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We illustrate the methods involved in screening and identification of the biosurfactant producing microbes. Methods for chromatographic characterization and chemical identification of the biosurfactants, determining the industrial applicability of the biosurfactant in enhancing residual oil recovery are also presented.
Biosurfactants are surface-active compounds capable of reducing the surface tension between two phases of different polarities. Biosurfactants have been emerging as promising alternatives to chemical surfactants due to less toxicity, high biodegradability, environmental compatibility and tolerance to extreme environmental conditions. Here, we illustrate the methods used for screening of microbes capable of producing biosurfactants. The biosurfactant producing microbes were identified using drop collapse, oil spreading, and emulsion index assays. Biosurfactant production was validated by determining the reduction in surface tension of the media due to growth of the microbial members. We also describe the methods involved in characterization and identification of biosurfactants. Thin layer chromatography of the extracted biosurfactant followed by differential staining of the plates was performed to determine the nature of the biosurfactant. LCMS, 1H NMR, and FT-IR were used to chemically identify the biosurfactant. We further illustrate the methods to evaluate the application of the combination of produced biosurfactants for enhancing residual oil recovery in a simulated sand pack column.
Biosurfactants are the amphipathic surface-active molecules produced by microorganisms that have the capacity to reduce the surface and the interfacial tension between two phases1. A typical biosurfactant contains a hydrophilic part that is usually composed of a sugar moiety or a peptide chain or hydrophilic amino acid and a hydrophobic part that is made up of a saturated or unsaturated fatty acid chain2. Due to their amphipathic nature, biosurfactants assemble at the interface between the two phases and reduce the interfacial tension at the boundary, which facilitates the dispersion of one phase into the other
1. Growth of microbial strains
Three bacterial strains (Rhodococcus sp. IITD102, Lysinibacillus sp. IITD104, and Paenibacillus sp. IITD108) were screened for the production of biosurfactants by various assays, which included drop collapse assay, oil displacement assay, emulsion index assay, and surface tension reduction. Cell-free supernatants of all the three bacterial strains and a solution of chemical surfactant resulted in a drop collapse and, therefore, were scored positive for the presence of the biosurfactants (
Biosurfactants are one of the most versatile group of biologically active components that are becoming attractive alternatives to chemical surfactants. They have a wide range of applications in numerous industries such as detergents, paints, cosmetics, food, pharmaceuticals, agriculture, petroleum, and water treatment due to their better wettability, lower CMC, diversified structure, and environmental friendliness18. This has led to an increased interest in discovering more microbial strains capab.......
The authors would like to thank the Department of Biotechnology, Government of India, for financial support.
....Name | Company | Catalog Number | Comments |
1 ml pipette | Eppendorf, Germany | G54412G | |
1H NMR | Bruker Avance AV-III type spectrometer,USA | ||
20 ul pipette | Thermo scientific, USA | H69820 | |
Autoclave | JAISBO, India | Ser no 5923 | Jain Scientific |
Blue flame burner | Rocker scientific, Taiwan | dragon 200 | |
Butanol | GLR inovations, India | GLR09.022930 | |
C18 column | Agilent Technologies, USA | 770995-902 | |
Centrifuge | Eppendorf, Germany | 5810R | |
Chloroform | Merck, India | 1.94506.2521 | |
Chloroform-d | SRL, India | 57034 | |
Falcon tubes | Tarsons, India | 546041 | Radiation sterilized polypropylene |
FT-IR | Thermo Fisher Scientific, USA | Â Nicolet iS50 | |
Fume hood | Khera, India | 47408 | Customied |
glacial acetic acid | Merck, India | 1.93002 | |
Glass beads | Merck, India | 104014 | |
Glass slides | Polar industrial Corporation, USA | Blue Star | 75 mm * 25 mm |
Glass wool | Merk, India | 104086 | |
Hydrochloric acid | Merck, India | 1003170510 | |
Incubator | Thermo Scientific, USA | MaxQ600 | Shaking incubator |
Incubator | Khera, India | Sunbim | |
Iodine resublimed | Merck, India | 231-442-4 | Â resublimed Granules |
K12 –Kruss tensiometer | Kruss Scientific, Germany | K100 | |
Laminar air flow cabnet | Thermo Scientific, China | 1300 Series A2 | |
LCMS | Agilent Technologies, USA | 1260 Infinity II | |
Luria Broth | HIMEDIA, India | M575-500G | Powder |
Methanol | Merck, India | 107018 | |
Ninhydrin | Titan Biotech Limited, India | 1608 | |
p- anisaldehyde | Sigma, USA | 204-602-6 | |
Petri plate | Tarsons, India | 460090-90 MM | Radiation sterilized polypropylene |
Saponin | Merck, India | 232-462-6 | |
Sodium chloride | Merck, India | 231-598-3 | |
Test tubes | Borosil, India | 9800U06 | Glass tubes |
TLC plates | Merck, India | 1055540007 | |
Vortex | GeNei, India | 2006114318 | |
Water Bath | Julabo, India | SW21C |
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