Biosurfactants are the empathic surface active molecules produced by microorganisms that have the capacity to reduce the surface tension of the liquid and the interficial tension between two different phases. The empathic nature of these molecules enables them to align themselves at the interface between two different phases, and enhance the solubilization of one phase into another. Biosurfactants have been gaining a lot of attention as compared to their chemical counterparts because of various advantages that they offer.
These include higher specificity, lower toxicity, greater structural diversity, ease of preparation, higher biodegradability, their higher environmental compatibility, and their activity under extreme conditions of environment. In this video, we demonstrate the methods involved in screening, characterization, and applicational biosurfactants, produced by different strains of rodococcus, lysinibacillus and paenibacillus. We further illustrate the methods to evaluate the application of combination of biosurfactants, produced by these three microbial strains in the recovery of residual oil by a sand pack column technique.
The work has been carried out under the guidance of Dr.Preeti Srivastava, and Dr.Manoj Kumar. For the growth of microbes, inoculate the flasks containing hundred milliliters of autoclaved Luria broth, by adding one milliliter of overnight grown seed culture to the flask inside the laminar airflow cabinet. Incubate the flasks in a rotary incubator at 30 degrees Celsius and 180 RPM for seven days.
After the completion of the incubation period, harvest the flasks, and transfer the culture broth to the centrifuge tubes. Centrifuge the culture at 4, 500 G for 20 minutes in a refrigerated centrifuge at four degrees Celsius. Gently pour the cell-free supernatant into a fresh beaker, and use it in screening assays for biosurfactant production.
For screening the biosurfactant production by a drop collapse assay, take a clean glass slide and coat the surface of the slide with 200 microliters of oil. Now add 20 microliters of cell-free supernatant to the center of the oil, and leave it undisturbed for two to three minutes. If the drop collapses, score the supernatant positive for the presence of biosurfactant.
In oil spreading assay, take 20 milliliters of double distilled water in a Petri plate and add 200 microliters of crude oil on the surface of water. Now add 20 microliters of cell-free supernatant to the center of the oil, and leave it undisturbed for one minute. If a clearing zone is formed as a result of oil displacement, score the supernatant positive for the presence of biosurfactant.
In emulsion index assay, add four milliliters of petrol and cell-free supernatant each into a clean glass tube. Then what the mixture vigorously for three minutes and leave it undisturbed for next 24 hours. For measuring the surface tension of the cell-free supernatant, clean the glass vessel with the liquid whose surface tension is to be determined.
Add the liquid into the vessel and mount the vessel on the vessel holder. Unlock the probe holder and mount the probe on it. Using the manual controller adjust the height of the platform, such that the probe is two to three millimeters away from the surface of the liquid.
Then go to softwares and follow the steps mentioned in the paper to measure the surface tension. After completion of the measurement, lower the height of the platform and unlock and unmount the probe and the vessel from the instrument. For extraction of biosurfactant, adjust the pH of cell-free supernatant to two, using two normal hydrochloric acid.
Store the mixture at four degrees Celsius overnight. Next day, add equal volume of chloroform methanol mixture, and mix vigorously for 20 minutes. Leave the mixture undisturbed for free separation to occur.
Remove the upper face, containing water and methanol, and leave the lower face containing biosurfactant to evaporate in a fume hood. After evaporation of the organic phase, redissolve the honey colored biosurfactant in three milliliters of chloroform, and use this mixture for characterization and identification of the biosurfactant. For chromatographic characterization of the extracted biosurfactant, spot 20 microliters of biosurfactant on TLC plates.
After drying the plates, place the TLC plate inside the chamber saturated with chloroform methanol mixture and run the TLC. For lipid detection, add some granules of iodine into the fresh chamber and saturate the chamber for five to 10 minutes. Place the TLC inside the chamber and observe for the development of yellow spots.
For protein and carbohydrate detection, spray the TLC plate with non hydrogen or anisaldehyde, and heat at 110 degrees Celsius and observe for the development of color. For NMR analysis, insert the tube in the spanner. Adjust the height of the tube using adjusted tube.
Place the NMR tube, along with spanner in an MR machine and follow the steps mentioned in the paper to get an NMR spectrum. For enhanced oil recovery experiment, take a glass column and seal the bottom outlet with glass wool and glass beads. Back the column with Sandy soil in such a way that some liquid can be added at the top of the soil.
Mount the column on the holder and add some glass beads on top of the soil. Flood the column with 50 milliliters of brine solution and collect the flow through to determine the poor volume. Remove the brine from the column by forcing crude oil to pass through it.
Collect the volume of brine and oil coming out of the column to determine initial oil saturation volume. After 24 hours, flood the column with 10 poor volumes of brine, and collect the oil coming out of the column to estimate secondary oil recovery. The oil left in the column after secondary oil recovery corresponds to the residual oil.
Now prepare a mixture of biosurfactants by adding the extracted biosurfactant to a glass beaker. And add the biosurfactant and mixture to the column, and leave it undisturbed for next 24 hours. After 24 hours, measure the amount of oil and water that has alluded out of the column to determine additional or enhanced oil recovery.
Cell-free supernatants of the three microbial strains were scored positive for the presence of biosurfactant as they resulted in drop collapse, oil spreading and emulsion formation. Confirmation of the biosurfactant production was provided by measurement of the surface tension of the cell-free broth. Due to growth of rhodococcus and lysinibacillus, the surface tension of the medium reduced from 59 to 45 mN per meter.
Due to growth of paenibacillus, the surface tension of the medium reduced from 59 to 28.4 mN per meter. Emulsion stability assays showed that the biosurfactants produced by the three microbial strains exhibited a great stability under diverse and environmental conditions. The emulsion indices did not show any significant change when the incubation temperature, pH, and the salt concentration of the cell-free broth were changed.
DLC characterization of the extracted biosurfactant showed that biosurfactants produced by all three strains were glycolipeds. Chemical identification of biosurfactants showed that rodococcus and lysinbacillus produced the same biosurfactant, which was identified as rhamnolipid, with a mass of around four 80 daltons. Paenibaccilus on the other hand, produces a rhamnolipid containing three lipid chains with a mass of around 802 daltons.
The combination of biosurfactants was capable of recovering 56.5%of the residual oil from the sand column. Biosurfactant production from the strains of rodococcus, lsyinibaccilus, and paenibacillus was assayed, and the strains were scored positive for the production of biosurfactant. The biosurfactants produced were characterized and identifies as rhamnolipids.
Since the biosurfactant combination was successful in recovering 56%of the residual oil in a sand pack technique, the biosurfactant combination can be used in field applications to recover the residual oil trapped inside the oil reservoirs.
