by a solution which is made up of chlororform, methanol and water. Ninhydrin reagent and Throne reagent was sprayed on the silica plate for detection of lipopeptide biosurfactant as red mark and glycolipid biosurfactant as yellow mark respectively.
Molecular weight of biosurfactant
The sediment of biosurfactant mixed with chloroform and given for GC-MC

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