Enhanced Oil Recovery using a Combination of Biosurfactants

Summary

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.

Abstract

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.

Introduction

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

Protocol

1. Growth of microbial strains

1. Weigh 2 g of Luria Broth powder and add to 50 mL of distilled water in a 250 mL conical flask. Mix the contents until the powder dissolves completely and make up the volume to 100 mL using distilled water.
2. Similarly, prepare two more flasks of 100 mL of Luria Broth and place cotton plugs on the neck of the flasks.
3. Cover the cotton plugs with aluminum foil and autoclave the flasks for 15 min at 121 °C and 15 psi to sterilize the media.

Discussion

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.......

Materials

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	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	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