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^1,3. Various types of biosurfactants that have been reported so far include glycolipids in which carbohydrates are linked to long chain aliphatic or hydroxy-aliphatic acids via ester bonds (e.g., rhamnolipids, trehalolipids and sophorolipids), lipopeptides in which lipids are attached to polypeptide chains (e.g., surfactin and lichenysin), and polymeric biosurfactants that are usually composed of polysaccharide- protein complexes (e.g., emulsan, liposan, alasan and lipomannan)⁴. Other types of biosurfactants produced by the microorganisms include fatty acids, phospholipids, neutral lipids, and particulate biosurfactants⁵. The most studied class of biosurfactants is glycolipids and among them most of the studies have been reported on rhamnolipids⁶. Rhamnolipids contain one or two molecules of rhamnose (which form the hydrophilic part) linked to one or two molecules of long chain fatty acid (usually hydroxy-decanoic acid). Rhamnolipids are primary glycolipids reported first from Pseudomonas aeruginosa⁷.

Biosurfactants have been gaining increasing focus as compared to their chemical counterparts due to various unique and distinctive properties that they offer⁸. These include higher specificity, lower toxicity, greater diversity, ease of preparation, higher biodegradability, better foaming, environmental compatibility and activity under extreme conditions⁹. Structural diversity of the biosurfactants (Figure S1) is another advantage that gives them an edge over the chemical counterparts¹⁰. They are generally more effective and efficient at lower concentrations as their critical micelle concentration (CMC) is usually several times lower than chemical surfactants¹¹. They have been reported to be highly thermostable (up to 100 °C) and can tolerate higher pH (up to 9) and high salt concentrations (up to 50 g/L)¹² thereby offer several advantages in industrial processes, which require exposure to extreme conditions¹³. Biodegradability and lower toxicity make them suitable for environmental applications such as bioremediation. Because of the advantages that they offer, they have been getting increased attention in various industries like food, agricultural, detergent, cosmetic and petroleum industry¹¹. Biosurfactants have also gained a lot of attention in oil remediation for removal of petroleum contaminants and toxic pollutants¹⁴.

Here we report the production, characterization, and application of biosurfactants produced by Rhodococcus sp. IITD102, Lysinibacillus sp. IITD104, and Paenibacillus sp. IITD108. The steps involved in screening, characterization, and application of a combination of biosurfactants for enhanced oil recovery are outlined in Figure 1.

Figure 1: A method for enhanced oil recovery using a combination of Biosurfactants. The stepwise work flow is shown. The work was carried out in four steps. First the microbial strains were cultured and screened for the production of biosurfactant by various assays, which included drop collapse assay, oil spreading assay, emulsion index assay, and surface tension measurement. Then, the biosurfactants were extracted from the cell-free broth and their nature was identified using thin layer chromatography and they were further identified using LCMS, NMR, and FT-IR. In the next step, the extracted biosurfactants were mixed together and the potential of the resulting mixture for enhanced oil recovery was determined using the sand pack column technique. Please click here to view a larger version of this figure.

Screening of these microbial strains to produce biosurfactants was done by drop collapse, oil spreading, emulsion index assay and determination of reduction in the surface tension of the cell-free medium due to growth of the microbes. The biosurfactants were extracted, characterized, and chemically identified by LCMS, ¹H NMR, and FT-IR. Finally, a mixture of biosurfactants produced by these microbes was prepared and was used to recover the residual oil in a simulated sand pack column.

The present study only illustrates the methods involved in screening, identification, structural characterization, and application of the biosurfactant combination on enhancing residual oil recovery. It does not provide a detailed functional characterization of the biosurfactants produced by the microbial strains^15,16. Various experiments such as critical micelle determination, thermogravimetric analysis, surface wettability, and biodegradability are performed for detailed functional characterization of any biosurfactant. But since this paper is a methods paper, the focus is on screening, identification, structural characterization, and application of the biosurfactant combination on enhancing residual oil recovery; these experiments have not been included in this study.

Protocol

1. Growth of microbial strains 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. Similarly, prepare two more flasks of 100 mL of Luria Broth and place cotton plugs on the neck of the flasks. Cover the cotton plugs with aluminum foil and autoclave the flasks for 15 min at 121 °C and 15 psi to sterilize the media.</l…

Representative Results

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 (<stron…

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

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